Bibliographies: 'Fos protein 3' – Grafiati (2024)

Abstract:

The requirements for activation of the transformation potential of the human c-fos proto-oncogene were investigated. Recombinant plasmids containing the Moloney murine leukemia virus long terminal repeat directing transcription of the c-fos coding region and either the authentic c-fos 3' untranslated region (UTR) or the 3' UTR from human c-myc were inefficient at inducing transformation. In contrast, a recombinant that substituted most of the c-fos 3' UTR with the 3' portion of the simian virus 40 T-antigen gene transformed cells well. This difference in transformation efficiency appeared to be due to significantly higher levels of fos mRNA and protein expressed from the transforming recombinant. This, in turn, was due to the much greater stability of its mRNA compared with those from the poorly transforming recombinants containing the c-fos or c-myc 3' UTR. Thus, the 3' UTR of the human c-fos mRNA is responsible for its rapid degradation and limits the steady-state levels of transcript and protein. Cells transformed by the activated human c-fos plasmids contained increased amounts of partially modified c-fos protein (c-Fos). This form of c-Fos turned over much more rapidly than the highly modified form of c-Fos induced by serum stimulation.

Abstract:

The requirements for activation of the transformation potential of the human c-fos proto-oncogene were investigated. Recombinant plasmids containing the Moloney murine leukemia virus long terminal repeat directing transcription of the c-fos coding region and either the authentic c-fos 3' untranslated region (UTR) or the 3' UTR from human c-myc were inefficient at inducing transformation. In contrast, a recombinant that substituted most of the c-fos 3' UTR with the 3' portion of the simian virus 40 T-antigen gene transformed cells well. This difference in transformation efficiency appeared to be due to significantly higher levels of fos mRNA and protein expressed from the transforming recombinant. This, in turn, was due to the much greater stability of its mRNA compared with those from the poorly transforming recombinants containing the c-fos or c-myc 3' UTR. Thus, the 3' UTR of the human c-fos mRNA is responsible for its rapid degradation and limits the steady-state levels of transcript and protein. Cells transformed by the activated human c-fos plasmids contained increased amounts of partially modified c-fos protein (c-Fos). This form of c-Fos turned over much more rapidly than the highly modified form of c-Fos induced by serum stimulation.

Abstract:

Mitogen-activated protein kinase (MAPK) pathways constitute key regulatory elements linking extracellular stimuli to nuclear gene expression. Immediate-early responsive genes (IEGs) of the activator protein 1 (AP-1) family, such as fos, achieve peak expression levels shortly after cells are stimulated with growth factors and sharply decrease thereafter. Several AU-rich binding proteins (AUBPs), including HuR (Hu-antigen R, Elav-like protein 1, ELAVL1) and KSRP (far upstream element-binding protein 2, KHSRP) bind to a fos AU-rich element (ARE) present in the 3′-UTR (untranslated region) of fos mRNA regulating its stability by a still poorly defined mechanism. We show in the present study that, whereas HuR binds and stabilizes transcribed reporter mRNAs bearing the fos 3′-UTR, KSRP counteracts this effect. Furthermore, we found that fos mRNA stability and HuR phosphorylation status are dependent on the activity of p38 MAPK in both epithelial cells and fibroblasts upon proliferative stimulation. Analysing PPI (protein–protein interaction) networks, we performed a thorough query of interacting proteins for p38 MAPKs, HuR and other AUBPs upon growth factor stimulation. This revealed novel HuR interactors including inhibitors of protein phosphatase 2 (PP2A) activity. Over-expression of two of these interactors, pp32 and APRIL (acidic leucine-rich nuclear phosphoprotein 32 family member B, ANP32B) and pharmacological inhibition of PP2A stabilized a fos reporter mRNA. Our results indicate that p38 MAPK regulates fos mRNA decay by affecting the state of phosphorylation of HuR while controlling yet to be fully elucidated PP regulatory networks.

Abstract:

Rapid decay of the c-fos transcript plays a critical role in controlling transforming potential of the c-fos proto-oncogene. One of the mRNA instability determinants is a 75-nucleotide AU-rich element (ARE) present in the 3' untranslated region of the c-fos transcript. It appears to control two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require the AUUUA motifs, and subsequent degradation of deadenylated mRNA, which appears to be dependent on the AUUUA motifs. In this study, we report the identification of four U-rich sequence binding proteins (URBPs) that specifically interact with a 20-nucleotide U-rich sequence within the c-fos ARE. Gel mobility shift assay and competition experiments showed that these protein factors form three specific band-shifted complexes with the c-fos ARE. Binding activity of one of the protein factors, a 37-kDa protein, is significantly affected by serum induction and by pretreatment of cells with drugs known to stabilize many of the immediate-early gene mRNAs. Combining UV cross-linking with a new approach, designated sequential RNase digestion, we were able to better determine the molecular masses of these cellular proteins. The binding sites for the four proteins were all mapped to a 20-nucleotide U-rich sequence located at the 3' half of the c-fos ARE, which contains no AUUUA pentanucleotides but stretches of uridylate residues. Single U-to-A point mutations in each of the three AUUUA motifs within the c-fos ARE have little effect on formation of the mobility-shifted complexes. Our data indicate c-fos ARE-protein interaction involves recognition of U stretches rather than recognition of the AUUUA motifs. We propose that UTBP binding may be involved in the first step, removal of the Poly(A) tail, in the c-fos ARE-mediated decay pathway.

Abstract:

Rapid decay of the c-fos transcript plays a critical role in controlling transforming potential of the c-fos proto-oncogene. One of the mRNA instability determinants is a 75-nucleotide AU-rich element (ARE) present in the 3' untranslated region of the c-fos transcript. It appears to control two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require the AUUUA motifs, and subsequent degradation of deadenylated mRNA, which appears to be dependent on the AUUUA motifs. In this study, we report the identification of four U-rich sequence binding proteins (URBPs) that specifically interact with a 20-nucleotide U-rich sequence within the c-fos ARE. Gel mobility shift assay and competition experiments showed that these protein factors form three specific band-shifted complexes with the c-fos ARE. Binding activity of one of the protein factors, a 37-kDa protein, is significantly affected by serum induction and by pretreatment of cells with drugs known to stabilize many of the immediate-early gene mRNAs. Combining UV cross-linking with a new approach, designated sequential RNase digestion, we were able to better determine the molecular masses of these cellular proteins. The binding sites for the four proteins were all mapped to a 20-nucleotide U-rich sequence located at the 3' half of the c-fos ARE, which contains no AUUUA pentanucleotides but stretches of uridylate residues. Single U-to-A point mutations in each of the three AUUUA motifs within the c-fos ARE have little effect on formation of the mobility-shifted complexes. Our data indicate c-fos ARE-protein interaction involves recognition of U stretches rather than recognition of the AUUUA motifs. We propose that UTBP binding may be involved in the first step, removal of the Poly(A) tail, in the c-fos ARE-mediated decay pathway.

