Anti-IgM induces transforming growth factor-beta sensitivity in a human B-lymphoma cell line: inhibition of growth is associated with a downregulation of mutant p53

We wished to examine the role of transforming growth factor-beta (TGF- beta) in the regulation of human lymphoma cell growth. The RL cell line is an immunoglobulin M (IgM)+, IgD+ B lymphoma cell line, which does not constitutively express receptors for TGF-beta, and thus has lost the ability to respond to the inhibitory effects of TGF-beta. We demonstrate here that anti-Ig antibodies can efficiently upregulate the expression of TGF-beta receptors and promote sensitivity to growth inhibition by TGF-beta. Furthermore, because TGF-beta has been shown to function in late G1 of the cell cycle, we examined the ability of TGF- beta to modulate two tumor suppressor proteins known to be critical regulators of the G1/S transition, Rb and p53. Rb is a 105- to 110-kD phosphoprotein, which has been shown to maintain its growth suppressive function when it is found in the hypophosphorylated state. Wild-type p53 is a 53-kD phosphoprotein that appears to be important in preventing cell-cycle progression and promoting apoptosis in cells with DNA damage, whereas mutant p53 can overcome those functions. We show here that TGF-beta treatment of phorbol myristate acetate (PMA) or anti- Ig-activated RL cells results in growth inhibition through a dual effect on Rb and mutant p53. After TGF-beta treatment, we observe a predominance of Rb in the hypophosphorylated, growth suppressive form. In addition, we show a decrease in levels of mRNA and protein for mutant p53. We also show that, although these changes are sufficient to halt progression through the cell cycle, the cells do not appear to undergo extensive programmed cell death following 72 hours of TGF-beta treatment. Thus, although these lymphoma cells maintain the capacity to be negatively growth regulated by TGF-beta, the ability of TGF-beta to induce apoptosis must be independently controlled.     

The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm

Thioredoxin 1 is a major thiol-disulfide oxidoreductase in the cytoplasm of Escherichia coli. One of its functions is presumed to be the reduction of the disulfide bond in the active site of the essential enzyme ribonucleotide reductase. Thioredoxin 1 is kept in a reduced state by thioredoxin reductase. In a thioredoxin reductase null mutant however, most of thioredoxin 1 is in the oxidized form; recent reports have suggested that this oxidized form might promote disulfide bond formation in vivo. In the Escherichia coli periplasm, the protein disulfide isomerase DsbC is maintained in the reduced and active state by the membrane protein DsbD. In a dsbD null mutant, DsbC accumulates in the oxidized form. This oxidized form is then able to promote disulfide bond formation. In both these cases, the inversion of the function of these thiol oxidoreductases appears to be due to an altered redox balance of the environment in which they find themselves. Here, we show that thioredoxin 1 attached to the alkaline phosphatase signal sequence can be exported into the E. coli periplasm. In this new environment for thioredoxin 1, we show that thioredoxin 1 can promote disulfide bond formation and, therefore, partially complement a dsbA strain defective for disulfide bond formation. Thus, we provide evidence that by changing the location of thioredoxin 1 from cytoplasm to periplasm, we change its function from a reductant to an oxidant. We conclude that the in vivo redox function of thioredoxin 1 depends on the redox environment in which it is localized.     

A mutant hunt for defects in membrane protein assembly yields mutations affecting the bacterial signal recognition particle and Sec machinery

We describe an Escherichia coli genetic screen that yields mutations affecting two different cellular processes: disulfide bond formation and membrane protein assembly. The mutants defective in disulfide bond formation include additional classes of dsbA and dsbB mutations. The membrane protein assembly defective mutants contain a mutation in the secA operon and three mutations in the ffs gene, which encodes 4.5S RNA. These latter mutations are the only ones to be isolated in a gene encoding a component of the bacterial signal recognition particle by screening in vivo for defects in membrane protein insertion. A sensitive method for examining membrane protein localization shows that the ffs and secA locus mutations affect membrane assembly of the polytopic membrane protein, MalF. The ffs mutations also affect the membrane insertion of the FtsQ and the AcrB proteins. Although both the ffs and the secA locus mutations interfere with membrane protein assembly, only the latter also reduces export of a protein containing a cleavable signal sequence.     

