Characterization of acetate uptake by the colonic epithelial cells of the rat

The characteristics of acetate uptake by colonic epithelial cells of the rat were studied. Clear saturation kinetics of acetate uptake were not observed in these experiments at either 0° C or 30° C. A decrease in the pH of the medium markedly increased the acetate uptake. The activation energy for acetete uptake derived from an Arrhenius plot was about 6.1 kcal/mole. Among the inhibitors tested, no effective inhibition of acetate uptake at 0° C was observer. Metabolic inhibitors severely inhibited transport at 30° C. Inhibition of acetate uptake by other short chain fatty acids, which was non-competitive, was observed. The finding that efflux from the cells was stimulated in the presence of compounds such as pyruvate and bicarbonate supported the notion of a close interrelationship between weak electrolyte transports in vivo. Although the H⁺ gradient across the cell membrane is suggested to be one of the factors determining the uptake rate, it seems difficult to explain all the results in this way.     

The finite element model for the propagation of light in scattering media: a direct method for domains with nonscattering regions

We present a method for handling nonscattering regions within diffusing domains. The method develops from an iterative radiosity-diffusion approach using Green's functions that was computationally slow. Here we present an improved implementation using a finite element method (FEM) that is direct. The fundamental idea is to introduce extra equations into the standard diffusion FEM to represent nondiffusive light propagation across a nonscattering region. By appropriate mesh node ordering the computational time is not much greater than for diffusion alone. We compare results from this method with those from a discrete ordinate transport code, and with Monte Carlo calculations. The agreement is very good, and, in addition, our scheme allows us to easily model time-dependent and frequency domain problems.     

Effects of anoxia and recovery on the neurotransmission and level of high-energy phosphates in thin hippocampal slices from the guinea-pig

The effects of anoxia and recovery on the neuronal transmission and the levels of high-energy phosphates such as ATP and phosphocreatine were studied using thin hippocampal slices from the guinea-pig. For the index of neuronal activity, postsynaptic field potentials were recorded in the CA3 and CA4 regions after electrical stimulation to the dentate gyrus during deprivation of oxygen and glucose from the perfusion medium at 36.5 degrees C. With deprivation of both oxygen and glucose from the medium, neuronal activity was abolished in 6-8 min. When the deprivation period was extended longer than 15 min, no recovery in the postsynaptic field potentials was observed. The concentrations of ATP and phosphocreatine in the slices decreased to 30-40% of original levels after 10 min deprivation of oxygen and glucose. ATP and phosphocreatine recovered to the original levels with the readmission of oxygen and glucose after 10 min anoxia, but the recovery of the ATP was worsened by the longer period of deprivation. Deprivation of oxygen only slowly decreased the amplitude of postsynaptic field potentials and blocked the neuronal activity after 70 min deprivation. The postsynaptic field potentials did not reappear after 180 min deprivation of oxygen. Even 120 min after deprivation of oxygen, the ATP and phosphocreatine levels were maintained at 60-70% of originals, whereas they both decreased to 30% after 150 min anoxia. The recovery of ATP even after 150 min anoxia was 64% and the recovery of phosphocreatine was over 100% even after 180 min anoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of phospholipase inhibitors in long-term potentiation in the rat hippocampal mossy fiber synapses and Schaffer/commissural synapses

Bath application of the inhibitors of phospholipases, nordihydroguaiaretic acid (NDGA) and p-bromophenacyl bromide (BPB), to the rat hippocampal slices suppressed long-term potentiation (LTP) in Schaffer/commissural-CA1 pyramidal synapses. On the other hand, neither of the two inhibitors suppressed LTP in mossy fiber-CA3 pyramidal cell synapses. BPB did not suppress phosphatidylinositol-specific phospholipase C (PI-PLC) activity of the slices. These results suggested that the mechanisms of LTP were quite different in the CA1 and CA3 subfields of rat hippocampus: in CA1, the involvement of an arachidonate metabolism was strongly suggested, whereas in CA3, an arachidonic acid cascade may not be necessary for LTP.     

