K252a is a potent and selective inhibitor of phosphorylase kinase

The inhibition of phosphorylase kinase by a number of protein kinase inhibitors was examined. Both K252a and staurosporine are potent inhibitors of phosphorylase kinase with IC50 values of 1.7 nM and 0.5 nM respectively. K252a shows a 300-fold selectivity for this enzyme over protein kinase C whereas staurosporine shows only a 20-fold selectivity for phosphorylase kinase. In contrast, the Roche bis-indolyl maleimides inhibit phosphorylase kinase with IC50 values of approximately 1 microM and are highly selective for protein kinase C.     

Mechanism of energization of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation [DMP+] into an acrA strain of Escherichia coli.

The mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) into cells and vesicles of the acrA strain AS-1 of Escherichia coli was examined. Uptake was energized by substrate oxidation and discharged by uncouplers. Uptake was enhanced by the presence of tetraphenylphosphonium cation, tetraphenylboron anion and tributyltin chloride, which may inhibit the efflux system for DMP+. Uptake was inhibited by 5-methoxyindole-2-carboxylic acid (MIC). By the use of ionophores with right-side-out vesicles loaded with monovalent cations it was shown that DMP+ uptake could be driven both by the establishment of a membrane potential across the vesicle membrane and by a H+/DMP+ antiport system. Attempts to demonstrate the latter mechanism in everted membrane vesicles were unsuccessful.     

pH probes respond to redox changes in cytochrome o

N-Phenylnaphthylamine (NPN) has been used previously to probe the fluidity or microviscosity of membrane lipids. We have shown (Sedgwick, E. G., and Bragg, P.D. (1988) FEBS Lett. 229, 127-130) that the fluorescence intensity of this probe abruptly increases upon depletion of the oxygen content of a medium by respiring cytochrome o of Escherichia coli that has been incorporated into soybean phospholipid vesicles. We now show that the pH probes pyranine and quinacrine behave similarly to NPN. The fluorescence change is not due to changes in the pH gradient across the membrane or to a change in the distribution of probe between the vesicles and the external medium. It is insensitive to uncouplers. The fluorescence change with pyranine and quinacrine occurs also with soluble cytochrome o in the absence of added phospholipid. The NPN response requires added phospholipid. Alteration of the redox state of cytochrome o with cyanide suggests that these probes respond to a change in the redox state of the cytochrome, either by alterations in binding of the probe to the cytochrome or by a change in the environment of the probe bound to the cytochrome. This behavior should be considered when pyranine or quinacrine are used to measure changes in the internal pH of membrane vesicles containing redox proteins.     

Antigen-specific damage to brain vascular endothelial cells mediated by encephalitogenic and nonencephalitogenic CD4+ T cell lines in vitro.

Experimentally induced and naturally occurring inflammatory diseases of the central nervous system (CNS) are often associated with a breakdown of the blood-brain barrier and edema within the CNS itself. CD4+ T cells are now clearly implicated in the pathogenesis of the induced CNS disease, experimental autoimmune encephalomyelitis, and previous in vivo experiments had indicated that these cells may be capable of directly damaging the CNS vasculature. To assess the capacity of CD4+ T cells to damage brain vascular endothelial cells (EC) in vitro, two lines with specificity for myelin basic protein and OVA were prepared and added to cultures of EC. We show here that both lines, when added in a resting state, severely disrupt the EC monolayers in an Ag-specific manner. The interaction is dependent on the recognition of Ag in the context of MHC class II and is blocked in the presence of mAb specific for CD4. Addition of T cell lines preactivated on irradiated thymocyte APC caused a high level of Ag nonspecific damage to the EC, which was not blocked by the addition of anti-CD4 mAb. Supernatants derived from these latter cells did not alone damage the EC monolayers despite the presence of TNF activity suggesting that T cell-EC contact may be required for these cell lines to mediate their effector function. Both resting and preactivated lines adhered strongly to the EC in the absence of Ag. The capacity of CD4+ T cells to strongly adhere to, and disrupt the integrity of, brain vascular EC may be important in the early stages of CNS disease mediated by this cell type.     

