Arachidonic and oleic acids stimulate zygote formation in the presence of mating pheromone in the yeast,Saccharomyces cerevisiae

Effect of exogenous fatty acids on zygote formation in Saccharomyces cerevisiae was studied. Arachidonic and oleic acids considerably stimulated zygote formation, but other fatty acids tested, linoleic, linolenic, stearic and palmitic acids, did not. Pretreatment experiments with arachidonic acid showed that the stimulation of zygote formation by the fatty acid required the presence of mating pheromone.Abbreviations YPD yeast-peptone-dextrose medium - A₅₃₀ absorbance at 530 nm     

Mapping and disruption of the cybB gene coding for cytochrome b561 in Escherichia coli

Abstract The cybB gene on a plasmid encoding cytochrome b ₅₆₁ in Escherichia coli was disrupted by insertion of Km^rl determinant DNA. The cromosomal cybB gene was replaced by the inactivated cybB gene on the plasmid by homologous recombination using λ phage lysogenization and heat-induction. The replacement was confirmed by Southern and Western blotting analyses. Deficiency on the cybB gene product did not affect the growth properties of the cells, and the oxidase activities of the cells dependent on various substrates were similar to those of the parental strain. Cytochrome b ₅₆₁ is concluded to be expressed in E. coli , but may not play a major role in cell growth. In the genetic map of E. coli , the cybB gene was determined by conjugational and transductional crosses to be at 31 min between trg and terC .     

Digit development and embryonic weight in mice: analysis of sex-related time difference and mating period-related interlitter variability

The embryonic growth and digit formation in limb buds were more advanced in male embryos than in female embryos at a specific time (day 12.0) of midgestation. Furthermore, when the number of digits was compared between the sexes according to their body weight, male embryos were found to be more advanced than females in the differentiation of the digit in limb buds. This is the first demonstration of the presence of a time difference in digit development between the sexes of mouse embryos. In the short-period, morning-mating group, embryonic weights at day 12.0 were lower than those in the overnight-mating group. However, the digit development was not very much delayed in proportion to the difference in body weights, and some "catch-up" phenomena were observed in this group. Interlitter and intralitter variability in body weights of mouse embryos at day 12.0 was greater in the overnight-mating group than in the short-period-mating group. These findings suggest that, in embryonic stage-related teratological experiments in mice, a short-period-mating schedule is advised and that the incidence of developmental anomalies should be analyzed separately for male and female fetuses.     

Effects of subacute treatment with cocaine on activities of N-demethylase, UDP-glucuronyltransferase and sulfotransferase in WKY and SHR rat liver--sex and strain differences

The effects of subacute treatment with cocaine on activities of cocaine N-demethylase, UDP-glucuronyltransferase (GT) toward 4-nitrophenol and phenolphthalein and sulfotransferase (ST) toward androsterone and 4-nitrophenol in livers from Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were investigated. Hepatic metabolism of cocaine was different between the sexes (with males having higher N-demethylase activity) and the strains (with WKY rats having higher activity). The effects of subacute cocaine administration on the activity of cocaine N-demethylase were also sex- and strain-related. Whereas cocaine administration increased activity of hepatic N-demethylase in both female strains, it decreased activity in male WKY and had no effect on activity in male SHR. Sex and strain-related as well as cocaine-induced differences were also found in activities of hepatic GT toward 4-nitrophenol and phenolphthalein as well as in activity of hepatic ST towards andersterone and 4-nitrophenol. These results suggest that some of the individual variation in the effects of cocaine may be due to sex and genetic differences in the hepatic metabolism of cocaine and/or in sexually and/or/genetically-determined differences in how cocaine affects hepatic metabolism of other xenobiotics.     

Nucleotide sequence of the cybB gene encoding cytochrome b561 in Escherichia coli K12

Summary The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b ₅₆₁ and its flanking region was determined. The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20160. From its deduced amino acid sequence, cytochrome b ₅₆₁ is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions. Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane. No significant homology of the primary structure of cytochrome b ₅₆₁ with those of other bacterial b-type cytochromes was observed.     

A simple method for calculation of free metal ion concentrations in the presence of a chelating agent and multiple metal ions by successive approximation

Calculation of free metal ion concentrations in the presence of a chelating agent (ligand) and multiple metal ions is complicated. In this paper we describe a simple method for calculation of free ion concentrations from given total ion concentrations. The outline of this method is as follows: i) Using an arbitrarily chosen provisional value of free ligand concentration (p-Lf), calculate a total ligand concentration (Lt). ii) Divide the p-Lf by the ratio, the calculated Lt (c-Lt)/the specified Lt (s-Lt). Take the resulting value as the next p-Lf and repeat the calculation until c-Lt is close enough to s-Lt. At this point, p-Lf is supposed to be set to a good approximation of true free ligand concentration. iii) Finally, calculate free metal concentrations from the above approximation.     

