Identification of alpha 2- and alpha 3-subunits of the GABAA-benzodiazepine receptor complex purified from the brains of young rats.

Polyclonal antibodies were raised to synthetic amino acid sequences of the bovine GABAA receptor alpha 2- and alpha 3-subunits and purified by affinity chromatography on a column coupled with the respective peptide. Anti-peptide alpha 2(416-424) and anti-peptide alpha 3(459-467) antibodies immunoprecipitated GABAA receptors and recognized a protein of 53 kDa (P53) and 59 kDa (P59), respectively, in Western blots of GABAA receptors purified from the brains of 5-10 day old rats. P53 as well as P59 are specifically photolabeled by [3H]flunitrazepam and are recognized by the alpha-subunit specific monoclonal antibody bd 28.

N-deglycosylation and immunological identification indicates the existence of beta-subunit isoforms of the rat GABAA receptor.

beta 2- and beta 3-subunits of GABAA receptors purified from the brains of 5-10-day-old rats were investigated in the intact or completely N-deglycosylated state using the beta-subunit-specific monoclonal antibody bd-17 and polyclonal antibodies directed against synthetic amino acid sequences specific for the GABAA receptor beta 2- or beta 3-subunits. The present results seem to indicate the existence of two different isoforms of the beta 3-subunit and several different isoforms of the beta 2-subunit of the GABAA receptor which probably are produced by alternative splicing.

New continuous cell-line from human medullary-thyroid carcinoma - sinj - phenotypic analysis and invivo carcinogenesis.

SINJ is a new continuous human cell line derived from a lymph node metastasis of a probably sporadic medullary thyroid carcinoma. It is compared to MTC-SK, another medullary thyroid carcinoma cell line, established earlier (1). SINJ has been continuously cultivated in vitro for two years. The cells grow as a suspension of single cells and cell clusters. Repeated immunocytochemistry showed positive immunoreactivity with antibodies to CT, CGRP and GRP. The maintenance of NSE and chromogranins were proved. Northern blot analysis confirmed endocrine activity at mRNA level. Flow cytometry of 27 SINJ - clones showed 25 diploid and two tetraploid subpopulations. Cytogenetic analyses strengthened these findings. According to DNA analysis the cells are free of SV40 sequences. Tumorigenicity was proved in nude mice. The new cell line SINJ has potential use for in vitro studies of medullary thyroid carcinomas in sporadic as well as hereditary forms - the MEN2A syndrome.

Establishment of a continuous cell line from a human carcinoid of the small intestine (KRJ-I).

A new continuous cell line from a human malignant carcinoid of the small intestine (KRJ-I) was established. The cells showed morphological and immunocytochemical features of the tumor of origin and expressed estrogen receptors. The cells are growing as a suspension, forming multicellular aggregates and spheroids. Electron microscopy confirmed the presence of neuroendocrine granules. Dose-dependent growth inhibition was observed after incubation with 5-azacytidine. Cytogenetic analyses of the tumor of origin, the cell line KRJ-I and a liver metastasis KRJ-II revealed clonal tetraploidy and clonal loss of the Y-chromosome and chromosome 19.

Structure and subunit composition of GABA(A) receptors.

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain and are the site of action of many clinically important drugs. These receptors are composed of five subunits that can belong to eight different subunit classes. If all GABA(A) receptor subunits could randomly combine with each other, an extremely large number of GABA(A) receptor subtypes with distinct subunit composition and arrangement would be formed. Depending on their subunit composition, these receptors would exhibit distinct pharmacological and electrophysiological properties. Recent evidence, however, indicates that not all subunits can assemble efficiently with each other and form functional homo- or hetero-oligomeric receptors. In addition, the efficiency of formation of hetero-oligomeric assembly intermediates determines the subunit stoichiometry and subunit arrangement for each receptor and thus further reduces the possible heterogeneity of GABA(A) receptors in the brain. Studies investigating the subunit composition of native GABA(A) receptors support this conclusion, but also indicate that receptors composed of one, two, three, four, or five different subunits might exist in the brain. Using a recently established immunodepletion technique, the subunit composition and quantitative importance of native GABA(A) receptor subtypes can be determined. This information, together with studies on the regional, cellular and subcellular distribution of these receptor subtypes, will be the basis for a rational development of drugs that specifically affect the GABAergic system.