Abstract:

Abstract The immediate early genes are regulated by a variety of extracellular signals, including pleiotropic cytokines. The effects of the testicular cytokines, interleukin-6 (IL-6) and interferon-γ (IFN-γ), on signal transducers and activators of transcription 3 and 1 (STAT-3 and STAT-1) and on c-fos gene expression in primary Sertoli cells are suggestive of their roles in differential function. Using the tyrosine phosphorylation inhibitor, genistein, and electrophoretic mobility shift assay, we show that IL-6 and IFN-γ induce nuclear factor STAT-3 and STAT-1 DNA-binding activity to the sis-inducible element of c-fos in a genistein-dependent pathway. Quantitative solution hybridization, Northern blot, and nuclear run-on analysis show that differential induction of c-fos, junB, and c-myc messenger RNA (mRNA) by these cytokines occur at transcriptional levels. IL-6 stimulates c-fos mRNA levels by 6-fold while increasing junB levels by 2-fold. IFN-γ increases c-fos message 2-fold, but has no effect on junB mRNA levels. Furthermore, genistein treatment blocks the induction of c-fos and junB gene expression, demonstrating that tyrosine phosphorylation of STAT proteins is involved in the cytokine regulation of the Sertoli immediate early genes. H7, a serine/threonine phosphorylation inhibitor, also blocks c-fos gene induction by IL-6 and IFN-γ, but does not affect the DNA-binding activities of STAT-3 and STAT-1. Finally, IL-6 treatment of Sertoli cells (3–6 h) increases the amounts of activating protein-1 binding to activating protein-1 element and c-myc transcription.

Abstract:

ABSTRACT The inducible transcriptional complex AP-1, composed of c-Fos and c-Jun proteins, is crucial for cell adaptation to many environmental changes. While its mechanisms of activation have been extensively studied, how its activity is restrained is poorly understood. We report here that lysine 265 of c-Fos is conjugated by the peptidic posttranslational modifiers SUMO-1, SUMO-2, and SUMO-3 and that c-Jun can be sumoylated on lysine 257 as well as on the previously described lysine 229. Sumoylation of c-Fos preferentially occurs in the context of c-Jun/c-Fos heterodimers. Using nonsumoylatable mutants of c-Fos and c-Jun as well as a chimeric protein mimicking sumoylated c-Fos, we show that sumoylation entails lower AP-1 transactivation activity. Interestingly, single sumoylation at any of the three acceptor sites of the c-Fos/c-Jun dimer is sufficient to substantially reduce transcription activation. The lower activity of sumoylated c-Fos is not due to inhibition of protein entry into the nucleus, accelerated turnover, and intrinsic inability to dimerize or to bind to DNA. Instead, cell fractionation experiments suggest that decreased transcriptional activity of sumoylated c-Fos is associated with specific intranuclear distribution. Interestingly, the phosphorylation of threonine 232 observed upon expression of oncogenically activated Ha-Ras is known to superactivate c-Fos transcriptional activity. We show here that it also inhibits c-Fos sumoylation, revealing a functional antagonism between two posttranslational modifications, each occurring within a different moiety of a bipartite transactivation domain of c-Fos. Finally we report that the sumoylation of c-Fos is a dynamic process that can be reversed via multiple mechanisms. This supports the idea that this modification does not constitute a final inactivation step that necessarily precedes protein degradation.

Abstract:

ObjectivesThe chief objective of this study was to identify the miRNAs targeting Fos, a well-recognized proto-oncogene that is commonly overexpressed in cervical cancer, and its biological significance on the cellular behaviors of HeLa, a cervical cancer cell.Materials and MethodsWe initially analyzed the 3′untranslated region (3′UTR) of Fos and screened the potential miRNAs targeting Fos using 3 bioinformatical Web sites. Luciferase reporter assay, real-time polymerase chain reaction, and Western blotting were used to validate the binding of chosen miRNA (miR-101) on the 3′UTR of Fos and the downstream regulation on its mRNA and protein levels. Furthermore, flow cytometry along with the Fos rescue strategy was applied to analyze the modulation of cell cycle of HeLa cells by miR-101.ResultsAmong these predicted candidate miRNAs, miR-101 was the miRNAs preferred by all the 3 used Web sites. The results of luciferase reporter assay, real-time polymerase chain reaction, and Western blotting demonstrated that miR-101 directly targeted on the 3′UTR of Fos and down-regulated the expression of Fos at mRNA and protein levels. Furthermore, cell cycle analysis showed that miR-101 arrests G1-to-S phase transition of HeLa cells, at least partially by targeting Fos.ConclusionsWe concluded that by targeting the proto-oncogene Fos, miR-101 is involved in G1-to-S phase transition in cervical cancer cells in vitro and might provide a new approach for the pharmacological interference node in cervical cancer treatment.

Abstract:

1. Studies of cells transfected with specific expression vectors allow functions of specific gene products to be investigated. We first established optimum conditions for transfection of neonatal cardiac myocytes in primary culture. Increased proto-oncogene expression has been implicated in the development of cardiac hypertrophy; we therefore wished to examine the effects of overexpression of the c-myc and c-fos proto-oncogenes induced by cell transfection on cell growth. 2. Neonatal rat ventricular myocytes were transfected by electroporation, voltage and capacitance settings were optimized and these conditions were then used to transfect expression vectors encoding MYC and FOS proteins into this cell type. Effects on cell number, DNA synthesis and protein content were determined. 3. Increased expression of c-myc and c-fos in cells transfected with proto-oncogene expression vectors was confirmed by Northern and Western blotting. Increases in cardiac myocyte cell number, DNA synthesis and total cellular protein were observed in cells transfected with either c-myc or c-fos expression vectors compared with cells transfected with non-coding vectors. 4. Overexpression of MYC and FOS proteins results in hyperplastic rather than hypertrophic growth of the neonatal rat heart, providing evidence for a role of these proteins in the control of myocardial growth.

Abstract:

O labirinto em cruz elevado é um teste comportamental sensível à iluminação ambiental. Na ausência de luz, ratos testados neste modelo exibem aumento da exploração dos braços abertos, quando comparados com animais testados em ambientes iluminados. No presente trabalho investigou-se a expressão da proteína Fos em cérebro de ratos expostos ao labirinto em cruz elevado na presença e ausência de iluminação. Duas horas depois do teste no labirinto em cruz elevado, ratos foram perfundidos com paraformaldeído juntamente com outros que somente permaneceram em uma gaiola enquanto os primeiros eram testados no labirinto. Para cada par (labirinto e gaiola) manteve-se a mesma condição de iluminação: claro ou escuro. Ainda, ratos de um terceiro grupo que permanecia no biotério eram perfundidos juntamente com cada dupla. Os cérebros foram retirados e preparados para inicio do procedimento imunoistoquímico da marcação da proteína Fos e posterior contagem de células marcadas. De um modo geral, animais expostos ao labirinto em cruz elevado sem iluminação, exibiram aumento na porcentagem de entradas e tempo de permanência nos braços abertos, assim como aumento da expressão de Fos em diferentes regiões do cérebro. A comparação entre os grupos sugere que a lâmina intergeniculada se relaciona provavelmente com a detecção de iluminação. A exploração de ambientes novos ou familiares envolve a participação do córtex cingulado e do locus coeruleus. Animais testados no labirinto em cruz elevado no escuro exibiram aumentos significativos na expressão de Fos nos núcleos da amígdala lateral, basolateral e medial, núcleos do hipotálamo lateral anterior e dorsomedial, quando comparados com os animais testados no mesmo modelo na presença de luz e com os que somente permaneceram na gaiola no escuro. Além disso, na ausência de luz, correlações significativas entre medidas comportamentais no labirinto em cruz elevado e número de neurônios marcados por Fos mostraram uma relação entre o aumento da exploração dos braços abertos e ativação de neurônios pertencentes à maioria dos núcleos descritos. Os resultados sugerem que a detecção de luminosidade em ambientes novos inibe a ativação neuronal e comportamental inicial. Esse processo induziria uma diminuição do número de neurônios ativos e comportamentos relacionados com ansiedade. A ausência de luz, pelo contrario, manteria a ativação inicial gerada pela novidade e envolveria comportamentos de exploração subjacentes ao aumento da expressão de Fos sobretudo no complexo amigdalóide e córtex piriforme.
The elevated plus-maze is a behavioral test sensitive to environmental illumination. In the absence of light, rats tested in with this model exhibit increases in the exploration of the open arms, when compared to animals tested in illuminated environments. The present work investigated Fos protein expression in the brain of rats exposed to the elevated plus-maze in the presence and absence of illumination. Two hours after the test in the plus-maze, the rats were perfused with paraformaldehyde together with others that just remained in a cage while the first were tested in the maze. For each pair (maze and cage) the same illumination condition was maintained: light or dark. Also, rats from a third group that only remained in the vivarium were perfused together with each pair. The brains were removed and prepared for the procedure of immunohistochemical staining for Fos followed by cell counting. In general, rats exposed to the elevated plus-maze in the dark exhibited increases in the percentage of entries and time spent in the open arms, as well as increases in Fos expression in different brain areas. Comparisons among groups suggests that intergeniculate leaflet is probably related to illumination detection. The exploration of novel of familiar environments involves the cingulate cortex and the locus coeruleus. Rats tested inn the elevated plus-maze in the dark exhibited significant increases in Fos expression in the lateral, basolateral, medial and central amygdala nuclei, lateral anterior and dorsomedial hypothalamic nuclei when compared to rats tested in this model under illumination and rats that remained in the cage in the dark. Besides, in the dark, significant correlations between behavioral measurements in the maze and amount of Fos-stained cells indicates a relationship between open arm exploration and neuron activation in most of the studied nuclei. The results suggest that light detection in novel environments inhibits the initial neuronal and behavioral activation. This process induces a decrease in the number of active neurons and behaviors related to anxiety. The absence of light, on the other hand, keeps the initial activation generated by novelty and involves the exploratory behaviors subserved by the increases in the expression of Fos protein, mainly in the amygdaloid complex and piriform cortex.

