cAMP mediates transepithelial K+ and Na+ transport in a strial marginal cell line

The recently described gastrointestinal glutathione peroxidase (GI-GPx) is the fourth member of the family of the selenoenzymes glutathione peroxidases (GPx). In contrast to the more uniform distribution of, for example, the classical glutathione peroxidase (cGPx), it is expressed exclusively in the gastrointestinal tract and has, therefore, been suggested to function as a primary barrier against alimentary hydroperoxides. In order to get an idea of its relative importance we investigated its position in the hierarchy of selenoprotein expression. The selenium-dependent expression of GI-GPx was analyzed in comparison with that of other GPx types at the level of mRNA and protein in HepG2 and CaCo-2 cells. Furthermore, the selenocysteine insertion sequence (SECIS) efficiencies of GI-GPx, phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cGPx in response to selenium were determined by a reporter-gene assay in human hepatoma cells and baby hamster kidney cells. GI-GPx mRNA levels increased during selenium deficiency, whereas cGPx mRNA levels decreased and PHGPx mRNA levels remained almost unaffected. In cells grown in selenium-poor media, all GPx-types were low in both activity and immunochemical reactivity. Upon selenium repletion immunoreactive GI-GPx protein reached a plateau after 10 h, whereas cGPx started to be expressed at 24 h and did not reach its maximum level before 3 days. SECIS efficiencies decreased in the order PHGPx > cGPx > GI-GPx. The augmentation of SECIS efficiencies by selenium was highest for cGPx and intermediate for PHGPx, whereas it was marginal for GI-GPx. The high mRNA stability under selenium restriction, the speed of biosynthesis upon selenium repletion and the marginal effect of selenium on the SECIS efficiency indicate that of the GPx isotypes, GI-GPx ranks highest in the hierarchy of selenoproteins and point to a vital role of GI-GPx in the gastrointestinal tract.     

Repeated immunization with recombinant gp160 human immunodeficiency virus (HIV) envelope protein in early HIV-1 infection: evaluation of the T cell proliferative response

This longitudinal study was designed to evaluate cellular immunity in early-stage, asymptomatic human immunodeficiency virus (HIV)-1-infected persons (CD4 cell count,>400/mm3; median, 625/mm3) who were immunized with either recombinant (r) gp160 or placebo every 2 months for 5 years. Proliferative responses were assessed against rgp160, rp24, and a panel of recall antigens and mitogens. Despite good reactivity to recall antigens, at baseline approximately 33% had proliferative responses to gp160, and approximately 42% showed p24 gag responses. There was no statistical difference between vaccine and placebo groups for antigens or mitogens. After 1 year, approximately 73% of the subjects in the vaccine arm had new or boosted responses to gp160, versus approximately 18% in the placebo arm. Statistical significance was maintained throughout the study. Recurrent vaccination with recombinant gp160 was proven to be persistently immunogenic, increasing significantly the ability of HIV-1-infected persons to mount new proliferative responses to the vaccine.     

Kinematics of cervical spine discectomy with and without bone grafting: quantitative evaluation of late fusion in a sheep model

OBJECTIVE: This study was conducted to evaluate the kinematic response of late fusion results for cervical spine discectomies with and without bone grafting. MATERIALS AND METHODS: Fifteen Barbados Black Belly sheep underwent sham operations (Group A, n = 5), C2-C3 discectomies only (Group B, n = 5), and C2-C3 discectomies with autologous iliac bone grafting (Group C, n = 5). Ten months after surgery, the animals were killed. Fresh ligamentous spines (C1-C5) were subjected to the relevantly applied loads through a loading frame attached to the C1. Each vertebra (from C2 to C4) was attached with a set of three infrared light-emitting diodes to record the spatial location relating to each load application using a Selspot II system (Selcom Selective Electronics, Inc., Valdese, NC). The load-deformation data of the C2-C3 and C3-C4 motion segments were recorded and analyzed for the three groups. RESULTS: At the C2-C3 motion segment, the results indicated that Group B displayed larger motion ranges of rotation and lateral bending loads than did the other two groups. Significantly larger motion ranges of rotation loads were found in Group B than in Group C (P<0.05, for both comparisons). In contrast, Group C had the smallest motion ranges of flexion, lateral bending, and rotation loads. At the C3-C4 motion segment, both groups that had undergone discectomies had a significantly larger motion range of flexion load compared with Group A (P<0.05, for both comparisons). A significant increase in the motion range of right axial rotation was found in Group B (P<0.05), but not in Group C, compared with Group A. Group B exhibited larger motion ranges responding to all six tested loads than did Group C. CONCLUSION: The results indicate that anterior fusion after C2-C3 cervical discectomies, regardless of the presence or absence of bone grafting, decreases the motion range of flexion load at the C2-C3 motion segment, and contrary data were seen at the C3-C4 motion segment. For axial rotation loads, discectomies without bone grafting resulted in increased motion ranges of both C2-C3 and C3-C4 motion segments whereas discectomies with bone grafting did not. The data may have clinical relevance regarding the role of bone grafting in cases of cervical spine disease.     