Inherited seborrheic dermatitis--a new mutant in mice.

A new mutation, affecting skin and hair, occurred in an expansion colony of Him:OF1 mice. Test crosses showed that a single autosomal recessive gene was responsible for this trait. Homozygotes have sparse greasy fur and lower viability and fertility than normal littermates. Histological observations showed hypertrophy of sebaceous glands, hyperkeratosis, parakeratosis, acanthosis and signs of inflammation. The disease was named 'inherited seborrheic dermatitis' and the gene name seb is proposed.

Reduced tumour incidence in mice with inherited seborrhoeic dermatitis.

121 mice homozygous for the gene seb (inherited seborrhoeic dermatitis) and their 142 unaffected heterozygous littermates were kept for their natural lifespan. Heterozygotes showed 84.1% total tumour incidence in males and 95.9% in females. The most common neoplasms were lymphomas, osteomas, lung tumours and neoplasms of the female genital tract. Homozygotes showed a tumour incidence of 36.1% in males and 45.0% in females. The reduction in incidence included all types of neoplasms except epithelial tumours of the skin: skin tumours were detected in 11 homozygous but only in one heterozygous animal. Life expectancy was not affected significantly by genotype. Homozygous mice showed rough and greasy fur and became alopecic with age. Energy intake was increased but growth and depository fat was reduced compared with heterozygous mice. Higher heat loss may incompletely be compensated by higher metabolic rate and thus 'dietary restriction' results in decreased tumour rates. As females show small gonads and a higher increase in food consumption hormonal factors may also be involved.

The effect of a cartilage bone marrow extract on experimentally induced osteoarthrosis in the knee joints of rabbits. A scanning electron microscopic study.

The development of osteoarthrosis following a partial meniscectomy on the knee cartilage of rabbits (Chinchilla hybrids) was monitored with a scanning electron microscope. Simultaneously, a study was made of the effect of the cartilage bone marrow extract Rumalon trademark on the development of the osteoarthrotic changes. Twelve days after the operation, osteoarthrotic changes were evident in the untreated operated joints. After 36 days the damage caused to the cartilage was already radical. The immobility of the operated joint also gave rise to obvious changes in the cartilage of the knee joints which had not undergone an operation. The irregular weight distribution due to the fixation of one joint was apparently enough to provoke degenerative processes on the other side. When the cartilage bone marrow extract Rumalon trademark was administered three times weekly (0.5 mg/kg body weight i.m.) a distinct retardation of the osteoarthrotic development in the early stages was observed. Where the changes had penetrated to the inner cartilage layers, no difference could be established compared to the untreated animals.

Induction of arthritic processes by synovial fluids of rheumatoid arthritis patients in long-term experiments with mice.

Diluted synovial fluids derived from three patients with rheumatoid arthritis (RA) and applied subcutaneously once as a single dose in Swiss mice caused arthritic processes after a long period of latency (10 months). The arthritis-inducing effect was enhanced by storing the original synovial fluids at 4 degrees C for three months. The effects were followed histologically for at least 18 months. In contrast to the theory that RA is based on immunological processes, these preliminary observations support the finding of other authors that a transmissible agent which is rather slow-acting exists in the synovial fluid of RA patients.

[The tumor spectrum of Him:OFA rats]. / Das Tumorspektrum von Him: OFA-Ratten.

The longevity and incidence of spontaneous tumors was investigated in 92 male and 182 female rats of the Sprague-Dawley (SD) derived stock Him: OFA. The overall tumour incidence was 85.9% in males and 97.8% in females with 32 different types of tumors in males and 30 in females. The most frequent neoplasms were mammary tumours in females with 84.6% incidence, followed in this sex by adrenal (36.8%), pituitary (32.9%) and thyroid neoplasms (10.9%). The incidence of all neoplasms in female genital tract was 12.6%. In male rats tumours of the adrenals have the highest incidence (53.2%, most of them cortical) followed by pituitary tumours (31.5%) and neoplasm of the mesenteric lymph nodes (14.1%, which is uncommonly high compared with other Sprague-Dawley stocks). All other tumours are below 10% incidence. The mean lifespan of females is with 719 +/- 142 d shorter than that of males with 752 +/- 108 d because of the high incidence of mammary tumours between 16 to 18 months of age.

