Multimeric bivalent immunogens from recombinant tetanus toxin H<Subscript>C</Subscript> fragment,synthetic hexasaccharides,and a glycopeptide adjuvant

Using recombinant tetanus toxin H_C fragment (rTT-H_C) as carrier, we prepared multimeric bivalent immunogens featuring the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Ogawa, in combination with either the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Inaba, or a synthetic disaccharide tetrapeptide peptidoglycan fragment as adjuvant. The conjugation reaction was effected by squaric acid chemistry and monitored in virtually real time by SELDI-TOF MS. In this way, we could prepare well-defined immunogens with predictable carbohydrate–carrier ratio, whose molecular mass and the amount of each saccharide attached could be independently determined. The ability to prepare such neoglycoconjugates opens unprecedented possibilities for preparation of conjugate vaccines for bacterial diseases from synthetic carbohydrates.     

Immunocytochemical localization of thrombomodulin in the aqueous humor passage of the rat eye

 This report describes the distribution and localization of thrombomodulin (TM) in the rat eye by light and electron microscopic immunocytochemistry. In addition to the endothelium of the entire vasculature, TM was found on the non-vascular structures lining the cavities of the posterior and anterior chambers and the limbus. TM was localized on the basal, lateral, and apical plasma membranes of the inner and outer ciliary epithelium, and the posterior iris epithelium in which there was no polarized expression of TM. In the anterior chamber, TM was localized on the luminal surface of the corneal endothelium, but was negative on the anterior border layer of the iris, which is composed of a discontinuous layer of fibroblasts and collagen fibers. Thus, TM was present at sites of cell-to-cell contact. TM was also present on the endothelia of the trabecular meshwork and the Schlemm’s canal in the limbus. TM was localized not only on the luminal plasma membrane, but also on the cytoplasmic giant vacuoles in the endothelial cells of the Schlemm’s canal. These findings extend the importance of anticoagulant mechanisms to the systems of secretion, circulation, and drainage of the aqueous humor. Accepted: 18 March 1997     

Differences between Lactobacillus casei subsp. casei 2206 and Citrate-Positive Lactococcus lactis subsp. lactis 3022 in the Characteristics of Diacetyl Production

Lactobacillus casei subsp. casei 2206 exhibited much lower levels of diacetyl reductase activity than Citr⁺Lactococcus lactis subsp. lactis 3022 but two-, three-, and more than eightfold-higher levels of diacetyl synthase, lactate dehydrogenase, and NADH oxidase activities, respectively. A requirement for metal ions by the diacetyl synthases in both species was observed. The extracts of strain 2206 but not strain 3022 produced more diacetyl from pyruvate when the reaction for diacetyl synthase was aerated than when it was conducted statically.     

Biochemical synthesis of novel,self-assembling glycolipids from ricinoleic acid by a recombinant α-glucosidase from <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

Purpose of work  

To explore a novel glycolipid, we performed biochemical reactions using a recombinant α-glucosidase from Geobacillus sp. which shows excellent transglycosylation reaction to hydroxyl groups in a variety of compounds.     

Genome 3'-end repair in dengue virus type 2

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication.     

Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of α2,6-sialyltransferase derived from Chinese hamster ovary cells

Improvement of glycosylation is one of the most important topics in the industrial production of therapeutic antibodies. We have focused on terminal sialylation with alpha-2,6 linkage, which is crucial for anti-inflammatory activity. In the present study, we have successfully cloned cDNA of beta-galactosyl alpha-2,6 sialyltransferase (ST6Gal I) derived from Chinese hamster ovary (CHO) cells regardless of reports that stated this was not endogenously expressed in CHO cells. After expressing cloned ST6Gal I in Escherichia coli, the transferase activity was confirmed by HPLC and lectin binding assay. Then, we applied ST6Gal I to alpha-2,6 sialylation of the recombinant antibody; the ST6Gal I expression vector was transfected into the CHO cell line producing a bispecific antibody. The N-glycosylation pattern of the antibody was estimated by HPLC and sialidase digestion. About 70% of the total N-linked oligosaccharide was alpha-2,6 sialylated in the transfected cell line whereas no sialylation was observed in the non-transfected cell line. The improvement of sialylation would be of practical importance for the industrial production of therapeutic antibodies.     

Characteristics of microbial fermentation and potential digestibility of fiber in the hindgut of dugongs (Dugong dugon)

The number of microorganisms in the hindgut of dugongs (Dugong dugon) were estimated and their in vitro volatile fatty acid (VFA) production and degradation of eelgrass measured. Scanning electron microscopy showed that some rod bacteria attached to the surface of plant tissue degraded and eroded the cell walls. Number of starch-, lactate-, cellobiose-, pectin-, xylan- and cellulose-utilizing bacteria, sulfate-reducing bacteria and methane-producing bacteria were estimated at 10⁹ ～ 10¹⁰ colony forming units g^?1. Microorganisms degraded the cellulose and noncellulolytic components of the eelgrass, and about 47.3% of dry matter was degraded after 36?h in vitro incubation. The total VFA concentration was 10.5?mmol?dL^?1 at 36?h incubation, which included 55.7?mol% acetate, 18.0?mol% n-butyrate and 15.1?mol% propionate. The gas composition of in vitro fermentation was 68.4% carbon dioxide, 22.2% methane and 9.4% hydrogen.     

