c-erbB-3: a nuclear protein in mammary epithelial cells

c-erbB receptors are usually located in cell membranes and are activated by extracellular binding of EGF-like growth factors. Unexpectedly, using immunofluorescence we found high levels of c-erbB-3 within the nuclei of MTSV1-7 immortalized nonmalignant human mammary epithelial cells. Nuclear localization was mediated by the COOH terminus of c-erbB-3, and a nuclear localization signal was identified by site-directed mutagenesis and by transfer of the signal to chicken pyruvate kinase. A nuclear export inhibitor caused accumulation of c-erbB-3 in the nuclei of other mammary epithelial cell lines as demonstrated by immunofluorescence and biochemical cell fractionation, suggesting that c-erbB-3 shuttles between nuclear and nonnuclear compartments in these cells. Growth of MTSV1-7 on permeable filters induced epithelial polarity and concentration of c-erbB-3 within the nucleoli. However, the c-erbB-3 ligand heregulin beta1 shifted c-erbB-3 from the nucleolus into the nucleoplasm and then into the cytoplasm. The subcellular localization of c-erbB-3 obviously depends on exogenous stimuli and on the stage of epithelial polarity and challenges the specific function of c-erbB-3 as a transmembrane receptor protein arguing for additional, as yet unidentified, roles of c-erbB-3 within the nucle(ol)us of mammary epithelial cells.     

Follicular dendritic cell-mediated up-regulation of CXCR4 expression on CD4 T cells and HIV pathogenesis

Follicular dendritic cells (FDCs) represent a major reservoir of HIV, and active infection occurs surrounding these cells, suggesting that this microenvironment is highly conducive to virus transmission. Because CD4 T cells around FDCs in germinal centers express the HIV coreceptor, CXCR4, whereas CD4 lymphocytes in many other sites do not, it prompted the hypothesis that FDCs may increase CXCR4 expression on CD4 T cells, thereby facilitating infection. To test this, HIV receptor/coreceptor expression was determined on CD4 T cells cultured with or without FDCs, and its consequence to infection was assessed by measuring virus binding and entry. FDCs had little effect on CCR5 or CD4 expression but increased CXCR4 expression on CD4 T cells. FDC-mediated up-regulation of CXCR4 on CD4 T cells occurred by 24 h and was sustained for at least 96 h in vitro, and FDC-CD4 T cell contact was necessary. Importantly, increased CXCR4 expression directly correlated with increased binding and entry of HIV-1 X4 isolates. Furthermore, CD4(+)CD57(+) germinal center T cells expressed high levels of CXCR4 and supported enhanced entry of X4 HIV compared with other CD4 T cells from the same tissue. Thus, in addition to serving as a reservoir of infectious virus, FDCs render surrounding germinal center T cells highly susceptible to infection with X4 isolates of HIV-1.     

BAG4/SODD protein contains a short BAG domain

BAG (Bcl-2-associated athanogene) proteins are molecular chaperone regulators that affect diverse cellular pathways. All members share a conserved motif, called the BAG domain (BD), which binds to Hsp70/Hsc70 family proteins and modulates their activity. We have determined the solution structure of BD from BAG4/SODD (silencer of death domains) by multidimensional nuclear magnetic resonance methods and compared it to the corresponding domain in BAG1 (Briknarová, K., Takayama, S., Brive, L., Havert, M. L., Knee, D. A., Velasco, J., Homma, S., Cabezas, E., Stuart, J., Hoyt, D. W., Satterthwait, A. C., Llinás, M., Reed, J. C., and Ely, K. R. (2001) Nat. Struct. Biol. 8, 349-352). The difference between BDs from these two BAG proteins is striking, and the structural comparison defines two subfamilies of mammalian BD-containing proteins. One subfamily includes the closely related BAG3, BAG4, and BAG5 proteins, and the other is represented by BAG1, which contains a structurally and evolutionarily distinct BD. BDs from both BAG1 and BAG4 are three-helix bundles; however, in BAG4, each helix in this bundle is three to four turns shorter than its counterpart in BAG1, which reduces the length of the domain by one-third. BAG4 BD thus represents a prototype of the minimal functional fragment that is capable of binding to Hsc70 and modulating its chaperone activity.     

Viral infection of infants and children with benign transient hyperphosphatasemia

In this study, we screened serum samples for transient hyperphosphatasemia (TH) using cellulose acetate membrane electrophoresis over a period of 3 years. In the patients found to suffer from TH, we examined the relationship between the clinical condition and viral infection. The frequency of TH was 0.26%, and all of the cases detected were in infants or young children. The female to male ratio of TH was 1.29/1. While there was no clear seasonal fluctuation or periodicity in the appearance of TH, two peaks were recognized in spring and autumn. Research on the clinical manifestations clarified that most of the TH cases had infectious diseases of the upper airways accompanied by symptoms of fever and diarrhea. We examined antibody titers for viruses causing upper airway infectious diseases and identified antibodies for enteroviruses such as Echo 22, Entero 71, and Coxsackie B4. Our results suggested that TH might be caused by an infection of the enterovirus group.     

Chronic treatment with Carvedilol improves ventricular function and reducesmyocyte apoptosis in an animal model of heart failure

Background

β-blocker treatmenthas emerged as an effective treatment modality for heart failure.Interestingly, β-blockers can activate both pro-apoptotic and anti-apoptoticpathways. Nevertheless, the mechanism for improved cardiac functionseen with β-blocker treatment remains largely unknown. Carvedilolis a non-selective β-blocker with α-receptor blockade and antioxidantproperties. We therefore studied the impact of the effects of carvedilolin an animal model of end-stage heart failure.

