Melatonin administration prevents lipopolysaccharide-induced oxidative damage in phenobarbital-treated animals

The protective effect of melatonin on lipopolysaccharide (LPS)-induced oxidative damage in phenobarbital-treated rats was measured using the following parameters: changes in total glutathione (tGSH) concentration, levels of oxidized glutathione (GSSG), the activity of the antioxidant enzyme glutathione peroxidase (GSH-PX) in both brain and liver, and the content of cytochrome P450 reductase in liver. Melatonin was injected intraperitoneally (ip, 4mg/kg BW) every hour for 4 h after LPS administration; control animals received 4 injections of diluent. LPS was given (ip, 4 mg/kg) 6 h before the animals were killed. Prior to the LPS injection, animals were pretreated with phenobarbital (PB), a stimulator of cytochrome P450 reductase, at a dose 80 mg/kg BW ip for 3 consecutive days. One group of animals received LPS together with N^w-nitro-L-arginine methyl ester (L-NAME), a blocker of nitric oxide synthase (NOS) (for 4 days given in drinking water at a concentration of 50 mM). In liver, PB, in all groups, increased significantly both the concentration of tGSH and the activity of GSH-PX. When the animals were injected with LPS the levels of tGSH and GSSG were significantly higher compared with other groups while melatonin and L-NAME significantly enhanced tGSH when compared with that in the LPS-treated rats. Melatonin alone reduced GSSG levels and enhanced the activity of GSH-PX in LPS-treated animals. Additionally, LPS diminished the content of cytochrome P450 reductase with this effect being largely prevented by L-NAME administration. Melatonin did not change the content of P450 either in PB- or LPS-treated animals. In brain, melatonin and L-NAME increased both tGSH levels and the activity of GSH-PX in LPS-treated animals. The results suggest that melatonin protects against LPS-induced oxidative toxicity in PB-treated animals in both liver and brain, and the findings are consistent with previously published observations related to the antioxidant activity of the pineal hormone.     

The Relationship between Systemic Resistance Induced by Pruning and Accumulation of Antifungal Substances in Barley Seedlings

Infection by a compatible race of Erysiphe graminis f. sp. hordei on barley secondary leaves was significantly suppressed upon pruning of the primary leaves when E. graminis hordei was inoculated 3–12 h after the pruning, but it, was rather enhanced during 15–21 h. The accumulation of antifungal substances was detected in hot ethanol extracts of barley seedlings from 15–27 h after pruning the primary leaves. Taking the time of the infection process of a challenger (E. graminis, hordei) into consideration, timing of systemic resistance induced upon pruning coincided with the accumulation of antifungal substances.     

Specific phases of root hair attachment in the Rhizobium trifolii-clover symbiosis

The time course and orientation of attachment of Rhizobium trifolii 0403 to white clover root hairs was examined in slide cultures by light and electron microscopy. Inocula were grown for 5 days on defined BIII agar medium and represented the large subpopulation of fully encapsulated single cells which uniformly bind the clover lectin trifoliin A. When 10(7) cells or more were added per seedling, bacteria attached within minutes, forming randomly oriented clumps at the root hair tips. Several hours later, single cells attached polarly to the sides of the root hair. This sequence of attachment to clover root hairs was selective for R. trifolii at inoculum sizes of 10(7) to 4 X 10(8) per seedling, specifically inhibited if 2-deoxy-D-glucose, a hapten for trifoliin A, was present in the inoculum, and not observed when 4 X 10(8) cells were added to alfalfa seedling roots or to large clover root cell wall fragments which lacked trifoliin A but still had trifoliin A receptors. Once attached, R. trifolii 0403 became progressively less detachable with 2-deoxy-D-glucose. At smaller inoculum sizes (10(5) to 10(6) cells per seedling), there was no immediate clumping of R. trifolii at clover root hair tips, although polar binding of bacteria along the root hair surface was observed after 4 h. The interface between polarly attached bacteria and the root hair cell wall was shown to contain trifoliin A by immunofluorescence microscopy. Also, this interface was shown by transmission electron microscopy to contain electron-dense granules of host origin. Scanning electron microscopy revealed an accumulation of extracellular microfibrils associated with the lateral and polar surfaces of the attached bacteria, detectable after 12 h of incubation with seedling roots. At this same time, there was a significant reduction in the effectiveness of 2-deoxy-D-glucose in dislodging bacteria already attached to root hairs and an increase in firm attachment of bacteria to the root hair surface, which withstood the hydrodynamic shear forces of high-speed vortexing. These results are interpreted as a sequence of phases in attachment, beginning with specific reversible interactions between bacterial and plant surfaces (phase I attachment), followed by production of extracellular microfibrils which firmly anchor the bacterium to the root hair (phase 2 adhesion). Thus, attachment of R. trifolii to clover root hairs is a specific process requiring more than just the inherent adhesiveness of the bacteria to the plant cell wall.(ABSTRACT TRUNCATED AT 400 WORDS)     

