Calmodulin plays a dominant role in determining neurotransmitter regulation of neuronal adenylate cyclase

Ca2+, through the mediation of calmodulin, stimulates the activity of brain adenylate cyclase. The growing awareness that fluctuating Ca2+ concentrations play a major role in intracellular signalling prompted the present study, which aimed to investigate the implications for neurotransmitter (receptor) regulation of enzymatic activity of this calmodulin regulation. The role of Ca2+/calmodulin in regulating neurotransmitter-mediated inhibition and stimulation was assessed in a number of rat brain areas. Ca2+/calmodulin stimulated adenylate cyclase activity in EGTA-washed plasma preparations from each region studied--from 1.3-fold (in striatum) to 3.4-fold (in cerebral cortex). The fold-stimulation produced by Ca2+/calmodulin was decreased in the presence of GTP, forskolin, or Mn2+. In EGTA-washed membranes, receptor-mediated inhibition of adenylate cyclase was strictly dependent upon Ca2+/calmodulin stimulation in all regions, except striatum. A requirement for Mg2+ in combination with Ca2+/calmodulin to observe neurotransmitter-mediated inhibition was also observed. In contrast, receptor-mediated stimulation of activity was much greater in the absence of Ca2+/calmodulin. The findings demonstrate that ambient Ca2+ concentrations, in concert with endogenous calmodulin, may play a central role in dictating whether inhibition or stimulation of adenylate cyclase by neurotransmitters may proceed.     

The cytogenetic and hepatotoxic effects of dioxin on mouse liver cells

The cytogenetic and hepatotoxic effects of 2,3,7,8-tetrachlorodibenzo p-dioxin (TCDD) on mouse liver cells were investigated. Male C57BL/6J strain mice, which have TCDD receptors, were given single intraperitoneal injections of 25, 37.5, 75 and 150 g of TCDD/kg body weight or corn oil carrier alone. Two-thirds hepatectomies were carried out at 1 or 7 days after injection and chromosomal aberrations and mitotic indexes of the regenerating hepatocytes were scored 54 hr after hepatectomy. Liver sections from additional intact mice were studied for TCDD-hepatotoxicity at 1, 7 and 30 days after injection. The three high doses of TCDD caused hepatotoxicity with necrosis of liver cells and focal architectural collapse by 30 days after injection. No evidence was obtained of an increase in the frequency of chromosomal structural aberrations at doses that allowed sufficient mitotic activity for cytogenetic evaluation. We conclude that TCDD is not a clastogen for mouse hepatocytes, although high doses cause marked hepatocellular necrosis.Abbreviations CSD chromosome deletion - META metacentric chromosome - TCDD 2,3,7,8-tetrachlorobenzo-p-dioxin     

Transforming activities of sodium fluoride in cultured Syrian hamster embryo and BALB/3T3 cells

The transforming activity of sodium fluoride was studied in the SHE and the BALBl3T3 cell culture systems. Initiating and promoting activities were then investigated by means of the orthogonal methodology. Sodium fluoride was found to induce morphological transformation of SHE cells seeded on a feeder layer of X-irradiated cells at high concentrations (75–125 g/ ml). When the cells were seeded in the absence of a feeder-layer, the transformation frequencies increased in a dose-dependent manner with the concentrations of sodium fluoride ranging from 0 to the highly toxic concentration of 200 g/ml. In the BALBl3T3 cell system, sodium fluoride was negative in the standard Kakunaga procedure, while through the experiment designed by table L₈ (2⁷) of the orthogonal method, an initiating-like effect and a weak promoting activity were detected within the concentrations ranging from a 25 g/ ml to a 50 g/ ml concentration which is highly toxic for BALBl3T3 cells. From these results, it is suggested that, besides a genetic mode of action, sodium fluoride could possibly act through a non-genotoxic mechanism.Abbreviations CE cloning efficiency - NaF sodium fluoride - SHE Syrian hamster embryo - TF transformation frequency     

Human hepatocyte and kidney cell metabolism of 2-acetylaminofluorene and comparison to the respective rat cells

