Mapping of the low pH-sensitive conformational epitope of rabies virus glycoprotein recognized by a monoclonal antibody #1-30-44

Monoclonal antibody (mAb) #1-30-44 recognized an acid-sensitive conformational epitope of rabies virus glycoprotein (G). The antigenicity of G protein exposed on the cell surface was lost when the infected cells were exposed to pH 5.8. By comparing the deduced amino acid sequence of G protein between the HEP-Flury strain and the epitope-negative CVS strain as well as the mAb-resistant escape mutants, two distant sites that contained Lys-202 and Asn-336 were shown to be involved in the epitope formation. Lys-202 is located in the so-called neurotoxin-like sequence, while Asn-336 is included in antigenic site III and is very near the amino acid at position 333, which is known to affect greatly the neuropathogenicity of rabies virus when changed. Consistent with this finding, antigenicity of a neurovirulent revertant of the HEP-Flury strain, in which Gln-333 of G protein was replaced by Arg, was also affected as shown by its greatly decreased reactivity with mAb #1-30-44 compared to that of the original avirulent HEP virus. Based on these results, we hypothesize that the neurotoxin-like domain and some amino acids in antigenic site III come into contact with each other to form a conformational epitope for mAb #1-30-44, and such a configuration would be lost when exposed to acidic conditions to perform a certain low pH-dependent function of G protein.     

Role of MAPK phosphorylation in cytoprotection by pro-vitamin C against oxidative stress-induced injuries in cultured cardiomyoblasts and perfused rat heart

The reactive oxygen species (ROS) are known to be generated upon post-ischemic reperfusion (I/R) of the heart, and to injure cardiac muscle cells. The hydrogen peroxide-induced mortality of rat cardiomyoblasts H2c9 was markedly inhibited by previous administration with auto-oxidation-resistant pro-vitamin C, the 2-O-phosphorylated derivative (Asc2P) of ascorbic acid (Asc). The cytoprotection was partially counteracted by an inhibitor of MAPK (mitogen-activated protein kinase) kinase (MEK) as shown by DNA strand cleavage assay and mitochondrial dehydrogenase assay. Immunostains indicated that phosphorylated MAPK increased in the hydrogen peroxide-treated cardiomyoblasts, and that this action was moderately inhibited by Asc2P and restored nearly to the initial, pretreatment level by combined administration of the MEK inhibitor and Asc2P. The I/R-induced cell injuries in perfused rat hearts as estimated by extracellular release of the cardiac enzyme CPK were inhibited by 2-O-alpha-glucosylascorbic acid (Asc2G) and Asc, whereas the observed cytoprotection for the cardiomyoblasts was partially counteracted by the MEK inhibitor. The increase in phosphorylated MAPK in I/R-operated hearts was moderately inhibited by pro-vitamin C, but restored nearly to the normal non-operated level by combined administration with the MEK inhibitor. This is in contrast to no alteration in levels of non-phosphorylated MAPK for all the cases examined as shown by Western blots, consistent with results of immunostains for the cardiomyoblasts. The inhibitory effect of the MEK inhibitor on MAPK phosphorylation was, therefore, suggested to counteract the cytoprotective effects of pro-vitamin C via a thorough interruption of the phosphorylated MAPK signaling pathway. This was not true of ROS-related events; the scavenging effects of Asc2G and Asc on hydroxyl radicals generated from I/R-operated heart were not affected by combined administration with the MEK inhibitor, as shown by the spin-trapping DMPO-based ESR method.     

Comparison of Three Methods for Epidemiological Typing of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">C. coli</Emphasis>

A total of 168 Campylobacter strains (154 C. jejuni and 14 C. coli) isolated from human clinical samples and chicken meat were typed using Penner serotyping, randomly amplified polymorphic DNA (RAPD), and pulsed-field gel electrophoresis (PFGE) with four restriction enzymes (Sac II, Sal I, Sma I, Kpn I).The 168 strains were found to represent 13 different Penner-types and 72 different RAPD-types. However, the discriminatory potential of PFGE was dependent on the restriction enzymes used. The 168 strains were divided into 74 (Sac II), 73 (Sal I), 72 (Sma I) and 69 (Kpn I) types. The DNA of some strains was not digested by Sal I, Sma I and Kpn I. Although three RAPD-types were further subdivided by PFGE, RAPD showed good discriminatory power and a high level of agreement with PFGE patterns in terms of strain differentiation.To compare the similarities of PFGE patterns (Sac II) among the strains, a dendrogram was constructed based on the unweighted pair group method with averages (UPGMA). In most cases, DNA types of C. coli were different from those of C. jejuni. The similarities between human and meat isolates were less than 0.42 except for one outbreak in which the isolates from both patients and chicken meat showed the same DNA types.     

