Suppression of glial glutamine release to the extracellular fluid studied in vivo by NMR and microdialysis in hyperammonemic rat brain

Release of glial glutamine (GLN) to the extracellular fluid (ECF), mainly mediated by the bidirectional system N transporter SN1, was studied in vivo in hyperammonemic rat brain, using (15)N-nuclear magnetic resonance (NMR) to monitor intracellular [5-(15)N]GLN and microdialysis/gradient (1)H-(15)N heteronuclear single-quantum correlation NMR to analyse extracellular [5-(15)N]GLN. GLN(ECF) was elevated to 2.4 +/- 0.2 mm after 4.5 h of intravenous ammonium acetate infusion. The [GLN(i)]/[GLN(ECF)] ratio (i = intracellular) was 9.6 +/- 0.9, compared with 17-20 in normal brain. GLN(ECF) then decreased substantially at t = 4.9 +/- 0.1 h. Comparison of the time-courses of intra- and extra-cellular [5-(15)N]GLN strongly suggested that the observed decrease reflects partial suppression of glial GLN release to ECF. Suppression also followed elevation of GLN(ECF) to 1.9 mM, resulting in a [GLN](i)/[GLN(ECF)] ratio of 8.4, upon perfusion of alpha-(methylamino)isobutyrate which inhibits neuronal uptake of GLN(ECF) mediated by sodium-coupled amino acid transporter (SAT). The results provide first evidence for bidirectional operation of SN1 in vivo, and clarify the effect of transmembrane GLN gradient on glial GLN release at physiological Na(+) gradient. Implications of the results for SN1 as an additional regulatory site in the glutamine/glutamate cycle and utility of this approach for examining the role of GLN in an experimental model of fulminant hepatic failure are discussed.     

Phylogenetic position of Rickettsia tsutsugamushi and the relationship among its antigenic variants by analyses of 16S rRNA gene sequences

Abstract The 16S rRNA gene sequences of Rickettsia tsutsugamushi and Rickettsia sibirica were determined by PCR and DNA sequencing. Phylogenetic analysis revealed that R. sibirica is positioned in a cluster of the genus Rickettsia with a similarity value of 98.1–99.6%, whereas R. tsutsugamushi is located apart from the cluster with a similarity value of 90.2–90.6%. This evidence suggests that R. tsutsugamushi should be excluded taxonomically from the genus Rickettsia . The phylogenetic classification of six antigenic variants in R. tsutsugamushi moderately reflected their antigenic relationship known in closely and distantly related strains.     

Isolation and characterization of sex chromosome rearrangements generating male muscle dystrophy and female abnormal oogenesis in the silkworm, Bombyx mori

In deletion-mapping of W-specific RAPD (W-RAPD) markers and putative female determinant gene (Fem), we used X-ray irradiation to break the translocation-carrying W chromosome (W ^Ze ). We succeeded in obtaining a fragment of the W ^Ze chromosome designated as Ze ^W, having 3 of 12 W-RAPD markers (W-Bonsai, W-Yukemuri-S, W-Yukemuri-L). Inheritance of the Ze ^W fragment by males indicates that it does not include the Fem gene. On the basis of these results, we determined the relative positions of W-Yukemuri-S and W-Yukemuri-L, and we narrowed down the region where Fem gene is located. In addition to the Ze ^W fragment, the Z chromosome was also broken into a large fragment (Z₁) having the + ^sch (1-21.5) and a small fragment (Z₂) having the + ^od (1-49.6). Moreover, a new chromosomal fragment (Ze ^WZ₂) was generated by a fusion event between the Ze ^W and the Z₂ fragments. We analyzed the genetic behavior of the Z₁ fragment and the Ze ^WZ₂ fragment during male (Z/Z₁ Ze ^WZ₂) and female (Z₁ Ze ^WZ₂/W) meiosis using phenotypic markers. It was observed that the Z₁ fragment and the Z or the W chromosomes separate without fail. On the other hand, non-disjunction between the Ze ^WZ₂ fragment and the Z chromosome and also between the Ze ^WZ₂ fragment and the W chromosome occurred. Furthermore, the females (2A: Z/Ze ^WZ₂/W) and males (2A: Z/Z₁) resulting from non-disjunction between the Ze ^WZ₂ fragment and the W chromosome had phenotypic defects: namely, females exhibited abnormal oogenesis and males were flapless due to abnormal indirect flight muscle structure. These results suggest that Z₂ region of the Z chromosome contains dose-sensitive gene(s), which are involved in oogenesis and indirect flight muscle development.     