The metabolism and mutagenic activation of 2-acetylaminofluorene by human and rat hepatocytes and kidney cells were measured. High performance liquid chromatography was used to separate the 2-acetylaminofluorene metabolites, and a cell-mediated Salmonella typhimurium mutagenesis assay was used to detect mutagenic intermediates. Rat and human differences were observed with cells from both organs and levels of metabolism and mutagenesis were higher in human cells. Within a species, liver and kidney cell differences were also evident, with levels of hepatocyte-mediated metabolism and mutagenesis being greater than kidney cells. Human inter-individual variation was apparent with cells from both organs, but the variation observed was significantly greater in hepatocytes than kidney cells. A knowledge of such differences, including an understanding that they may vary with the chemical being studied, should be useful in the extrapolation of rodent carcinogenesis data to humans.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethylsulfoxide - HPLC high performance liquid chromatography - N-OH-AAF N-hydroxy-2-acetylaminofluorene - 1-OH-AAF 1-hydroxy-2-acetylaminofluorene - 3-OH-AAF 3-hydroxy-2-acetylaminofluorene - 5/9-OH-AAF a combination of 5 and 9-hydroxy-2-acetylaminofluorene - 7-OH-AAF 7-hydroxy-2-acetylaminofluorene - 8-OH-AAF 8-hydroxy-2-acetylaminofluorene     

Growth and turnover of microbial biomass during the decomposition of organic matter(Polygonum cuspidatum) in vitro

Air-dried fresh and dead specimens ofPolygonum cuspidatum were incubated for 250 days in the laboratory, and the growth and turnover of microbial biomass-C in the organic matter were studied. The biomass-C in the fresh leaf and fresh stem attained maximum levels on day 14 and day 7, respectively, and then settled down to stable levels. In the dead leaf and dead stem, increase in biomass-C ceased by day 4 and the biomass-C levels did not change thereafter. The turnover time of the biomass-C was estimated from the amount of biomass-C and the release rate of CO₂-C. The turnover was rapid in the early period of incubation. Then the turnover time became longer and after incubation for 70 days the values approached those in natural soils (longer than 16 days). During the incubation period, nitrogen was not mineralized in any organic matter. In the dead leaf and dead stem, asymbiotic nitrogen fixation activity increased after incubation for about 40 days and disappeared by the end of the incubation period, whereas nitrogen fixation was hardly detected in the fresh leaf and fresh stem.     

Studies on haemosiderin and ferritin from iron-loaded rat liver

Summary Haemosiderin has been isolated from siderosomes and ferritin from the cytosol of livers of rats iron-loaded by intraperitoneal injections of iron-dextran. Siderosomal haermosiderin, like ferritin, was shown by electron diffraction to contain iron mainly in the form of small particles of ferrihydrite (5Fe₂O₃ · 9H₂O), with average particle diameter of 5.36±1.31 nm (SD), less than that of ferritin iron-cores (6.14±1.18 nm). Mössbauer spectra of both iron-storage complexes are also similar, except that the blocking temperature,T _B, for haemosiderin (23 K) is lower than that of ferritin (35 K). These values are consistent with their differences in particle volumes assuming identical magnetic anisotropy constants. Measurements of P/Fe ratios by electron probe microanalysis showed the presence of phosphorus in rat liver haemosiderin, but much of it was lost on extensive dialysis. The presence of peptides reacting with anti-ferritin antisera and the similarities in the structures of their iron components are consistent with the view that rat liver haemosiderin arises by degradation of ferritin polypeptides, but its peptide pattern is different from that found in human-thalassaemia haemosiderin. The blocking temperature, 35 K, for rat liver ferritin is near to that reported, 40 K, for human-thalassaemia spleen ferritin. However, the haemosiderin isolated from this tissue, in contrast to that from rat liver, had aT _B higher than that of ferritin. The iron availability of haemosiderins from rat liver and human-thalassaemic spleen to a hydroxypyridinone chelator also differed. That from rat liver was equal to or greater, and that from human spleen was markedly less, than the iron availability from either of the associated ferritins, which were equivalent. The differences in properties of the two types of haemosiderin may reflect their origins from primary or secondary iron overload and differences in the duration of the overload.     

