Solid-state 15N NMR of oriented lipid bilayer bound gramicidin A'

Highly oriented samples of lipid and gramicidin A' (8:1 molar ratio) have been prepared with the samples extensively hydrated (approximately 70% water v/w). These preparations have been shown to be completely in a bilayer phase with a transition temperature of 28 degrees C, and evidence is presented indicating that the gramicidin is in the channel conformation. An estimate of the disorder in the alignment of the bilayers parallel with the glass plates used to align the bilayers can be made from the asymmetry of the nuclear magnetic resonances (NMR). Such an analysis indicates a maximal range of disorder of +/- 3 degrees. Uniformly 15N-labeled gramicidin has been biosynthesized by Bacillus brevis grown in a media containing 15N-labeled Escherichia coli cells as the only nitrogen source. When prepared with labeled gramicidin, the oriented samples result in high-resolution 15N NMR spectra showing 12 resonances for the 20 nitrogen sites of the polypeptide. The frequency of the three major multiple resonance peaks has been interpreted to yield the approximate orientation of the N-H bonds in the peptide linkages with respect to the magnetic field. These bond orientations are only partially consistent with the extant structural models of gramicidin.     

The renin-angiotensin system in hypothyroidism of short duration

In six hypothyroid patients (2 male, 4 females, ages 22 through 59 years), plasma renin activity (PRA) and aldosterone (Aldo) were measured when the patients were euthyroid on levothyroxine therapy and one month after the therapy was stopped. Colonic mucosal potential differences were measured during the hypothyroid and euthyroid stages, and catecholamine sensitivity was determined by the blood pressure response to infused norepinephrine. Significant differences were observed in the PRA and aldosterone concentrations which were 4.1 +/- 2.5 ng/ml/h and 9.4 +/- 5.9 ng/dl, respectively in the hypothyroid stage and 6.9 +/- 2.3 ng/ml/h and 15.2 +/- 7.3 ng/dl, respectively when the patients were made euthyroid. The colonic mucosal potential differences (which reflect increased endogenous mineralocorticoid activity), became more electronegative after correction of hypothyroidism (-16.8 +/- 7.5 mV vs -32 +/- 18.2 mV; P less than 0.04) concentrations. Statistically significant decreases in norepinephrine pressor effects were observed in hypothyroid patients when compared to the euthyroid state (7.4 +/- 2.3 vs 10.9 +/- 1.9 micrograms/ng/min; P less than 0.01). It is concluded that patients with hypothyroidism have a hormonal pattern reminiscent of "low renin hypertension", and exhibit decreased sensitivity to catecholamines. Such changes are corrected when the patients become euthyroid on levothyroxine therapy.     

Morphological,biochemical, and molecular characterization of Oscillatoria kawamurae (Oscillatoriales,Cyanobacteria) isolated from different geographical regions

Oscillatoria kawamurae is an unusual freshwater cyanobacterium because of its large trichome and ambiguous gas vacuole. Because little is known about its phenotypic or genotypic characteristics, this study conducted morphological, biochemical, and genetic characterization of O. kawamurae strains isolated from Japan, Laos, and Myanmar. All strains displayed similar morphological characteristics; however some differences were observed in vegetative cell widths, trichome colors, and the distribution patterns of their gas vacuole‐like structures. The in vivo and phycobiliprotein absorption spectra revealed the two different trichome colors found in the four representative strains of O. kawamurae (Inle1, Lao7, Biwa6, and Inba3). These different trichome colors corresponded to the different ratios of phycoerythrin and phycocyanin, the two types of phycobilin pigments: 0.25 for olive‐green strain (Inle1) and 0.65–0.73 for brown‐green strains (Biwa6, Inba3, and Lao7). Cellular fatty acid compositions of the four strains were C14:0, C15:0, C16:0, C16:1c, C17:0, C18:0, C18:1c, C18:3α and C18:4, whereas two strains (Biwa6 and Inba3) lacked C17:0. Of the fatty acids, palmitic acid (C16:0) was predominant. PCR experiments using primers targeting a gas vesicle gene (gvpA) recovered gvpA fragments from all O. kawamurae strains, suggesting that this species has true gas vacuoles. The 16S rDNA sequences of all of the strains were identical regardless of their different trichome colors and/or geographic origins. Phylogenetic analyses based on the 16S rDNA sequences indicated that O. kawamurae forms a monophyletic clade with O. princeps CCALA 1115 clB1 and O. duplisecta ETS‐06. We discuss the taxonomy of O. kawamurae based on the data obtained in this study.     

