gamma-Aminobutyric acid receptors visualized in spinal cord cultures by [3H]muscimol autoradiography.

gamma-Aminobutyric acid (GABA) receptors were visualized in mouse spinal cord explant cultures by [3H]muscimol autoradiography over certain small neurons of the dorsal horn grey matter, in the interneuronal neuropil of dorsal and ventral horns, and (rarely) in small clusters on processes of anterior horn cells. Specificity was indicated in control experiments by inhibition of binding by [3H]muscimol after pretreatment with GABA or a GABA analogue, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, isoxazole 16), or with receptor antagonists bicuculline and picrotoxin. Unlabeled muscimol exchanged effectively with [3H]muscimol and produced autoradiographic label in locations indistinguishable from those found in experiments with [3H]muscimol alone. Pretreatment with the GABA uptake and transport inhibitors (-)-nipecotic acid and guvacine did not affect binding with [3H]muscimol. These experiments indicate that explant culture systems can be used for demonstration of functional receptors.     

Understanding the function-structure and function-mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis

Inactivation of the tumor suppressor p53 by missense mutations is the most frequent genetic alteration in human cancers. The common missense mutations in the TP53 gene disrupt the ability of p53 to bind to DNA and consequently to transactivate downstream genes. However, it is still not fully understood how a large number of the remaining mutations affect p53 structure and function. Here, we used a comprehensive site-directed mutagenesis technique and a yeast-based functional assay to construct, express, and evaluate 2,314 p53 mutants representing all possible amino acid substitutions caused by a point mutation throughout the protein (5.9 substitutions per residue), and correlated p53 function with structure- and tumor-derived mutations. This high-resolution mutation analysis allows evaluation of previous predictions and hypotheses through interrelation of function, structure and mutation.     

Oxygen metabolism of the liver during an HA clamp: HV saturation and free radicals

BACKGROUND/AIMS: To clarify changes in the hepatic oxygen metabolism and tissue damage resulting from oxygen-derived free radical generation from polymorphonuclear cells during a hepatic arterial clamp. METHODOLOGY: Subjects were 32 male Wistar rats. Hepatic tissue blood flow, and hepatic venous chemiluminescence, indicating oxygen-derived free radicals from polymorphonuclear cells, and liver lipid peroxide were measured, and hepatic and portal venous blood gas analysis were performed before and after 130 minutes of hepatic arterial clamping. RESULTS: Hepatic tissue blood flow decreased by hepatic arterial clamp. The values of hepatic arterial oxygen pressure (HTBF), hepatic venous oxygen saturation (ShvO2), and O2 contents after hepatic arterial clamp were lower than those before hepatic arterial clamp (P = 0.035, 0.024, and 0.028, respectively). Hepatic venous chemiluminescence decreased and the lipid peroxide level of the liver increased by hepatic arterial clamp (P = 0.001). CONCLUSIONS: ShvO2 is useful for the evaluation of hepatic oxygen metabolism and hepatic tissue blood flow during acute hepatic arterial clamp. This condition should prepare the following tissue damage due to oxygen-derived free radicals from polymorphonuclear cells.     

Large conformational changes of the epsilon subunit in the bacterial F1F0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme

The F(1)F(0) ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the gamma and epsilon subunits in the F(1) part and c subunit ring in the F(0) part, rotates relative to a stator composed of alpha(3)beta(3)deltaab(2) during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the epsilon subunit plays a key role by acting as a switch of this motor. Two different arrangements of the epsilon subunit have been visualized recently. The first has been observed in beef heart mitochondrial F(1)-ATPase where the C-terminal portion is arranged as a two-alpha-helix hairpin structure that extends away from the alpha(3)beta(3) region, and toward the position of the c subunit ring in the intact F(1)F(0). The second arrangement was observed in a structure determination of a complex of the gamma and epsilon subunits of the Escherichia coli F(1)-ATPase. In this, the two C-terminal helices are apart and extend along the gamma to interact with the alpha and beta subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F(1)F(0), confirming that both conformations of the epsilon subunit exist in the enzyme complex. With the C-terminal domain of epsilon toward the F(0), ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F(1) part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the epsilon subunit in the F(1)F(0) complex and argue for a ratchet function of this subunit.     

High concentration simvastatin induces apoptosis in fibroblast-like synoviocytes from patients with rheumatoid arthritis

OBJECTIVE: We previously reported that 10 mg/day of simvastatin significantly reduced clinical scores of rheumatoid arthritis (RA) in patients with active RA with hypercholesterolemia. We have also reported that a certain pharmacological concentration of simvastatin, i.e., 0.05-0.1 microM, inhibits the production of interleukin 6 (IL-6) and IL-8 and the cell proliferation induced by tumor necrosis factor-alpha (TNF-alpha) in fibroblast-like synoviocytes (FLS) derived from patients with RA in vitro. We investigated other effects of simvastatin on FLS from the standpoint of cell viability and apoptosis. METHODS: RA FLS were cultured with or without 0.05-50 microM simvastatin for 48 h. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was measured by flow cytometric analysis using propidium iodide and annexin-V. Caspase-3 and -9 activities were analyzed by colorimetric assays. RESULTS: High concentrations of simvastatin, i.e., 1.0-50 microM, reduced cell viability and induced prominent apoptosis in FLS in a dose-dependent manner. The apoptosis induced by simvastatin was caspase-3- and caspase-9-dependent. These effects were completely reversed in the presence of mevalonic acid or geranylgeranyl-pyrophosphate, but not in the presence of farnesyl-pyrophosphate. Further, a geranylgeranyl transferase inhibitor and a RhoA kinase inhibitor mimicked the effect of simvastatin. CONCLUSION: These data, together with our previous report, suggest that low (pharmacological range) and high concentrations of simvastatin affect FLS differently: (1) at a low concentration, it inhibits IL-6 and IL-8 production and the cell proliferation of FLS induced by TNF-alpha (2) at high concentrations, it induces apoptosis in FLS. Understanding this dose-dependent biphasic effect of simvastatin may prove important for its clinical applications in the treatment of RA.     

