Protease inhibitors (gebexate mesylate and ulinastatin) stimulate intracellular chemiluminescence in human neutrophils.

The effect of protease inhibitors on the intracellular production of free radicals was investigated by measuring chemiluminescence (CL) elicited from phagocytosed luminol-bound microspheres (Lumispheres) in human neutrophils stimulated with formylmethionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8), phorbol 12-myristate 13-acetate, or diacylglycerol. Both gabexate mesylate (Foy) and ulinastatin (Miraclid), urinary trypsin inhibitor, increased intracellular CL in a dose dependent manner. Compared to control buffer without protease inhibitor, gabexate mesylate (322 micrograms/ml) caused about a 10-fold increase in intracellular CL in stimulated neutrophils, and ulinastatin (3100 U/ml) a twofold increase in neutrophils stimulated with fMLP or IL-8. When the protease inhibitors were added to the cell suspension after the phagocytosis of lumispheres, CL responses rapidly increased again to the level which was observed when both protease inhibitors and neutrophil stimulants were incubated simultaneously. In contrast, extracellular release of oxygen metabolites from stimulated neutrophils, assayed by a conventional measurement of luminol-dependent CL, was reduced by the protease inhibitors in a dose dependent fashion. When luminol-unbound microspheres were incubated with neutrophils stimulated by fMLP in luminol solution, extracellular CL was almost completely inhibited by gabexate mesylate. These results indicate that the protease inhibitors enhance the generation of intracellular CL and suppress the extracellular release of free radicals.     

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.     

Uneven distribution of enzymatic alterations on the liver cell surface in experimental extrahepatic cholestasis of rat.

The effect of bile duct ligation on enzyme activities in the subfractions of the rat liver plasma membrane was investigated. Two subfractions were isolated from the rat liver plasma membrane by homogenization and subsequent centrifugation in a discontinuous sucrose gradient. The light subfraction contained fragments of the bile canalicular surface and the heavy fraction contained fragments of the sinusoidal and lateral surfaces of the hepatocyte. Bile duct ligation decreased NaK ATPase and Mg-ATPase activity in the light subfraction, whereas it had no significant effect on these enzyme activities in the heavy fraction. Leucyl-β-naphthylamidase activity was reduced and alkaline phosphatase activity was increased in both subfractions. These facts suggested that ligation of the bile duct led to loss of the secretory polarity of the liver cell. The in vitro effects of some bile acids on the membranebound enzymes in the light subfraction were investigated, and a possible involvement of chenodeoxycholic acid in the alteration of enzyme activities in the bile canalicular membrane was suggested.     

IgE antibody to fish gelatin (type I collagen) in patients with fish allergy

BACKGROUND: Most children with anaphylaxis to measles, mumps, and rubella vaccines had shown sensitivity to bovine gelatin that was included in the vaccines. Recently, it was found that bovine type I collagen, which is the main content in the gelatin, is a major allergen in bovine gelatin allergy. Fish meat and skin also contain type I collagen. OBJECTIVE: The present study was designed to investigate IgE antibody to fish gelatin in children with fish allergy. METHODS: Serum samples were taken from patients in 3 groups: (1) 10 patients with fish allergy and specific IgE to fish meat; (2) two patients with allergies to both fish meat and bovine gelatin and specific IgE to fish meat and bovine gelatin; and (3) 15 patients with atopic dermatitis and specific IgE to fish meat. Various fish gelatins (type I collagen) were prepared from fish skin. IgE antibody to fish gelatin was analyzed by using ELISA and immunoblotting. RESULTS: Of 10 patients with fish allergy, 3 had specific IgE to fish gelatin. Of two patients with fish allergy and bovine gelatin allergy, all had specific IgE to fish gelatin. Of 15 patients with atopic dermatitis and specific IgE to fish meat, 5 had specific IgE to fish gelatin. Furthermore, IgE from pooled serum of the patients reacted with both the alpha1 and alpha2 chains of fish type I collagen in immunoblots. There is cross-reactivity among gelatins from various fishes, but there is little cross-reactivity between fish and bovine gelatins. CONCLUSION: Some fish-sensitive patients possessed IgE antibody to fish gelatin. Fish gelatin (type I collagen) might be an allergen in subjects with fish allergy.     

Grading score system: A method for evaluation of the degree of senescence in Senescence Accelerated Mouse (SAM)

For evaluation of the degree of senescence in SAM-P, accelerated senescence prone mouse, formerly called SAM or prone series or P-series, consisting of SAM-P/1, SAM-P/2, SAM-P/3 and SAM-P/4 corresponding to P-1, P-2, P-3 and P-4 series, respectively, in the previous reports, and in SAM-R, accelerated senescence resistant mouse, formerly called resistant series or R-series, consisting of SAM-R/1, SAM-R/2 and SAM-R/3 corresponding to R-1, R-2 and R-3 series, respectively, in the previous reports, the grading score system was adopted. The items to be examined in this system include 11 categories selected from the clinical signs and gross lesions considered to be associated with the aging process. The degree of the senescence in each category was graded from 0 to 4 according to the detailed criteria devised in our laboratory. After 8 months of age each mouse was examined every 4 months, and some of the mice were examined after 2 months of age.In almost all categories, the grading score and incidence began to increase from 4 or 6 months of age and continued to increase with advancing age in both SAM-P and SAM-R. The increase, however, was more marked in SAM-P than in SAM-R. The slow but steady increase in the SAM-R levelled out at 24 months of age and was comparable to that of 12 months of age in SAM-P. In both SAM-P/1 at 8 months of age and SAM-R/2 at 12 months of age, there was a significant reverse correlation between total score of this grading score system and length of residual life after examination.Systematic and extensive studies using the grading score system showed that if the validity of the system is, based on “irreversibility” and “universality” of the changes in     