Change in Gene Expression in Radish Cotyledons during Dark-Induced Senescence

Cotyledons detached from light-grown radish (Raphanus sativusL. cv. Comet) seedlings were used as a model system to studythe changes in nuclear gene expression during dark-induced senescenceof green leaves. Polyadenylated RNA was prepared from the cotyledonsat different times and then translated in a wheat germ system.Approximately 1,000 different polypeptides of the translationproducts were separated from each other by two-dimensional gelelectrophoresis. As judged from the density of autoradiographicspots of the translation products, the induction of senescenceby dark treatment involved an increase in 26 species, a decreasein 11 species, and a temporary increase and subsequent decreasein 8 species of translatable mRNA. A similar pattern of changein protein synthesis was also observed in the dark-treated cotyledonswhen the cotyledons were pulse-labeled with ³⁵S-methionine andthe soluble proteins separated by two-dimensional gel electrophoresis,though the polypeptide pattern on the gel did not coincide exactlywith those of the cell-free translation products. These findingsstrongly suggest that the process of leaf senescence is notsimply a passive and gradual death of the tissue, but involvesa drastic and sequential response of the cells to environmentalstimuli with respect to the gene expression of the cells. (Received July 21, 1987; Accepted September 30, 1987)     

Respiration-Dependent Proton and Sodium Pumps in a Psychrophilic Bacterium, Vibrio sp. Strain ABE-1

Respiration-dependent proton and sodium flows in a psychrophilicbacterium, Vibrio sp. strain ABE-1, were examined. At alkalinepH, this bacterium grew without being affected by a proton conductor,carbonylcyanide m-chlorophenylhydrazone (CCCP). O₂-pulse intoanaerobic cell suspensions prepared with Na^$-free buffers inducedtransient alkalization in the presence of CCCP and acidificationat pH 8.5 and 6.5, respectively. However, using cells preparedwith Na^$-containing buffer, the transient pH changes of thecell suspension could be simultanously detected at both pHs.Several inhibitory experiments suggested that the acidificationand alkalization should be attributed to a respiration-dependentprimary H^$ pump and Na^$ pump, respectively, and that the latterwas similar to that first reported in a marine bacterium, Vibrioalginolyticus. This Na^$ pump may have supported the CCCP-resistantgrowth at alkaline pH. The H^$ and Na^$ pumps operated very actively at low temperatures,such as 5?C, and should markedly help sustain bacterial growthat low temperatures. (Received May 30, 1987; Accepted November 13, 1987)     

Senescence-specific increase in cytosolic glutamine synthetase and its mRNA in radish cotyledons

Changes in the levels of cytosolic and chloroplastic isoforms of glutamine synthetase were examined in senescing radish (Raphanus sativus L. cv Comet) cotyledons by immunoblotting analysis using antibodies raised separately against maize glutamine synthetase isoforms. Translatable mRNAs for these isoforms were also examined by analyzing translation products from poly(A)⁺ RNA in a wheat germ system with the antibodies. The relative content of cytosolic isoform (GS₁) increased twofold in the cotyledons that were placed in the dark for 72 hours to accelerate senescence, while that of chloroplastic isoform (GS₂) declined to half of its initial level. The dark-treatment also increased the relative level of translatable mRNA for GS₁ sevenfold after 72 hours, and decreased rapidly that for GS₂ and for other nuclear-coded chloroplast proteins as well. Cotyledons also accumulated GS₁ mRNA when they became senescent after a lengthy growth period under continuous light. These observations suggested that GS₁ genes were activated, while those for GS₂ were repressed, and eventually the population of the enzyme was altered in senescent cotyledonary cells. The role of increased cytosolic enzyme is discussed in relation to the nitrogen metabolism in senescent leaves.     

Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis

Proximal tubular epithelial cells from mice which develop autoimmune interstitial nephritis were found to express the nephritogenic target antigen, 3M-1. Anti-3M-1 mAbs (alpha 3M-1-Ab) were used to positively select for 3M-1-secreting tubular epithelium and, after stabilization in culture, this new cell line (MCT) was examined for the production of several moieties important to either immune interactions or to the development of extracellular matrix. Alkaline phosphatase-staining MCT cells also express epithelial growth factor receptors with a Kd of 0.87 nM and an epithelial growth factor receptor constant (Ro) of 2.1 X 10(4) receptors/cell. MCT culture supernatants contain greater amounts of laminin, and types IV and V procollagens compared to types I and III procollagens, and growing MCT cells on type I collagen matrix causes them to preferentially secrete even more type IV and V procollagen. The 30,000-Mr 3M-1 antigen could be immunoprecipitated from biosynthetically labeled MCT cell supernatants with alpha 3M-1-Ab. An identical-sized moiety was isolated by immunoaffinity chromatography from collagenase-solubilized mouse kidney tubular basement membranes. The 3M-1 antigen can be found on the MCT cell surface by radioimmunoassay, or deposited in a linear array in the extracellular matrix surrounding the MCT cells in culture by immunofluorescence. Mature messenger RNA species for both class I and class II major histocompatibility complex (MHC) molecules were detected by Northern hybridization, and their corresponding cell surface gene products were detected by cytofluorography of MCT cells stained with haplotype-specific antibodies. Both the cell surface 3M-1 and the small amounts of detected class II MHC molecules appear to be biologically functional, as MCT cells can support the proliferation of 3M-1-specific, class II MHC-restricted helper T cells in culture. These findings suggest that MCT cells provide all the necessary biological parameters for interfacing both as the target of a nephritogenic immune response, and as a potential source for new extracellular matrix which develops as a fibrogenic response to interstitial nephritis.