Expression of tumor necrosis factor ligand superfamily costimulatory molecules CD27L, CD30L, OX40L and 4-1BBL in the heart of patients with acute myocarditis and dilated cardiomyopathy.

BACKGROUND: T-cell-mediated myocardial damage is known to be involved in acute myocarditis and dilated cardiomyopathy. Recently, we found that tumor necrosis factor (TNF) ligand superfamily costimulatory molecules, especially 4-1BBL, played an important role in the myocardial damage of murine acute viral myocarditis. METHODS AND RESULTS: To investigate the roles for CD27L, CD30L, OX40L and 4-1BBL, which belong to TNF ligand superfamily, in the development of acute myocarditis and dilated cardiomyopathy, we analyzed the expression of these antigens in the myocardial tissues of patients with acute myocarditis and dilated cardiomyopathy. We also examined expression of the receptors for these molecules, CD27, CD30, OX40 and 4-1BB, which belong to TNF receptor superfamily, on the infiltrating cells. Strong expression of CD27L, CD30L and 4-1BBL and weak to moderate expression of OX40L was found in the cardiac myocytes of patients with acute myocarditis. Moderate expression of CD27L, CD30L and 4-1BBL and weak expression of OX40L was found on the cardiac myocytes of patients with dilated cardiomyopathy. Most of the infiltrating cells expressed CD27, CD30 and 4-1BB and a part of the infiltrating cells expressed OX40. CONCLUSIONS: Our findings suggest that expression of TNF ligand superfamily costimulatory molecules on cardiac myocytes may play a role in the cell-mediated myocardial damage in patients with acute myocarditis and dilated cardiomyopathy as in murine viral myocarditis.     

Specific Interaction of a 25-kilodalton Cellular Protein,a 40S Ribosomal Subunit Protein,with the Internal Ribosome Entry Site of Hepatitis C Virus Genome

Translation initiation of hepatitis C virus (HCV) RNA is controlled by an internal ribosome entry site (IRES) contained in 5 noncoding region (NCR) and in several nucleotides of the coding region. The ability of a 25-kilodalton cellular protein (p25) to bind the HCV 5 NCR is correlated with the efficiency of translation initiation of HCV RNA, indicating that this protein plays a critical role in HCV translation (S. Fukushi, C. Kurihara, N. Ishiyama, F. B. Hoshino, A. Oya, and K. Katayama, J Virol 71, 1662–1666, 1997). We have extended the study for identification of the IRES region required for p25 binding. For this purpose, we have performed UV cross-linking competition analyses using 5- or 3- deleted mutants of the HCV 5 NCR as competitor RNAs for binding of p25 to wild-type HCV 5 NCR. Competitor RNAs lacking nucleotides (nt) 47–74 or nt 279–331 did not inhibit p25 binding to the HCV IRES, indicating that these regions are necessary for interaction of the p25 and HCV IRES. Since p25 binding was not observed in the IRES elements of encephalomyocarditis virus and poliovirus in UV cross-linking competition analyses, the p25 binding may be specific for the HCV IRES. p25 bound to the HCV IRES was detected when a purified 40S ribosomal subunit was used for UV cross-linking experiment, indicating that p25 is one of 40S ribosomal subunit proteins. These results reveal an unique interaction between the 40S ribosomal subunit and HCV IRES to contribute to translation initiation of the HCV genome.     

Identification of Goodpasture antigens in human alveolar basement membrane.

Goodpasture (GP) antigens, protein components reactive with human autoantibodies against glomerular basement membrane (GBM), were identified in human alveolar basement membrane (ABM) using an enzyme-linked immunoassay (ELISA), Western blotting and immunoprecipitation. All six anti-GBM antisera studied, three obtained from patients with glomerulonephritis and pulmonary haemorrhages (i.e. GP syndrome), and three from patients with glomerulonephritis alone, distinctively reacted with collagenase-digested (CD) ABM. Very cationic 22-28 kD and 40-48 kD components were detected by blot analysis combined with two-dimensional gel electrophoresis. These proteins showed some similarities to GP antigens in human GBM with respect to the monomer-dimer composition and charge distribution. Inhibition ELISA revealed that the binding of anti-GBM antisera to CDGBM decreased when they were pre-incubated with CDABM, suggesting that the anti-GBM antisera recognized the same epitope(s) on the GBM and ABM. Heterogeneity of the GP antigens in human ABM was demonstrated by blotting; monomeric antigens were absent or at low levels in the CDABM of three out of 10 normal individuals. In immunoprecipitation, anti-GBM antisera from patients with and without pulmonary haemorrhage showed different reactivities with CDABM. The former antisera precipitated both monomeric and dimeric components, but the latter did not. The observations of variation in monomer-dimer composition of ABM, and the different binding of anti-GBM antisera to it may explain why only some patients with anti-GBM nephritis have lung involvement.     

