Showing papers by "University of Córdoba (Spain) published in 1993"

The growth and signalling defects of the ggs1 (fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII

Abstract: Yeast cells defective in the GGS1 (FDP1/BYP1) gene are unable to adapt to fermentative metabolism. When glucose is added to derepressed ggs1 cells, growth is arrested due to an overloading of glycolysis with sugar phosphates which eventually leads to a depletion of phosphate in the cytosol. Ggs1 mutants lack all glucose-induced regulatory effects investigated so far. We reduced hexokinase activity in ggs1 strains by deleting the gene HXK2 encoding hexokinase PII. The double mutant ggs1Δ, hxk2Δ grew on glucose. This is in agreement with the idea that an inability of the ggs1 mutants to regulate the initiation of glycolysis causes the growth deficiency. However, the ggs1Δ, hxk2Δ double mutant still displayed a high level of glucose-6-phosphate as well as the rapid appearance of free intracellular glucose. This is consistent with our previous model suggesting an involvement of GGS1 in transport-associated sugar phosphorylation. Glucose induction of pyruvate decarboxylase, glucoseinduced cAMP-signalling, glucose-induced inactivation of fructose-1,6-bisphosphatase, and glucose-induced activation of the potassium transport system, all deficient in ggs1 mutants, were restored by the delection of HXK2. However, both the ggs1Δ and the ggs1Δ, hk2Δ mutant lack detectable trehalose and trehalose-6-phosphate synthase activity. Trehalose is undetectable even in ggs1Δ strains with strongly reduced activity of protein kinase A which normally causes a very high trehalose content. These data fit with the recent cloning of GGS1 as a subunit of the trehalose-6-phosphate synthase/phosphatase complex. We discuss a possible requirement of trehalose synthesis for a metabolic balance of sugar phosphates and free inorganic phosphate during the transition from derepressed to fermentative metabolism.

Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae

Abstract: Summary Cells of the yeast Saccharomyces cerevisiae display a wide range of glucose-induced regulatory phenomena, including glucose-induced activation of the RAS-adenylate cyclase pathway and phosphatidylinositol turnover, rapid post-translational effects on the activity of different enzymes as well as long-term effects at the transcriptional level. A gene called GGS1 (for General Glucose Sensor) that is apparently required for the glucose-induced regulatory effects and several ggs1 alleles (fdp1, byp1 and cif1) has been cloned and characterized. A GGS1 homologue is present in Methanobacterium thermoautotrophicum. Yeast ggs1 mutants are unable to grow on glucose or related readily fermentable sugars, apparently owing to unrestricted influx of sugar into glycolysis, resulting in its rapid deregulation. Levels of intracellular free glucose and metabolites measured over a period of a few minutes after addition of glucose to cells of a ggsi1Δ strain are consistent with our previous suggestion of a functional interaction between a sugar transporter, a sugar kinase and the GGS1 gene product. Such a glucose-sensing system might both restrict the influx of glucose and activate several signal transduction pathways, leading to the wide range of glucose-induced regulatory phenomena. Deregulation of these pathways in ggs1 mutants might explain phenotypic defects observed in the absence of glucose, e.g. the inability of ggs1 diploids to sporulate.

Extraction and characterization of chitin from crustaceans

Abstract: Chitin was isolated from various natural sources including Cuban lobsters, Sanlucar prawns, Norway lobsters, Squills, Spanish crayfish, American crayfish and Fusarium oxysporum with a yield of 14–25% on a dry basis. The physico-chemical properties of chitin from the different sources were studied by IR spectroscopy and scanning electron microscopy, and its degree of acetylation was determined. The chitin thus obtained is suitable for biotechnological applications (e.g. as supporting material for immobilizing enzymes).

