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.     

PD-1 Expression and Cytokine Secretion Profiles of Mycobacterium tuberculosis-Specific CD4+ T-Cell Subsets; Potential Correlates of Containment in HIV-TB Co-Infection

HIV co-infection is an important risk factor for tuberculosis (TB) providing a powerful model in which to dissect out defective, protective and dysfunctional Mycobacterium tuberculosis (MTB)-specific immune responses. To identify the changes induced by HIV co-infection we compared MTB-specific CD4+ responses in subjects with active TB and latent TB infection (LTBI), with and without HIV co-infection. CD4+ T-cell subsets producing interferon-gamma (IFN-γ), interleukin-2 (IL-2) and tumour necrosis factor-alpha (TNF-α) and expressing CD279 (PD-1) were measured using polychromatic flow-cytometry. HIV-TB co-infection was consistently and independently associated with a reduced frequency of CD4+ IFN-γ and IL-2-dual secreting T-cells and the proportion correlated inversely with HIV viral load (VL). The impact of HIV co-infection on this key MTB-specific T-cell subset identifies them as a potential correlate of mycobacterial immune containment. The percentage of MTB-specific IFN-γ-secreting T-cell subsets that expressed PD-1 was increased in active TB with HIV co-infection and correlated with VL. This identifies a novel correlate of dysregulated immunity to MTB, which may in part explain the paucity of inflammatory response in the face of mycobacterial dissemination that characterizes active TB with HIV co-infection.     

Laboratory Studies with a Selective Enterococcus Medium

Lancefield group D streptococci are involved with appreciable frequency in a variety of infectious processes. The presumptive recognition of these bacteria on initial culturing of clinical specimens is an objective not attained readily by selective media available in the clinical laboratory. Selective Enterococcus agar was evaluated with emphasis on its ability to sequester enterococci from specimens with many microbial components. In addition, the sensitivity of this new agar was compared with Trypticase Soy agar containing sheep blood and Mitis Salivarius agar. All enterococci isolated from clinical material were classified in accordance with accepted biochemical and immunochemical criteria. The enterococci grew on the new medium as distinctive colonies surrounded by a black zone. Only Listeria monocytogenes presented similar colonial morphology after 48 hr. Most other bacteria did not grow at all or appeared markedly different. The sensitivity of the new agar was of the same order of magnitude as on blood or Mitis-Salivarius agars, but its selectivity was superior.     

Segmental motion in catalysis: investigation of a hydrogen bond critical for loop closure in the reaction of triosephosphate isomerase.

A residue essential for proper closure of the active-site loop in the reaction catalyzed by triosephosphate isomerase is tyrosine-208, the hydroxyl group of which forms a hydrogen bond with the amide nitrogen of alanine-176, a component of the loop. Both residues are conserved, and mutagenesis of the tyrosine to phenylalanine results in a 2000-fold drop in the catalytic activity (kcat/Km) of the enzyme compared to the wild-type isomerase. The nature of the closure process has been elucidated from both viscosity dependence and primary isotope effects. The reaction catalyzed by the mutant enzyme shows a viscosity dependence using glycerol as the viscosogen. This dependence can be attributed to the rate-limiting motion of the active-site loop between the "open" and the "closed" conformations. Furthermore, a large primary isotope effect is observed with [1-2H]dihydroxyacetone phosphate as substrate [(kcat/Km)H/(kcat/Km)D = 6 +/- 1]. The range of isotopic experiments that were earlier used to delineate the energetics of the wild-type isomerase has provided the free energy profile of the mutant enzyme. Comparison of the energetics of the wild-type and mutant enzymes shows that only the transition states flanking the enediol intermediate have been substantially affected. The results suggest either that loop closure and deprotonation are coupled and occur in the same rate-limiting step or that these two processes happen sequentially but interdependently. This finding is consistent with structural information that indicates that the catalytic base glutamate-165 moves 2 A toward the substrate upon loop closure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytokine regulation of complement receptor-mediated ingestion by mouse peritoneal macrophages. M-CSF and IL-4 activate phagocytosis by a common mechanism requiring autostimulation by IFN-beta

Macrophage CSF (M-CSF, CSF-1) and IL-4 are two cytokines known to have effects on mature monocytic phagocytes in vitro. In this report we show that M-CSF and IL-4 activate resident mouse peritoneal macrophages to ingest particles via their C3b and C3bi receptors, which are not capable of mediating ingestion in resting cells. IgG-mediated ingestion was also increased by IL-4 and M-CSF. IL-1, IL-2, TNF-alpha, and IFN-gamma were not able to stimulate C receptor-mediated ingestion. Stimulation by IL-4 and M-CSF is dependent upon high cell density and greater than 24-h exposure to the cytokine. Interestingly, antibody to IFN-alpha/beta and mAb to IFN-beta inhibited the enhanced ingestion caused by both M-CSF and IL-4. However, neither IFN-alpha nor IFN-beta alone stimulated C receptor-mediated ingestion. M-CSF did not affect the ligand-independent distribution of CR3 on the macrophage surface. We conclude that two apparently unrelated cytokines, M-CSF and IL-4, both enhance macrophage phagocytosis of C and IgG-coated targets via a common pathway in which autocrine stimulation with IFN-alpha/beta is necessary but not sufficient.     

Paracloacal glands of Alligator mississippiensis: A histological and histochemical study

The histology of the paracloacal 'musk' glands of adult American alligators ( Alligator mississippiensis ) is described. The gland is a single secretory sac with a single duct and a central lumen partially occluded by a central, cylindrical conglomerate of cells and secretion product. The capsule of the gland consists of an outer layer of smooth muscle and an inner layer of connective tissue containing collagen and elastin fibres. Septa carrying blood vessels radiate from the connective tissue layer of the capsule to the border of the central conglomerate. Parenchymal cells containing lipid droplets enlarge from the periphery to the centre of the gland. Secretions formed by degeneration of cells in the central cylinder are concentrated near the secretory duct. Histochemical tests indicate lipids but not mucopolysaccharides in the glandular exudate.     

Detecting proteoglycans immobilized on positively charged nylon

Proteoglycans (PG) immobilized on positively charged Nylon 66 are detected readily by staining with Alcian blue. With the exception of hyaluronic acid, free glycosaminoglycans appear unreactive when treated similarly. Immobilization was performed by dot blotting or by electrophoretic transblotting from various gel supports. When transblotted to positively charged Nylon 66 from large-pore agarose-acrylamide gels, levels of 10-50 ng of PG could be detected by Alcian blue staining. This procedure appeared to be nearly 10(2) times more sensitive than staining of gels with toluidine blue. The transblot and staining procedure also appears to be effective with PG separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was applied to a preparation enriched in basement membrane components.     

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution^1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions ^7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin ¹⁸, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH < 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.