A mechanism for the development of Clara cell lesions in the mouse lung after exposure to trichloroethylene.

Female CD-1 mice exposed to trichloroethylene (6 h/day) at concentrations from 20-2000 ppm developed a highly specific lung lesion after a single exposure, characterised by vacuolation of the Clara cells, the number of cells affected increasing with increasing dose level. At the highest dose levels pyknosis of the Clara cells was apparent. After 5 days of repeated exposures the lesion had resolved but exposure of mice following a 2-day break resulted in recurrence of the lesion. The changes in mouse lung Clara cells were accompanied by a marked loss of cytochrome P-450 activities. No morphological changes were seen in the lungs of rats exposed to either 500 or 1000 ppm trichloroethylene. Isolated mouse lung Clara cells were shown to metabolize trichloroethylene to chloral, trichloroethanol and trichloroacetic acid. Chloral was the major metabolite. Trichloroethanol glucuronide was not detected. In comparative experiments using mouse hepatocytes the major metabolites were trichloroethanol and its glucuronide conjugate. The activity of UDP-glucuronosyltransferase was compared in mouse lung Clara cells and hepatocytes using two phenolic substrates and trichloroethanol. Hepatocytes readily formed glucuronides from all three substrates whereas Clara cells were only active with the two phenolic substrates. The three major metabolites of trichloroethylene, chloral, trichloroethanol and trichloroacetic acid were each dosed to mice and of these metabolites, only chloral had an effect on mouse lung causing a lesion (Clara cell) identical to that seen with trichloroethylene. It is proposed that the failure of Clara cells to conjugate trichloroethanol leads to an accumulation of chloral which results in cytotoxicity. The known genotoxicity of chloral suggests that this lesion may be related to the development of lung tumours in mice exposed to trichloroethylene by inhalation.     

Immunolocation analysis of glycosaminoglycans in the human growth plate.

Monoclonal antibodies were used in this study to immunolocate glycosaminoglycans throughout the human growth plate. Chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate were observed in the extracellular matrix of all zones of the growth plate and persisted into the cartilage trabeculae of newly formed metaphyseal bone. Also present in the extracellular matrix was an oversulfated chondroitin/dermatan sulfate glycosaminoglycan which appeared to be specific to the proliferative and hypertrophic zones of the growth plate. As with the other extracellular matrix molecules, this epitope persisted into the cartilage trabeculae of the metaphyseal bone. Zonal differences between the extracellular and pericellular or lacunae matrix were also observed. The hypertrophic chondrocytes appeared to synthesize chondroitin sulfate chains containing a non-reducing terminal 6-sulfated disaccharide, which were located in areas immediately adjacent to the cells. This epitope was not found to any significant extent in the other zones. The pericellular region around hypertrophic chondrocytes also contained a keratan sulfate epitope which was also observed in the resting zone but not in the proliferative zone. These cell-associated glycosaminoglycans were not found in the cartilage trabeculae of metaphyseal bone, indicating their removal as the terminal hypertrophic chondrocytes and their lacunae are removed by invading blood vessels. These changes in matrix glycosaminoglycan content, both in the different zones and within zones, indicate constant subtle alterations in chondrocyte metabolic products as they proceed through their life cycle of proliferation, maturation, and hypertrophy.     

The thermal and energetic significance of clustering in the speckled mousebird,Colius striatus

Summary The effect of clustering behaviour on metabolism, body temperature, thermal conductance and evaporative water loss was investigated in speckled mousebirds at temperatures between 5 and 36°C. Within the thermal neutral zone (approximately 30–35 °C) basal metabolic rate of clusters of two birds (32.5 J·g^-1·h^-1) and four birds (28.5 J·g^-1·h^-1) was significantly lower by about 11% and 22%, respectively, than that of individuals (36.4 J·g^-1·h^-1). Similarly, below the lower critical temperature, the metabolism of clusters of two and four birds was about 14% and 31% lower, respectively, than for individual birds as a result of significantly lower total thermal conductance in clustered birds. Body temperature ranged from about 36 to 41°C and was positively correlated with ambient temperature in both individuals and clusters, but was less variable in clusters. Total evaporative water loss was similar in individuals and clusters and averaged 5–6% of body weight per day below 30°C in individuals and below 25°C in clusters. Above these temperatures total evaporative water loss increased and mousebirds could dissipate between 80 and 90% of their metabolic heat production at ambient temperatures between 36 and 39°C. Mousebirds not only clustered to sleep between sunset and sunrise but were also observed to cluster during the day, even at high ambient temperature. Whereas clustering at night and during cold, wet weather serves a thermoregulatory function, in that it allows the brrds to maintain body temperature at a reduced metabolic cost, clustering during the day is probably related to maintenance of social bonds within the flock.Abbreviations BMR basal metabolic rate - bw body weight - C _totab total thermal conductance - EWI evaporative water loss - M metabolism - RH relative humidity - T _a ambient temperature - T _b body temperature - T _ch chamber temperature - T _cl cluster temperature - TEWL total evaporative water loss - LCT lower critical temperature - TNZ thermal neutral zone     

Initiation of the extrinsic pathway of blood coagulation: evidence for the tissue factor dependent autoactivation of human coagulation factor VII