Five nitrate assimilation-related loci are clustered in Chlamydomonas reinhardtii

Abstract: Three overlapping clones covering a Chlarnydomonas reinhardtii genomic region of about 32 kb appear to contain five genes potentially involved in nitrate assimilation in addition to the nitrate reductase structural locus nit-1 These new loci produced transcripts of 28, 22, 18 and 17 kb in nitrate-induced wild-type cells that, like the 34 kb transcript of nit-1, were undetectable in cells grown in ammonium In addition, in a mutant defective at the regulatory locus, nit-2 for nitrate assimilation, which does not express the nit-1 gene transcript, accumulation of the four other transcripts was also blocked They have been named nar (nitrate assimilation related) genes The nar-1 and nar-2 loci are transcribed in the same orientation as nit-1 The nar-3 and nar-4 loci are transcribed divergently from nit-1 DNA and RNA sequences from both nar-3 and nar-4 cross-hybridized with each other indicating that they share similar sequences Four nitrate assimilation-deficient mutants (C2, D2, F6 and G1) were characterized These mutants lack nar transcripts and have major deletions and/or rearrangements in the nar gene cluster In contrast to other nitrate reductase-deficient mutants and to wild type, deletion mutants and the regulatory mutant nit-2 were incapable of accumulating intracellular nitrate Two of the mutants in which expression of all of the nar loci did not occur, C2 and D2, grew in nitrite medium and showed wild-type levels of both nitrite uptake and nitrite reductase activities Thus the nar loci cannot be required for nitrite assimilation Mutants F6 and G1 were unable to grow in either nitrite- or nitrate-containing medium, and lacked nitrate reductase, nitrite reductase, nitrate uptake and nitrite uptake activities The inability to assimilate nitrite co-segregated with nit-1 in crosses between these mutants and wild type These results indicate that a complex gene cluster responsible for the assimilation of nitrate has been identified in C reinhardtii, and that, in addition, at least one locus necessary for nitrite assimilation is genetically linked to this cluster

The perfume flowers of Cyphomandra (Solanaceae): pollination by euglossine bees, bellows mechanism, osmophores, and volatiles.

Abstract: The perfume syndrome and pollination by fragrance-collecting euglossine bees in the neotropic solanaceous genusCyphomandra was confirmed by field observations. In SE Brazil,C. sciadostylis was visited byEufriesea violaceae, andC. diploconos byEuglossa mandibularis; C. hartwegii was pollinated byEulaema meriana in Costa Rica. The primary attractant, fragrant droplets that ooze from the dorsally bulged connectives, is mopped up by the males with the forebasitarsi. Thereby, the poricidal thecae are inadvertently pushed causing the dry pollen to dust the bee's sternum. The number and direction of the pollen jets are related to pollinator size and stigma structure. The flowers are homogamous, selfsterile, and last three days. The androecium is optically non-contrasting or has cryptic colour. Flowers ofC. sciadostylis andC. diploconos undergo a colour change and an almost three-fold increase in corolla size when scent production and visits cease. The dorsal papillar epidermis of the connective is underlain by a glandular parenchyma typical of osmophores. GC techniques revealed germacrene D as the main component in the mentholic scent ofC. sciadostylis, ipsdienol, heneicosane, and tricosane as dominant in the nutmeg-like scent ofC. diploconos, and benzyl acetate and benzyl alcohol in the sweet fragrance ofC. hartwegii. In all cases, these were accompanied by numerous minor components of heterogeneous chemical nature.—Pollen release by means of a peculiar pneumatic bellows mechanism appears as a necessary and probably ubiquitous feature ofCyphomandra. Even a slight pressure exerted upon the thin, elastic thecal walls blows pollen jets through the pores. Unusual anatomic changes accompany anther maturation. Initially voluminous parenchymatic locular intumescences (placentoids) contract completely during meiosis, then expand once more when the pollen is ripe, pushing the grains against the locular wall, and contract a second time, allowing air to enter the thecae.—Cyphomandra pinetorum was found to be exceptional in exhibiting a pollen flower syndrome, and not cryptical but optically contrasting yellow anthers, as known forSolanum.

Muscle fiber type composition and fiber size in successfully and unsuccessfully endurance-raced horses.