Sequences of E/NS1 Gene Junction from Four Dengue-2 Viruses of Northeastern Thailand and Their Evolutionary Relationships with Other Dengue-2 Viruses

We determined the 240-nueleotide sequences of the E/NS1 gene junction of four dengue-2 viruses by the primer extension dideoxy chain termination method. These viruses were isolated from dengue patients with different clinical severities in Nakhon Phanom, Northeastern Thailand in 1993. The results were compared with the 52 published dengue-2 sequences of the same gene region. Sequence divergence of four new isolates varied from 4.17% to 5.42% compared with dengue-2 prototype New Guinea C strain whereas it varied from 5.42% to 6.67% and from 6.67% to 7.09% when compared with Jamaica 1409 strain and PR159/S1 strain, respectively. All nucleotide substitutions were found at the 3rd position of the codons which were silent mutations. All 56 isolates studied were classified into five genotypic groups by constructing the dendrogram. The results indicated that four new isolates from Northeastern Thailand belong to genotype II of dengue virus serotype 2, and were most closely related to prototype New Guinea C strain. We also observed the variation in nucleotide and amino acid sequences among clusters of isolates (Thailand-1980, Malaysia-1989 and Thailand-1993) which were obtained from the dengue patients with different clinical severities. The significance of these genetic differences have been discussed in terms of the possible correlation between genetic variability and virulence.     

Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

Background  

Control genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples.     

Biohydrogen generation by mesophilic anaerobic fermentation of microcrystalline cellulose

Sixteen batch experiments were performed to evaluate the stability, kinetics, and metabolic paths of heat-shocked digester (HSD) sludge that transforms microcrystalline cellulose into hydrogen. Highly reproducible kinetic and metabolic data confirmed that HSD sludge could stably convert microcrystalline cellulose to hydrogen and volatile fatty acids (VFA) and induce metabolic shift to produce alcohols. We concluded that clostridia predominated the hydrogen-producing bacteria in the HSD sludge. Throughout this study the hydrogen percentage in the headspace of the digesters was greater than 50% and no methanogenesis was observed. The results emphasize that hydrogen significantly inhibited the hydrogen-producing activity of sludge when initial microcrystalline cellulose concentrations exceeded 25.0 g/L. A further 25 batch experiments performed with full factorial design incorporating multivariate analysis suggested that the ability of the sludge to convert cellulose into hydrogen was influenced mainly by the ratio of initial cellulose concentration (So) to initial sludge density (Xo), but not by interaction between the variables. The hydrogen-producing activity depended highly on interaction of So x (So/Xo). Through response surface analysis it was found that a maximum hydrogen yield of 3.2 mmol/g cellulose occurred at So = 40 g/L and So/Xo = 8 g cellulose/g VSS. A high specific rate of 18 mmol/(g VSS-d) occurred at So = 28 g/L and So/Xo = 9 g cellulose/g VSS. These experimental results suggest that high hydrogen generation from cellulose was accompanied by low So/Xo.     

Differential effects of carboxyfullerene on MPP+/MPTP-induced neurotoxicity

The effects of carboxyfullerene on a well-known neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenyl-pyridinium (MPP+) were investigated. In chloral hydrate-anesthetized rats, cytosolic cytochrome c was elevated in the infused substantia nigra 4 h after an intranigral infusion of MPP+. Five days after local application of MPP+, lipid peroxidation (LP) was elevated in the infused substantia nigra. Furthermore, dopamine content and tyrosine hydroxylase (TH)-positive axons were reduced in the ipsilateral striatum. Concomitant intranigral infusion of carboxyfullerene abolished the elevation in cytochrome c and oxidative injuries induced by MPP+. In contrast, systemic application of carboxyfullerene did not prevent neurotoxicity induced by intraperitoneal injection of MPTP. In mice, systemic administration of MPTP induced a dose-dependent depletion in striatal dopamine content. Simultaneous injection of carboxyfullerene (10 mg/kg) actually potentiated MPTP-induced reduction in striatal dopamine content. Furthermore, systemic administration of carboxyfullerene (30 mg/kg) caused death in the MPTP-treated mice. An increase in the striatal MPP+ level and reduction in hepatic P450 level were observed in the carboxyfullerene co-treated mice. These data showed that systemic application of carboxyfullerene appears to potentiate MPTP-induced neurotoxicity while local carboxyfullerene has been suggested as a neuroprotective agent. Furthermore, an increase in striatal MPP+ level may contribute to the potentiation by carboxyfullerene of MPTP-induced neurotoxicity.