Inhibition of cholesterol crystallization under bilirubin deconjugation: partial characterization of mechanisms whereby infected bile accelerates pigment stone formation

Pigment gallstones have been reported to be closely associated with biliary tract infection. We previously reported that addition of unconjugated bilirubin (UCB), which is deconjugated by beta-glucuronidase in infected bile, could enhance cholesterol crystal formation in supersaturated model bile (MB). The present study evaluated the effect of beta-glucuronidase on the processes of pigment gallstone formation and cholesterol crystallization. Supersaturated MB (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 10.0 g/dl and a cholesterol saturation index (CSI) of 2.0) and native rat bile were mixed at a ratio of 3:1. Then, mixed bile was incubated with or without beta-glucuronidase and changes of the following parameters were investigated over time: (1) the UCB/total bilirubin ratio; (2) cholesterol crystal formation; (3) the precipitate weight and the cholesterol concentration in the precipitate and supernatant; and (4) the lipid distribution of vesicles in the supernatant. Compared with beta-glucuronidase-free bile, (1) beta-glucuronidase-containing bile showed a significant increase of the UCB/total bilirubin ratio, (2) as well as a significantly longer nucleation time (96+/-17.0 vs. 114+/-20.0) and fewer cholesterol crystals. (3) The precipitate weight and the cholesterol concentration in the precipitate were significantly increased, while the cholesterol concentration in supernatant was decreased. (4) When mixed bile was incubated with beta-glucuronidase, the cholesterol concentration in the vesicles was lower than in bile without beta-glucuronidase. The precipitate weight and the cholesterol concentration in the precipitate was increased by incubation with beta-glucuronidase, while cholesterol concentration was decreased in the supernatant (especially in the vesicles). This means that bile vesicles were more stable and it was more difficult for cholesterol crystals to form. Thus, the presence of beta-glucuronidase may inhibit the formation of pure cholesterol stones even in the presence of cholesterol supersaturation.     

Hyaluronic acid: separation and biological implications

Hyaluronic acid (hyaluronan) is a ubiquitous extracellular matrix component, and present at high concentrations in skin, joints and cornea. In the skin, it is synthesized primarily by dermal fibroblasts and by epidermal keratinocytes. Hyaluronic acid usually exists as a high molecular mass (600,000-1,000,000) and non-sulfated glycosaminoglycan composed of a disaccharide unit of [bond]3GlcNAc beta 1[bond]4GlcA beta 1[bond]. Hyaluronic acid has been widely used not only for osteoarthritis and ophthalmology but also for cosmetics for skin care. To examine the biological activities of hyaluronic acid, we have to accurately determine the quantity and molecular masses in biological samples. We review recent development in the analysis of hyaluronic acid having various molecular sizes using electrophoretic and chromatographic techniques. Recently, interactions between hyaluronic acid oligomers and hyaluronic acid-binding proteins have attracted the interest for understanding the biological functions. We show some interesting reports on biological interactions of hyaluronic acid and its oligomers with some proteins.     

Cobalt(III) promoted ligand fusion reactions of thiobarbituric acid and 4,6-diamino-2-thiouracil (or 4-amino-2-thiouracil)

Novel cobalt(III) complexes containing three kinds of assembled ligands, L₁L₂=dapymt-tbba(2-), tbba-dapymt(1-) and apymt-tbba(1-) (H₃tbba=thiobarbituric acid; Hdapymt=4,6-diamino-2-thiouracil; Hapymt=4-amino-2-thiouracil), were prepared from the mixed ligand systems, where L₂ indicates the coordinated ligand to the Co(III) ion and L₁ is a pendant ligand bonded to L₂. These complexes were characterized by UV-Vis absorption spectra and NMR spectroscopy. The crystal structures of [Co(Htbba)(en)₂]ClO₄·2H₂O (2) (en=ethane-1,2-diamine), [Co{dapymt-tbba(2-)}(en)₂]ClO₄·3H₂O (3) and [Co{apymt-tbba(1-)}(en)₂](ClO₄)Cl·3H₂O (5′) revealed that coordination occurs through the S(1) and N(1) donors of tbba and the latter complexes 3 and 5′ have an assembled ligand; a new bond is formed between the C(5) atom of tbba and the S(2) atom of dapymt or apymt. An intramolecular hydrogen bond between O(1) of tbba and NH of en was found in all crystals. An interesting intermolecular π-π stacking interaction was found in 5′.