Synthesis of Chiral NADH Model Compounds Containing a Sulfur Atom and Their Asymmetric Reduction

Oryzalexins A, B and C were isolated as a group of novel phytoalexins from rice (Oryza sativa) blast leaves infected with Pyricularia oryzae. The basis of the structures of Oryzalexins A, B and C was laid by spectroscopic methods and their inhibitory activities against spore germination and germ tube growth of P. oryzae were assayed.

Oryzalexins A, B and C, isolated from rice leaves infected with P. oryzae as a group of novel phytoalexins, were confirmed to be (+)-sandaracopimaradiene derivatives by chemical and spectroscopic studies, i.e. A: 3-oxy-7-oxo- (I); B: 3-oxo-7-oxy- (II) and C: 3,7-dioxo-(+)-sandaracopimaradiene (III).     

Effective production of anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies by serum-free culture of hybridomas

Two hybridoma systems, mouse·human-human (m·h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m·h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semicontinuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.     

Synthesis of Substituted Aminomalonates by the Reaction of Chloramine with Malonate Carbanions

Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice.     

A New Lysine-containing Lipid Isolated from Agrobacterium tumefaciens

Fermentative production of 3aα-H-4α-(3′-propionic acid)-5α-hydroxy-7aβ-methylhexa-hydro-1-indanone-δ-lactone (HIL) from soybean sterol was studied in order to use it as an intermediate for chemical synthesis of 19-norsteroids. A mutant of Nocardia corallina converted 20 g/liter of soybean sterol into 2.8 g/liter of HIL with a 25% yield on a molar basis. The dominant factors improving the productivity were the use of an amino acid mixture as a nitrogen source and the preparation of the sterol suspension by sonication or with surface-active agents.     

Bimodal response in a chemotactic behaviour of Drosophila larvae to monovalent salts

A chemotactic behaviour in Drosophila larvae to monovalent salts was investigated. Larvae showed clear-cut bimodal responses; they preferred low concentration of salts and rejected high. For example, they were attracted to solutions over the range of concentrations of NaCl from 10^?4 M to 10^?1 M but avoided concentrations above 2 × 10^?1 M.The responses to KCl, KBr and Kl showed differences in attraction whereas avoidance thresholds were similar. On the other hand, when the responses to ChCl and ChBr were compared with those to KCl and KBr respectively, only the avoidance responses were depressed. Furthermore, from choice experiments between KCl, NaCl and ChCl, it was shown that cations did not affect the attraction responses.The results observed strongly suggest the existence of two salt receptors (or receptor sites) which act antagonistically on the chemotaxis.     

Specific assay for endotoxin using immobilized histidine and Limulus amebocyte lysate.

The LAL test is inhibited or enhanced by many substances. To overcome these problems, we have developed a specific endotoxin assay method using an ultrafiltration unit, a fluorometric LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This method is composed of two steps. The first step is the adsorption of endotoxins. Using immobilized histidine, endotoxins are quantitatively adsorbed on the adsorbent, and the adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the ultrafiltration unit. The second step is the reaction of adsorbed endotoxins with the LAL reagent. The endotoxins adsorbed on immobilized histidine are directly reacted with the LAL reagent in a filter cup and show enough activity for assay. The reproducibility and the accuracy of this method are high, and the recovery of endotoxins from a sample solution is more than 95%. The new endotoxin assay method using immobilized histidine can be utilized for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics instead of requiring employment of the more common gel-clot technique.     

Detection of Specific RNAs by in situ Hybridization in Plant Protoplasts

Combined exposure of fixed tomato protoplasts to 0.2 N HCl atroom temperature, heating at 70?C in 2 ? SSC for 30 min, anddigestion with 10 µg/ml proteinase K allowed localizationof viroid RNA by in situ hybridization, without destructionof cellular structures. The technique also allowed localizationof viral RNA in tobacco protoplasts. ⁴Present address: National Institute of Animal Health, Tsukuba,Ibaraki, 305 Japan (Received March 1, 1989; Accepted February 21, 1990)     

Pharmacological targeting of HSP90 with 17-AAG induces apoptosis of myogenic cells through activation of the intrinsic pathway

Endothelial inflammation and monocyte plays an essential role in the initiation and progression of atherosclerosis. Ghrelin is beneficial for atherosclerosis progression. However, the detailed and precise molecular mechanisms of how ghrelin regulates endothelial inflammation are not clear. In this study, we investigated the regulation mechanism of ghrelin on TNF-α-activated endothelial inflammation and monocyte adhesion. It was found that TNF-α-induced monocyte adhesion on HUVEC was significantly attenuated by ghrelin. Furthermore, we found that ghrelin effectively suppressed TNF-α-induced inflammatory factors’ (including ICAM-1, VCAM-1, MCP-1, and IL-1β) expression through inhibiting AMPK phosphorylation and p65 expression both in HUVEC and THP-1. This phenomenon was further demonstrated by using AMPK agonist AICAR and inhibitor compound C, respectively. Our findings suggest that ghrelin may mediate TNF-α-induced endothelial inflammation and monocyte adhesion, in part via AMPK/NF-κB signaling pathway. These novel anti-inflammatory and immunoregulatory actions of ghrelin may play a certain role in understanding the formation and development of atherosclerosis.     

Electron microscopic study of immunoreactive LHRH perikarya with special reference to neuronal regulation

Summary In early postnatal rats, immunoreactive LHRH perikarya in the preoptic area were studied by light and electron microscopy. Synaptic junctions were found between the immunoreactive perikaryon or its process, and the immunonegative nerve fibers. The significance of these synapses is discussed in relation to possible mechanisms by which the activities of LHRH neurons are regulated.