Insulin and the in vitro protein synthesis of liver and skeletal muscle ribosomes in experimental acute uraemia.

Tissue dependent alteration of liver and muscle protein synthesis was previously observed in acute uraemia, when intracellular amino acids were used as substrate. Since protein synthesis depends also on substrate availability, it was of interest to investigate the role of isolated liver and skeletal muscle ribosomes of nephrectomized rats and their susceptibility to in vivo insulin stimulation. Under marked uraemic conditions, no difference was demonstrated between controls and uraemic animals; after insulin administration and in the presence of endogenous messenger both liver and muscle ribosomes showed an enhancement of [3H]-phenylalanine incorporation of 20% and 54% respectively in controls, and of 32% and 45% respectively in uraemic rats. This study may rule out the influence of acute uraemia on protein synthesis capacity of isolated ribosomes and emphasize the importance of intracellular amino acid availability in altered protein synthesis.

[Insulin and in vitro protein synthesis of isolated liver ribosomes in experimental acute uraemia (author's transl)]. / Insulin und in-vitro-Proteinsynthese isolierter Leberribosomen bei akuter experimenteller Urämie.

Liver protein synthesis was shown to be enhanced in acute experimental uraemia, when intracellular amino acids were used as substrate. Since protein synthesis also depends on substrate availability and amino acid concentration is higher in uraemic liver cells, it was of interest to investigate the in vitro protein synthesis capacity of isolated liver ribosomes in the presence of standardized amino acids with and without in vivo insulin stimulation. It was demonstrated that protein synthesis capacity of isolated liver ribosomes remains unaltered in acute uraemia and can be stimulated by in vivo insulin application. Our data suggest that in acute uraemia, intracellular substrate availability plays the essential role in impaired liver protein synthesis.

[Basal and LH-RH-stimulated gonadotropin release after transport stress in male rats]. / Basale und LH-RH stimulierbare Gonadotropin-freisetzung nach Transportstress bei der männlichen Ratte.

A total of 120 male rats of the Sprague-Dawley-strain (6 weeks old) were used in this experiment. 5 groups of 12 animals each were treated intraperitoneally with 200 ng gonadotropin releasing hormone (LH-RH) per animal. 30 minutes later blood was sampled by heart puncture. Group I were animals without transport, group II immediately after, group III one day, group IV one week and group V six weeks after a standardised transport. Another 5 groups were subjected to the same protocol but received saline i.p. instead of LH-RH. Serum levels of LH and FSH were estimated by radioimmunoassay. LH and FSH serum levels could be stimulated by LH-RH in all groups. A significant rise of basal and LH-RH stimulated LH levels was observed until the first day after transport. Thereafter a drop was registered. No consistent patterns of basal as well LH-RH stimulated FSH-levels were noted. These data combine to suggest an elevation of LH-RH secretion as response to the stress. This results in a sensibilisation of the pituitary to exogenous LH-RH.

Maturation of 17 beta-estradiol binding protein in the hypothalamus and anterior pituitary of male rats.

Levels of estrogen binding proteins in the cytosol of hypothalamic and pituitary tissue of male rats at different ages were estimated. Two classes of estrogen binding sites were detected in 13 and 20 days old animals. The number of high affinity estrogen binding sites (K = ca. 2 x 10(-10) mol) of the hypothalamus was slightly larger in 13 days old animals, then decreased to constant levels at the age of 20 days and no changes were observed thereafter. Levels of low affinity binding sites of the hypothalamus (K = ca. 5 x 10(-9) mol) decreased with age and were no longer detectable in 30 days old animals. Similarly, a decrease of low affinity estrogen binding sites with age was recorded for pituitary cytosol samples and this class of receptor sites was no longer observable in 30 days old male rat. At 13 days high levels of high affinity 17 beta-estradiol binding protein was registered and minimal numbers of binding sites were found at 20, 30 and 40 days. Thereafter, the levels remained constant until 100 days and were similar to values observed in the 13 days old male animals. Apparent affinity constants of two classes of binding sites in hypothalamus and pituitary were similar...

[Histologic studies of the effect of superoxide dismutase on an arthrosis model in rabbits]. / Histologische Untersuchungen über die Wirkung der Superoxid-Dismutase (SOD) an einem Arthrosemodell beim Kaninchen.

