Cloning and characterization of a gene which determines osmotic stability in Saccharomyces cerevisiae.

The srb1-1 mutation of Saccharomyces cerevisiae is an ochre allele which renders the yeast dependent on an osmotic stabilizer for growth and gives the cells the ability to lyse on transfer to hypotonic conditions. A DNA fragment which complements both of these phenotypic effects has been cloned. This clone contains a functional gene which is transcribed into a 2.3-kb polyadenylated mRNA molecule. Transformation of yeast strains carrying defined suppressible alleles demonstrated that the cloned fragment does not contain a nonsense suppressor. Integrative transformation and gene disruption experiments, when combined with classical genetic analysis, confirmed that the cloned fragment contained the wild-type SRB1 gene. The integrated marker was used to map SRB1 to chromosome XV by Southern hybridization and pulsed-field gel electrophoresis. A disruption mutant created by the insertion of a TRP1 marker into SRB1 displayed only the lysis ability phenotype and was not dependent on an osmotic stabilizer for growth. Lysis ability was acquired by growth in (or transfer to) an osmotically stabilized environment, but only under conditions which permitted budding. It is inferred that budding cells lyse with a higher probability and that weak points in the wall at the site of budding are involved in the process. The biotechnological potential of the cloned gene and the disruption mutant is discussed.     

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)     

Thermogenic effect of adrenaline: interaction with insulin

The contribution of insulin (3.6 pmol.kg body mass-1.min-1) to adrenaline-induced (0.164 nmol.kg fat free mass-1.min-1) thermogenesis was studied in ten postabsorptive healthy volunteers using two sequential protocols. Variables considered were oxygen consumption as well as carbon dioxide production, heart rate, blood pressure, plasma concentrations of glucose, insulin, glycerol, free fatty acids, beta-HO-butyrate and lactate. Adrenaline increased plasma concentrations of glucose, glycerol, free fatty acids, and beta-HO-butyrate, and heart rate and metabolic rate during normo-insulinaemia [61.3 (SEM 6.6) pmol.l-1]. Similar effects were observed during hyperinsulinaemia [167.9 (SEM 18.7) pmol.l-1], but the effect of adrenaline on oxygen consumption was reduced. On average, metabolic rate increased by 12.9% during normo-insulinaemia and by 8.9% during hyperinsulinaemia. We concluded that relative hyperinsulinaemia resulted in decreased adrenaline-induced thermogenesis and therefore increased whole body anabolism.     

Growth of Wolinella succinogenes with polysulphide as terminal acceptor of phosphorylative electron transport

Polysulphide was formed according to reaction (1), when tetrathionate was (1) $${\text{S}}_4 {\text{O}}_6^{2 - } + {\text{HS}}^ - \to 2{\text{S}}_2 {\text{O}}_3^{2 - } + {\text{S(O)}} + {\text{H}}^ + $$ added to an anaerobic buffer (pH 8.5) containing excess sulphide. S(O) denotes the zero oxidation state sulphur in the polysulphide mixture S _infn ^sup2- . The addition of formate to the polysulphide solution in the presence of Wolinella succinogenes caused the reduction of polysulphide according to reaction (2). The bacteria grew in a medium containing formate and sulphide, (2) $${\text{HCO}}_2^ - + {\text{S(O)}} + {\text{H}}2{\text{O}} \to {\text{HCO}}_3^ - + {\text{HS}}^ - + {\text{H}}^ + $$ when tetrathionate was continuously added. The cell density increased proportional to reaction (3) which represents the sum of reactions (1) and (3) $${\text{HCO}}_2^ - + {\text{S}}_{\text{4}} {\text{O}}_6^{2 - } + {\text{H}}2{\text{O}} \to {\text{HCO}}_3^ - + 2{\text{S}}_{\text{2}} {\text{O}}_3^{2 - } + 2{\text{H}}^ + $$ (2). The cell yield per mol formate was nearly the same as during growth on formate and elemental sulphur, while the velocity of growth was greater. The specific activities of polysulphide reduction by formate measured with bacteria grown with tetrathionate or with elemental sulphur were consistent with the growth parameters. The results suggest that W. succinogenes grow at the expense of formate oxidation by polysulphide and that polysulphide is an intermediate during growth on formate and elemental sulphur.     