Effect of magnesium deficiency on ultrastructural changes in coronary arteries of swine

The effects of magnesium (Mg) deficiency on the coronary arteries of 27 Yorkshire swine were studied by light and electron microscopy. The experimental animals were divided into 4 groups which received the following supplements: Group I, basal ration with adequate Mg (540 mg/kg diet), Group II, basal ration with insufficient Mg (270 mg/kg diet) Group III, 10% milk powder with adequate Mg (540 mg/kg diet), Group IV, 10% milk powder with insufficient Mg (270 mg/kg diet). Serum analysis indicated that dietary low Mg supplementation decreased cholesterol levels and increased phospholipid concentrations significantly. The highest magnitude and incidence of intimal thickening were observed in the coronary arteries of Group IV (p less than 0.003). No significant intimal thickening was detected in any of the other groups. Ultrastructural studies revealed a greater frequency of degenerated cells in Group III and IV (p less than 0.01). Numerous calcifications were observed in only Group IV. These data suggest that moderate Mg deficiency can promote atherosclerosis in combination with some atherogenic diet, and that the presence of smooth muscle cell degeneration is important in order for a magnesium deficiency to exert an effect on the coronary artery of swine.     

Molecular interactions of fission yeast Skp1 and its role in the DNA damage checkpoint

Skp1 is a central component of the E3 ubiquitin ligase SCF (Skp1-Cullin-1-F-box). It forms an adapter bridge between Cullin-1 and the substrate-determining component, the F-box protein. In order to establish the role of Skp1, a temperature sensitive (ts) screen was carried out using mutagenic PCR (polymerase chain reaction) and 9 independent ts mutants were isolated. Mapping the mutated residues on the 3-D structure of human Skp1 suggested that the mutants would be compromised in binding to F-box proteins but not Cullin-1 (Pcu1). In order to assess the binding properties of ts Skp1, 12 F-box proteins and Pcu1 were epitope-tagged, and co-immunoprecipitation performed. This systematic analysis showed that ts Skp1 retains binding to Pcu1. However, binding to three specific F-box proteins, essential Pof1, Pof3 involved in maintaining genome integrity, and nonessential Pof10, was reduced. skp1ts cells exhibit a G2 cell cycle delay, which is attributable to activation of the DNA damage checkpoint. Intriguingly, contrary to pof3 mutants, in which this checkpoint is required for survival, checkpoint abrogation in skp1(ts) suppresses a G2 delay and furthermore almost rescues the ts phenotype. The activation mechanism of the DNA damage checkpoint therefore differs between pof3Delta and skp1(ts), implicating a novel role for Skp1 in the checkpoint-signalling cascade.     

Detection of genes expressed in primary colon cancers by in situ hybridisation: overexpression of RACK 1.

AIMS: The isolation of various genes that are expressed in a region specific manner is considered useful for research in molecular pathology. In situ hybridisation (ISH) was used in a screening procedure to isolate these genes efficiently, using colon cancer as a model. METHODS: Suppression subtractive hybridisation (SSH) between colon cancer tissue samples and corresponding non-cancerous tissues was performed. Genes showing high expression in the cancers were selected using macro-DNA array analysis. As a final screening procedure, conventional ISH was performed to isolate genes expressed specifically in colon cancers. RESULTS: Sixty nine clones were selected by SSH and macro-DNA array analyses. These clones were then analysed by ISH to examine their expression patterns. ISH screening revealed that all the clones screened showed more intense signals in colon cancers than in non-cancerous tissues. Among them, RACK 1, which is a protein kinase C receptor and a homologue of the G protein beta subunit, was expressed intensely in colon cancer cells. RACK 1 expression was evaluated in multiple samples by ISH, and the results confirmed that RACK 1 was universally overexpressed in cells of all 11 colon cancers examined. CONCLUSIONS: Many genes, including RACK 1, expressed in colon cancer cells can be isolated efficiently by this method, and their precise expression pattern can be evaluated. These results indicate that ISH is an excellent technique for systemic screening of genes expressed in a region specific manner.     

Altered EphA5 mRNA expression in rat brain with a single methamphetamine treatment

Methamphetamine is a potent and indirect dopaminergic agonist which can cause chronic brain dysfunctions including drug abuse, drug dependence and drug-induced psychosis. Methamphetamine is known to trigger molecular mechanisms involved in associative learning and memory, and thereby alter patterns of synaptic connectivity. The persistent risk of relapse in methamphetamine abuse, dependence and psychosis may be caused by such alterations in synaptic connectivity. EphA5 receptors constitute large families of tyrosine kinase receptor and are expressed almost exclusively in the nervous system, especially in the limbic structures. Recent studies suggest EphA5 to be important in the topographic projection, development, and plasticity of limbic structures, and to be involved in dopaminergic neurotransmission. We used in situ hybridization to examine whether methamphetamine alters EphA5 mRNA expression in the brains of adult male Wister rats. EphA5 mRNA was widely distributed in the medial frontal cortex, cingulate cortex, piriform cortex, hippocampus, habenular nucleus and amygdala. Compared to baseline expression at 0 h, EphA5 mRNA was significantly decreased (by 20%) in the medial frontal cortex at 24 h, significantly increased (by 30%) in the amygdala at 9 and 24 h, significantly but transiently decreased (by 30%) in the habenular nucleus at 1 h after a single injection of methamphetamine. Methamphetamine did not change EphA5 mRNA expression in the cingulate cortex, piriform cortex or hippocampus. Our results that methamphetamine altered EphA5 mRNA expression in rat brain suggest methamphetamine could affect patterns of synaptic connectivity, which might be responsible for methamphetamine-induced chronic brain dysfunctions.