Previous studies demonstrated proteolytic activation of human blood coagulation factor VII by an unidentified protease following complex formation with tissue factor expressed on the surface of a human bladder carcinoma cell line (J82). In the present study, an active-site mutant human factor VII cDNA (Ser344----Ala) has been constructed, subcloned, and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity in a single step from serum-free culture supernatants by immunoaffinity column chromatography. Mutant factor VII was fully carboxylated, possessed no apparent clotting activity, and was indistinguishable from plasma factor VII by SDS-PAGE. Cell binding studies indicated that mutant factor VII bound to J82 tissue factor with essentially the same affinity as plasma factor VII and was cleaved by factor Xa at the same rate as plasma factor VII. In contrast to radiolabeled single-chain plasma factor VII that was progressively converted to two-chain factor VIIa on J82 monolayers, mutant factor VII was not cleaved following complex formation with J82 tissue factor. Incubation of radiolabeled mutant factor VII with J82 cells in the presence of recombinant factor VIIa resulted in the time-dependent and tissue factor dependent conversion of single-chain mutant factor VII to two-chain mutant factor VIIa. Plasma levels of antithrombin III had no discernible effect on the factor VIIa catalyzed activation of factor VII on J82 cell-surface tissue factor but completely blocked this reaction catalyzed by factor Xa. These results are consistent with an autocatalytic mechanism of factor VII activation following complex formation with cell-surface tissue factor, which may play an important role in the initiation of extrinsic coagulation in normal hemostasis.     

5-Azacytidine increases the total cellular copper content and basal level metallothionein mRNA accumulation of human Hep G2 cells.

In this study we have demonstrated the ability of 5-azacytidine to elevate the basal level expression of the metallothionein (MT)-IF and MT-IG genes and increase the basal level expression of the MT-IIA gene in Hep G2 cells, a cell line which exhibits heavy metal inducible MT gene expression. Atomic absorption analysis of 5-azacytidine treated Hep G2 cells detected a 2-fold increase in the total cellular copper content. Pretreatment of 5-azacytidine exposed cells with hydroxyurea and cycloheximide indicated that the increase in total cellular copper content was a direct response to 5-azacytidine treatment. S1 nuclease analysis illustrated that pretreatment of Hep G2 cells with KCN, a copper specific chelator and uptake inhibitor, suppressed 5-azacytidine- and copper-inducible MT-IG gene expression. Thus, the increase in MT gene expression in response to 5-azacytidine treatment can be correlated to an increase in the total cellular copper content. Possible mechanisms on how 5-azacytidine could alter the influx/efflux of copper in Hep G2 cells are discussed.     

Sex differences in nutritional modulation of gonadotropin secretion during development: studies in the growth-retarded lamb

This study determined whether changes in nutrition during development alter LH secretion in males in a manner similar to that in females; sheep were used as an experimental model. Studies were conducted in the absence of gonadal steroid negative feedback. First, we compared the effect of chronic growth restriction on LH secretion in male and female lambs. Second, we determined whether the gonadotropic response to acute increases and decreases in nutrition is sexually differentiated. Seven male and 8 female Suffolk lambs, gonadectomized, and weaned by 8 wk of age were maintained at a target weight of 20 kg by level of nutrition. After 7 wk of chronic low nutrition (15 wk of age), LH pulse frequency was equally low in males (2.0 +/- 0.7 pulses/4 h) and females (2.0 +/- 0.4 pulses/4 h) relative to that (ca. hourly pulses) in normally growing gonadectomized lambs. Seven weeks later, at 22 wk of age, LH pulse frequency dropped further (males 0.9 +/- 0.3/4 h; females 0.9 +/- 0.4 pulses/4 h). The results of this first experiment, in which we observed no sex difference in gonadotropin secretion under chronic growth restriction, imply equal neuroendocrine sensitivity in males and females to long-term low nutrition. In the second experiment, however, a sex difference was evident in the response to increased and decreased nutrition. Both sexes responded to feeding ad libitum with a rapid increase in LH pulse frequency, but the response was greater in the males than in the females.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paradoxical shortening of scrapie incubation times by expression of prion protein transgenes derived from long incubation period mice.

Prolonged incubation times for experimental scrapie in I/LnJ mice are dictated by a dominant gene linked to the prion protein gene (Prn-p). Transgenic mice were analyzed to discriminate between an effect of the I/LnJ Prn-pb allele and a distinct incubation time locus designated Prn-i. Paradoxically, 4 independent Prn-pb transgenic mouse lines had scrapie incubation times shorter than nontransgenic controls, instead of the anticipated prolonged incubation periods. Aberrant or overexpression of the Prn-pb transgenes may dictate abbreviated incubation times, masking genuine Prn-p/Prn-i congruence; alternatively, a discrete Prn-i gene lies adjacent to Prn-p.     

Variable region sequence analysis of anti-DNA antibodies: evidence for a family of closely related germ-line VH genes encoding lupus autoantibodies.

Cloning and sequencing of the V regions of the anti-DNA monoclonal antibodies (mAbs), H438 and H130, indicate that H438 is encoded by a J558 VH gene, a single D region nucleotide, and unmutated JH1, V kappa-1C and J kappa 1 genes, and the H130 L chain is encoded by a V kappa-21 subgroup gene J kappa 1 gene. Identification of VH438, which shared VH hybridization pattern with 6% of a panel of 352 MRL/lpr hybridomas, suggests that the frequency of J558 use among spontaneously activated B cells in MRL/lpr mice is greater than previously reported. The VHH438 J558 family gene is identical to VHPAR, which encodes the independently derived MRL/lpr autoantibody, MRP-2, and is highly homologous to the previously reported VHH130, which is identical to a BALB/c germ-line VH gene. Comparison of consensus sequences of homologous autoantibodies and previously reported restriction mapping suggest that a minimum of three highly related J558 germ-line genes encode lupus autoantibodies.