Radioimmunoassay for non-enzymatically glycated serum proteins

We have developed a radioimmunoassay (RIA) for nonenzymatically glycated serum proteins. The polyclonal antibodies prepared against reduced glycated human albumin were specific for the glucitollysine residues of serum proteins. Serum proteins from diabetic patients (n = 25) contained 5.3 +/- 2.8 nmoles of glucitollysine/mg protein, compared to 2.0 +/- 0.2 in controls (n = 20). The intra- and inter-assay variables were 3.2-6.2% and 4.4-8.6%, respectively. Results from this assay procedure correlated well with those from the boronate affinity chromatography procedure (r = 0.94; P less than 0.001). The data suggested that diabetic serum proteins contained at least 2.5 times as much immunochemically detectable glucitollysine residures as normal serum proteins after reduction of the proteins with sodium borohydride.     

Possible involvement of calcium ions in the hatching of Schistosoma mansoni eggs in water

The possibility of involvement of calcium ions in the hatching of Schistosoma mansoni eggs in water is described. The hatching of S. mansoni eggs under low osmotic pressure was partially inhibited by EGTA (5 mM), lanthanum chloride (1-5 mM), and ruthenium red (0.1-1 mM). The reagents used in these experiments were not toxic to the eggs however, because miracidia hatched normally when the reagents were removed.     

Anti-shock effects of human superoxide dismutase in splanchnic artery occlusion (SAO) shock

We studied the effects of human superoxide dismutase (h-SOD) in splanchnic artery occlusion (SAO) shock. Pentobarbital anesthetized rats subjected to total occlusion of the superior mesenteric and the celiac arteries for 40 min developed a severe shock state usually resulting in a fatal outcome within 20 min after the release of the occlusion. h-SOD (10 mg/kg) was infused intravenously starting at reperfusion and lasting for 10 min. SAO shock rats treated with h-SOD maintained postreperfusion MABP at significantly higher values compared to rats receiving the vehicle (final MABP 84 +/- 6 vs 46 +/- 1 mm Hg, P less than 0.01, respectively). Treatment with h-SOD attenuated the plasma accumulation of free amino-nitrogen compounds (P less than 0.01 from vehicle) as well as the activity of the lysosomal protease cathepsin D (P less than 0.05 from vehicle). Furthermore, the plasma activity of a myocardial depressant factor was significantly lower in h-SOD-treated rats than in SAO rats receiving only the vehicle (27 +/- 1 vs 64 +/- 3 U/ml, P less than 0.01). SAO shock rats treated with h-SOD also exhibited a significantly higher survival rate than the SAO shock +/- vehicle group (88% vs 11%, P less than 0.01, respectively). These results support the role of oxygen-derived radicals in the pathophysiology of SAO shock, and indicate that h-SOD effectively ameliorates the deleterious effects of oxygen radicals in this severe model of ischemia and reperfusion.     

Efficacy and safety of foscarnet for recurrent orolabial herpes: a multicentre randomized double-blind study.

Foscarnet sodium (trisodium phosphonoformate hexahydrate) has been shown to inhibit herpes simplex virus (HSV) in vitro and to be efficacious for topical treatment of experimental HSV infection in animals. To assess its clinical efficacy in the treatment of recurrent orolabial herpes a multicentre collaborative, double-blind, placebo-controlled trial was conducted. The study patients were randomly assigned to receive either 3% foscarnet cream (78 patients) or placebo (cream vehicle) (75 patients) and were asked to start treatment at the earliest indication of a recurrence. Efficacy was evaluated in 143 patients (74 in the foscarnet group and 69 in the placebo group). There was no significant difference in time to healing or duration of virus shedding between the two groups. However, in the subgroup of patients who started treatment before vesicles appeared, the duration of virus shedding was shorter in the foscarnet group than in the placebo group (p = 0.04), and the proportion of lesions that evolved to the vesicular stage was smaller (p = 0.03). No significant difference in the incidence of local or systemic adverse effects was noted between the two groups. We conclude that the beneficial effect of foscarnet was limited to a subgroup of patients who started treatment in the prevesicular stage.     

Effects of chronic bifemelane hydrochloride administration on receptors for N-methyl-d-aspartate in the aged-rat brain

We assayed N-methyl-d-aspartate (NMDA) receptors [³H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([³H]CPP) bindings) and evaluated their distribution in the brain by quantitative autoradiography in young adult and aged rats. In the young adult rats, NMDA receptors were present at relatively high concentrations in the cerebral cortex and hippocampus. In the aged rats, NMDA receptors were decreased in the nealy all areas of the brain, especially in the cerebral cortex and hippocampus. Chronic administration of bifemelane hydrochloride, a drug for sequela of cerebrovascular diseased, at a dose of 15 mg/kg/day for 14 days, markedly attenuated these decrease in NMDA receptors. Since NMDA receptors are considered to be involved in memory and learning processes, our results suggest that bifemelane hydrochloride may be applicable to the treatment of disturbed memory and learning.     