Abstract: Triplicate biopsies from three different depths of the gluteus medius muscle were obtained in 36 endurance-raced horses, aged 8.42 +/- 2.85 yr. Twenty of the horses were considered excellent endurance performers according to the mean speed of their three fastest records in endurance events for the past 2 or 3 years, whereas 16 were moderate performers, with a mean racing speed < 12.5 km/h (in 120- to 180-km endurance rides), < 14 km/h (in 80- to 120-km endurance rides), or < 13.5 km/h (in 40- to 60-km endurance rides). Significant differences in muscle fiber type composition and fiber size were recorded; excellent performers had a higher percentage and a larger size of type I and type IIa fibers (high and low myosin adenosinetriphosphatase activity at pH 4.5, respectively) and a lower percentage of type IIb fibers (moderate myosin adenosinetriphosphatase activity at pH 4.5), including both type IIb oxidative (moderate to high NADH-tetrazolium reductase activity) and IIb nonoxidative (low NADH-tetrazolium reductase activity). The differences in distribution of myofiber types and in fiber sizes were more marked in the deeper parts compared with the superficial regions of muscle. Our results also imply a greater homogeneity among the fiber type sizes across the muscle in horses with a superior endurance performance than in horses that had been poorly or moderately endurance raced. Thus the results show that fiber type proportions and fiber size in equine skeletal muscle are directly related to the athletic ability of the horse for endurance events.

Clinical and hemodynamic predictors of survival in patients aged <65 years with severe congestive heart failure secondary to ischemic or nonischemic dilated cardiomyopathy

Abstract: To identify which clinical or hemodynamic parameters predict survival in patients with end-stage heart failure due to dilated cardiomyopathy,130 consecutive patients aged <65 years (mean 46 ± 13) assessed for heart transplantation from May 1986 to April 1991 were studied. Mean follow-up was 15 ± 11 months. Left ventricular ejection fraction was 22 ± 7%. Left ventricular end-diastolic pressure was 27 ± 9 mm Hg, and cardiac index was 2.2 ± 0.6 liter/min/m2. Symptom class was IV in 91% of patients and III in 9%. Etiology was ischemic in 40% of patients and idiopathic in 60%. After intensive medical therapy, heart transplantation was considered indicated in 53% of patients, contraindicated in 20% and not indicated in 27%. Transplantation was performed in 36% of patients during follow-up, and 35% died and 29% were alive without transplantation. A comparison, excluding patients with transplantation, was performed between those who were alive and had survived ≥6 months after assessment, and those who died. On multivariate analysis, the following 3 parameters were independent predictors of prognosis: intravenous Inotropic requirement (p <0.001), maximal, tolerated captopril dose (p = 0.013) and systolic blood pressure (p = 0.003). When patients with transplantation were considered as deaths, stabilization on medical therapy also reached statistical significance (p = 0.009). Classic prognostic markers including ventricular anhythmias, left ventricular enddiastolic pressure, cardiac index, amiodarone therapy and etiology were not associated with prognosis in this homogeneous population of severely ill patients.

Regulation of CD69 expression on human natural killer cells: differential involvement of protein kinase C and protein tyrosine kinases

Abstract: Human peripheral blood natural killer (NK) cells (CD56+, CD16+, CD3 epsilon- lymphocytes) express CD69 after their stimulation by interleukin-2 (IL-2) or interferon-alpha (IFN-alpha). This activation antigen represents a triggering surface molecule in NK cell clones as its stimulation triggers the cytolytic machinery of these cells. However, the mechanisms regulating the expression of CD69 in NK cells are unknown despite the functional relevance of CD69 in NK cell activation. Thus, we have analyzed the role of protein kinase C (PKC) and protein tyrosine kinases (PTK) in the expression of CD69 on purified NK cells activated by IL-2, IFN-alpha, anti Fc gamma RIII (CD16) monoclonal antibodies or by K562 target cells. We found that CD69 is induced on NK cells not only by IL-2 and IFN-alpha but also by activation of the CD16 pathway, the interaction with NK target cells and the direct activation of PKC by phorbol 12-myristate 13-acetate (PMA), indicating that CD69 induction is associated to different NK activation pathways. The treatment with the PKC inhibitor staurosporine abolished the induction of CD69 induced by PMA or K562. However, it did not significantly affect CD69 induction by IL-2, IFN-alpha or CD16 cross-linking. This demonstrates that whereas PKC can play a central role in the regulation of CD69 expression in some instances (response to K562 cells or PMA), it does not participate in others (response to IL-2, IFN-alpha or anti CD16 monoclonal antibodies). On the other hand genistein, a competitive inhibitor of PTK enzymes, blocked the expression of CD69 induced by activation of NK cells via IL-2 or IFN-alpha receptors, CD16 and K562 receptor(s), indicating that stimulation of PTK is a common step in the signal transduction events leading to the induction of CD69 antigens after the activation of NK cells via these receptors.