Abstract:

La myélofibrose primitive (MFP) est un néoplasme myéloprolifératif (NMP) chronique BCR-ABL1-négatif associant une dérégulation de l'hématopoïèse (myéloprolifération, dysmégacaryopoïèse et migration des cellules souches et progéniteurs hématopoïétiques (CSH/PH)) à une altération du stroma médullaire et splénique (fibrose ostéomyélosclérose, néoangiogenèse). Le mégacaryocyte (MK) est un acteur majeur de sa pathogenèse, via la production de cytokines et facteurs fibrosants, dans un contexte inflammatoire. Plusieurs arguments suggèrent que les mutations JAK2V617F et MPL515L/K qui caractérisent les NMP ne sont pas les événements initiaux de la MFP car elles ne sont retrouvées que chez la moitié des patients. L'objectif de mon travail a été de rechercher si d'autres anomalies, géniques ou non, pouvaient expliquer la pathogenèse de la MFP. Pour cela, parallèlement à une démarche génomique (transcriptome et CGH array), nous avons développé une approche de biologie cellulaire ciblée sur le rôle du stroma hématopoïétique. Bien que n'ayant pas identifié d'autres anomalies génomiques que celles décrites dans la littérature et en particulier, la délétion 13q, les approches génomiques que nous avons développées nous ont permis de préciser les bornes de cette délétion dans les PH CD34+ et les polynucléaires des patients. Cette délétion (région chromosomique minimale 13q14-13q21) est située à 2 mégabases (télomérique) du cluster FLT où est localisé le gène FLT3. Plusieurs arguments nous ont ensuite conduits à rechercher si le couple Flt3-ligand/Flt3 était impliqué dans la dérégulation de l'hématopoïèse et plus particulièrement dans la dysmégacaryopoïèse observée chez les patients. Parmi ceux-ci, citons : 1) l'existence d'une modulation d'expression de gènes inclus dans la zone de délétion 13q et dans le cluster FLT, dont le gène FLT3 et 2) le fait que Flt3, un récepteur clé de la régulation de l'hématopoïèse primitive, soit souvent impliqué dans la pathogenèse d'hémopathies malignes et que son ligand, Flt3-ligand, soit majoritairement produit par le stroma hématopoïétique. Notre étude montre une dérégulation de Flt3 et des MAPKs p38 dans les PH CD34+ et les MK des patients atteints de MFP et ceci, quelque soit leur statut mutationnel Jak2. Elle démontre également que la persistance de la stimulation de l'axe Flt3/p38 en réponse à une production accrue de Flt3 ligand, participe à la dysmégacaryopoïèse qui caractérise la maladie. En effet, nous avons mis en évidence : 1) une augmentation du taux sérique de Flt3 ligand et de son expression par les cellules du stroma médullaire et splénique ainsi que par les PH des patients atteints de MFP, 2) une surexpression spécifique de son récepteur Flt3 et de sa phosphorylation dans les CSH/PH CD34+ et les progéniteurs mégacaryocytaires (MK), qui persistent au cours de la différenciation MK, quelque soit le statut mutationnel de Jak2 des patients, 3) une activation de Flt3 dans les progéniteurs MK en réponse au Flt3 ligand conduisant à la phosphorylation en cascade de la voie de signalisation des MAPKs p38 et à l'expression de ses gènes cibles tels que AP-1, p53, NFATc4, ATF2, IL-8, 4) une restauration de la mégacaryopoïèse et une inhibition de la migration (Flt3-ligand)-dépendante des progéniteurs MK des patients après inhibition de Flt3 ou de p38.Nos résultats confirment l'importance d'une altération des MAPKs dans une dérégulation de l'hématopoïèse et soulignent le rôle d'une activation persistante de la voie p38, via le couple Flt3-ligand/Flt3, dans la dysmégacaryopoïèse qui caractérise la myélofibrose primitive. Ils suggèrent également que cette dérégulation participe au processus inflammatoire à l'origine de la réaction stromale et " lit " d'une transformation leucémique potentielle. Ce dialogue altéré entre les cellules hématopoïétiques pathologiques (Bad seeds), en particulier mégacaryocytaires et les cellules stromales (Bad soil), conforte notre concept " Bad seeds in Bad soil ". Ce travail pourrait contribuer à l'amélioration de ce dialogue par des approches thérapeutiques ciblées sur l'axe Flt3-ligand/Flt3 médié par l'activation de p38 qui, en réduisant le processus inflammatoire, rétablirait un lien entre le " Seed " et le " Soil ".

Abstract:

