Sister-chromatid exchanges in lymphocytes of workers exposed to trichloroethylene

To detect mutagenic effects of trichloroethylene (TCE) on humans, sister-chromatid exchanges (SCEs) were analyzed in lymphocytes of 22 workers occupationally exposed to TCE and 22 matched controls. Although urinalysis in the workers revealed their obvious exposure to TCE, no increase in SCE frequencies was found in lymphocytes of the workers. SCE analysis in lymphocytes could not detect mutagenic effects by occupational exposure to TCE on humans.     

Correction of congenital microtia using the tissue expander

We attempted auricular reconstruction using Radovan-type inflatable silicone expanders in six children and one adult, with the complete hypoplastic, the conchal remnant, and constricted type of microtia. Ear frameworks, including the helix, anthelix, concha, and tragus, were prepared using autologous rib cartilage. Based on the surface area of the normal adult auricle, the silicone expander was tentatively shaped and sized into a rotated semiellipse and expanded with 70 cc saline. Auricular reconstruction on the framework was completed at the time of insertion in four of the seven patients, requiring no elevation of the ear. The reconstructed auricle was satisfactory in both color and texture and had nearly normal sensation. Mild complications were noted in three of the seven patients. However, no resorption of the inserted rib cartilage has been observed 14 months to 2 years and 5 months after the operation. Slight shrinkage of the expanded skin was noted in each patient.     

Immunocytochemical determination of estrogen and progesterone receptors in human endometrial adenocarcinoma cells (Ishikawa cells).

The regulation of both estrogen and progesterone receptor levels in human endometrial adenocarcinoma cells of the Ishikawa line was investigated immunocytochemically by using monoclonal antibodies. Positive staining for estrogen and progesterone receptors was observed in the nuclei of Ishikawa cells. Intercellular heterogeneity in receptor content was evident from the presence of receptor-positive or -negative cells and from differences in staining intensity of positive cells. Quantitative analysis was performed by scoring the staining intensity and the proportion of positively stained cells. The time and dose-dependent stimulatory effect of estradiol added to culture media on progesterone receptor levels was studied by applying both immunocytochemical and biochemical methods. Estradiol at 10 nM (optimal concentration) increased the intensity score for PR from an initial value of 10.1 to 78.3 after 72 h incubation, and the proportion of the positive staining cells from 6.7 to 42.7%. Promegestone (R5020) was effective at 1 microM concentration in decreasing the intensity score for ER from 31.1 to 14.6 after 72 h exposure and the proportion of positive cells from 19.0 to 11.4%.     

Isolation and characterization of the human ornithine transcarbamylase gene: structure of the 5'-end region

We isolated over 20 phage clones carrying the ornithine transcarbamylase (OTC) [EC 2.1.3.3] gene, from two independently constructed human genomic DNA libraries, using as probes either a rat OTC cDNA or several nuclear DNA fragments derived from some of these isolated clones. These clones, classified into 10 different groups, overlapped and spanned a region of more than 85 kilobase pairs of the human genomic DNA. Restriction mapping and Southern blot analyses demonstrated that one of the clones covers the 5'-end region of the OTC gene. We sequenced the 5'-end region of the OTC gene and found that it covered 665 base pairs of the 5'-flanking region, the complete first exon and a part of the first intron (150 base pairs). In the 5'-flanking region, there were two pairs of putative CAAT and TATA boxes and one enhancer core-like sequence, GTGGAAAG. The first exon contained a coding region for most of the OTC presequence, i.e. 26 out of 32 amino acid residues of the presequence, including the initiation methionine.     

Regulation of Histamine Turnover via Muscarinic and Nicotinic Receptors in the Brain

To clarify the regulation of central histaminergic (HAergic) activity by cholinergic receptors, the effects of drugs that stimulate the cholinergic system on brain histamine (HA) turnover were examined, in vivo, in mice and rats. The HA turnover was estimated from the accumulation of tele-methylhistamine (t-MH) during the 90-min period after administration of pargyline (65 mg/kg, i.p.). In the whole brain of mice, oxotremorine, at doses higher than 0.05 mg/kg, s.c., significantly inhibited the HA turnover, this effect being completely antagonized by atropine but not by methylatropine. A large dose of nicotine (10 mg/kg, s.c.) also significantly inhibited the HA turnover. This inhibitory effect was antagonized by mecamylamine but not by atropine or hexamethonium. A cholinesterase inhibitor, physostigmine, at doses higher than 0.1 mg/kg, s.c., significantly inhibited the HA turnover. This effect was antagonized by atropine but not at all by mecamylamine. None of these cholinergic antagonists used affected the steady-state t-MH level or HA turnover by themselves. In the rat brain, physostigmine (0.1 and 0.3 mg/kg, s.c.) also decreased the HA turnover. This inhibitory effect of physostigmine was especially marked in the striatum and cerebral cortex where muscarinic receptors are present in high density. Oxotremorine (0.2 mg/kg, s.c.) and nicotine (1 mg/kg, s.c.) also decreased the HA turnover in the rat brain. However, these effects showed no marked regional differences. These results suggest that the stimulation of central muscarinic receptors potently inhibits the HAergic activity in the brain and that strong stimulation of central nicotinic receptors can also induce a similar effect.     

Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B

The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5). The crystals are hexagonal, space group P6(2)22 or P6(4)22. The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees. The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600). The crystals diffract to at least 2.5 A resolution.     

L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts

Proliferation of human skin fibroblasts was stimulated significantly by the presence of L-ascorbic acid 2-phosphate (Asc 2-P). The presence of Asc 2-P (0.1-1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2-fold as well as cell growth 4-fold. Coexistence of L-azetidine 2-carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2-P in a dose-dependent manner. Supplementation of the medium with Asc 2-P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. Electron microscopic observations showed multilayered, rough endoplasmic reticulum-rich cells surrounded by dense ECM. These results indicate that Asc 2-P is useful in culture systems as a long-acting vitamin C derivative and also that it promotes reorganization of a three-dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.     

Dimer Formation of a Cell-Cell Adhesion Protein, gp64 of the Cellular Slime Mold, Polysphondylium pallidum

The cellular slime mold, Polysphondylium pallidum, has two EDTA-resistanttypes of cell-cell adhesion. The major component of them hasbeen identified as a glycoprotein with a molecular mass of 64kDa on SDS-PAGE (referred to as gp64). We found that a substantialamount of the gp64 run as dimer, when gp64 was dissolved inSDS-sample buffer without 2-mercaptoethanol and then subjectedto electrophoresis. The occurrence of a homophilic dimer wasdemonstrated by analyzing the dimer-like band on a gel for itsamino acid sequence and amino acid composition. The dimer-likeband also was analyzed by three sorts of monoclonal antibodies,two of which recognize respectively a conforniational epitopeand a denatured epitope of the protein moiety of gp64. The dataindicate that the native conformation of gp64 is necessary fordimer formation. ²Present address: Institute of Immunological Science, HokkaidoUniversity, Sapporo, 060 Japan