Present status of verticillium wilt of olive in Andalucía (southern Spain)

Abstract: Verticillium wilt caused by Verticillium dahliae is a disease highly prevalent in newly established olive orchards in Andalucia, southern Spain. Two syndromes of the disease occur in Andalucia, namely apoplexy and slow decline. Apoplexy is characterized by quick dieback of twigs and branches while slow decline consists of rapid drying out of inflorescences together with leaf chlorosis and necrosis. Systematic disease observations carried out in two experimental orchards planted with susceptible cv. Picual indicated that natural recovery of diseased trees occurred over time. Infection and vascular colonization of olive plants by V. dahliae were studied in susceptible (Picual) and resistant (Oblonga) cultivars inoculated with a mildly virulent or a highly virulent cotton-defoliating isolate of V. dahliae. Disease symptoms developed 24–32 days after inoculation in cv. Picual, but at that time plants of cv. Oblonga remained free from symptoms. However anatomical observations and isolations indicated that systemic infections by the two isolates had occurred to a large extent in both cultivars.

Leaf expansion, photosynthesis, and water relations of sunflower plants grown on compacted soil

Abstract: Leaf expansion and growth response of sunflower (Helianthus annuus, L.) to soil compaction were investigated in relation to compaction effects on water relations, nitrogen nutrition, and photosynthesis. A series of field experiments were conducted with plants grown in 20 cm-diameter cylinders with soil bulk densities ranging from 1.2 to 1.7 g cm−3 at the 0–20 cm depth (equivalent to 0.8 to 2.4 MPa soil strength measured with a soil penetrometer). Relative leaf expansion rate (RLER) decreased linearly with increasing soil strength. Smaller plant size in compacted treatments was due not only to slower expansion rates, but also smaller maximum size of individual leaves. Sensitivity of leaf expansion to soil strength was best illustrated by a reduction in RLER and maximum size of the first leaf to emerge in a treatment with only the lower 10–20 cm of the profile compacted (bulk density of 1.7 g cm−3). Root growth was less affected than shoot growth by compaction and root:shoot ratios of compacted treatments were significantly higher than the control. Soil compaction had no significant effect on pre-dawn or midday leaf water potential, osmotic potential or leaf turgor. Specific leaf weight was usually higher in plants grown on compacted soil, and leaf nitrogen and photosynthesis per unit leaf area were either unaffected by treatment or significantly higher in compacted treatments. The results suggest that early growth reduction of sunflower plants grown on compacted soil was more sink- than source-limited with regard to water, nitrogen, and carbon supply. Further evaluation of this hypothesis will require verification that these whole-leaf measurements provided a sufficiently accurate approximation of treatment effects on the dynamic equilibria of expanding cells.

Factors determining late success after mitral balloon valvulotomy

Abstract: Mitral balloon valvulotomy (MBV) has proved to be an effective method in the treatment of patients with mitral stenosis. Although several factors determining an optimal immediate result have been described, there is little information regarding long-term follow-up, as well as factors influencing late success after MBV. This study analyzes 350 patients (mean age 46 +/- 12 years) treated by MBV who were clinically followed up between 6 months and 6 years. At least 1 echo-Doppler follow-up study was obtained in 298 patients 28 +/- 14 months after MBV; hemodynamic reevaluations were performed in 66 patients after 23 +/- months. Late success was considered if the patient was in functional class I to II and free of major events (death, restenosis and valve surgery). Restenosis was defined as a 50% loss of initial gain with regard to valve area by echocardiography, which was confirmed hemodynamically. After a mean follow-up of 38 +/- 15 months, 296 patients (84%) remained in functional class I to II, without surgery or the need for an increase in medical treatment. The 5-year Kaplan-Meier survival rate was 94 +/- 1%, whereas restenosis, valve surgery and major event-free probability were 90 +/- 3%, 91 +/- 2% and 85 +/- 2%, respectively. Multivariate study (Cox regression model) identified the presence of sinus rhythm (p < 0.001) and the absence of calcium at fluoroscopy (p < 0.003) as the only independent factors of late success.(ABSTRACT TRUNCATED AT 250 WORDS)

Near infrared calibrations for goat's milk components: protein, total casein, α s -, β- and κ-caseins, fat and lactose

Abstract: A fast and inexpensive method to quantify the main constituents of goat's milk using near infrared (NIR) spectroscopy, is proposed. Since the efficiency of milk-to-cheese transformation depends on ...