The effect of the enzyme superoxide dismutase (SOD) on experimentally induced osteoarthritis was investigated. Immobilization of the right knee of rabbits over variable periods (1-4 weeks) was used as the model for the study. SOD was applied intraarticularly. Distinct histological changes were detected in the immobilized joints in contrast to the freely mobile joints. In the group of rabbits treated with SOD, erosions, necroses, and inflammatory symptoms were more frequent than in the placebo group. The changes found in animals after SOD application indicate the enzyme has an intensifying effect on osteoarthritis.

[Effect of superoxide dismutase on an arthritis model in the rabbit. A scanning electron microscopy study]. / Einfluss von Superoxid-Dismutase (SOD) auf ein Arthrosemodell beim Kaninchen. Eine rasterelektronenmikroskopische Studie.

The effect of superoxide dismutase (SOD) was studied in an experimental arthrosis model (fixation arthrosis in rabbits). Out of a total of 40 animals, 20 of which were to be treated with SOD and 20 with placebos, 5 animals each were given padded stiffened bandages to immobilize their right knee joints for 1 week, 2 weeks, 3 weeks and 4 weeks respectively. After removing the bandage and a two-week recovery period the SOD or placebo treatment respectively was begun. During this period the animals were allowed to move freely. In 20 animals 0.5 mg SOD in 0.2 ml of a 0.5% xylocaine solution were applied intraarticularly in both popliteal spaces once a week for a total of 4 weeks. 20 control animals likewise received 0.2 ml of a 0.5% xylocaine solution (placebo) in both popliteal spaces in the same time intervals. After treatment the animals were killed and examined. A further 5 animals were not given a stiffened bandage, but for 4 weeks they received intraarticular injections once a week of 0.2 ml of a 0.9% saline to act as controls. The evaluation of the changes in the joints was by histological examination and examination by scanning electron microscope. In comparison to the placebo treatment the SOD treatment in this animal experiment did not lead to a significant change in the arthrotic process.

The effect of substance L on glucose-mediated cross-links of collagen in the diabetic db/db mouse.

Genetically diabetic mice (db/db) were given 50 mg/kg body weight/day substance L, a nontoxic basic amino acid and compared to control diabetic mice without treatment. The oral administration of the compound was started at the age of 3 months and the animals were sacrificed at the age of 7 months. No adverse effects were observed in animals given the substance L. Total food consumption, drinking water intake and body weight were comparable between the groups. Nonenzymatic glycosylation of serum proteins and hemoglobin was not significantly different in the groups. Renal pathological lesions in the control diabetic mice showed glomerular mesangial expansion and on electron microscopy thickened glomerular basement membranes with a mean thickness of 3,204 +/- 186 A. Treated animals showed significantly less mesangial crescents and thinner glomerular basement membrane thickness of 2,520 +/- 252 A (p less than 0.01). The experimental animals showed in addition a lower mean kidney weight. Glomerular but not tubular proteinuria was reduced in the treated group. Basement membrane collagen type IV isolated from kidneys of experimental animals was more soluble in acidity and showed a lower degree of cross-linking as evaluated by SDS-polyacrylamide gel electrophoresis. We conclude that substance L is beneficial to diabetic renal changes. We suggest that this positive effect could be due to the inhibition of glucose-mediated abnormal cross-linking of collagenous structures by the interaction of substance L with reactive carbonyl residues of glycosylation adducts of collagen. Other possible mechanisms are discussed.

Evidence for the existence of differential O-glycosylated alpha 5-subunits of the gamma-aminobutyric acidA receptor in the rat brain.

Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the gamma-aminobutyric acidA (GABAA) receptor alpha 5-subunit. These anti-peptide alpha 5(2-10) or anti-peptide alpha 5(427-433) antibodies reacted specifically with GABAA receptors purified from the brains of 5-10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABAA receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABAA receptors with N-Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABAA receptors with neuraminidase and O-Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide alpha 5(2-10) and the anti-peptide alpha 5(427-433) antibody. These results indicate the existence of at least two different alpha 5-subunits of the GABAA receptor that differ in their carbohydrate content. In contrast to other alpha- or beta-subunits of GABAA receptors so far investigated, at least one of these two alpha 5-subunits contains O-linked carbohydrates.