Characterization of acute and latent herpes simplex virus infection of dorsal root ganglia in rats.

The characteristics of HSV type-1 infection following subcutaneous inoculation in the dorsum of one hind paw of Sprague-Dawley rats were studied to determine whether infection in rats might more closely parallel the infection in man than is seen in other animals. The serologic and virologic characteristics of acute and latent ganglion infection conformed to those of human infection. Immunohistochemical studies suggested that sensory ganglion infection arose via centripetal axonal migration of virus as is hypothesized in man. In rat, small type B neuronal cell bodies appeared central to the maintenance of latent infection and reactivation observed during cocultivation of lumbar ganglia. Acute and latent lumbar sensory ganglion infection in rats after subcutaneous hind paw injection of HSV-1 appears to be another suitable model of this infection in man.     

Conformational changes of papain induced on interaction with thiol proteinase inhibitors from newborn rat epidermis

The conformational changes of the papain molecular on interaction with two thiol proteinase inhibitors (TPI(1) and TPI(2] from newborn rat epidermis were studied by measuring circular dichroism (CD), the difference absorption spectrum, and the fluorescence spectrum due to tryptophan residues in papain. The far-ultraviolet CD band of papain between 210 and 230 nm was distinctly reduced on interaction with both inhibitors. Also, the near-ultraviolet CD spectrum of TPI(1)-bound papain changed between 285 and 320 nm as well as that of the TPI(2)-bound enzyme. The difference absorption spectrum for TPI(1)-bound papain exhibited two distinct peaks at 276.5 and 282 nm, indicating perturbation of aromatic amino acid residues. The fluorescence intensity of papain was significantly decreased on interaction with both inhibitors, which showed pH-dependency on an ionizable group, with pK values of 8.5 and 7.9 for TPI(1) and TPI(2), respectively. The complex formation of papain with both inhibitors caused a reduction of the susceptibility of a tryptophan residue, probably tryptophan-177, to chemical modification with N-bromosuccinimide. These results suggest that the active site involving histidine-159 in the papain molecule was much influenced by the alteration of the microenvironment of tryptophan-177 as a part of the interaction site for these two thiol proteinase inhibitors.     

Cross-linking site in fibrinogen for alpha 2-plasmin inhibitor

A plasma proteinase inhibitor, alpha 2-plasmin inhibitor (alpha 2PI), is cross-linked with alpha chain of fibrin(ogen) by activated coagulation Factor XIII (plasma transglutaminase). alpha 2PI serves only as a glutamine substrate (amine acceptor) for activated Factor XIII in the cross-linking reaction, and the cross-linking occurs between Gln-2 of the alpha 2PI molecule and a lysine residue (amine donor) of fibrin(ogen) alpha chain, whose position was investigated. alpha 2PI and fibrinogen were reacted by activated Factor XIII. The resulting alpha 2PI fibrinogen A alpha chain complex was separated and subjected to two cycles of Edman degradation using phenyl isothiocyanate for the first cycle and dimethylaminoazobenzene-isothiocyanate for the second cycle. The aqueous phase after the cleavage stage of the second cycle, containing dimethylaminoazobenzene-thiohydantoin-Gln cross-linked with A alpha chain, was subjected to CNBr fragmentation and tryptic digestion. Only one of the peptides was found to have the peak of absorbance at 420 nm, indicating the presence of dimethylaminoazobenzene-thiohydantoin-Gln in that peptide. The peptide was identified as corresponding to residues Asn-290-Arg-348 of A alpha chain by analyses of the NH2-terminal amino acid sequence and amino acid composition. The peptide contains a single lysine at position 303, indicating that Lys-303 of fibrinogen A alpha chain is the lysine residue that forms a cross-link with Gln-2 of alpha 2PI.     

Hg2+-induced contracture in mechanically skinned fibers of frog skeletal muscle

In mechanically skinned fibers of the semitendinosus muscle of bullfrogs, we examined the role of membrane sulfhydryl groups on Ca2+ release from the sarcoplasmic reticulum (SR). Hg2+, a sulfhydryl reagent (20-100 microM), induced a repetitive contracture of skinned fibers, and this contracture did not occur in skinned fibers in which the SR had been disrupted by treatment with a detergent (Brij 58). Procaine (10 mM), Mg2+ (5 mM), or dithiothreitol (1 mM) blocked the Hg2+-induced contracture. Ag+ or p-chloromercuribenzenesulfonic acid produced similar contractures to that induced by Hg2+. We conclude that Hg2+ releases Ca2+ from SR of a skinned fiber by modifying sulfhydryl groups on the SR membrane, and suggest that the Ca2+ released by Hg2+ may trigger a greater release of Ca2+ from SR to develop tension.