Variations in the concentrations of airborne Olea pollen and associated pollinosis in Córdoba (Spain): a study of the 10-year period 1982-1991.

Abstract: The amount and the seasonal and daily changes in the concentrations of Olea pollen grains in the atmosphere of Cordoba (Spain) have been studied over a 10-year period. The year by year seasonal variation pattern and the theoretical intradiurnal variation model are presented. The data show a high annual variability, median concentrations varying from 75 g/m3/h in 1983 to 1413 g/m3/h in 1986. A steady increase in the total amount of pollen is attributed to the climatological characteristics of the period studied. On the other hand, the clinical data show that the number of cases of Olea allergy has increased considerably during this period, probably more because of changes in the quality of the atmosphere than because of the increase in the amount of antigen present in the air.

Leaf Expansion in Field-Grown Sunflower in Response to Soil and Leaf Water Status

Abstract: Leaf responses to soil water deficits in controlled environments may be mediated by nonhydraulic root signals (RS). The aim of this study was to evaluate in the field the effects of RS and leaf water potential (ψ) on leaf expansion rate (LER) of sunflower (Helianthus annuus L.). Two experiments were performed on a deep sandy-loam soil (Typic Xerofluvent) in a Mediterranean environment under spring (Exp. 1) and summer conditions (Exp. 2). WET and DRY treatments were established at the 16th-leaf stage in both experiments- WET plots were irrigated daily with an amount of water equal to the reference evapotranspiration of the previous day [...]

Screening techniques and sources of resistance to parasitic angiosperms

Abstract: Parasitic angiosperms cause great losses in many important crops under different climatic conditions and soil types. The most widespread and important parasitic angiosperms belong to the genera Orobanche, Striga, and Cuscuta. The most important economical hosts belong to the Poaceae, Asteraceae, Solanaceae, Cucurbitaceae, and Fabaceae. Although some resistant cultivars have been identified in several crops, great gaps exist in our knowledge of the parasites and the genetic basis of the resistance, as well as the availability of in vitro screening techniques. Screening techniques are based on reactions of the host root or foliage. In vitro or greenhouse screening methods based on the reaction of root and/or foliar tissues are usually superior to field screenings and can be used with many species. To utilize them in plant breeding, it is necessary to demonstrate a strong correlation between in vitro and field data. The correlation should be calculated for every environment in which selection is practiced. Using biochemical analysis as a screening technique has had limited success. The reason seems to be the complex host-parasite interactions which lead to germination, rhizotropism, infection, and growth of the parasite. Germination results from chemicals produced by the host. Resistance is only available in a small group of crops. Resistance has been found in cultivated, primitive and wild forms, depending on the specific host-parasite system. An additional problem is the existence of pathotypes in the parasites. Inheritance of host resistance is usually polygenic and its transfer is slow and tedious. Molecular techniques have yet to be used to locate resistance to parasitic angiosperms. While intensifying the search for genes that control resistance to specific parasitic angiosperms, the best strategy to screen for resistance is to improve the already existing in vitro or greenhouse screening techniques.

Changes in pituitary secretion during the early postnatal period and anovulatory syndrome induced by neonatal oestrogen or androgen in rats

Abstract: The following experiments were performed: (i) concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and prolactin in plasma were measured at 2, 5, 8, 10 and 15 days in female Wistar rats treated on the first day of life with 100 micrograms oestradiol benzoate or vehicle; (ii) females injected on day 1 with 100 micrograms of oestradiol benzoate or 1 mg of testosterone propionate and from day 1 to day 10 or 15 with FSH and LH were killed on day 90; (iii) females injected from day 1 to day 10 or 15 with prolactin or vehicle were killed on day 90; (iv) females injected on day 1 with oestradiol benzoate and from day 1 to day 15 with a luteinizing-hormone-releasing hormone (LHRH) agonist were killed on day 90; (v) groups of females injected on days 1, 4, 7, 10, 13 and 16 with an LHRH antagonist were killed on day 90. Onset of puberty, vaginal cycles, organ weights and hormonal plasma concentrations were measured. Females treated on the first day of life with 100 micrograms oestradiol showed inhibition of gonadotrophin secretion and stimulation of prolactin secretion during the neonatal period. Females injected on the first day of life with oestradiol benzoate or testosterone propionate showed, in adulthood, anovulation, ovarian atrophy, reduced FSH plasma concentrations, increased prolactin plasma concentrations and reduced pituitary prolactin content.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of enzyme-immunohistochemistry for the diagnosis of aspergillosis, candidiasis, and zygomycosis in three lovebirds.