Les Cinases de Proteïna (PKs) estan implicades en processos fonamentals de la regulació del cicle cel•lular. L’acumulació d’anomalies als mecanismes de control i el comportament disfuncional que se’n deriva han estat detectats a cèl•lules de diferents teixits afectades per càncer, desordres immunològics, endocrins, nerviosos, neurodegenaratius, cardiovasculars, malalties infeccioses, diabetis, Alzheimer, asma, restenosi, arteriosclerosi, leucèmia, artritis, etc. Però d’entre totes les PKs, les Cinases de Tirosina (TKs) han estat destacades com a l’element central de tots aquests processos i, per tant, han estat objecte d’una gegantina tasca investigadora que n’ha augmentat el seu innegable interès com a diana terapèutica. És per aquest motiu, que el desenvolupament d’inhibidors selectius de TKs ha esdevingut una àrea d’investigació molt activa.El Laboratori de Síntesi de l’IQS disposa d’una dilatada experiència de síntesi de compostos heterocíclics, especialment pirido[2,3-d]pirimidines, de gran semblança amb coneguts inhibidors de TKs. Malauradament, d’entre tots els building blocks emprats per les estratègies sintétiques desenvolupades, les guanidines (especialment les arilguanidines) sempre han limitat l’espai químic assequible per culpa de la poca diversitat d’aquests reactius comercialment disponibles. Per tant, l’objectiu fonamental d’aquest treball és desenvolupar una metodologia com més genèrica millor que permeti obtenir guanidines i, especialment arilguanidines, i que sigui compatible amb les eines sintètiques disponibles emprades per a l’obtenció massiva de pirido[2,3-d]pirimidines.Amb aquest objectiu s’optimitza un procediment de guanidinació d’amines amb àcid aminoiminometanosulfònic (AIMSOA, acrònim de l’anglès aminoiminomethanesulfonic acid) en metanol procurant acoblar-lo amb la reacció multicomponent de Victory. Desgraciadament els rendiments amb arilguanidines són baixos com a resultat de llur baixa nucleofília, llur degradació causada pel metanol i per la competència amb el dissolvent de reacció. Com a alternativa, s’assagen protocols de condensació de piridones en 1,4-dioxà per tal d’afavorir la nucleofília d’aquestes arilguanidines. Sorprenentment no s’obtenen directament les piridopirimidines sinó uns intermedis que després d’un procés de transposició de Dimroth donen lloc als heterobicicles esperats amb rendiments força superiors als descrits mitjançant altres metodologies.En darrer lloc, es planteja i estudia una nova proposta estratègica alternativa al tradicional plantejament del Laboratori de Síntesi de l’IQS per a la síntesi orientada a diversitat. Aquesta proposta implica la construcció de l’esquelet pirido[2,3-d]pirimidínic, seguida de la seva activació mitjançant bromació i diazotizació, i finalment introducció de la diversitat química desitjada. Arran d’aquest estudi s’ha obtingut i aïllat un intermedi de Wheland bicíclic mai descrit fins ara i que posteriorment és transformat per tractament amb DMSO en un terme piropirimidínic dibromat i deshidrogenat a l’anell piridònic. A més a més, s’han desenvolupat eines sintètiques per obtenir sistemes 4-oxopirido[2,3-d]pirimidínics partint de llurs anàlegs 4-amino, metodologies que són una alternativa molt atractiva de les estratègies ja desenvolupades.
Las Quinasas de Proteína (PKs) se hallan implicadas en procesos fundamentales de la regulación del ciclo celular. La acumulación de anomalías en los mecanismos de control y el consiguiente comportamiento disfuncional han sido detectados en células de diferentes tejidos afectadas por cáncer, desórdenes inmunológicos, endocrinos, nerviosos, neurodegenarativos, cardiovasculares, enfermedades infecciosas, diabetes, Alzheimer, asma, restenosis, arteriosclerosis, leucemia, artritis, etc. Pero de entre todas las PKs, las Quinasas de Tirosina (TKs) han demostrado ser un elemento central en todos estos procesos y, por tanto, han atraído sobre sí un enorme esfuerzo investigador que ha remarcado, su innegable interés como diana terapéutica. Así pues, el desarrollo de inhibidores selectivos de TKs se ha convertido en un área muy activa de investigación.El Laboratorio de Síntesis del IQS posee amplia experiencia en la síntesis de compuestos heterocíclicos, en especial pirido[2,3-d]pirimidinas, de gran similitud con inhibidores conocidos de TKs. Ahora bien, de todos los building blocks empleados en las estrategias sintéticas desarrolladas, las guanidinas (especialmente las arilguanidinas) siempre han limitado el espacio químico asequible por la poca diversidad de estos reactivos comercialmente asequibles. Por consiguiente, el objetivo fundamental del presente estudio es desarrollar una metodología para la obtención de guanidinas y, en especial de arilguanidinas, que sea compatible con las herramientas sintéticas disponibles para la obtención masiva de pirido[2,3-d]pirimidinas.A tal efecto se optimiza una guanidinación de aminas con ácido aminoiminometanosulfónico en metanol con vistas a poder acoplarlo con la reacción multicomponente de Victory. Desgraciadamente los rendimientos con arilguanidinas son bajos como consecuencia de su baja nucleofilia, su degradación por efecto del metanol y el efecto competente del disolvente de reacción. Para circunvalar este contratiempo se ensayan reacciones de condensación de piridonas en 1,4-dioxano para favorecer la nucleofilia de estas guanidinas. Sorpredentemente no se obtienen directamente las piridopirimidinas sino unos intermedios que tras un proceso de transposición de Dimroth rinden los heterobiciclos deseados con rendimientos bastante superiores a los referidos para otras metodologías.Por último, se propone y estudia una estrategia alternativa de síntesis orientado a diversidad. En este sentido, se construye el esqueleto pirido[2,3-d]pirimidínico para después activarlo mediante bromación y diazotización, y finalmente introducir la diversidad deseada. Fruto de este estudio se ha obtenido y aislado un intermedio de Wheland bicíclico que posteriormente rinde un término piropirimidínico dibromado y deshidrogenado en el anillo piridónico. Adicionalmente, se han desarrollado herramientas sintéticas para la obtención de sistemas 4-oxopirido[2,3-d]pirimidínicos a partir de sus análogos 4-amino.
Protein Kinases (PKs) are involved in basic cellular cycle regulatory mechanisms. Deregulation of those has been found on cells of different tissues with cancer, immunological disorders, endocrine disorders, nervous disorders, neurodegenerative disorders, cardiovascular disorders, infectious diseases, diabetes, Alzheimer syndrome, asthma, restenosis, atherosclerosis, leukemia, arthritis, and more. Among all the PKs huge family, Tyrosine Kinases (TKs) have been described as key point of those regulatory mechanisms and so stated as promising drug targets for treating such diseases. As a result of this biological knowledge, there have been a lot of developments in this field, resulting in some interesting and commercial TKs selective inhibitors.The Laboratori de Síntesi de l’IQS has developed some highly efficient heterocyclic synthetic procedures, especially for the synthesis of pyrido[2,3-d]pyrimidines that are structurally closely related to some well stated TKs inhibitors. Unfortunately, some of the building blocks used in those methodologies have a very narrow commercial variety and are only available from unusual vendors. This is the case for arylguanidines. As a result, the accessible chemical space is shortened. So then, the present work deals with the establishment of general procedures for the synthesis of arylguanidines and how to couple them with our previous described methodologies in order to obtain pyrido[2,3-d]pyrimidine libraries.Aminoiminomethanesulfonic acid (AIMSOA) is selected as guanidination agent and a protocol is optimized by Experimental Design. The coupling of this guanidination with the one-pot multicomponent Victory reaction is also studied. Unfortunately, coupling reaction yields with arylguanidines are very low as a result of lack of nucleophilicity, methanol mediated degradation and nucleophilic competition with this reaction solvent. Pyridone condensation with arylguanidines in 1,4-dioxane is stated as methodological alternative in order to improve nucleophilicity of the arylguanidines. Surprisingly, this procedure does not yield the expected pyridopyrimidines but a family of new, not previously described, heterobicyclic compunds that can be converted to the desired pyridopyrimidines through Dimroth rearrangement. The overall yields for the final pyridopyrimidines are higher with this new procedure than with the previous methodologies.Finally, a new global strategy is developed for the diversity oriented synthesis of 2-arylamino substituted pirido[2,3-d]pyrimidines. Firstly, the heterobicyclic skeleton is build and, secondly, this skeleton is activated by bromination and diazotization. Finally, diversity is introduced by substitution reactions. During this development a pyridopyrimidine Wheland intermediate, never described before ,has been isolated and its structure spectroscopically confirmed. The subsequent treatment of this compound with DMSO yields a new dibrominated pyridopyrimidine dehydrogenated on the pyridone ring. In addition, some synthetic procedures for the conversion of 4-aminopirido[2,3-d]pyrimidines into their 4-oxo analogues have been established. Such methodologies are auseful alternative to our old strategies for the synthesis of this kind of compounds.