Isolation, amino acid sequence determination and binding properties of two fatty-acid-binding proteins from axolotl (Ambistoma mexicanum) liver. Evolutionary relationship

Up until now, the primary structure of fatty-acid-binding proteins (FABPs) from the livers of four mammalian (rat, human, cow and pig) and three nonmammalian (chicken, catfish and iguana) species has been determined. Based on amino acid sequence comparisons, it has been suggested that mammalian and nonmammalian liver FABPs may be paralogous proteins that originated by gene duplication, rather than as a consequence of mutations of the same gene. In this paper we report the isolation and amino acid sequence determination of two FABPs from axolotl (Ambistoma mexicanum) liver. One of them is similar to mammalian liver FABPs (L-FABPs) and the other to chicken, catfish and iguana liver FABPs (Lb-FABPs). The finding of both L-FABP and Lb-FABP in a single species, as reported here, indicates that they are paralogous proteins. The time of divergence of these two liver FABP types is estimated to be of approximately 694 million years ago. The ligand-binding properties of axolotl liver FABPs were studied by means of parinaric-acid-binding and parinaric-acid-displacement assays. L-FABP binds two fatty acids per molecule but Lb-FABP displays a fatty-acid-conformation-dependent binding stoichiometry; L-FABP shows a higher affinity for fatty acids, especially oleic acid, while Lb-FABP has a higher affinity for other hydrophobic ligands, especially retinoic acid. In addition, the tissue-expression pattern is different, L-FABP is present in liver and intestinal mucosa while the expression of Lb-FABP is restricted to liver. Data indicate distinct functional properties of both liver FABP types.     

Analysis of lichen thin sections by PIXE and STIM using a proton microprobe

In order to better understand the distribution pattern of mineral elements in lichen tissues, thin sections (15 μm) of the foliose, vagrant soil lichen Xanthoparmelia chlorochroa were examined using proton microprobe Particle induced X-ray emission (PIXE). This technique was used to make two-dimensional scans, with 5 μm resolution, across tissue cross sections of the test species. Element maps for Si, P, S, Cl, K, Ca, Ti, Mn, Fe, Cu, Zn, and As have been prepared. Several elements are strongly localized in the element maps. PIXE data are complimented with STIM, light micrographs, and SEM images. Preliminary data suggest that nuclear microprobe techniques may be useful in elucidating element absorption and transport mechanisms in lichens.     

In vivo microdialysis of 2-deoxyglucose 6-phosphate into brain: a novel method for the measurement of interstitial pH using 31P-NMR

A unique method for simultaneously measuring interstitial (pHe) as well as intracellular (pHi) pH in the brains of lightly anesthetized rats is described. A 4-mm microdialysis probe was inserted acutely into the right frontal lobe in the center of the area sampled by a surface coil tuned for the collection of 31P-NMR spectra. 2-Deoxyglucose 6-phosphate (2-DG-6-P) was microdialyzed into the rat until a single NMR peak was detected in the phosphomonoester region of the 31P spectrum. pHe and pHi values were calculated from the chemical shift of 2-DG-6-P and inorganic phosphate, respectively, relative to the phosphocreatine peak. The average in vivo pHe was 7.24+/-0.01, whereas the average pHi was 7.05+/-0.01 (n = 7). The average pHe value and the average CSF bicarbonate value (23.5+/-0.1 mEq/L) were used to calculate an interstitial Pco2 of 55 mm Hg. Rats were then subjected to a 15-min period of either hypercapnia, by addition of CO2 (2.5, 5, or 10%) to the ventilator gases, or hypocapnia (PCO2 < 30 mm Hg), by increasing the ventilation rate and volume. pHe responded inversely to arterial Pco2 and was well described (r2 = 0.91) by the Henderson-Hasselbalch equation, assuming a pKa for the bicarbonate buffer system of 6.1 and a solubility coefficient for CO2 of 0.031. This confirms the view that the bicarbonate buffer system is dominant in the interstitial space. pHi responded inversely and linearly to arterial PCO2. The intracellular effect was muted as compared with pHe (slope = -0.0025, r2 = 0.60). pHe and pHi values were also monitored during the first 12 min of ischemia produced by cardiac arrest. pHe decreases more rapidly than pHi during the first 5 min of ischemia. After 12 min of ischemia, pHe and pHi values were not significantly different (6.44+/-0.02 and 6.44+/-0.03, respectively). The limitations, advantages, and future uses of the combined microdialysis/31P-NMR method for measurement of pHe and pHi are discussed.     

