Jamil, Sarwat

Growth factor withdrawal from hemopoietic cells results in activation of the mitochondrial pathway of apoptosis. Members of the Bcl-2 family regulate this pathway, with anti-apoptotic members counteracting the effects of pro-apoptotic members. We investigated the effect on Mcl-1 function of mutation at a conserved threonine 163 residue (T163) in its proline, glutamate, serine, and threonine rich (PEST) region. Under normal growth conditions, Mcl-1 half-life increased with alteration of T163 to glutamic acid, but decreased with mutation to alanine. However, both T163 mutants exhibited greater pro-survival effects compared with the wild type, which can be explained by an increased stability of the T163A mutant in cytokine-starved conditions. Both the mutant forms exhibited prolonged binding to pro-apoptotic Bim in cytokine-deprived cells. The extent to which Mcl-1 mutants were able to exert their anti-apoptotic effects correlated with their ability to associate with Bim. We further observed that primary bone marrow derived macrophages survived following cytokine withdrawal as long as Bim and Mcl-1 remained associated. In our study, we were unable to detect a role for GSK-3-mediated regulation of Mcl-1 expression. Based on these results we propose that upon cytokine withdrawal, survival of hemopoietic cells depends on association between Mcl-1 and Bim. Furthermore, alteration of T163 of Mcl-1 may change the protein such that its association with Bim is affected, resulting in prolonged association and increased survival. Le retrait de facteurs de croissance du milieu de croissance des cellules hémopoïétiques résulte en l'activation de la voie mitochondriale de l'apoptose. Les membres de la famille Bcl-2 régulent cette voie, les membres anti-apoptotiques contrecarrant les effets des membres pro-apoptotiques. Nous avons examiné l'effet de la mutation d'un résidu thréonine conservé (T163), présent dans la région riche en résidus proline, glutamate, sérine et thréonine (PEST), sur la fonction de Mcl-1. Le remplacement de la T163 par un acide glutamique augmentait la demi-vie de Mcl-1 en conditions de croissance normale, alors que la mutation en alanine la diminuait. Cependant, les deux types de mutations de la T163 favorisaient la survie comparativement au type sauvage, ce qui pouvait s'expliquer par une stabilité accrue des protéines mutantes en conditions de privation en cytokines. Les deux formes mutantes se liaient plus longtemps à Bim, un effecteur pro-apoptotique, chez les cellules privées de cytokines. La mesure avec laquelle les protéines Mcl-1 mutantes étaient capables d'exercer leurs effets anti-apoptotiques étaient corrélés avec leur capacité de s'associer à Bim. Nous avons ensuite observé que les macrophages primaires dérivés de la moelle osseuse survivaient après le retrait des cytokines tant et aussi longtemps que Bim et Mcl-1 restaient associés. Dans notre étude, nous avons été incapables de détecter un rôle de la régulation de l'expression de Mcl-1 par GSK-3. Nous proposons à partir de ces résultats qu'à la suite du retrait des cytokines, la survie des cellules hémopoïtiques dépend de l'association de Mcl-1 à Bim. De plus, la modification de la T163 de Mcl-1 pouvait changer la protéine de telle sorte que son association à Bim était affectée, résultant en une association prolongée et en une survie accrue. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase2 has been implicated in tendon pathology caused by repetitive movements. However, its activity in the early stages of the tendon's response to overuse, and its presence in the circulation as a possible indicator of tendon degradation, remain unknown. Human tendon cells were repetitively stretched for 5 days, and the rabbit Achilles tendon complex underwent repetitive motion 3× per week for 2 weeks. Quantitative polymer chain reaction analysis was performed to detect matrix metalloproteinase2/14 and tissue inhibitor of matrix metalloproteinase2 messenger ribonucleic acid of cells and rabbit tissue, and matrix metalloproteinase2 protein levels were determined with an enzyme linked immunoassay. Matrix metalloproteinase2 activity was examined using zymography of the conditioned media, tendon and serum. Immunohistochemistry was used to localize matrix metalloproteinase2 in tendon tissue, and the density of fibrillar collagen in tendons was examined using second harmonic generation microscopy. Tendon cells stretched with high strain or high frequency demonstrated increased matrix metalloproteinase2 messenger ribonucleic acid and protein levels. Matrix metalloproteinase2 activity was increased in the rabbit Achilles tendon tissue at weeks 1 and 2; however, serum activity was only increased at week 1. After 2 weeks of exercise, the collagen density was lower in specific regions of the exercised rabbit Achilles tendon complex. Matrix metalloproteinase2 expression in exercised rabbit Achilles tendons was detected surrounding tendon fibroblasts. Repetitive mechanical stimulation of tendon cells results in a small increase in matrix metalloproteinase2 levels, but it appears unlikely that serum matrix metalloproteinase2 will be a useful indicator of tendon overuse injury.