Abstract:

O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal.
The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.

Abstract:

Extracellular vesicles (EVs) are released by almost all types of cells, including tumor, immune and stem cells, and are also present in body fluids like saliva, urine, breast milk and malignant ascites. EVs have a unique cargo of proteins, lipids and nucleic acids, and conserve characteristics from donor cells.

Abstract:

Thesis (M.S.)--South Dakota State University, 2005.
Includes bibliographical references (leaves 42-49). Also available online (PDF file) by a subscription to the set or by purchasing the individual file.

Abstract:

Abstract The third edition of this book contains a total of 20 chapters (including 3 new chapters), including the implementation of an effective animal welfare programme; the importance of measurement to improve the welfare of livestock, poultry and fish; the social and ethical importance of agricultural animal welfare; the implementation of effective animal-based measurements for assessing animal welfare on farms and slaughter plants; how to improve livestock handling and reduce stress; painful husbandry procedures in livestock and poultry; the importance of good stockmanship and its benefits to animals; in-farm considerations of animal behaviour and emotions; improving livestock, poultry and fish welfare in slaughter plants with auditing programmes and animal-based measures; recommended on-farm euthanasia practices; welfare during transport of livestock and poultry; animal well-being on organic farms; a practical approach on sustainability for supply chain managers of meat, dairy and other animal proteins; the effect of economic factors on the welfare of livestock and poultry; practical approaches for changing and improving animal care and welfare; successful technology transfer of behavioural and animal welfare research to the farm and slaughter plant; technological innovations for individualized animal care and welfare; technology designed to enhance poultry welfare; precision livestock farming and technology in swine welfare and practical methods for improving the welfare of horses, donkeys and mules. There is also a list of videos that will allow students to see different types of farms and technology for raising broiler chickens, cattle, laying hens and pigs. This book provides practical information which will enable veterinarians, managers, animal scientists and policy makers to improve welfare. It will be especially useful for training animal welfare specialists.

Abstract:

About 14 per cent of the human body is protein, so a growing child, or pregnant woman must have protein intake to increase the total amount of protein in the body, or foetus, as it grows. But why does an adult, whose body weight does not change, require protein in the diet? ‘Protein nutrition’ explains that proteins contain the element nitrogen in their constituent amino acids. Nitrogen balance is the difference between the intake of nitrogen-containing compounds in the diet and the excretion of nitrogen-containing compounds from the body. There is a requirement for dietary protein as the continual breakdown of tissue proteins in the body needs replacement by newly synthesized protein.

Abstract:

‘Movements, parties, policies’ explains the ‘machinery’ of environmental politics—the movement that nurtures and expresses the politics, the parties that are the electoral vehicles for getting the politics into the system, and the array of policy instruments that are available for putting society on a more sustainable footing. The organizations that make up the environmental movement can be categorized into different groups: public interest lobbies, professional protest organizations, participatory pressure groups, participatory protest organizations, and intentional communities. Green political parties are considered along with environmental policy-making. What are the challenges and what are the policy tools that politicians have at their disposal to meet them?

Abstract:

Ostracism—ignoring and excluding—is an evolutionarily adaptive response that protects groups from burdensome members either by correcting the misbehavior while promoting sameness and civility, or, if correction is not achieved, then ejecting the member, resulting again in a hom*ogeneous, albeit smaller, group. Over 20 years of research demonstrates that ostracism is a powerful tool of social influence. Being the target of ostracism activates brain regions associated with pain, threatens fundamental needs, worsens mood, and causes behavior changes aimed at fortifying threatened needs. We review research showing three functions of ostracism: (1) to protect—shielding groups from threatening members; (2) to correct—signaling to individuals that their behavior needs modification to remain in the group; and (3) to eject—permanently removing deviant individuals who resist correction. Although ostracism is a powerful and effective social influence tool, it can cause unintended and potentially dangerous consequences for those who employ it.

Abstract:

Hepatitis E virus (HEV) is a small, non-enveloped, single-strand, positive-sense RNA virus of approximately 7.2 kb in size. HEV is classified in the family Hepeviridae consisting of four recognized major genotypes that infect humans and other animals. Genotypes 1 and 2 HEV are restricted to humans and often associated with large outbreaks and epidemics in developing countries with poor sanitation conditions, whereas genotypes 3 and 4 HEV infect humans, pigs and other animal species and are responsible for sporadic cases of hepatitis E in both developing and industrialized countries. The avian HEV associated with Hepatitis-Splenomegaly syndrome in chickens is genetically and antigenically related to mammalian HEV, and likely represents a new genus in the family. There exist three open reading frames in HEV genome: ORF1 encodes non-structural proteins, ORF2 encodes the capsid protein, and the ORF3 encodes a small phosphoprotein. ORF2 and ORF3 are translated from a single bicistronic mRNA, and overlap each other but neither overlaps ORF1. Due to the lack of an efficient cell culture system and a practical animal model for HEV, the mechanisms of HEV replication and pathogenesis are poorly understood. The recent identification and characterization of animal strains of HEV from pigs and chickens and the demonstrated ability of cross-species infection by these animal strains raise potential public health concerns for zoonotic HEV transmission. It has been shown that the genotypes 3 and 4 HEV strains from pigs can infect humans, and vice versa. Accumulating evidence indicated that hepatitis E is a zoonotic disease, and swine and perhaps other animal species are reservoirs for HEV. A vaccine against HEV is not yet available.

Abstract:

Nephrotic syndrome is a clinical syndrome of heavy proteinuria (greater than 3.5 g per 24 hours), oedema, and hypoalbuminaemia, which is associated with hyperlipidaemia and a procoagulant state. Causes of nephrotic syndrome are traditionally classified by their histopathological descriptions. In most cases, the histological picture can have a primary (idiopathic) or secondary cause. Minimal change, membranous nephropathy, and focal segmental glomerulosclerosis account for over 60% of cases. Diabetic nephropathy and renal amyloidosis are common secondary causes of nephrotic syndrome. Nephrotic-range proteinuria will show up as at least 3+ protein on urinalysis. The diagnosis is confirmed by a urinary protein-to-creatinine ratio over 300 mg/mmol, and hypalbuminaemia. In adults, renal biopsy is the diagnostic test. This chapter addresses the causes, diagnosis, and management of nephrotic syndrome in adults.

Abstract:

This chapter examines the state of customary international law governing international investments, that is, the law that exists in the absence of an applicable treaty. Following World War II, such law for most investors was incomplete, vague, contested, and without an effective enforcement mechanism, meaning that investors and their home governments needed to find another way to protect investments of their nationals. This would lie in negotiating investment treaties. Topics covered include state and investor interests shaping international investment law; the sources of international law; customary international law and general principles of law governing international investment; customary international law on expropriation and breach of state contracts; challenges to Western views on international investment law; and deficiencies of customary international law on investment.

Abstract:

Chapter 3 addresses the institutional legacy (that is, the set of formal and informal rules that regulate the exercise of power in a political regime) of the transition to democracy, particularly those institutional dimensions that are more relevant for social movements—what social movement studies have defined as political opportunities. After setting the theoretical framework by specifying the main qualities of democracy the research has addressed, the chapter covers the legal and constitutional provisions on civil (especially protest) rights, political rights (right to resistance, majoritarian versus consensual assets), and social rights as well as practices—particularly with regard to protest, citizens’ participation, protest policing, and concertation.