Increased NAD(P)H oxidase-mediated superoxide production in renovascular hypertension: evidence for an involvement of protein kinase C

BACKGROUND: Angiotensin II infusion has been shown to cause hypertension and endothelial dysfunction and to increase superoxide (O-.2) production in vascular tissue, mainly via an activation of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-dependent oxidase, the most significant O-.2 source in endothelial and/or smooth muscle cells. With these studies, we sought to determine whether endothelial dysfunction in renovascular hypertension is secondary to an activation of these oxidases. METHODS: Endothelial function in aortas from rats with two kidney-one clip (2K-1C) hypertension and age-matched controls was assessed using isometric tension studies in organ chambers. Changes in vascular O-.2 production were measured using lucigenin-enhanced chemiluminescence and electron spin resonance spectroscopy. RESULTS: In hypertensive animals, relaxation to endothelium-dependent (acetylcholine) and endothelium-independent nitrovasodilators (nitroglycerin) was impaired. Constriction to a direct activator of protein kinase C (PKC) phorbol ester 12,13 dibutyrate (PDBu) was enhanced, and vascular O-.2 was significantly increased compared with controls. Vascular O-.2 was normalized by the PKC inhibitor calphostin C, by the inhibitor of flavin-dependent oxidases, diphenylene iodonium, and recombinant heparin-binding superoxide dismutase, whereas inhibitors of the xanthine oxidase (oxypurinol), nitric oxide synthase (NG-nitro-l-arginine) and mitochondrial NADH dehydrogenase (rotenone) were ineffective. Studies of vascular homogenates demonstrated that the major source of O-.2 was a NAD(P)H-dependent oxidase. Incubation of intact tissue with PDBu markedly increased O-. 2, the increase being significantly stronger in vessels from hypertensive animals as compared with vessels from controls. Endothelial dysfunction was improved by preincubation of vascular tissue with superoxide dismutase and calphostin C. CONCLUSIONS: We therefore conclude that renovascular hypertension in 2K-1C rats is associated with increased vascular O-.2 leading to impaired vasodilator responses to endogenous and exogenous nitrovasodilators. Increased vascular O-.2 is likely secondary to a PKC-mediated activation of a membrane-associated NAD(P)H-dependent oxidase.     

Evaluation of the natural virucidal activity of teas for use in the phage amplification assay

Many natural products have intrinsic antimicrobial activity. In this study we have examined infusions from nine types of loose-leaf tea for their ability to inactivate bacteriophage, for use as an alternative to plant extract in a phage-based Salmonella detection assay. The results demonstrated that tea infusions, either freshly prepared or stored at 4 degrees C had virucidal action against two phages, Felix 01 and P22. Crucially, for use in the detection assay, there was no antibacterial effect of the virucide on the target bacteria. Therefore, tea was a good candidate to replace pomegranate as the virucidal agent in the phage amplification assay.     

The effect of conducting bounding plates on the onset of Horton–Rogers–Lapwood convection

We present an analytical and numerical stability analysis of the onset of natural convection in a horizontal fluid-saturated porous cavity. The cavity is bounded by thin horizontal plates with uniform thickness whose outer surfaces are subject to a constant heat flux. The main aim is to determine the effect of the presence of the bounding plates on the onset of convection. The onset criterion is found to be sensitively dependent on the relative thickness of the plates and the porous layer, δ, and their relative conductivities, d. For the long wavelength mode it is precisely Ra_c = 12(1 + 2δd).