Abstract: Aspergillosis, candidiasis, and zygomycosis were diagnosed in tissues from three lovebirds (Agapornis roseicollis) using indirect enzyme-immunohistochemical techniques. In these techniques, the first antibody was raised against fungal antigen. A second antibody, which was raised in another animal species, was added to link the first antibody to enzyme-immunocomplexes. The reactivity of specific monoclonal and polyclonal antibodies was visualized by immunoreactivity of alkaline phosphatase anti-alkaline phosphatase and peroxidase anti-peroxidase immunocomplexes. All three birds examined had dermal candidiasis. In addition, one of the birds was diagnosed with concomitant acute ocular aspergillosis, and another bird was diagnosed with chronic zygomycotic myocarditis.

Purification of Cu, Zn-Superoxide Dismutase Isoenzymes from Fish Liver: Appearance of New Isoforms as a Consequence of Pollution

Abstract: Liver cell-free extracts of fish (Mugil sp.) from polluted environments show new Cu, Zn-SOD isoenzymes when analyzed by polyacrylamide gel electrophoresis or isoelectrofocusing followed by in situ staining for SOD activity. The most active isoenzymes, with pI 6.1 and 5.1, were present both in control and problem samples while the isoenzymes of intermediate pI value showed significant differences. Fish from control areas showed three intermediate isoenzymes with pI 5.7, 5.5 and 5.4 (the last one quite faint) while polluted animals showed three bands of pI 5.9, 5.45 and 5.35, this last very intense. To further characterize their utility as biomarkers, Cu, Zn-SOD isoenzymes from polluted fish livers were purified to homogeneity. Five superoxide dismutase peaks were purified, named thereafter I (pI 6.1) to V (pI 5.1) respectively. Isoenzymes I and V displayed the highest specific activity. Upon incubation with moderate H2O2 concentrations, pure isoenzyme I yielded more acidic bands with pI 5.5, 5.45 and 5.35,...

Characterization of a nitrophenol reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1.

Abstract: The phototrophic bacterium Rhodobacter capsulatus E1F1 photoreduced 2,4-dinitrophenol to 2-amino-4-nitrophenol by a nitrophenol reductase activity which was induced in the presence of nitrophenols and was repressed in ammonium-grown cells. The enzyme was located in the cytosol, required NAD(P)H as an electron donor, and used several nitrophenol derivatives as alternative substrates. The nitrophenol reductase was purified to electrophoretic homogeneity by a simple method. The enzyme was composed of two 27-kDa subunits, was inhibited by metal chelators, mercurial compounds, and Cu2+, and contained flavin mononucleotide and possibly nonheme iron as prosthetic groups. Purified enzyme also exhibited NAD(P)H diaphorase activity which used tetrazolium salt as an electron acceptor.

Nucleotide sequence and genetic analysis of the Rhodobacter capsulatus ORF6-nifUI SVW gene region: possible role of NifW in homocitrate processing.

Abstract: DNA sequence analysis of a 3494-bp HindIII-BclI fragment of the Rhodobacter capsulatus nif region A revealed genes that are homologous to ORF6, nifU, nifS, nifV and nifW from Azotobacter vinelandii and Klebsiella pneumoniae. R. capsulatus nifU, which is present in two copies, encodes a novel type of NifU protein. The deduced amino acid sequences of NifUI and NifUII share homology only with the C-terminal domain of NifU from A. vinelandii and K. pneumoniae. In contrast to nifA and nifB, which are almost perfectly duplicated, the predicted amino acid sequences of the two NifU proteins showed only 39% sequence identity. Expression of the ORF6-nifUISVW operon, which is preceded by a putative sigma 54-dependent promoter, required the function of NifA and the nif-specific rpoN gene product encoded by nifR4. Analysis of defined insertion and deletion mutants demonstrated that only nifS was absolutely essential for nitrogen fixation in R. capsulatus. Strains carrying mutations in nifV were capable of very slow diazotrophic growth, whereas ORF6, nifUI and nifW mutants as well as a nifUI/nifUII double mutant exhibited a Nif+ phenotype. Interestingly, R. capsulatus nifV mutants were able to reduce acetylene not only to ethylene but also to ethane under conditions preventing the expression of the alternative nitrogenase system. Homocitrate added to the growth medium repressed ethane formation and cured the NifV phenotype in R. capsulatus. Higher concentrations of homocitrate were necessary to complement the NifV phenotype of a polar nifV mutant (NifV-NifW-), indicating a possible role of NifW either in homocitrate transport or in the incorporation of this compound into the iron-molybdenum cofactor of nitrogenase.

