Isolation and characterization of an asparagine-linked keratan sulfate from the skin of a marine teleost, Scomber japobicus

Keratan sulfate was isolated from the skin of Pacific mackerel (Scomber japonicus) after exhaustive digestion with pronase followed by ethanol precipitation and fractionation on a cellulose column with 0.3% recovery of dried material. The keratan sulfate preparation was separated into four major fractions by Dowex-1 column chromatrography. The chemical and infrared spectrum analyses of the four fractions showed a high degree of heterogeneity in sulfation. Since the carbohydrate-peptide linkage in the teleost skin keratan sulfate was found to be stable in alkali, and asparagine was the predominant amino acid, the asparagine residue in the peptide backbone was most likely to be involved in the N-glycosyl linkage with the carbohydrate moiety. Besides the type of carbohydrate-peptide linkage, the teleost skin keratan sulfate is very similar to corneal keratan sulfate, (keretan sulfate I) in two respects: (1) The teleost skin and bovine corneal keratan sulfates were hydrolyzed much faster by endo-β-galactosidase that the whale nasal cartilage keratan sulfate (keratan sulfate II). (2) Although the teleost skin keratan sulfate showed considerable polydispersity, the molecular weight was in the same range as the corneal keratan sulfate, and it was relatively higher than that of the cartilage keratan sulfate.     

High resolution proton NMR studies of gangliosides. Structure of two types of GD3 lactones and their reactivity with monoclonal antibody R24

Ganglioside GD3 was converted at room temperature to two stable lactones, denoted as GD3 lactones I and II. The reaction sequence was presumed to be GD3----GD3 lactone I----GD3 lactone II based on the time course of their production. Lactone I behaved as a monosialoganglioside and lactone II as a neutral species. The two lactones were isolated by DEAE-Sephadex column chromatography. The positions of the inner ester linkages were investigated by two-dimensional J-correlated proton NMR spectroscopy. An ester linkage was most likely formed between the carboxyl group of the external sialic acid residue and C9-OH of the internal sialic acid residue in lactone I. In addition to this ester linkage, a second ester linkage between the carboxyl group of the internal sialic acid and C2-OH of the galactose residue was likely formed in lactone II. The structural changes induced by lactonization were further examined by their reactivity with the monoclonal antibody R24 (Puckel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., and Old, L. J. (1982) J. Exp. Med. 155, 1133-1147), which reacted with GD3. R24 was found to bind weakly to GD3 lactone I, but not to GD3 lactone II. The results suggest that the monoclonal antibody requires both sialic acid residues for high affinity binding, and the complete lactonization results in a loss of negative charges and/or a change in the overall conformation of the oligosaccharide moiety which may account for the loss of binding.     

Antibodies against ganglioside complexes in Guillain-Barré syndrome and related disorders

Guillain-Barré syndrome (GBS) is acute autoimmune neuropathy, often subsequent to an infection. Serum anti-ganglioside antibodies are frequently elevated in titer. Those antibodies are useful diagnostic markers and possible pathogenetic factors. Recent data demonstrated that sera from some patients with GBS react with ganglioside complexes (GSCs) consisting of two different gangliosides, but not with each constituent ganglioside. Those antibodies may specifically recognize a new conformational epitope formed by two gangliosides. In particular, the antibodies against GD1a/GD1b and/or GD1b/GT1b complexes are associated with severe GBS requiring artificial ventilation. The antibodies to GM1/GalNAc-GD1a and those to GSCs containing GQ1b or GT1a are associated with pure motor GBS and Fisher syndrome, respectively. In contrast, the binding activities of the antibodies highly specific to GD1b are strongly inhibited by the addition of GD1a to GD1b. Gangliosides along with other components as cholesterol are known to form lipid rafts, in which two different gangliosides may form a new conformational epitope. Future investigation is necessary to elucidate the roles of GSCs in the plasma membrane and of the clinical relevance of the anti-GSCs antibodies.     

Phenylalanine 171 is a molecular brake for translesion synthesis across benzo[a]pyrene-guanine adducts by human DNA polymerase kappa

Human cells possess multiple specialized DNA polymerases (Pols) that bypass a variety of DNA lesions which otherwise would block chromosome replication. Human polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. To better understand the relationship between the structural features in the active site and lesion bypass by Pol κ, we mutated codons corresponding to amino acids appearing close to the adducts in the active site, and compared bypass efficiencies. Remarkably, the substitution of alanine for phenylalanine 171 (F171), an amino acid conserved between Pol κ and its bacterial counterpart Escherichia coli DinB, enhanced the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG 18-fold. This substitution affected neither the fidelity of TLS nor the efficiency of dCMP incorporation opposite normal guanine. This amino acid change also enhanced the binding affinity of Pol κ to template/primer DNA containing (-)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 functions as a molecular brake for TLS across BPDE-N(2)-dG by Pol κ and that the F171A derivative of Pol κ bypasses these DNA lesions more actively than does the wild-type enzyme.     