Ricinus communis agglutinin I blocks the binding of human anti-renal basement membrane antibodies to the kidney.

Whether or not glycosyl moieties of glycoproteins present in human renal basement membranes are related to the sites where anti-basement membrane antibodies bind was examined by blocking experiments using several kinds of lectin. Ricinus communis agglutinin I (RCA I), specific for galactose, blocked the binding of human anti-glomerular basement membrane (GBM) and anti-tubular basement membrane (TBM) antibodies to renal basement membranes. This lectin also diminished the binding of rabbit anti-laminin antibody, but did not inhibit the binding of mouse anti-fibronectin or rabbit anti-human TBM antibodies. These findings suggest that the binding sites of human anti-GBM and anti-TBM antibodies and heteroantibodies to laminin are closely related to the galactose moieties in glycoproteins of human renal basement membranes. Whether the galactose-containing branches are associated with the nephritogenicity of human anti-GBM and anti-TBM antibodies or simply exist adjacently to the antibody binding sites remains to be discerned.     

Poliovirus infection of established human blood cell lines: relationship between the differentiation stage and susceptibility of cell killing

The replication of type 1 poliovirus in 13 established human blood cell lines differing in the differentiation stage and cell lineage was investigated. Three T (CCRF-CEM, CCRF-HSB-2, and Molt-3) and three B (Raji, CCRF-SB, and RPMI 8226) cell lines showed no cytopathic effects (CPE) or virus production. CPE associated with virus production were detected in the other seven cell lines: HL-60, ML-1, and KG-1 (granulocytic lineage), U-937 and THP-1 (monocytic lineage), K-562 (erythroid lineage), and Molt-4 (T cell lineage). These susceptible cell lines greatly differed in the speed at which the CPE progressed. The progression of CPE was faster in relatively well-differentiated cell lines such as HL-60 and U-937, independently of the multiplicity of infection, than in less differentiated cell lines such as K-562, KG-1, and THP-1. Thus, for the same lineage, the speed at which CPE progressed became proportionally higher with subsequent differentiation stages. In the K-562 cell culture, CPE were not observed until at least 5 days postinfection (p.i.), while more than 80% of HL-60 cells were killed within 3 days p.i. There were no significant differences between infected HL-60 and K-562 cells in the efficiency of infection determined at 8 hr p.i. by the indirect immunofluorescent technique, the rate of virus growth, or the amount of viral capsid protein synthesized. This indicated that there were similar viral replication cycles in the two cell lines. These observations suggest that the killing function of the virus is expressed more slowly in K-562 cells than in HL-60 cells.     

Immunohistochemical analysis of centromere protein F expression in buccal and gingival squamous cell carcinoma

Centromere protein F (CENP-F) expression (localization and characteristics) in relation to tumor clinicopathological parameters was immunohistochemically examined and evaluated in 47 archival biopsy specimens of buccal and gingival squamous cell carcinomas (SCC). Centromere protein F expression was detected in 79% of the samples. An increase in the labeling index (LI) with WHO grading was obtained ( P  < 0.05). Correlations were obtained between the CENP-F LI and tumor size ( P  < 0.05). Immunoelectron microscopy showed CENP-F nuclear staining as punctate or fine dots. The present study shows that CENP-F expression and detection of a more specific cell subpopulation presents a theoretical advantage for the analysis of the precise cell cycle of G₂ to M cells, compared to Ki-67.