gltF, a member of the gltBDF operon of Escherichia coli, is involved in nitrogen-regulated gene expression

We report here the construction and analysis of insertional mutations in each of the three genes of the gltBDF operon and the nucleotide sequence of the region downstream from gltD. Two open reading frames were identified, the first of which corresponds to gltF. The gltB and gltD genes code for the large and small subunits, respectively, of the enzyme glutamate synthase (GOGAT). gltF codes for a protein, with a molecular mass of 26,350 Da, which is required for Ntr induction. Histidase synthesis was determined as a measure of Ntr function. First, insertions in gltB, gltD or gltF all prevent Ntr induction. Second, complementation analysis indicates that high-level expression of both the gltD and gltF genes is required for the induction of the Ntr enzymes under nitrogen-limiting conditions, indicating that the phenotype of the gltB insertion probably results from polarity on gltD and gltF. Third, glutamate-dependent repression of the glt operon appears to be mediated by the product of the gltF gene. Thus, the gltBDF operon of Escherichia coli is involved in induction of the so-called Ntr enzymes in response to nitrogen deprivation, as well as in glutamate biosynthesis.     

Identification of two glutaminases inRhizobium etli

We present evidence thatRhizobium etli has two glutaminases differentiated by their thermostability and electrophoretic mobility. The thermostable glutaminase (B) is constitutive, in contrast with the thermolabile glutaminase (A), which is positively regulated by glutamine and negatively regulated by ammonium and by the carbon source. In distinction to glutaminase A, glutaminase B plays a minor role in the utilization of glutamine as a carbon source, but it may play a role in maintaining the balance of glutamine and glutamate. By complementation of theRhizobium etli LM16 mutant that lacks glutaminase A, we have cloned the gene that codes for this enzyme.     

Phytochrome-mediated effects on extracellular peroxidase activity, lignin content and bending resistance in etiolated Vicia faba epicotyls

Etiolated Vicia faba seedlings were exposed to continuous red light to investigate whether changes in extracellular peroxidase activity were correlated in time and localization with changes in extension growth and/or lignin content in the subapical region of the epicotyl. Continuous red light: (a) increased extracellular peroxidase activity after a lag of ca 0.5 h, followed by a maximum peak after 2.5 h due to slightly acidic isoforms (pI = 6–6.5, according to isoelectrofocusing gels), a minimum after 4 h and a second maximum after 8 h due to acidic isoforms (pI=4–5), (b) increased lignin content and epicotyl resistance to bending after a lag of ca 4 h, i.e. simultaneously with changes in acidic extracellular peroxidase activity, and (c) reduced extension growth to a stable rate after a lag of ca 1 h, not coinciding with the kinetics of any of the extracellular peroxidase isoforms. These effects of continuous red light were at least partially mediated by phytochrome. Tissue printing and anatomical studies revealed red light effects on extracellular peroxidase activity and lignin content mainly in the outer cortical parenchyma. The results are consistent with the involvement of phyto-chrome-mediated effects on extracellular peroxidases (acidic isoforms) in the transduction chain leading to lignin responses to red light.     

Follicle-stimulating hormone and human chorionic gonadotropin induced changes in granulosa cell glycosyl-phosphatidylinositol concentration

In the present investigation, a hCG sensitive glycosyl-phosphatidylinositol (GPI) was isolated from cultured rat granulosa cells obtained from the ovaries of diethylstilbestrol (DES) implanted immature rats. The inositol-phosphoglycan (IPG) moiety of the GPI-lipid contains galactose, glucosamine, and myoinositol as demonstrated by metabolic labelling of granulosa cells for different time periods (5–96 h) with [³H]galactose, [³H]glucosamine, or [³H]myoinositol and treatment of the purified [³H]GPI with phosphatidylinositol-specific phospholipase C. Labelling equilibrium of the GPI-lipid was achieved after 24 h ([³H]galactose and [³H]myoinositol) or 72 h ([³H]glucosamine) incubation, whereas incorporation of other labelled carbohydrates tested ([³H]galactosamine, [³H]mannose, and [³H]sorbitol) was negligible throughout the time period studied. The glucosamine C-1 appears to be linked through a glycosidic bond to the myoinositol molecule of the IPG moiety as revealed by the generation of phosphatidylinositol (PtdIns) after nitrous acid deamination of dual labelled ([³H]glucosamine/[¹⁴C]palmitate or [³H]glucosamine/[¹⁴C]myristate) glycosyl-phosphatidylinositol. To investigate the fatty acid composition of the diacylglycerol (DAG) backbone of the GPI, granulosa cells were also labelled (5–72 hr) with [¹⁴C]linoleate, [³H]myristate, [³H]-oleate, [³H]palmitate, or [³H]stearate and the radioactivity associated with the purified glycosyl-phosphatidylinositol determined. Incorporation of [³H]palmitate and [³H]myristate into the GPI-lipid peaked after 8 h and 24 h of labelling, respectively, and both fatty acids were partially released after PLA₂ treatment of the dual labelled ([³H]glucosamine/[¹⁴C]palmitate or [³H]glucosamine/[¹⁴C]myristate) GPI. In parallel experiments no significant incorporation of labelled stearate, oleate, or linoleic acid into the DAG backbone of the glycosylphosphatidylinositol could be detected. Granulosa cells were also labelled with [³H]glucosamine in the presence of FSH (30 ng/ml), cholera toxin (1 μg/ml), or the membrane permeable cAMP analog (but)₂ cAMP (1 mM). Time related increases in GPI-labelling were apparent after 48 h and reached a maximum level (3-, 5-, and 7-fold for FSH, CT, and (but)₂ cAMP, respectively) after 72 h in culture. In another set of experiments, granulosa cells were labelled for 72 h with [³H]glucosamine in the presence of (but)₂cAMP (1 mM), TPA (10^?7 M), or combination thereof. The effect of treatment with the membrane permeable cAMP analog on GPI labelling was prevented in the presence of TPA, whereas no differences in [³H]GPI content could be observed in untreated granulosa cells or cells cultured in the presence of the protein kinase C-activating phorbol ester alone. In cells differentiated with FSH (30 ng/ml for 3 days) to induce LH receptors, treatment with hCG (100 ng/ml) induced a rapid (60 sec) and transient (5 min) decrease in the GPI content, whereas no efect of the hormone on undifferentiated granulosa cells could be observed. The rapid effect elicited by hCG on GPI content and turnover may be an early transduction mechanism involved in the biological effects of LH/hCG in differentiated granulosa cells. © 1993 Wiley-Liss, Inc.     