Results

To test whetherchronic treatment with β-blockade decreases apoptosis, we treated myopathicturkeys with two dosages of carvedilol, 1 mg/kg (DCM₁)and 20 mg/kg (DCM₂₀), for four weeks and compared themto non-treated DCM animals (DCM₀) and to control turkeys(CON). Echocardiographic measurements showed that non-treated DCManimals had a significantly lower fractional shortening (FS) whencompared to CON (68.73 ± 1.37 vs. 18.76 ± 0.59%, p < 0.001). Bothdoses of carvedilol significantly improved FS (33.83 ± 10.11 and27.73 ± 6.18% vs. 18.76 ± 0.59 % for untreated DCM, p < 0.001).DCM left ventricles were characterized by a higher percentage ofapoptotic nuclei when compared to CON (5.64 ± 0.49 vs. 1.72 ± 0.12%,respectively p < 0.001). Both doses of carvedilol significantlyreduced the number of apoptotic nuclei (2.32 ± 0.23% and 2.36 ±0.26% 1 mg and 20 mg/kg respectively).

Conclusions

Carvedilol improvesventricular function. Furthermore, treatment with carvedilol decreasedthe incidence of apoptosis in cardiac myocytes from failing heartsat both doses. These data suggest that the inhibition of apoptosiswith carvedilol may lead to improvement in ventricular functionand may underlie a beneficial effect of β-blockade independent ofheart rate lowering effects.

     

Vitamin C (ascorbic acid) induced hydroxyl radical formation in copper contaminated household drinking water: role of bicarbonate concentration

We have previously shown that Vitamin C (ascorbic acid) can trigger hydroxyl radical formation in copper contaminated household drinking water. We report here that the capacity of ascorbic acid to catalyze hydroxyl radical generation in the drinking water samples is strongly dependent on the bicarbonate concentration (buffer capacity and pH) of the samples. We found that at least 50 mg/l bicarbonate was required in the water samples to maintain the pH over 5.0 after ascorbic acid addition. At this pH, that is higher than the pKa

1

4.25 of ascorbic acid, a hydroxyl radical generating redox cycling reaction involving the mono-anion of vitamin C and copper could take place. The ascorbic acid induced hydroxyl radical generating reaction could easily be mimicked in Milli-Q water by supplementing the water with copper and bicarbonate. Our results demonstrate that ascorbic acid can induce a pH dependent hydroxyl radical generating reaction in copper contaminated household tap water that is buffered with bicarbonate. The impact of consuming ascorbic acid together with copper and bicarbonate containing drinking water on human health is discussed.     

Effect of humoral immune responses on controlling viremia during primary infection of rhesus monkeys with simian immunodeficiency virus

Cellular immune responses mediated by CD8+ lymphocytes exert efficient control of virus replication during primary simian immunodeficiency virus (SIV) infection. However, the role that antibodies may play in the early control of virus replication remains unclear. To evaluate how antibody responses may affect virus replication during primary SIVmac infection, we depleted rhesus monkeys of B cells with anti-CD20 antibody. In normal rhesus monkeys immunized with tetanus toxoid, anti-CD20 treatment and resulting depletion of B cells inhibited the generation of antitetanus antibodies, while tetanus-specific T-cell responses were preserved. During the first 4 weeks after inoculation with SIVmac251, development of SIV-specific neutralizing antibody was delayed, and titers were significantly lower in B-cell-depleted monkeys than control-antibody-treated monkeys. Despite the lower neutralizing antibody titers, the levels of plasma SIV RNA and the linear slope of the decline seen in B-cell-depleted monkeys did not differ from that observed in monkeys treated with control antibody. However, beginning at day 28 after SIV infection, the B-cell-depleted monkeys showed a significant inverse correlation between neutralizing antibody titers and plasma virus level. These results suggest that the rapid decline of peak viremia that typically occurs during the first 3 weeks of infection was not significantly affected by SIV-specific antibodies. However, the inverse correlation between neutralizing antibodies and plasma virus level during the postacute phases of infection suggests that humoral immune responses may contribute to the control of SIV replication.     

EDEM contributes to maintenance of protein folding efficiency and secretory capacity

A stringent quality control process selects misfolded polypeptides generated in the endoplasmic reticulum (ER) for ER-associated degradation (ERAD). Here we assessed the maintenance of efficient glycoprotein folding in cells with defective ERAD caused by lack of adaptation of the intralumenal level of ER degradation-enhancing alpha-mannosidase-like protein (EDEM) to an increase in the ER cargo load. When these cells were converted into factories for production of high levels of human beta-secretase, maturation of this N-glycosylated aspartic protease progressed as in wild-type cells initially to gradually become less efficient. Up-regulation of EDEM to strengthen the ERAD machinery (but not up-regulation of calnexin to reinforce the folding machinery) was instrumental in maintaining folding efficiency and secretory capacity. Our data underscore the important role that the degradation machinery plays in maintaining a functional folding environment in the ER.     

Artificial genetic selection for an efficient translation initiation site for expression of human RACK1 gene in Escherichia coli

In bacterial expression systems, translation initiation is usually the rate limiting and the least predictable stage of protein synthesis. Efficiency of a translation initiation site can vary dramatically depending on the sequence context. This is why many standard expression vectors provide very poor expression levels of some genes. This notion persuaded us to develop an artificial genetic selection protocol, which allows one to find for a given target gene an individual efficient ribosome binding site from a random pool. In order to create Darwinian pressure necessary for the genetic selection, we designed a system based on translational coupling, in which microorganism survival in the presence of antibiotic depends on expression of the target gene, while putting no special requirements on this gene. Using this system we obtained superproducing constructs for the human protein RACK1 (receptor for activated C kinase).