A fluorimetric determination of the activity of glycolipid transfer protein and some properties of the protein purified from pig brain

The fluorimetric method of Correa-Freire et al. (Correa-Freire, M.C., Barenholz, Y. and Thompson, T.E. (1982) Biochemistry 21, 1244-1248) to measure glucosylceramide transfer between phospholipid bilayers has been applied to the determination of the activity of glycolipid transfer protein purified from pig brain. The transfer of pyrene-labeled galactosylceramide (PyrGalCer) from donor to acceptor vesicles was measured by a decrease in the intensity ratio of eximer (E) to excited monomer (M). A sensitive determination of the glycolipid transfer activity is possible by the fluorimetric method without separation of the donor and acceptor vesicles. The newly developed fluorimetric assay of glycolipid transfer protein was used to study the effects of N-ethylmaleimide, HgCl2 and sugars on the transfer activity. The treatment with N-ethylmaleimide inactivated the activity to about 40%. The activity was almost completely inactivated by the treatment with HgCl2. Monosaccharides and methyl-alpha-D-glucoside had no inhibitory effect on the transfer activity. A marked and immediate drop of the E/M ratio was observed by the addition of glycolipid transfer protein to vesicles containing PyrGalCer at a protein-to-PyrGalCer molar ratio of 1.56:1. The result suggests a complex formation of glycolipid transfer protein with PyrGalCer.     

Specific phases of root hair attachment in the Rhizobium trifolii-clover symbiosis.

The time course and orientation of attachment of Rhizobium trifolii 0403 to white clover root hairs was examined in slide cultures by light and electron microscopy. Inocula were grown for 5 days on defined BIII agar medium and represented the large subpopulation of fully encapsulated single cells which uniformly bind the clover lectin trifoliin A. When 10(7) cells or more were added per seedling, bacteria attached within minutes, forming randomly oriented clumps at the root hair tips. Several hours later, single cells attached polarly to the sides of the root hair. This sequence of attachment to clover root hairs was selective for R. trifolii at inoculum sizes of 10(7) to 4 X 10(8) per seedling, specifically inhibited if 2-deoxy-D-glucose, a hapten for trifoliin A, was present in the inoculum, and not observed when 4 X 10(8) cells were added to alfalfa seedling roots or to large clover root cell wall fragments which lacked trifoliin A but still had trifoliin A receptors. Once attached, R. trifolii 0403 became progressively less detachable with 2-deoxy-D-glucose. At smaller inoculum sizes (10(5) to 10(6) cells per seedling), there was no immediate clumping of R. trifolii at clover root hair tips, although polar binding of bacteria along the root hair surface was observed after 4 h. The interface between polarly attached bacteria and the root hair cell wall was shown to contain trifoliin A by immunofluorescence microscopy. Also, this interface was shown by transmission electron microscopy to contain electron-dense granules of host origin. Scanning electron microscopy revealed an accumulation of extracellular microfibrils associated with the lateral and polar surfaces of the attached bacteria, detectable after 12 h of incubation with seedling roots. At this same time, there was a significant reduction in the effectiveness of 2-deoxy-D-glucose in dislodging bacteria already attached to root hairs and an increase in firm attachment of bacteria to the root hair surface, which withstood the hydrodynamic shear forces of high-speed vortexing. These results are interpreted as a sequence of phases in attachment, beginning with specific reversible interactions between bacterial and plant surfaces (phase I attachment), followed by production of extracellular microfibrils which firmly anchor the bacterium to the root hair (phase 2 adhesion). Thus, attachment of R. trifolii to clover root hairs is a specific process requiring more than just the inherent adhesiveness of the bacteria to the plant cell wall.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic polymorphism of complement C6 and haplotype analysis between C6 and C7 in a Japanese population