p53 is a tumor suppressor protein which is either lost or inactivated in a large majority of tumors. The small molecule 2-phenylethynesulfonamide (PES) was originally identified as the inhibitor of p53 effects on the mitochondrial death pathway. In this report we demonstrate that p53 protein from PES-treated cells was detected in reduced mobility bands between molecular weights 95–220 kDa. Resolution of p53 aggregates on urea gel was unable to reduce the high molecular weight p53 aggregates, which were shown to be primarily located in the nucleus. Therefore, our data suggest that PES exerts its effects through covalent cross-linking and nuclear retention of p53. [ABSTRACT FROM AUTHOR]

Protection against Mycobacterium tuberculosis infection is dependent on T cell and macrophage activation regulated by cytokines. Cytokines and chemokines produced at disease sites may be released into circulation. Data available on circulating cytokines in tuberculosis (TB) is mostly on pulmonary TB (PTB) with limited information on extrapulmonary disease (EPul-TB). We measured interferon-gamma (IFN-γ), interkeukin-10 (IL-10), CXCL9 and CCL2 in sera of patients ( n = 80) including; PTB ( n = 42), EPul-TB ( n = 38) and BCG vaccinated healthy endemic controls (EC, n = 42). EPul-TB patients comprised those with less severe (LNTB) or severe (SevTB) disease. Serum IFN-γ, IL-10 and CXCL9 levels were significantly greater while CCL2 was reduced in TB patients as compared with EC. IFN-γ was significantly greater in PTB as compared with LNTB ( P = 0.002) and SevTB ( P = 0.029). CXCL9 was greater in PTB as compared with LNTB ( P = 0.009). In contrast, CCL2 levels were reduced in PTB as compared with LNTB ( P = 0.021) and SevTB ( P = 0.024). A Spearman’s rank correlation analysis determined a positive association between IFN-γ and IL-10 (rho = 0.473, P = 0.002) and IFN-γ and CXCL9 (rho = 0.403, P = 0.008) in the PTB group. However, in SevTB, only IFN-γ and CXCL9 were positively associated (rho = 0.529, P = 0.016). Systemic levels of cytokines are reflective of local responses at disease sites. Therefore, our data suggests that in PTB increased IFN-γ and CXCL9 balanced by IL-10 may result in a more effective cell mediated response in the host. However, elevated inflammatory chemokines CXCL9 and CCL2 in severe EPul-TB without concomitant down modulatory cytokines may exacerbate disease related pathology and hamper restriction of M. tuberculosis infection. [ABSTRACT FROM AUTHOR]

MCL-1, a pro-survival member of the BCL-2 family, was previously shown to have functions in ATR-dependent Chk1 phosphorylation following DNA damage. To further delineate these functions, we explored possible differences in DNA damage response caused by lack of MCL-1 in mouse embryo fibroblasts (MEFs). As expected, Mcl-1(-/-) MEFs had delayed Chk1 phosphorylation following etoposide treatment, compared to wild type MEFs. However, their response to hydroxyurea, which causes a G(1)/S checkpoint response, was not significantly different. In addition, appearance of gamma-H2AX was delayed in the Mcl-1(-/-) MEFs treated with etoposide. We next investigated whether MCL-1 is present, together with other DNA damage response proteins, at the sites of DNA damage. Immunoprecipitation of etoposide-treated extracts with anti-MCL-1 antibody showed association of MCL-1 with gamma-H2AX as well as NBS1. Immunofluorescent staining for MCL-1 further showed increased co-staining of MCL-1 and NBS1 following DNA damage. By using a system that creates DNA double strand breaks at specific sites in the genome, we demonstrated that MCL-1 is recruited directly adjacent to the sites of damage. Finally, in a direct demonstration of the importance of MCL-1 in allowing proper repair of DNA damage, we found that treatment for two brief exposures to etoposide , followed by periods of recovery, which mimics the clinical situation of etoposide use, resulted in greater accumulation of chromosomal abnormalities in the MEFs that lacked MCL-1. Together, these data indicate an important role for MCL-1 in coordinating DNA damage mediated checkpoint response, and have broad implications for the importance of MCL-1 in maintenance of genome integrity.