Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.     

Bacterial species and evolution: Theoretical and practical perspectives

A discussion of the species problem in modern evolutionary biology serves as the point of departure for an exploration of how the basic science aspects of this problem relate to efforts to map bacterial diversity for practical pursuits—for prospecting among the bacteria for useful genes and gene-products. Out of a confusing array of species concepts, the Cohesion Species Concept seems the most appropriate and useful for analyzing bacterial diversity. Techniques of allozyme analysis and DNA fingerprinting can be used to put this concept into practice to map bacterial genetic diversity, though the concept requires minor modification to encompass cases of complete asexuality. Examples from studies of phenetically definedBacillus species provide very partial maps of genetic population structure. A major conclusion is that such maps frequently reveal deep genetic subdivision within the phenetically defined specles; divisions that in some cases are clearly distinct genetic species. Knowledge of such subdivisions is bound to make prospecting within bacterial diversity more effective. Under the general concept of genetic cohesion a hypothetical framework for thinking about the full range of species conditions that might exist among bacteria is developed and the consequences of each such model for species delineation, and species identification are discussed. Modes of bacterial evolution, and a theory of bacterial speciation with and without genetic recombination, are examined. The essay concludes with thoughts about prospects for very extensive mapping of bacterial diversity in the service of future efforts to find useful products. In this context, evolutionary biology becomes the handmaiden of important industrial activities. A few examples of past success in commercializing bacterial gene-products from species ofBacillus and a few other bacteria are reviewed.     

Hormone-induced conformational changes in the hepatic insulin receptor

The insulin receptor can exist in either a lower or a higher affinity state. Hormone binding alters the equilibrium between the two states of the insulin receptor, favoring the formation of that of higher affinity (Corin, R.E., and Donner, D.B. (1982), J. Biol. Chem. 257, 104-110). After brief or extended incubations with hormone, during which the fraction of higher affinity receptors increased, 125I-insulin was covalently coupled to the alpha subunits of its receptor using disuccinimidyl suberate. Some 125I-insulin remained bound to higher affinity receptors after dissociation of hormone from lower affinity sites. This hormone could also be covalently coupled to the alpha subunit of the receptor. During extended incubations between 125I-insulin and liver plasma membranes, components of the receptor were cleaved to yield degradation products of 120,000 and 23,000 Da. The significance of this process remains undetermined. Unoccupied insulin receptors were cleaved by trypsin to produce fragments of 94,000 and 37,000 Da which remained membrane-bound and could be covalently coupled to 125I-insulin. Trypsin treatment after binding yielded an additional receptor fragment of 64,000 Da. As the incubation time between 125I-insulin and membranes was lengthened, components of the receptor became progressively less sensitive to trypsin. Higher affinity binding sites isolated after release of rapid dissociating insulin were less sensitive to trypsin than were mixtures of higher and lower affinity receptors. These observations suggest that hormone binding produces two conformational changes (alterations of tryptic lability) in the hepatic insulin receptor. The first change is rapid and exposes parts of the receptor to tryptic degradation. The second, slower conformational change renders the receptor less sensitive to trypsin and occurs with the same time course as the increase of receptor affinity mediated by site occupancy.     

TLR ligand-dependent activation of naive CD4 T cells by plasmacytoid dendritic cells is impaired in hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling, we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C), R-848, or CpG), in the presence or absence of cytokine (TNF-alpha or IL-3), and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-gamma. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons, the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-alpha did not differ between HCV-infected and healthy control subjects. In contrast, PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-alpha) expression upon R-848 stimulation, and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection.     

Selective impairments in dendritic cell-associated function distinguish hepatitis C virus and HIV infection

Impaired APC functions may play important roles in chronicity of hepatitis C virus (HCV) and HIV infections. To investigate the separate and combined effects of HCV and HIV infection on immature dendritic cells (DCs), we evaluated myeloid-derived DC (MDC) and plasmacytoid-derived DC (PDC) frequencies and functions, measured by Toll-like receptor ligand-induced IFN-alpha and IL-12, in healthy controls and subjects with chronic HCV, HIV, and HCV-HIV infection. To evaluate the relation between innate and adaptive immunity, we measured HCV-specific IFN-gamma-producing T cell frequency. MDC frequencies tended to be reduced in HIV infection (1.8-fold), while PDC frequencies were minimally reduced in HCV infection (1.4-fold). In contrast, a striking reduction in non-PDC-associated IFN-alpha production was observed in HIV-infected subjects (17-fold), while PDC-associated IFN-alpha production was markedly reduced in HCV-infected subjects (20-fold). Both non-PDC and PDC functions were impaired in HCV-HIV coinfection. MDC-associated IL-12 production was markedly reduced in both HCV and HIV-infected subjects (over 10-fold). Functional defects were attenuated with slowly progressive HIV infection. The proportion of subjects with HCV-specific T cell responses, and the number of Ags recognized were reduced in HCV-HIV subjects as compared with HCV singly infected subjects. A positive association was observed between MDC-associated IL-12 production and HCV-specific T cell frequency in HCV-infected subjects. These results indicate that immature DC function is dysregulated in HIV and HCV infections, but differentially, and that these defects are attenuated in slowly progressive HIV infection. These selectively different impairments may contribute to the reduced adaptive immune response to HCV in HCV-HIV coinfection.