Monoclonal antibodies against beta-galactoside-binding lectin of chick embryo recognizes conformational change of the antigen

Monoclonal antibodies against chick embryonic beta-galactoside-binding lectin were obtained. One of the monoclonal antibodies was ineffective in Western blotting and seemed to be unable to bind the SDS-denatured lectin. When the native lectin was dotted on a nitrocellulose filter and subjected to denaturation by treatment with SDS, urea or heat, binding of this antibody no longer occurred, though other monoclonal antibodies bound normally. This antibody seems to have been raised against an epitope which is destroyed upon denaturation.     

Hepatic drug-metabolizing enzyme system and endotoxin tolerance: structural requirement of LPS in induction of an early tolerance

The alteration of hepatic drug-metabolizing enzyme activities in mice given Salmonella endotoxin by single or multiple intraperitoneal injections was investigated. An essentially the same biphasic, early and late phase, endotoxin tolerance was observed in the animals receiving a single injection of endotoxin or repetitive daily injections. The results of reciprocal cross tolerance tests using lipopolysaccharide and free lipid A preparations derived from Salmonella minnesota, Salmonella typhimurium, E. coli, Pseudomonas aeruginosa, and Chromobacterium violaceum suggested that lipid A moiety plays an important role in the induction of early endotoxin tolerance to endotoxin response.     

Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

Formation of Hydrogen Peroxide by NAD(P)H Oxidation with Isolated Cell Wall-Associated Peroxidase from Cultured Liverwort Cells, Marchantia polymorpha L.

Hydrogen peroxide was formed in isolated cell walls from Marchantiapolymorpha L. in the presence of MnCl₂ by either NADH or NADPHoxidation. This reaction was stimulated by phenols such as 2,4-dichlorophenolor p-coumarate, suggesting a reaction similar to that proposedfor the last step of lignification in higher plant cells, althoughbryophytes have been reported to be devoid of lignin. (Received June 16, 1987; Accepted March 3, 1987)     

Optimum conditions for electric pulse-mediated gene transfer to mammalian cells in suspension

A pulse-generating machine which delivers exponentially decaying pulses over broad range of pulse lengths was used to determine the optimum pulse conditions for gene transfer to FM3A cells. In the transformation of tk- cells with pTK1, a single pulse of 100-2000 microseconds gave a high transformation frequency at 1.5-6 kV/cm and room temperature, the highest transformation frequency obtained being 3 X 10(-3). As the suspension buffer for cells exposed to the pulse, Saline G was better than PBS(-) for obtaining a large number of transformants because it ensured high cell viability.     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

A computer program that draws pedigree charts for inbred strains of animals

We produced a computer program that draws pedigree charts for inbred strains of animals such as mice or rats. This program is composed of four subprograms, which are (1) inputting the data, (2) drawing pedigree charts, (3) listing the data which have been input, and (4) backup of the system and the data. Pedigree charts and lists of data can be displayed on a TV screen and printed out on the papers. Using this program, we drew the pedigree charts of the inbred strains of rats which we are maintaining by brother-sister inbreeding in our institute and found that there were three sublines in one of the strains, WKAH/Hkm, because of unsuitable maintenance. This program is very convenient to draw the pedigree charts and useful for checking the maintenance of inbred strains or the strains of animal models of human diseases.     

Release of human epidermal growth factor from platelets in accordance with aggregation in vitro

We measured the intra-platelet content of human epidermal growth factor (hEGF) and beta-thromboglobulin (beta-TG) and the quantities of these released from platelets during in vitro aggregation. The intra-platelet amounts of hEGF and beta-TG in 10(8) platelets were 104.9 +/- 18.9 (Mean +/- SEM) pg and 2920.9 +/- 149.9 ng, respectively. During platelet aggregation elicited by 9, 11-epithio-11, 12-methano-thromboxane A2, a stable thromboxane A2 agonist, hEGF and beta-TG were released in amounts about 50% and 40% of the respective content in platelets. Also during arachidonate-induced aggregation, hEGF and beta-TG were released at about 60% and 50%, respectively. Various concentrations of thromboxane A2 antagonist, (9, 11), (11, 12)-di-deoxa-9, 11-dimethyl-methano-11, 12-methano-13, 14-dihydro-13-aza-14-oxo-15-cyclopentyl-16, 17, 18, 19, 20-pentanor-15-epi-thromboxane A2, suppressed both aggregation and release reactions in a dose-dependent manner. There were good correlations between the platelet aggregation rate and released beta-TG (r = 0.9368, p less than 0.01) or hEGF (r = 0.8931, p less than 0.01) and between released beta-TG and hEGF (r = 0.9385, p less than 0.01). These results suggest that hEGF is released from platelets in a similar fashion to beta-TG in vitro.     

Endogenous beta-galactoside-binding lectin expression is suppressed in retinol-induced mucous metaplasia of chick embryonic epidermis

The fate of endogenous beta-galactoside-binding lectin of chick embryo (14K type) was investigated during the course of skin differentiation. Lectin (14K) was found in keratinized epidermis and was localized mainly in the basal and intermediate cells. However, the protein lectin in the epidermis disappeared when the cultured skin was treated with vitamin A and mucous metaplasia was observed. The synthesis of lectin mRNA was also strongly suppressed by vitamin A in a concentration-dependent manner. On the other hand, in the dermis, in which the lectin was localized in the extracellular matrix, lectin expression was scarcely affected by vitamin A. These results indicated that the lectin was expressed in the keratinized epidermis but that its expression was suppressed in vitamin A-induced mucous-secreting epithelium. The suppression may be a result of a transition of the epidermal regulatory system to one of mucous-secreting epithelium. This is the first finding that 14K lectin expression might be regulated during the course of the epidermal differentiation.