A novel costimulatory signaling in human T lymphocytes by a splice variant of CD28

We have characterized a splice variant (isoform) of the human CD28 T cell costimulatory receptor. The nucleotide sequence of this CD28 isoform was identical to that of CD28 in the signal peptide, the transmembrane domain, and the cytoplasmic tail, but it was missing a large segment of the extracellular ligand-binding domain, which is encoded by the second exon. This isoform (CD28i), whose message level exceeded 25% of CD28, was a transmembrane homodimer. CD28i was found noncovalently associated with CD28 and was tyrosine-phosphorylated/PI3-kinase-complexed following the crosslinking of CD28, and the CD28 costimulatory signal was enhanced in T cells expressing CD28i. These data demonstrate that CD28i, via noncovalent association with CD28, plays a role as a costimulatory signal amplifier in human T cells.     

Spherical aggregates of beta-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase-3beta

beta-Amyloid (Abeta) acquires toxicity by self-aggregation. To identify and characterize the toxic form(s) of Abeta aggregates, we examined in vitro aggregation conditions by using large quantities of homogenous, chemically synthesized Abeta1-40 peptide. We found that slow rotation of Abeta1-40 solution reproducibly gave self-aggregated Abeta1-40 containing a stable and highly toxic moiety. Examination of the aggregates purified by glycerol-gradient centrifugation by atomic force microscopy and transmission electron microscopy revealed that the toxic moiety is a perfect sphere, which we call amylospheroid (ASPD). Other Abeta1-40 aggregates, including fibrils, were nontoxic. Correlation studies between toxicity and sphere size indicate that 10- to 15-nm ASPD was highly toxic, whereas ASPD <10 nm was nontoxic. A positive correlation between the toxicity and ASPD >10 nm also appeared to exist when Abeta1-42 formed ASPD by slow rotation. However, Abeta1-42-ASPD formed more rapidly, killed neurons at lower concentrations, and showed approximately 100-fold-higher toxicity than Abeta1-40-ASPD. The toxic ASPD was associated with SDS-resistant oligomeric bands in immunoblotting, which were absent in nontoxic ASPD. Because the formation of ASPD was not disturbed by pentapeptides that break beta-sheet interactions, Abeta may form ASPD through a pathway that is at least partly distinct from that of fibril formation. Inhibition experiments with lithium suggest the involvement of tau protein kinase I/glycogen synthase kinase-3beta in the early stages of ASPD-induced neurodegeneration. Here we describe the identification and characterization of ASPD and discuss its possible role in the neurodegeneration in Alzheimer's disease.     

A case of pneumonitis and hepatic injury caused by a herbal drug (sho-saiko-to)]

We report a case of pneumonitis and hepatic injury caused by Sho-saiko-to. A 56-year-old man was admitted to our hospital because of hepatic disorder. The levels of serum transaminases returned to normal within two months without specific treatment and he was discharged. Four weeks later, he was readmitted because of severe pneumonitis and mild hepatic disorder. Under the suspicion of drug-induced pneumonitis, all medications were discontinued and high-dose glucocorticoid including "pulse therapy" was given. Consequently, pneumonitis and hepatic function markedly improved. Careful history taking revealed the ingestion of Sho-saiko-to before both admissions. Lymphocyte stimulation test against Sho-saiko-to was positive. Challenge test using Sho-saiko-to resulted in decrease of PaO2 and elevation of serum transaminases. Based on these findings, the diagnosis of pneumonitis and hepatic injury induced by Sho-saiko-to was established.     

Countermeasures against non-specific reactions in the measurement of beta-glucan in plasma by alkaline treatment, chromogenic automated kinetic assay

We investigated the detection of non-specific reactions in the measurement of plasma (1-->3)-beta-D-glucan (beta-glucan) and countermeasures against them using alkaline treatment, chromogenic automated kinetic assay (alkaline-kinetic assay). In this study, we reexamined the values of beta-glucan using the alkaline-kinetic assay with and without laminaran oligosaccharides (LO) as a kind of beta-glucan that blocks the Limulus reaction. The materials for this study were 584 plasma samples in which beta-glucan had been measured. These were taken from 232 patients in Kawasaki Medical School Hospital between January 2002 and March 2002. Non-specific reactions were judged by a calculated value under a LO additive condition. Determination as to whether or not the each time course of the Limulus reaction was influenced by a non-specific reaction was also studied by applying a non-specific reaction index set up independently. Non-specific reactions were recognized in 51.9% of the samples (81/156). The amount of non-specific reaction was 9.9 pg/ml or less in major samples. On the other hand, when the cut off value of the index for detection of non-specific reactions was set at 0.5, the sensitivity was 88.9% and specificity was 73.7%. The positive and negative predictive values were 93.5% and 60.9% respectively. Non-specific reactions can be approximately distinguished by applying the non-specific reaction index. By so doing, unnecessary initiation of anti fugal therapy in response to non-specific reactions can be avoided. Further prospective and radical studies of non-specific reactions in the alkaline-kinetic assay are necessary.     

Treatment of paroxysmal supraventricular tachycardia with intravenous injection of adenosine triphosphate.

Intravenous adenosine triphosphate rapidly terminated all 11 episodes of paroxysmal supraventricular tachycardia in 10 patients. Eight patients reported side effects but these resolved within 20 seconds and did not require treatment. Adenosine triphosphate is a suitable agent for the rapid termination of paroxysmal supraventricular tachycardia.