The Glucose-Related Decrease in Polar Auxin Transport During Ripening and its Possible Role in Grapevine Berry Coloring

Journal of Plant Growth Regulation - Auxin is a hormone that delays ripening in part by reducing anthocyanin content and impairing color development. Auxin content declines during the ripening...     

Tunicamycin inhibits the initiation of DNA synthesis stimulated by prostaglandin F2α in Swiss mouse 3T3 cells

Tunicamycin, an inhibitor of the asparagine-linked protein N-glycosylation, blocks the initiation of DNA synthesis in Swiss 3T3 cells stimulated by prostaglandin F_2α alone or with insulin. This effect is exerted only when tunicamycin is added from 0 to 8 h after stimulation and it decreases the rate of entry into S phase. Blocking of labeled sugar incorporation to proteins occurs regardless of the time of PGF_2α stimulation. In contrast tunicamicin does not inhibit protein synthesis. These results suggest that N-glycoprotein synthesis early during the prereplicative phase is an important event controlling the mitogenic action of PGF_2α     

Creation of an allosteric phosphofructokinase starting with a nonallosteric enzyme. The case of dictyostelium discoideum phosphofructokinase.

An allosteric phosphofructokinase (PFK) was created by sequence manipulation of the nonallosteric enzyme from the slime mold Dictyostelium discoideum (DdPFK). Most amino acid residues proposed as important for catalytic and allosteric sites are conserved in DdPFK except for a few of them, and their reversion did not modify its kinetic behavior. However, deletions at the unique C-terminal extension of this PFK produced a markedly allosteric enzyme. Thus, a mutant lacking the last 26 C-terminal residues exhibited hysteresis in the time course, intense cooperativity (n(H) = 3.8), and a 200-fold decrease in the apparent affinity for fructose 6-phosphate (S(0.5) = 4500 microm), strong activation by fructose 2,6-bisphosphate (K(act) = 0.1 microm) and fructose 1,6-bisphosphate (K(act) = 40 microm), dependence on enzyme concentration, proton inhibition, and subunit association-dissociation in response to fructose 6-phosphate versus the nonhysteretic and hyperbolic wild-type enzyme (n(H) = 1.0; K(m) = 22 microm) that remained as a stable tetramer. Systematic deletions and point mutations at the C-tail region of DdPFK identified the last C-terminal residue, Leu(834), as critical to produce a nonallosteric enzyme. All allosteric mutants were practically insensitive to MgATP inhibition, suggesting that this effect does not involve the same allosteric transition as that responsible for fructose 6-phosphate cooperativity and fructose bisphosphate activation.     

First record of anomalous otoliths of Menticirrhus americanus in the South Atlantic

Sagitta otoliths are usually formed of calcium carbonate polymorphs as aragonite. The objective of this study was to verify which carbonate polymorph is predominant in the sagitta otolith of Menticirrhus americanus and check whether this pattern remains in otoliths with morphological alterations. Otoliths of M. americanus were obtained from five sites on the southeast‐south coast of Brazil (São Sebastião (SS) 23°45′S–45°24′O, n = 29; Cananéia‐Iguape Estuarine Complex (CI) 25°02′S–47°54′O, n = 30; Paranaguá Estuarine Complex (PEC) 25°28′S–48°20′O, n = 35; Itapoá (IT) 26°07′S–48°36′O, n = 31; Laguna (LA) 28°28′S–48°46′O, n = 13). The characterization of carbonate polymorphs of otoliths was performed through Raman spectroscopy, a photonic and non‐destructive technique that analyzes molecular vibrations induced by laser. We analyzed 138 pairs of M. americanus otoliths, of which eight otoliths from different pairs presented morphological alterations (SS n = 1, CEP n = 5, IT n = 1, LA n = 1). The Raman spectra show that normal otoliths, that is, without morphological alterations, presented only aragonite in their structure. Among the otoliths that presented morphological alterations, the Raman spectra allowed to identify in six otoliths the deposition of aragonite and in only two otoliths the deposition of vaterite (one specimen of the PEC and one of SS).