Disruption of the Kluyveromyces lactis GGS1 gene causes inability to grow on glucose and fructose and is suppressed by mutations that reduce sugar uptake

Abstract: In the yeast Saccharomyces cerevisiae the GGS1 gene is essential for growth on glucose or other readily fermentable sugars. GGS1 is the same gene as TPS1 which was identified as encoding a subunit of the trehalose-6-phosphate synthase/phosphatase complex and it is allelic to the fdp1, byp1, glc6 and cif1 mutations. Its precise function in the regulation of sugar catabolism is unknown. We have cloned the GGS1 homologue from the distantly related yeast Kluyveromyces lactis. The KlGGS1 gene is 74% and 79% identical at the nucleotide and amino acid sequence level, respectively, to the S. cerevisiae counterpart. We also compared the sequence with the partly homologous products of the S. cerevisiae genes TPS2 and TSL1 which code for the larger subunits of the trehalose synthase complex and with a TSL1 homologue, TPS3, of unknown function. Multiple alignment of these sequences revealed several particularly well conserved elements. Disruption of GGS1 in K. lactis caused the same pleiotropic phenotype as in S. cerevisiae, i.e. inability to grow on glucose or fructose and strongly reduced trehalose content. We have also studied short-term glucose-induced regulatory effects related to cAMP and cAMP-dependent protein kinase, i.e. the cAMP signal, trehalase activation, trehalose mobilization and inactivation of fructose-1,6-bisphosphatase. These effects occur very rapidly in S. cerevisiae and are absent in the Scggs1 mutant. In K. lactis all these effects were much slower and largely unaffected by the Klggs1 mutation. On the other hand, glucose strongly induced pyruvate decarboxylase and activated the potassium transport system in K. lactis and both effects were absent in the Klggs1 mutant. Addition of glucose to galactose-grown cells of the Klggs1 mutant caused, as in S. cerevisiae, intracellular accumulation of free glucose and of sugar phosphates and a rapid drop of the ATP and inorganic phosphate levels. Glucose transport kinetics were the same for the wild type and the Klggs1 mutant in both derepressed cells and in cells incubated with glucose. We have isolated phenotypic revertants of the Klggs1 mutant for growth on fructose. The suppressors that we characterized had, to different extents, diminished glucose uptake in derepressed cells but cells incubated in glucose showed very different characteristics. The suppressor mutations prevented deregulation of glycolysis in the Klggs1 mutant but not the accumulation of free glucose. The mutants with higher residual uptake activity showed partially restored induction of pyruvate decarboxylase and activation of potassium transport.(ABSTRACT TRUNCATED AT 400 WORDS)

Automatic gas chromatographic determination of N-methylcarbamates in milk with electron capture detection

Abstract: A new analytical method that combines on-line derivatization-extraction and gas-liquid chromatography for the determination of N-methylcarbamates is reported. The hydrolysis products of N-methylcarbamates (phenols) are derivatized and extracted in a continuous fashion using pentafluoropropionic anhydride (derivatizing reagent) in n-hexane as an extractant. The thermal instability of N-methylcarbamates is overcome by forming fluoro derivatives that can be identified and quantified at the nanogram per milliliter level with an ECD detector. Application of the proposed method to six mixtures of N-methylcarbamates yielded detection limits between 2 and 20 ng/mL and a relative standard deviation of 2.35-3.70%. Average recoveries of N-methylcarbamates added to cow's milk at concentrations between 50 ng/mL and 2 micrograms/mL ranged from 93.7 to 100.8%.