Summary Genetic polymorphism of C6 in the Japanese population has been described using polyacrylamide gel isoelectric focusing electrophoresis followed by the electrophoretic blotting technique, and haplotype analysis between C6 and C7 has also been investigated. In 565 plasma samples five different common patterns and three rare variant patterns were observed, and these were controlled by autosomal codominance at a single locus with three common and one rare alleles. These alleles were designated C6^*B, C6^*A, C6^*B2, and C6^*M, and gene frequencies were estimated to be 0.50265, 0.43186, 0.06018, and 0.00531 for C6^*B, C6^*A, C6^*B2, and C6^*M, respectively. It is noteworthy that C6^*B2 has a polymorphic frequency in the Japanese population. The distribution of phenotypes fitted the Hardy-Weinberg equilibrium. Two combinations between C6 and C7 alleles, namely C6B-C7B and C6M-C7B, were shown to be in significant positive linkage disequilibrium. The presence of allelic combinations showing linkage disequilibrium suggests the close proximity between the C6 and C7 loci.     

G6PD Ube, a glucose-6-phosphate dehydrogenase variant found in four unrelated Japanese families.

A total of 6,120 Japanese males were screened for glucose-6-phosphate dehydrogenase deficiency (G6PD). Five cases with the deficiency were discovered. Two of them and an additional two cases have the same variant, G6PD Ube, characterized by moderate enzyme deficiency, fast moving enzyme activity on electrophoresis, high Ki Nadph, utilization of substrate analogues, kinetics, pH optima, and stability. This variant was distinguished for G6PD A- and from other Oriental variants by biochemical parameters. Differences in the frequency and type of the variants between southern Asia and Japan, suggest that the Japanese who have been isolated on islands where malaria is not endemic, may have developed their own variant traits.     

Partial tetrasomy 9(9pter to 9q2101) due to an extra iso-dicentric chromosome.

A 3-year-old boy with partial No. 9 tetrasomy is described. The patient showed markedly retarded physical and mental development as well as multiple congenital anomalies. Routine chromosome analysis revealed an extra C-group chromosome. It had a pronounced secondary constriction at the proximal part of its long arm. Based on studies by a variety of banding techniques, the extra chromosome was identified to be an iso-dicentric No. 9 chromosome with inactivation of one of the two centromeres, the karyotype being 47,XY, + DIC (9)(Q2101). The value of BrdUrd treatment was emphasized in the detection of a very small piece of euchromatin within a long stretch of constitutive heterochromatin.     

Vitamin A receptors. I. Comparison of retinol binding to serum retinol-binding protein and to tissue receptors in chick retina and pigment epithelium.

1. A simple, efficient three-step method for purification of serum retinol-binding-protein is described with homogeneity obtained after chromatography on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. 2. Evidence is presented indicating that retinol receptors present in the cytosol fraction of chick retina and pigment epithelium are separate and distinct from purified retinol-binding protein. Fluorescence characteristics are different in tissue cytosol and serum as assessed by sucrose density gradient analysis. Tissue retinol receptors do not interact with human serum prealbumin although the prealbumin readily complexes with purified chicken retinol-binding protein. Likewise, no binding to serum retinol-binding protein antibody could be detected by sucrose density gradient analysis, in immunoprecipitation experiments or by double immunodiffusion. It thus appears that specific retinol receptors are present in neural retina and pigment epithelium that are different from serum retinol-binding protein.