Induction of SCEs in CHL cells by dichlorobiphenyl derivative water pollutants, 2-phenylbenzotriazole (PBTA) congeners and river water concentrates

We recently identified dichlorobiphenyl (DCB) derivatives and 2-phenylbenzotriazole (PBTA) congeners as major mutagenic constituents of the waters of the Waka River and the Yodo River system in Japan, respectively. In this study we examined sister chromatid exchange (SCE) induction by two dichlorobiphenyl derivatives, 3,3′-dichlorobenzidine (DCB, 4,4′-diamino-3,3′-dichlorobiphenyl) and 4,4′-diamino-3,3′-dichloro-5-nitrobiphenyl (5-nitro-DCB); three PBTA congeners, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1), 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), and 2-[2-(acetylamino)amino]-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6); and water concentrates from the Waka River in Chinese hamster lung (CHL) cells. Concentration-dependent induction of SCE was found for all DCBs and PBTAs examined in the presence of S9 mix, and statistically significant increases of SCEs were detected at 2 μg per ml of medium or higher concentrations. SCE induction of MeIQx was examined to compare genotoxic activities of these water pollutants. According to the results, a ranking of the SCE-inducing potency of these compounds is the following: 5-nitro-DCB ≈ MeIQx > PBTA6 > PBTA-1 ≈ PBTA-2 > DCB.Water samples collected at a site at the Waka River showed concentration-related increases in SCEs at 6.25–18.75 ml-equivalent of river water per ml of medium with S9 mix. The concentrations of 5-nitro-DCB and DCB in the river water samples were from 2.5 to 19.4 ng/l and from 4100 to 18,900 ng/l, respectively. However, these chemicals showed only small contribution to SCE induction by the Waka River water.     

How is autonomic nervous system activity in subjects who are sleepy but are unable to sleep in the daytime?

We investigated changes in the activity of the autonomic nervous system (ANS) in the relaxed condition in subjects who felt sleepy, but were unable to sleep. A total of 1021 subjects underwent daytime polysomnography. The sleep latency (SL) and the visual analog scale (VAS) were used to assess “immediate” objective and subjective sleepiness, respectively. The subjects were assigned to an “Alert-Alert” group (VAS ≤ 25 mm, SL ≥ 8 min), a “Sleepy-Alert” group (VAS ≥ 75 mm, SL ≥ 8 min), or a “Sleepy-Sleepy” group (VAS ≥ 75 mm, SL ≤ 4 min). In order to assess the ANS, the spectral analysis and the geometric method were used. The ANS data collected during the relaxed condition (after lights off, post-LO) was compared to that obtained during the control condition (before lights off, pre-LO). From the spectral analysis, a significant decrease of sympathetic function and an increase of parasympathetic function at post-LO in the Sleepy-Sleepy group, a tendency for sympathetic function decrease at post-LO in the Alert-Alert group, and no significant changes to sympathetic and parasympathetic function in the Sleepy-Alert group were observed. The results from the geometric method supported the results of the spectral analysis in the Alert-Alert group and the Sleepy-Sleepy group. The results of this study suggest that the ANS plays a role in individuals who are unable to sleep even though they feel sleepy and are given the opportunity to sleep.

     

Axenic culture of <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> using antibiotics

The rotifer Brachionus plicatilis culture is composed of complex microcosms including bacteria, protozoans, algae, and fungi. Previous studies reported methods to establish axenic rotifer cultures, but further refinement of these techniques is needed, for molecular biological research which requires pure culture to isolate nucleic acids from rotifers only. In order to render rotifer culture axenic, we tested five antibiotics: ampicillin (Amp), chloramphenicol (Cp), kanamycin (Km), nalidixic acid (Na), and streptomycin (Sm) at 30–100 μg/ml. Except for Cp, which reduces rotifer reproduction, all other antibiotics at the tested concentrations did not affect rotifer reproduction or show any toxic effects. A rotifer disinfection method was finally established by treating the resting eggs with 0.25% (w/v) sodium hypochlorite (NaOCl) for 3 min, washing with sterilized sea water, and then exposing the neonates to an Amp, Km, Na, and Sm mixture. Using four nutrient media, we confirmed that this protocol renders the rotifer culture bacterial and fungus free. The axenic rotifer culture generated here is useful not only for genetic analysis of Brachionus plicatilis, but for studying the rotifer life cycle without bacterial influence.