Abstract:

Enteral nutrition (EN) is an integral part of the patient care in the intensive care unit (ICU) in order to maintain gut integrity, to modulate stress and the systemic immune response, and to attenuate disease severity. The timing of commencing EN in critically-ill patients depends on patient status and should be initiated as soon as the patient is stabilized. The energy and protein targets should be estimated and the feed prescription should match the nutritional target. The rate of EN dose increment or the addition of supplemental parenteral nutrition (PN) to reach the nutritional target is still debatable and ranges between 3 days (ESPEN approach) and up to 8 days (ASPEN approach). Micronutrients should be supplemented to all patients. The role of pharmaconutrition is controversial due to recent negative trials, but the use of EN with supplemental omega-3 and GLA for acute respiratory distress syndrome patients is still advocated by ESPEN and ASPEN guidelines.

Abstract:

Vascular bypass graft failure is a significant clinical problem and is frequently due to the formation of intimal hyperplasia (IH) [1–3]. IH is characterized by the accumulation of smooth muscle cells (SMC) and extracellular matrix in the intima of the vessel, which occurs when the normal balance between vascular cell proliferation and apoptosis (regulated cell death) is altered [4]. The disturbed flow present at the anastomosis has been implicated in the formation of IH and the link between hemodynamics and graft failure is via a complex cascade of events whereby biomechanical forces cause biological responses [5, 6]. For example, immediate early genes (IEG) such as c-fos, c-jun and egr-1 are involved in the signaling pathways for proliferation and apoptosis. When extracellular biomechanical stimuli (e.g. shear stress) cause the expression of IEG, their protein products translocate to the nucleus. These proteins regulate the expression of a number of genes implicated in cardiovascular disease including growth factors, adhesion molecules, proapoptotic substrates and coagulation factors [7–9]. Because IEG are involved in both proliferation and apoptosis, their expression may upset the normal balance between cell proliferation and apoptosis and could play a vital role in the IH formation in vascular bypass grafts.

Abstract:

The stabilization of proteins is a priority for several important fields, most notably the pharmaceutical industry. Protein-based therapeutic drugs have demonstrated significant efficacy in controlling and curing disease. Unlike traditional small molecule-based drug therapies, a major hurdle in the development of protein drugs is the challenge of maintaining the protein in the folded state throughout processing and also during storage at the end point-of-use. When a protein is taken from its native environment, it is often unstable and unfolds. Because the protein’s 3-dimensional structure is responsible for its functional activity, much work has been dedicated to finding excipients that will stabilize proteins outside of their native environment.

Abstract:

Sugar (one of the critical nutrition elements for all life forms) transport across the cell membranes play essential roles in a wide range of living organism. One of the most important active transport (against the sugar concentration) mechanisms is facilitated by the transmembrane transporter proteins, such as the Escherichia coli lactose permease (LacY) proteins. Active transport of sugar molecules with LacY proteins requires a proton gradient and a sequence of complicated protein conformational changes. However, the exact molecular mechanisms and the protein structural information involved in the transport process are largely unknown. All atom atomistic simulations are able to provide full details but are limited to relative small length and time scales due to the computational cost. The protein conformational changes during sugar transport across LacY are large scale structural reorganization and inaccessible to all atom simulations. In this work, we investigate the molecular mechanisms and conformational changes during sugar transport using coarse-grained molecular dynamics (CGMD) simulations. In our coarse-grained force field, we follow the procedures developed by Han et al. [1, 2], in which the protein model is united-atom based and each heavy atom together with the attached hydrogen atoms is represented by one site, then the protein force filed is coupled with the MARTINI [3] water and lipid force fields. This hybrid force field takes the advantage of the efficiency of MARTINI force field for the environment (water and lipid), while retaining the detailed conformational information for the proteins. Specifically, we develop the new force fields for interactions between sugar molecules and protein by matching the potential of mean force between all-atom and coarse-grained models. Then we validate our force field by comparing the potential of mean force for a glucose interaction with a carbohydrate binding protein from our new force field, with the results from all atom simulations. After validation, we implement the force field for sugar transport across LacY proteins. Through our simulations we are able to capture the formation/breakage of the important hydrogen bonds and salt bridges, which are crucial to the overall conformational changes of LacY.

Abstract:

Protein C, free and bound protein S and C4 binding protein levels (C4bp), were measured by electroimmunoassay in 7 pregnant women aged 22-29 years at 16-18 weeks of gestation, immediately prior to termination of pregnancy for social reasons. Protein C and protein S levels were also measured in their foetuses from blood taken through the umbilical cord. In this group of pregnant women the mean levels for protein C were 104% of normal adult mean (range 80-128%), for C4bp 100% (52-150%), for free protein S 66% (43-89%). In the foetuses the mean value for protein C was 15.3% (10.5-21%) and for free protein S 36.85% (27-47%) of the normal adult mean. Bound protein S and C4bp levels were zero. Conclusions: (1) free protein S is significantly decreased (< 2SD below the normal adult mean) in women after the first trimester of gestation whereas no change is seen in protein C concentration; (2) C4bp levels are at zero in the foetus as also are the levels of bound protein S; (3) foetal blood protein S level is approximately 2.5 times higher than protein C. Since all other vitamin K-dependent factors have been observed to be in the range of 10-20% of normal at this stage of gestation, our findings may be further proof of a non hepatic (endothelial) source of plasma protein S.

Abstract:

Pore-forming cytolytic proteins distributed in a wide variety of eukaryotic and prokaryotic organisms have been intensively studied in terms of pathophysiological functions and molecular architecture of transmembrane pores. These proteins are also being developed for various analytical applications such as detector of proteins and DNA by engineering the structure of the pore. Staphylococcal gamma-hemolysin (Hlg), a pore-forming protein, which consists of two separate proteins, LukF and Hlg2, has potential to be a useful tool as a multifunctional biosensor. However, the fine structure of the Hlg pore has not been clarified. Our previous studies revealed that LukF and Hlg2 assemble alternately on the membrane in a molar ratio of 3:4 and 4:3 and form cylindrical heteroheptameric transmembrane pores. In the present study, we conducted quantitative analysis of the subunit arrangement of the pore by using two-dimensional (2-D) image analysis based on high-resolution transmission electron microscopy (TEM) images. Results of this study suggest a new aspect of the characteristic structure in two-component pore-forming protein and can contribute to the engineering of the Hlg pore.

Abstract:

The sequence of 20 types of amino acid residues in a heteropolymer chain of a protein is believed to be the basis for the 3-D conformation (folded structure) that a protein assumes to serve its functions. We present a deterministic optimization method to design the sequence of a simplified model of proteins for a desired conformation. A design methodology developed for the topology optimization of compliant mechanisms is adapted here by converting the discrete combinatorial problem of protein sequence design to a continuous optimization problem. It builds upon our recent work which used a minimum energy criterion on a deterministic approach to protein design using continuous models. This paper focuses on the energy gap criterion, which is argued to be one of the most important characteristics determining the stable folding of a protein chain. The concepts, methodology, and illustrative examples are presented using HP models of proteins where only two types (H: hydrophobic and P: polar) of monomers are considered instead of 20. The highlight of the method presented in this paper is the drastic reduction in computational costs.

