Brown adipose tissue thermogenesis and metabolic rate contribute to the variation in obesity among rats fed a high fat diet.

A high fat (HF) diet is known to induce obesity, but susceptibility to obesity induced by a HF diet differs not only among different strains of rats but also within the same strain. The present study revealed that the Lee index (an index of obesity) positively correlated with insulin, and inversely correlated with both the mitochondrial oxygen consumption in interscapular brown adipose tissue (BAT) and the resting metabolic rate (RMR) in Sprague-Dawley rats. This suggests the contribution of BAT thermogenesis and RMR, in addition to hyperphagia, to the intrastrain variation in susceptibility to HF diet-induced obesity.     

Down-regulation of antigen-specific antibody production by TCR antagonist peptides in vivo

The efficacy of TCR antagonist peptides in inhibition of antigen-specific antibody production and T cell responses in vivo was evaluated. Among amino acid-substituted analogs of a peptide corresponding to residues 119 - 133 of bovine beta-lactoglobulin (p119 - 133), pR124Q and pD129S, prepared by substitution of Gln and Ser for Arg(124) and Asp(129), respectively, have been shown to display TCR antagonist activity for three out of four distinct p119 - 133-specific T cell clones and for polyclonal T cells derived from p119 - 133-immunized C57BL / 6 mice. Both pD129S and pR124Q inhibited in vivo priming and subsequent activation of T cells by p119 - 133 when co-injected with p119 - 133 into mice, as shown by the decreased proliferation of T cells in response to p119-133 in vitro. pD129S significantly inhibited production of anti-p119 - 113 antibodies of IgG1, IgG2b and IgE isotype in vivo when co-injected into mice together with p119 - 133 at the time of the first immunization. However, pR124Q was totally ineffective in inhibition of the antibody responses. Anti-p119 - 133 antibodies from p119 - 133-immunized mice could bind to pR124Q but not to pD129S, suggesting that the difference in cross-reactivity is responsible for the different effect of these two peptides on specific antibody production. Our findings demonstrate that a single TCR antagonist peptide can inhibit antigen-specific polyclonal antibody production when this antagonist peptide does not cross-react with the antibody elicited in response to an antigenic peptide.     

Recombinant human interleukin 1 alpha is cytotoxic for and increases surface expression of HLA-A,B,C antigens of a human hepatoma cell line, PLC/PRF/5

Our study was undertaken to determine whether human recombinant interleukin 1 alpha (rIL 1 alpha) has any effect on the proliferation and expression of HLA-A,B,C antigens of human liver cell lines. The addition of rIL 1 alpha reduced the cell number of the human hepatoma cell line, PLC/PRF/5. This effect was determined to be cytotoxic, but not growth inhibitory, rIL 1 alpha did not change the number of Chang cell or SK-Hep-1 at a concentration as, high as 25,000 U/ml. rIL 1 alpha enhanced the expression of HLA-A,B,C antigens on PLC/PRF/5, but had no effect on Change cell or SK-Hep-1. Receptor binding studies showed that 125I-rIL 1 alpha bound to PLC/PRF/5 in a specific and saturable manner, but did not bind to Chang cell or SK-Hep-1. Scatchard plot analysis of the binding to PLC/PRF/5 revealed a single type of high affinity binding site with an apparent dissociation constant of approximately 5 x 10(-5) M and the presence of approximately 150 binding sites per cell. These findings suggest that IL 1 alpha may play a role in host defense against some hepatomas as cytotoxic factor and may be an enhancer of expression of HLA-A,B,C antigens on tumor cells.     

Effect of a spider toxin (JSTX) on excitatory postsynaptic current at neuromuscular synapse of spiny lobster

1. We studied the blocking properties of a spider (Nephila clavata) toxin (JSTX) purified from venom on the spiny lobster neuromuscular junction. 2. When a small amount of JSTX was applied to the neuromuscular junction, the excitatory postsynaptic potential (EPSP) was partially suppressed. The amplitude of EPSPs remained at a steady level for several hours during the washing of the preparation, showing that the action of JSTX is irreversible. 3. We recorded the excitatory postsynaptic current (EPSC) from synaptic site using a macro-patch electrode. The amplitude of EPSC increased linearly with hyperpolarization of the membrane potential in the presence and absence of JSTX. 4. The decay phase time constant of EPSC and spontaneous EPSC was decreased by hyperpolarizing the membrane potential both in the absence and in the presence of JSTX. The relationship between the decay time constant and the membrane potential was not modified by JSTX. 5. It is suggested that JSTX irreversibly blocks EPSC by acting on the site that is apart from the ionic channel of the glutamate receptor molecule.     