Serotype-specific inactivation of the cellular DNA damage response during adenovirus infection

Adenovirus type 5 (Ad5) inactivates the host cell DNA damage response by facilitating the degradation of Mre11, DNA ligase IV, and p53. In the case of p53, this is achieved through polyubiquitylation by Ad5E1B55K and Ad5E4orf6, which recruit a Cul5-based E3 ubiquitin ligase. Recent evidence indicates that this paradigm does not apply to other adenovirus serotypes, since Ad12, but not Ad5, causes the degradation of TOPBP1 through the action of E4orf6 alone and a Cul2-based E3 ubiquitin ligase. We now have extended these studies to adenovirus groups A to E. While infection by Ad4, Ad5, and Ad12 (groups E, C, and A, respectively) cause the degradation of Mre11, DNA ligase IV, and p53, infection with Ad3, Ad7, Ad9, and Ad11 (groups B1, B1, D, and B2, respectively) only affects DNA ligase IV levels. Indeed, Ad3, Ad7, and Ad11 cause the marked accumulation of p53. Despite this, MDM2 levels were very low following infection with all of the viruses examined here, regardless of whether they increase p53 expression. In addition, we found that only Ad12 causes the degradation of TOPBP1, and, like Ad5, Ad4 recruits a Cul5-based E3 ubiquitin ligase to degrade p53. Surprisingly, Mre11 and DNA ligase IV degradation do not appear to be significantly affected in Ad4-, Ad5-, or Ad12-infected cells depleted of Cul2 or Cul5, indicating that E1B55K and E4orf6 recruit multiple ubiquitin ligases to target cellular proteins. Finally, although Mre11 is not degraded by Ad3, Ad7, Ad9, and Ad11, no viral DNA concatemers could be detected. We suggest that group B and D adenoviruses have evolved mechanisms based on the loss of DNA ligase IV and perhaps other unknown molecules to disable the host cell DNA damage response to promote viral replication.     

A direct role of Mad1 in the spindle assembly checkpoint beyond Mad2 kinetochore recruitment

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying entry into anaphase until all sister chromatids have become bi‐oriented. A key component of the SAC is the Mad2 protein, which can adopt either an inactive open (O‐Mad2) or active closed (C‐Mad2) conformation. The conversion of O‐Mad2 into C‐Mad2 at unattached kinetochores is thought to be a key step in activating the SAC. The “template model” proposes that this is achieved by the recruitment of soluble O‐Mad2 to C‐Mad2 bound at kinetochores through its interaction with Mad1. Whether Mad1 has additional roles in the SAC beyond recruitment of C‐Mad2 to kinetochores has not yet been addressed. Here, we show that Mad1 is required for mitotic arrest even when C‐Mad2 is artificially recruited to kinetochores, indicating that it has indeed an additional function in promoting the checkpoint. The C‐terminal globular domain of Mad1 and conserved residues in this region are required for this unexpected function of Mad1.     

Perceived Stigma towards Leprosy among Community Members Living Close to Nonsomboon Leprosy Colony in Thailand

Background

Interpretation of Leprosy as a sickness differs among society. The set of beliefs, knowledge and perceptions towards a disease play a vital role in the construction of stigma towards a disease. The main purpose of this study was to explore the extent and correlates of the perceived stigma towards leprosy in the community living close to the leprosy colony in Non Somboon region of Khon Kaen Province of Thailand.

Methods

A cross-sectional study was conducted among 257 leprosy unaffected community participants, above the age of 18 who were living close to the Leprosy colony in Non Somboon region of Thailand. Each participant was asked a questionnaire containing characteristics of the participants in terms of socio-demographic background and knowledge regarding the disease. In addition perceived stigma towards leprosy was measured using EMIC (Explanatory Model Interview Catalogue) questionnaire.

Results

Among EMIC items, shame or embarrassment in the community due to leprosy was felt by 54.5%, dislike to buy food from leprosy affected persons were 49.8% and difficulty to find work for leprosy affected persons were perceived by 47.1%. Higher total EMIC score was found in participants age 61 years or older (p = 0.021), staying longer in the community (p = 0.005), attending fewer years of education (p = 0.024) and who were unemployed (p = 0.08). Similarly, perceptions about leprosy such as difficult to treat (p = 0.015), severe disease (p = 0.004) and punishment by God (p = 0.011) were significantly associated with higher perceived stigma.

Conclusions

Perceived stigma towards leprosy was found highest among participants with age 61 years or older, longer duration of stay in community close to the leprosy colony, lower duration of education and participants who were unemployed had higher perceived stigma. Similarly, participants with perceptions of leprosy such as difficult to treat, severe disease and punishment by God had higher perceived stigma towards leprosy. There is an urgent need of stigma reduction strategies focused on education and awareness concerning leprosy.