Abstract:

The interaction of the complement inhibitor S protein, which is identical to the serum spreading factor, vitronectin, with cultured human endothelial cells of macro- and microvas- cular origin was investigated. Purified S protein, coated for 2 h on polystyrene petri dishes, induced concentration- and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVEC) as well as human omental tissqe microvasular endothelial cells (HOTMEC) at 37°C. With 3 × 105 cells/ml (final concentration) more than 50% of the cells attached within 2 h incubation at 0.3 - 3 μg/ml S protein. The effect of S protein was specific, since only monospecific antibodies against S protein prevented attachment of cells, while antibodies against fibronectin, fibrinogen or von Wille-brand factor were uneffective. The pentapeptide Gly-Arg-Gly-Asp-Ser, which contains the cell-attachment site of these adhesive proteins including S protein, inhibited the activity of S protein to promote attachment of endothelial cells in a concentration-dependent fashion; at 200 μM peptide, less than 10% of the cells became attached. Direct binding of S protein to HUVEC and HOTMEC was studied with cells in suspension at a concentration of 1 × 106 cells/ml in the presence of 1% (w/v) human serum albumin and 1 mM CaCl2 and was maximal after 120 min. Both cell types bound S protein in a concentration-dependent fashion with an estimated dissociation constant KD=0.2pM. More than 80% of bound radiolabelled S protein was displaced by unlabelled S protein, whereas binding was reduced to about 50% by the addition in excess of either fibronectin, fibrinogen, von Willebrand factor or the pentapeptide. These findings provide evidence for the specific association of S protein with endothelial cells, ultimately leading to attachment and spreading of cells. Although the promotion of attachment was highly specific for S protein, other adhesive proteins than S protein, also known to associate with endothelial cells, may in part compete with direct S protein binding.

Abstract:

Genetic engineering of protein-based materials provides material scientists with high levels of control in material microstructures, properties, and functions [1]. For example, multi-block protein copolymers in which individual block may possess distinct mechanical or biological properties have been biosynthesized [2, 3]. Polypeptide sequences derived from well-studied structural proteins (e.g., collagen, silk, elastin) are often used as motifs in the design and synthesis of new protein-based material, in which new functional groups may be incorporated. In this fashion, we have produced a series of silk-elastin-like proteins (SELPs) consisting of polypeptide sequences derived from silk of superior mechanical strength and elastin that is extremely durable and resilient [2, 4]. Notably, the silk-like blocks are capable of crystallizing to form virtual cross-links between elastin-mimetic sequences, which, in turn, lower the crystallinity of the silk-like blocks and thus enhance the solubility of SELPs. Consequently, SELPs may be fabricated into useful structures for biomedical applications, including drug delivery. In this study, we will characterize viscoelastic properties of SELPs, which are particularly relevant to tissue engineering applications.

Abstract:

Abstract Even though silkworm are the most dominant type of silk fibers used for commercial applications, spider silk has a definitive role in biomedical applications due to its biocompatibility and excellent mechanical properties as biomaterials. In recent years, recombinant production of the silk proteins at a larger scale has found new interest. Spider silk composites with a combination of a variety of other biomaterials have also been used to improve properties such as bio-compatibility, mechanical strength and controlled degradation. [1] A major constituent of spider silk fibers, are spidroin proteins. These are made up of repetitive segments flanked by conserved non-repetitive domains. The fiber proteins consist of a light chain and a heavy chain that are connected via a single disulfide bond. [2] Present paper employed steered molecular dynamics (SMD) as the principal method of investigating the mechanical properties of these nanoscale spider silk protein 3LR2, with a residual count of 134 amino acids. [3]. SMD simulations were performed by pulling on β-chain of the protein in the x-direction, while holding the other fixed. The focus of this paper is to investigate the mechanical properties of the nanoscale spider silk proteins with lengths of about 4.5nm in a folded state, leading to understanding of their feasibility in bio-printing of a composite spider silk biomaterial with a blend of various other biomaterials such as collagen. An in-depth insight into the fraying and tensile deformation and structural properties of the spider silk proteins are of innovative significance for a multitude of biomedical engineering applications. A calculated Gibbs free energy value of 18.59 kCal/mol via umbrella sampling corresponds with a complete separation of a single chain from a spider silk protein in case of fraying. Force needed for complete separation of the chain from the spider silk protein is analyzed, and discussed in this paper. It is found that the protein molecule undergoes a tensile stretch at strain rates of ≅ 11.65. An elastic modulus of 20.136 GPa, calculated via simple SMD simulations by subjecting the silk β-chain to a tensile stretch is also presented.

Abstract:

PADGEM (Platelet Activation-Dependent Granule ⇒ External Membrane) glycoprotein, a platelet alpha granule integral membrane protein with a molecular weight of 140,000, is translocated to the plasma membrane during granule secretion. PADGEM protein is expressed solely on activated platelets, but is not on the surface of resting platelets. Because HEL cells contain platelet alpha granule-like organelles and proteins (e.g. platelet factor 4, von Villebrands factor, β-thromboglobulin) and express certain platelet membrane proteins (e.g. GP IIb/IIIa, GPIb), we evaluated induced and uninduced HEL cells for the synthesis and expression of PADGEM protein. HEL cells were induced with 1.25% DMSO for 3-4 days, then grown in the absence of DMSO for 1-3 weeks. After eight cycles of DMSO exposure, the induced HEL cells were found to increase the expression of PADGEM, in contrast to the uninduced cells. Intact fixed and unfixed induced HEL cells were observed by immunofluorescence, utilizing KC4, a monoclonal anti-PADGEM antibody, to express PADGEM while non-induced HFT. cells expressed low levels of PADGEM. Both induced and uninduced HEL cells bound A2A9, an anti—GP Ilb/IIIa monoclonal antibody. Quantitative analysis by fluorescence activated cell sorting demonstrated a 2.5—fold increase in mean surface expression of PADGEM and 3.3—fold mean increase in GP IIb/IIIa surface expression compared to uninduced cells. By fluoresence microscopy, 70% of induced HEL cells expressed PADGEM protein versus 20% of the uninduced cells. GP-IIb/IIIa expression inoreased from 40% in noninduced cells to 90% in induced cells. The induced HEL cells contained PADGEM with a molecular weight identical to that of platelets, as demonstrated by Western blotting using the KC4 antibody. Direct binding experiments with 125I-KC4 antibody demonstrated that surface binding was specific, saturable, and time-dependent. Surface expression of PADGEM protein was not increased with platelet agonists (thrombin, epinephrine, ADP) nor cytokines (IL-1, IL-2, tissue necrosis factor). The surface density of PADGEM protein on induced HEL cells and activated platelets appears similar. HEL cells should provide a useful model to assist in the elucidation of the structure, function and biology of PADGEM protein.