Regulation of inwardly rectifying K(+) channel in cultured opossum proximal tubule cells by protein phosphatases 1 and 2A

The inwardly rectifying ATP-regulated K(+) channel with an inward conductance of about 90 pS in the surface membrane of cultured opossum kidney proximal tubule (OKP) cells is activated at least in part by protein kinase A (PKA). In this study, we examined the effects of protein serine/threonine phosphatase types 1 (PP-1) and 2A (PP-2A) on activity of the K(+) channel using the patch-clamp technique. In cell-attached patches, channel activity was enhanced by the application of okadaic acid (OA, 1 microM), a membrane-permeable inhibitor of PP-1 and PP-2A, to the bath solution. This enhancement was abolished by the pretreatment of cells with KT5720 (200 nM), a specific inhibitor of PKA. In inside-out patches, channel activity which could be maintained in the presence of ATP (3 mM) in the bath solution was also increased by the addition of OA (1 microM), and the OA-induced increase in channel activity was partially prevented in the presence of KT5720 (200 nM). Direct application of either PP-1 (1 U/ml) or PP-2A (1 U/ml) to the cytoplasmic surface of the patch membrane inhibited channel activity maintained by ATP (3 mM) in inside-out patches. Moreover, channel activity stimulated by PKA (20 nM) in the presence of ATP (3 mM) was also inhibited by the application of either PP-1 (1 U/ml) or PP-2A (1 U/ml). These results indicate that the OA-sensitive protein phosphatase is involved in the regulation of channel activity, and suggest that both PP-1 and PP-2A are candidates responsible for the inhibition of channel activity through dephosphorylation of the PKA-mediated protein phosphorylation.     

Endogenous adenosine regulates the effects of low-frequency stimulation on the induction of long-term potentiation in CA1 neurons of guinea pig hippocampal slices

A train of low-frequency afferent stimuli (LFS, 1 Hz, 1000 pulses), given 60 min prior to a tetanus (100 Hz, 100 pulses), suppresses the induction of long-term potentiation (LTP) in which a short-term potentiation decreases gradually back to the pre-tetanic level within 40-50 min (LTP suppression). We investigated the effects of adenosine A1 or A2 receptor antagonists (8-cyclopentyltheophylline (8-CPT) and CP-66713, respectively) on LTP suppression in CA1 neurons of guinea pig hippocampal slices. When the LFS was delivered in the presence of 8-CPT (1 microM), LTP suppression was not significantly affected. However, when LFS was delivered in the presence of CP-66713 (10 microM), LTP suppression was inhibited, leading to successful LTP induction. These results indicate that endogenous adenosine, acting via A2 receptors, is involved in the mechanism of LTP suppression.     

Self-splicing of the Tetrahymena group I ribozyme without conserved base-triples

BACKGROUND: Group I introns share a conserved core region consisting of two domains, P8-P3-P7 and P4-P6, joined by four base-triples. We showed previously that the T4 td intron can perform phosphoester transfer reactions at two splice sites in the absence of both P4-P6 and the conserved base-triples, whereas it is barely able to perform the intact splicing reaction due to the difficulty of conducting the sequential reactions. RESULTS: Based on previous findings, we constructed a bimolecular ribozyme lacking a large portion of P4-P6 and the base-triples from the Tetrahymena intron, on the assumption that the long-range interactions of the peripheral regions in the two RNAs can compensate for the deteriorated core. The bimolecular ribozyme performed the intact splicing reaction. CONCLUSION: The present analysis indicates that the base-triples are nonessential, but that L4 and the distal part of P4 in P4-P6 are important for conducting the splicing reaction. The reconstituted self-splicing ribozyme provides an amenable system for analysing the role(s) of elements in the core region in the self-splicing reaction mechanism.     

Retinoblastoma protein prevents staurosporine-induced cell death in a retinoblastoma-defective human glioma cell line.

OBJECTIVE: To investigate the mechanism of staurosporine-induced glioma cell death and cell cycle arrest using adenovirus-mediated gene transfection, as well as the function of retinoblastoma (Rb) and genetic instability induced by staurosporine. METHODS: Cell cycle regulation, cell death and nuclear abnormalities induced by staurosporine were examined using an adenovirus vector expressing Rb, p16 or p21 genes in human glioma cell lines. RESULTS: The Rb-defective SF-539 cell line was resistant to staurosporine compared with cell lines expressing intact Rb. SF-539 glioma cells exposed to staurosporine became multinucleated and then died. Multinucleation was prevented in SF-539 cells transfected with the Rb gene, thus decreasing the death rate of these cells. CONCLUSIONS: These results imply that enforced Rb expression protects cells from genomic instability induced by staurosporine regardless of its upstream molecular effects.     

Tissue-type plasminogen activator and its inhibitor in human glomerulonephritis.

We carried out an immunohistochemical study of tissue-type plasminogen activator (PA) and urokinase-type PA, and their inhibitors, PA inhibitor-1 and PA inhibitor-2, using renal biopsy specimens obtained from 86 patients with various forms of glomerulonephritis. The controls were four normal renal tissue specimens. On immunofluorescent observation, granular staining for tissue-type PA was found to be distributed along the glomerular capillary walls. The fluorescence was weak in the normal renal tissue and occasionally intense in the tissues of patients with IgA nephritis, minimal change nephrotic syndrome, and lupus nephritis. PA inhibitor-1 was abundant in the glomerular epithelial cells and scarce in the mesangial area and glomerular capillary lumens of the normal renal tissues. This was confirmed by immunoelectron microscopy using gold staining. The fluorescence of PA inhibitor-1 was weaker in some specimens of nephritic tissues than in the normal renal tissues. Urokinase-type PA and PA inhibitor-2 were negative within the glomeruli in all the specimens. In the glomerulonephritic tissues which were fibrin deposition-positive, tissue-type PA expression in the glomeruli tended to be strong. An association between fibrin deposition and PA inhibitor-1 staining was not clear. These data suggest that expression of tissue-type PA in the glomeruli increases in association with fibrin deposition.