Purification and properties ofl-serine dehydratase fromLactobacillus fermentum ATCC 14931

l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM l-cysteine in 50 mM phosphate buffer. M_r=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (M_r=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent K_m forl-serine was 65 mM. Fe⁺⁺ was required for the enzymatic activity, and the apparent K_m value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol^–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.     

Limnologic consequences of the decline in hemlock 4800 years ago in three Southern Ontario lakes

Four thousand eight hundred years ago hemlock (Tsuga canadensis) populations were decimated throughout eastern North America. We have studied the effects of this loss from the terrestrial community on three Southern Ontario lakes: Little Round Lake, Sunfish Lake, and McKay Lake. This study includes the use of cladocerans, diatoms, chrysophytes, and bacterial pigments to assess the limnologic changes that occurred in these lakes. Each lake experienced a change in trophic status that coincided with the loss of hemlock from its catchment, but the change in the aquatic biota was different in each lake. The lakes' size may have been the most influential factor governing the response to this terrestrial disturbance.     

Correlation between ventilation and brain blood flow during hypoxic sleep

Ventilation and brain blood flow (BBF) were simultaneously measured during carbon monoxide (CO) inhalation in awake and sleeping goats up to HbCO levels of 40%. Unilateral BBF, which was continuously measured with an electromagnetic flow probe placed around the internal maxillary artery, progressively increased with CO inhalation in the awake and both sleep stages. The increase in BBF with CO inhalation during rapid-eye-movement (REM) sleep (delta BBF/delta arterial O2 saturation = 1.34 +/- 0.27 ml X min-1 X %-1) was significantly greater than that manifested during wakefulness (0.87 +/- 0.14) or slow-wave sleep (0.92 +/- 0.13). Ventilation was depressed by CO inhalation during both sleep stages but was unchanged from base-line values in awake goats. In contrast to slow-wave (non-REM) sleep, the ventilatory depression of REM sleep was primarily due to a reduction in tidal volume. Since tidal volume is more closely linked to central chemoreceptor function, we believe that these data suggest a possible role of the increased cerebral perfusion during hypoxic REM sleep. Induction of relative tissue alkalosis at the vicinity of the medullary chemoreceptor may contribute to the ventilatory depression exhibited during this sleep period.     

Alternate substrates and inhibitors of bacterial 4-hydroxyphenylpyruvate dioxygenase

A variety of analogues of (4-hydroxyphenyl)pyruvic acid were synthesized, and the reactions of these compounds with the 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874 were examined. Several of the ring-substituted substrate analogues are reversible inhibitors of the enzyme, the most potent being the competitive inhibitor (2,6-difluoro-4-hydroxyphenyl) pyruvate (Ki = 1.3 microM). Two substrate analogues (2-fluoro-4-hydroxyphenyl)pyruvate and [(4-hydroxyphenyl)thio]pyruvate proved to be alternate substrates for the enzyme. The former compound is converted to (3-fluoro-2,5-dihydroxyphenyl)acetate in an essentially normal catalytic sequence including oxidative decarboxylation, ring hydroxylation, and side-chain migration. The latter compound, however, undergoes oxidative decarboxylation and sulfoxidation to give [(4-hydroxyphenyl)sulfinyl]acetate; ring oxidation is not observed. The implications of these results with regard to the catalytic mechanism of 4-hydroxyphenylpyruvate dioxygenase are discussed.     

Effect of Drying Medium on Residual Moisture Content and Viability of Freeze-Dried Lactic Acid Bacteria

The effect of various substances on the relationship between residual moisture content and the viability of freeze-dried lactic acid bacteria has been studied. Compounds such as polymers, which display considerable ability in displacing water, showed no protective action during freeze-drying. Adonitol, on the other hand, produced the smallest change in water content at various times during drying and allowed the highest rate of survival.     

Effect of the rehydration medium on the recovery of freeze-dried lactic acid bacteria.

Sixteen cultures of lactic acid bacteria were freeze-dried in 10% nonfat skim milk plus 0.75 M adonitol and rehydrated by using different rehydration media. Marked variations in their capacity to repair cellular damage after freeze-drying were observed among the species and strains under consideration.