Abstract:

Recent laboratory results confirm that it is possible to protect concrete from freezing solely using chemical admixtures and indicate that the amount of admixture required may be significantly less than previously recommended. Researchers have also verified that admixture-based freeze protection can produce concrete that is durable to winter exposure for a minimum of 20 years, through petrographic examination of core specimens obtained from past field demonstrations. Freeze protection for concrete using chemical admixtures alone has been an area of active research for 3 decades; however, the most recent methodology recommends very high addition rates of accelerating and corrosion inhibiting admixtures, which result in significant challenges, including slump loss, rapid setting, and potentially excessive temperature rise. As part of a laboratory study, researchers systematically varied the dosage of freeze protection admixtures used in concrete cured in a 23 °F environment. Preliminary findings indicate that a 50% reduction in admixture dose maintained adequate freeze protection and resulted in compressive strengths exceeding those of room-temperature controls at 7 and 28 days. The combination of improved handling, reduced cost, and verified durability associated with the use of admixtures for freeze protection makes a compelling case for broader adoption of this technique in winter operations

Abstract:

This report maps the African landscape of Open Science – with a focus on Open Data as a sub-set of Open Science. Data to inform the landscape study were collected through a variety of methods, including surveys, desk research, engagement with a community of practice, networking with stakeholders, participation in conferences, case study presentations, and workshops hosted. Although the majority of African countries (35 of 54) demonstrates commitment to science through its investment in research and development (R&D), academies of science, ministries of science and technology, policies, recognition of research, and participation in the Science Granting Councils Initiative (SGCI), the following countries demonstrate the highest commitment and political willingness to invest in science: Botswana, Ethiopia, Kenya, Senegal, South Africa, Tanzania, and Uganda. In addition to existing policies in Science, Technology and Innovation (STI), the following countries have made progress towards Open Data policies: Botswana, Kenya, Madagascar, Mauritius, South Africa and Uganda. Only two African countries (Kenya and South Africa) at this stage contribute 0.8% of its GDP (Gross Domestic Product) to R&D (Research and Development), which is the closest to the AU’s (African Union’s) suggested 1%. Countries such as Lesotho and Madagascar ranked as 0%, while the R&D expenditure for 24 African countries is unknown. In addition to this, science globally has become fully dependent on stable ICT (Information and Communication Technologies) infrastructure, which includes connectivity/bandwidth, high performance computing facilities and data services. This is especially applicable since countries globally are finding themselves in the midst of the 4th Industrial Revolution (4IR), which is not only “about” data, but which “is” data. According to an article1 by Alan Marcus (2015) (Senior Director, Head of Information Technology and Telecommunications Industries, World Economic Forum), “At its core, data represents a post-industrial opportunity. Its uses have unprecedented complexity, velocity and global reach. As digital communications become ubiquitous, data will rule in a world where nearly everyone and everything is connected in real time. That will require a highly reliable, secure and available infrastructure at its core, and innovation at the edge.” Every industry is affected as part of this revolution – also science. An important component of the digital transformation is “trust” – people must be able to trust that governments and all other industries (including the science sector), adequately handle and protect their data. This requires accountability on a global level, and digital industries must embrace the change and go for a higher standard of protection. “This will reassure consumers and citizens, benefitting the whole digital economy”, says Marcus. A stable and secure information and communication technologies (ICT) infrastructure – currently provided by the National Research and Education Networks (NRENs) – is key to advance collaboration in science. The AfricaConnect2 project (AfricaConnect (2012–2014) and AfricaConnect2 (2016–2018)) through establishing connectivity between National Research and Education Networks (NRENs), is planning to roll out AfricaConnect3 by the end of 2019. The concern however is that selected African governments (with the exception of a few countries such as South Africa, Mozambique, Ethiopia and others) have low awareness of the impact the Internet has today on all societal levels, how much ICT (and the 4th Industrial Revolution) have affected research, and the added value an NREN can bring to higher education and research in addressing the respective needs, which is far more complex than simply providing connectivity. Apart from more commitment and investment in R&D, African governments – to become and remain part of the 4th Industrial Revolution – have no option other than to acknowledge and commit to the role NRENs play in advancing science towards addressing the SDG (Sustainable Development Goals). For successful collaboration and direction, it is fundamental that policies within one country are aligned with one another. Alignment on continental level is crucial for the future Pan-African African Open Science Platform to be successful. Both the HIPSSA ((Harmonization of ICT Policies in Sub-Saharan Africa)3 project and WATRA (the West Africa Telecommunications Regulators Assembly)4, have made progress towards the regulation of the telecom sector, and in particular of bottlenecks which curb the development of competition among ISPs. A study under HIPSSA identified potential bottlenecks in access at an affordable price to the international capacity of submarine cables and suggested means and tools used by regulators to remedy them. Work on the recommended measures and making them operational continues in collaboration with WATRA. In addition to sufficient bandwidth and connectivity, high-performance computing facilities and services in support of data sharing are also required. The South African National Integrated Cyberinfrastructure System5 (NICIS) has made great progress in planning and setting up a cyberinfrastructure ecosystem in support of collaborative science and data sharing. The regional Southern African Development Community6 (SADC) Cyber-infrastructure Framework provides a valuable roadmap towards high-speed Internet, developing human capacity and skills in ICT technologies, high- performance computing and more. The following countries have been identified as having high-performance computing facilities, some as a result of the Square Kilometre Array7 (SKA) partnership: Botswana, Ghana, Kenya, Madagascar, Mozambique, Mauritius, Namibia, South Africa, Tunisia, and Zambia. More and more NRENs – especially the Level 6 NRENs 8 (Algeria, Egypt, Kenya, South Africa, and recently Zambia) – are exploring offering additional services; also in support of data sharing and transfer. The following NRENs already allow for running data-intensive applications and sharing of high-end computing assets, bio-modelling and computation on high-performance/ supercomputers: KENET (Kenya), TENET (South Africa), RENU (Uganda), ZAMREN (Zambia), EUN (Egypt) and ARN (Algeria). Fifteen higher education training institutions from eight African countries (Botswana, Benin, Kenya, Nigeria, Rwanda, South Africa, Sudan, and Tanzania) have been identified as offering formal courses on data science. In addition to formal degrees, a number of international short courses have been developed and free international online courses are also available as an option to build capacity and integrate as part of curricula. The small number of higher education or research intensive institutions offering data science is however insufficient, and there is a desperate need for more training in data science. The CODATA-RDA Schools of Research Data Science aim at addressing the continental need for foundational data skills across all disciplines, along with training conducted by The Carpentries 9 programme (specifically Data Carpentry 10 ). Thus far, CODATA-RDA schools in collaboration with AOSP, integrating content from Data Carpentry, were presented in Rwanda (in 2018), and during17-29 June 2019, in Ethiopia. Awareness regarding Open Science (including Open Data) is evident through the 12 Open Science-related Open Access/Open Data/Open Science declarations and agreements endorsed or signed by African governments; 200 Open Access journals from Africa registered on the Directory of Open Access Journals (DOAJ); 174 Open Access institutional research repositories registered on openDOAR (Directory of Open Access Repositories); 33 Open Access/Open Science policies registered on ROARMAP (Registry of Open Access Repository Mandates and Policies); 24 data repositories registered with the Registry of Data Repositories (re3data.org) (although the pilot project identified 66 research data repositories); and one data repository assigned the CoreTrustSeal. Although this is a start, far more needs to be done to align African data curation and research practices with global standards. Funding to conduct research remains a challenge. African researchers mostly fund their own research, and there are little incentives for them to make their research and accompanying data sets openly accessible. Funding and peer recognition, along with an enabling research environment conducive for research, are regarded as major incentives. The landscape report concludes with a number of concerns towards sharing research data openly, as well as challenges in terms of Open Data policy, ICT infrastructure supportive of data sharing, capacity building, lack of skills, and the need for incentives. Although great progress has been made in terms of Open Science and Open Data practices, more awareness needs to be created and further advocacy efforts are required for buy-in from African governments. A federated African Open Science Platform (AOSP) will not only encourage more collaboration among researchers in addressing the SDGs, but it will also benefit the many stakeholders identified as part of the pilot phase. The time is now, for governments in Africa, to acknowledge the important role of science in general, but specifically Open Science and Open Data, through developing and aligning the relevant policies, investing in an ICT infrastructure conducive for data sharing through committing funding to making NRENs financially sustainable, incentivising open research practices by scientists, and creating opportunities for more scientists and stakeholders across all disciplines to be trained in data management.