Endogenous cholesterol synthesis is sufficient for ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary To define the role of endogenously synthesized cholesterol in the differentiation of adrenocortical cells in primary culture, fetal rat adrenal cells were cultured in the presence of exogenous cholesterol (serum-supplemented medium) or in the absence of it (serum-free medium or lipoprotein-free medium). Ultrastructurally the cells had features of glomerulosa cells: mitochondria were oval or rod shaped with lamellar inner membranes. The amount of smooth endoplasmic reticulum was small, and lipid droplets were few. When the cells were cultured in serum-free medium some intracytoplasmic vacuoles were seen. The undifferentiated zona glomerulosa-like cells secreted low amounts of corticosterone and 18-OH-deoxycorticosterone (18-OH-DOC) in all three media (serum-supplemented medium, serum-free medium, and lipoprotein-free medium). Stimulation of the adrenocortical cells with ACTH induced the ultrastructural features of differentiated zona fasciculata-like cells. Mitochondrial inner membranes were well developed in lipoprotein-free medium, but not in serum-free medium. The amount of intracellular lipids was increased in both media devoid of cholesterol. In the ACTH stimulated cultures the presence of exogenous cholesterol resulted in increased secretions of corticosterone and 18-OH-DOC. In the absence of an exogenous source of cholesterol, the amounts of steroids secreted were only half of that secreted in the presence of serum-supplemented medium. Endogenously synthesized cholesterol is sufficient for the morphologic differentiation of fetal rat adrenocortical cells under ACTH stimulation. However, without exogenously provided cholesterol, the steroid production accounts only for half of the maximal output achieved using serum-supplemented medium. This work was supported by Finnish Culture Foundation.     

马齿苋叶片PEP羧化酶的分子特性

马齿苋叶片PEPCase由四个相同的亚基组成,亚基分子量为83kD。远紫外CD光谱分析表明,此酶含有36.6％α—螺旋结构。马齿苋叶片PEPCase可被G6P激活,但不能被Gly、Ser激活。G6P可防止酶的尿素变性和枯草杆菌蛋白酶的作用。这种保护效应与G6P诱导的酶构象变化有关。 从酶对低温、高温及尿素的反应来看,马齿苋叶片PEPCase的稳定性高于高粱叶片PEP—Case,两者的免疫特性和电泳特性亦不同。     

Subcellular localization of acid carboxypeptidase in rat liver and human fibroblasts.

The subcellular distribution of acid carboxypeptidase was investigated in rat liver, normal human skin (CRL 1501) and lung (WI-38) fibroblasts, galactosialidosis skin fibroblasts (GM 00806) and transformed lung fibroblasts (WI-38 VA 13). Results of differential and isopycnic centrifugations and osmotic activation experiments clearly indicate that the enzyme is located in lysosomes, in agreement with observations suggesting that carboxypeptidase is the protective protein of the 'Galjaard complex' which is defective in galactosialidosis.     

Synthesis and secretion of lipids by long-term cultures of female rat hepatocytes.

The objective of this work was to characterize lipid metabolism in long-term cultures of adult rat hepatocytes from female rats and explore the potential use of this culture system to study the effect of hormones, drugs and toxic chemicals on it. Hepatocytes, seeded on a feeder layer of 3T3 cells, maintained for 2 weeks their typical morphology. The cultures were able to take up [14C]acetic and [14C]oleic acid from the culture medium and incorporate them into lipids. The synthesis and secretion of lipids by [14C]acetic acid-labeled cultures had a maximum value after 11 and 13 days in culture. Triacylglycerols were the main lipidic species synthesized and secreted by hepatocytes (up to 67% of the total lipids); they also synthesized and secreted phospholipids, cholesterol and cholesterol esters from [14C]acetic acid. Similarly, [14C]oleic acid-labeled cultures synthesized and secreted mostly triacylglycerols (up to 60-70% of the total lipids), but they were also able to incorporate the labeled precursor into both cellular and secreted phospholipids and cholesterol esters. The activity of glycerol-phosphate-dehydrogenase, marker enzyme of glycerolipid synthesis, decreased slightly during the culture time whereas the activity of malic enzyme, marker of fatty acid synthesis, increased. Our results show that long-term cultures of female rat hepatocytes are able to synthesize and secrete several lipids, specially triacylglycerols, from both [14C]acetic and [14C]oleic acid for at least 2 weeks and that they maintain enzyme activities related with the synthetic pathways of glycerolipids and fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)