Adenovirus vector-mediated assay system for hepatitis C virus replication

The efficient delivery of the hepatitis C virus (HCV) RNA subgenomic replicon into cells is useful for basic and pharmaceutical studies. The adenovirus (Ad) vector is a convenient and efficient tool for the transduction of foreign genes into cells in vitro and in vivo. However, an Ad vector expressing the HCV replicon has never been developed. In the present study, we developed Ad vector containing an RNA polymerase (pol) I-dependent expression cassette and a tetracycline-controllable RNA pol I-dependent expression system. We prepared a hybrid promoter from the tetracycline-responsive element and the RNA pol I promoter. Ad vector particles coding the hybrid promoter-driven HCV replicon could be amplified, and interferon, an inhibitor of HCV replication, reduced HCV replication in cells transduced with the Ad vector coding HCV replicon. This is the first report of the development of an Ad vector-mediated HCV replicon system.     

Daily fecal sex steroid hormonal changes and mating success in captive female cheetahs (Acinonyx jubatus) in Japan

Daily fecal estrogen and progestin concentrations were measured by enzyme immunoassay in five female cheetahs (Acinonyx jubatus) for 4-6 months. The animals were housed under different conditions: (1) a female always housed in a group including one or more males; (2) two females isolated individually for short or long periods; (3) the other two females housed together. These females were separately housed with males for mating around the time of the estrogen peaks. The hormone profiles were similar in all five females regardless of the housing conditions. However, only the female that had been isolated from other cheetahs for over a year mated and reproduce cubs successfully, whereas the remaining four did not (one was isolated for only 6 weeks, another was always housed with males and the other two were housed together). In all females, the estrogen peaks were obtained at regular intervals of approximately 8-15 days. Unlike estrogen, the progestin concentrations were always low in all females except during pregnancy and they did not increase following the estrogen surges. These results showed that female cheetahs are typically reflex ovulators and female receptiveness may not be reflected to her hormonal states. It was also suspected that individual housing and long-term separation are advantageous for breeding this wild cat in captivity, mimicking the ecological/behavioral patterns in the wild, though housing condition might have no effect on the estrous cycle.     

A non-NadB type L-aspartate dehydrogenase from Ralstonia eutropha strain JMP134: molecular characterization and physiological functions

We report the molecular characterization and physiological function of a novel L-aspartate dehydrogenase (AspDH). The purified enzyme was a 28-kDa dimeric protein, exhibiting high catalytic activity for L-aspartate (L-Asp) oxidation using NAD and/or NADP as cofactors. Quantitative real-time PCR analysis indicated that the genes involved in the AspDH gene cluster, poly-3-hydroxyalkanoate (PHA) biosynthesis, and the TCA cycle were substantially induced by L-Asp in wild-type cells. In contrast, expression of the aspartase and aspartate aminotransferase genes was substantially induced in the AspDH gene knockout mutant (ΔB3576) but not in the wild type. GC-MS analyses revealed that the wild-type strain synthesized poly-3-hydroxybutyrate from fructose or L-Asp, whereas the ΔB3576 mutant did not synthesize PHA from L-Asp. AspDH gene cluster products might be involved in the biosynthesis of the PHA precursor, revealing that AspDH was a non-NadB type enzyme, and thus entirely different from the previously reported NadB type enzymes working in NAD biosynthesis.     

Genome profiling (GP) method based classification of insects: congruence with that of classical phenotype-based one

Background

Ribosomal RNAs have been widely used for identification and classification of species, and have produced data giving new insights into phylogenetic relationships. Recently, multilocus genotyping and even whole genome sequencing-based technologies have been adopted in ambitious comparative biology studies. However, such technologies are still far from routine-use in species classification studies due to their high costs in terms of labor, equipment and consumables.

Methodology/Principal Findings

Here, we describe a simple and powerful approach for species classification called genome profiling (GP). The GP method composed of random PCR, temperature gradient gel electrophoresis (TGGE) and computer-aided gel image processing is highly informative and less laborious. For demonstration, we classified 26 species of insects using GP and 18S rDNA-sequencing approaches. The GP method was found to give a better correspondence to the classical phenotype-based approach than did 18S rDNA sequencing employing a congruence value. To our surprise, use of a single probe in GP was sufficient to identify the relationships between the insect species, making this approach more straightforward.

Conclusion/Significance

The data gathered here, together with those of previous studies show that GP is a simple and powerful method that can be applied for actually universally identifying and classifying species. The current success supported our previous proposal that GP-based web database can be constructible and effective for the global identification/classification of species.