Antimicrobial activity of lipoprotein particles containing apolipoprotein Al

Human plasmain vitro inhibits the growth of coagulase negative staphylococci,S. epidermidis, which may be pathogenic in the immunocompromised host. To determine the antimicrobial components, serum was fractionated by column chromatography, which revealed that elution areas where lipoproteins can be yielded had high antimicrobial activity againstS. epidermidis. Therefore, lipoprotein fractions, including very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL), were separated by ultracentrifugation and incubated withS. epidermidis. All 3 lipoprotein fractions suppressed bacterial growth within the first 3 h but VLDL enhanced bacterial growth after 9 h of incubation compared with the control. HDL, however, inhibited bacterial growth throughout 21 h of incubation.To confirm these results, serum from healthy volunteers was separated by ion exchange column chromatography and again by HPLC to purify the antimicrobial fraction. In the protein analysis with gradient polyacrylamide-SDS gel, apolipoprotein Al (apo Al), which is a major apolipoprotein of HDL, was detected in the antimicrobial fraction. Therefore, this fraction was loaded onto an immunoaffinity column coupled with the anti-apo Al monoclonal antibody (Mab). Unbound fraction had no antimicrobial activity, but anti-S. epidermidis activity was recovered from the bound fraction which consisted mainly of apo Al, All and apo C in protein composition.These results indicated that the antimicrobial activity was associated with the apo Al-containing lipoprotein particles (HDL). This property of HDL may directly affect bacterial growth and promote the self-defense mechanisms of normal and immunocompromised individuals.     

Determination of Reduced, Protein-Unbound, and Total Concentrations of N-Acetyl- -cysteine and -Cysteine in Rat Plasma by Postcolumn Ligand Substitution High-Performance Liquid Chromatography

A high-performance liquid chromatographic assay was developed for the quantitative determination of the sulfur-containing amino acids N-acetyl- -cysteine (NAC) and -cysteine (Cys) in rat plasma. The thiols were separated by reverse-phase ion-pair chromatography, and the column eluent was continuously mixed with an iodoplatinate-containing solution. The substitution of sulfur of the thiol compound with iodide was quantitatively determined by measuring changes in the absorption at 500 nm. The low-molecular-weight disulfides and mixed disulfide conjugates of thiols with proteins were entirely reduced to the original reduced compounds by dithiothreitol. By reducing these two types of disulfides separately during sample pretreatment, the reduced, protein-unbound, and total thiol concentrations could also be determined. Validation testing was performed, and no problems were encountered. The limit of detection was approximately 20 pmol of thiol on the column. The present method was used to measure the plasma concentrations of NAC and Cys in the rat after a bolus intravenous administration of NAC, focusing on disulfide formation. The binding of NAC to protein through mixed disulfide formation proceeds in a time-dependent and reversible manner. Moreover, this “stable” covalent binding might limit total drug elimination, while the unbound NAC is rapidly eliminated. Consequently, the analytical method described in this study is very useful for the determination of plasma NAC and Cys, including disulfide conjugates derived from them.     

Clone-specific responses in leaf phenolics of willows exposed to enhanced UVB radiation and drought stress

The effects of enhanced UVB radiation and drought stress on willow secondary phenolics were studied using the leaves of 8‐week‐old micropropagated plantlets from interspecific hybrids (Salix myrsinites L. ×S. myrsinifolia Salisb.) and pure species (S. myrsinifolia). The plantlets were subjected for 4 weeks to two levels of UVB radiation (ambient, enhanced) and two levels of watering (well‐watered, drought‐stressed) according to a 2 × 2 factorial design. Enhanced UVB radiation increased the total concentration of flavonoids and phenolic acids in all plantlets, while the total concentration of salicylates remained unaffected. Drought stress reduced the total concentration of salicylates and phenolic acids in S. myrsinifolia plantlets, while in hybrids only phenolic acids were affected. The response of phenolic acids to enhanced UVB in drought‐stressed plantlets was different from that in well‐watered ones, indicating that drought stress limited the accumulation of phenolic acids under enhanced UVB radiation. Flavonoids increased in response to enhanced UVB radiation in drought‐stressed plantlets, although drought caused serious physiological stress on growth. There were significant differences between hybrid and S. myrsinifolia plantlets with respect to the composition of phenolics and between families and clones with respect to their concentration. In addition, the response of salicylates, flavonoids and phenolic acids to enhanced UVB and drought stress was clone‐specific, which may indicate that climatic changes will alter the genetic composition of northern forests.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.