Evidence that the pathway of disulfide bond formation in Escherichia coli involves interactions between the cysteines of DsbB and DsbA.

Disulfide bond formation is catalyzed in the periplasm of Escherichia coli. This process involves at least two proteins: DsbA and DsbB. Recent evidence suggests that DsbA, a soluble periplasmic protein directly catalyzes disulfide bond formation in proteins, whereas DsbB, an inner membrane protein, is involved in the reoxidation of DsbA. Here we present direct evidence of an interaction between DsbA and DsbB. (Kishigami et al. [Kishigami, S., Kanaya, E., Kikuchi, M. & Ito, K. (1995) J. Biol. Chem. 270, 17072-17074] have described similar findings.) We isolated a dominant negative mutant of dsbA, dsbAd, where Cys-33 of the DsbA active site is changed to tyrosine. Both DsbAd and DsbA are able to form a mixed disulfide with DsbB, which may be an intermediate in the reoxidation of DsbA. This complex is more stable with DsbAd. The dominance can be suppressed by increasing the production of DsbB. By using mutants of DsbB in which one or two cysteines have been changed to alanine, we show that only Cys-104 is important for complex formation. Therefore, we suggest that in vivo, reduced DsbA forms a complex with DsbB in which Cys-30 of DsbA is disulfide-bonded to Cys-104 of DsbB. Cys-104 is rapidly replaced by Cys-33 of DsbA to generate the oxidized form of this protein.     

Syndemic effect of mental illness and substance use on viral suppression among recently-incarcerated,HIV-infected individuals in the CARE+ Corrections study

Few studies on HIV-related syndemics of co-occurring and mutually reinforcing psychosocial conditions have assessed clinical outcomes in criminal justice (CJ)-involved populations. Baseline data from the CARE+ Corrections study were used to quantify co-occurring mental illness and substance use and examine syndemic effects on viral suppression among 106 CJ-involved HIV-infected individuals. Ninety-one (86%) reported a mental illness diagnosis, 30 (28%) reported hazardous alcohol use, and 61 (58%) were drug dependent. Eighteen (17%) experienced all three conditions. Drug dependence was clustered with mental illness (prevalence odds ratio [POR] 3.20, 95% CI 1.01–10.14) and hazardous alcohol use (POR 2.61, 95% CI 1.03–6.56). The association between syndemic score, representing the number of conditions reported by each individual, and viral suppression was not statistically significant, although 86% of participants with none of these conditions were virally suppressed, compared to 56% of those with all three (p?=?0.56). Mental illness and substance use were concentrated in this sample, indicating a need for integrated care services.     

Estimated Brain Tissue Response Following Impacts Associated With and Without Diagnosed Concussion

Kinematic measurements of head impacts are sensitive to sports concussion, but not highly specific. One potential reason is these measures reflect input conditions only and may have varying degrees of correlation to regional brain tissue deformation. In this study, previously reported head impact data recorded in the field from high school and collegiate football players were analyzed using two finite element head models (FEHM). Forty-five impacts associated with immediately diagnosed concussion were simulated along with 532 control impacts without identified concussion obtained from the same players. For each simulation, intracranial response measures (max principal strain, strain rate, von Mises stress, and pressure) were obtained for the whole brain and within four regions of interest (ROI; cerebrum, cerebellum, brain stem, corpus callosum). All response measures were sensitive to diagnosed concussion; however, large inter-athlete variability was observed and sensitivity strength depended on measure, ROI, and FEHM. Interestingly, peak linear acceleration was more sensitive to diagnosed concussion than all intracranial response measures except pressure. These findings suggest FEHM may provide unique and potentially important information on brain injury mechanisms, but estimations of concussion risk based on individual intracranial response measures evaluated in this study did not improve upon those derived from input kinematics alone.