Implication of a repression system, homologous to those of other bacteria, in the control of arginine biosynthesis genes inStreptomyces coelicolor

As with most amino acid biosynthetic pathways in streptomycetes, enzymes of arginine biosynthesis inStreptomyces coelicolor show only slight derepression in minimal medium without, as opposed to with, exogenous arginine. However, when an arginine auxotroph was cultured in limiting arginine, ornithine carbamoyltransferase (OCT) activities rose by as much as 100-fold. The response was not due to a general starvation effect. To elucidate the repression-derepression mechanism, a DNA fragment containing the upstream region of the previously isolatedS. coelicolor argCJB cluster was cloned into a multicopy vector and transformed into wild-typeS. coelicolor; a slight transient derepression of OCT was observed in minimal medium without, though not with, added arginine, consistent with titration by the insert of a negatively acting macromolecule such as a repressor. A sub-fragment carrying the 5 end ofargC and the region immediately upstream showed specific binding, in mobility shift assays, to purified AhrC, the repressor/activator of genes of arginine metabolism inBacillus subtilis. It is therefore likely that inS. coelicolor, expression of arginine biosynthesis genes is controlled by a protein homologous to the well-characterisedB. subtilis andEscherichia coli repressors.     

Temporal and ontogenetic aspects of protein induction in foliage of the tomato, Lycopersicon esculentum

Damage to foliage of the tomato, Lycopersicon esculentum, causes the induction of proteinase inhibitors and of the oxidative enzymes polyphenol oxidase, peroxidase, and lipoxygenase. The time courses of induction of these proteins by feeding of two caterpillar species (Manduca sexta and Helicoverpa zea) were studied in a series of experiments. In another series of experiments, the effects of plant age on the inducibility of these proteins were studied. In the time course experiments, induction of proteinase inhibitors and oxidative enzymes in the damaged leaflet was rapid, with higher protein activities evident in damaged leaflets within 12–24 h of damage, depending on the enzyme and the species of insect used to damage the plant. Systemic induction of proteinase inhibitors was also rapid, but systemic induction of polyphenol oxidase was delayed relative to systemic induction of proteinase inhibitors, possibly because high constitutive polyphenol oxidase activities obscured expression of systemic induction at earlier time points. Lipoxygenase and peroxidase were not induced systemically. Induction of all proteins persisted for at least 21 days. In the phenology experiments, inducibility of all proteins decreased in magnitude and was less consistent as plants aged. The results of these experiments exemplify the numerous constraints on induction in tomato plants. Knowledge of these physiological constraints is important to an understanding of the ecological role and causal basis of induced resistance.     

Biosynthesisin vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core

The galactosyltransferase, GalT-4, which catalyses the biosynthesisin vitro of neolactotetraosylceramide, nLcOse4Cer (Gal1-4GleNAc1-3Gal1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcNAc1-3Gal1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn²⁺). TheK _m values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 µm, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-1-acid glycoprotein orN-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc1-1-3Gal1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for anN-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH₂1-3Gal1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while theK _m andV _max of the reacetylated substrate (GlcNac1-3Gal1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.Abbreviations EDTA ethylenediamine tetraacetate - ME -mercaptoethanol - PEG polyethylene glycol - PBS phosphate buffered saline - Suc sucrose - Mn²⁺ manganese - Gal galactose - GlcNAc N-acetylglucosamine - UDP-Gal Uridine diphosphate galactose - Ab antibody - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ECB embryonic chicken brain - Cer ceramide - nLc4 or NlcOse4Cer Gal1-4GleNac1-3Gal1-4Glc-Cer, neoLactotetraosylceramide - Lc3 or LcOse3Cer GlcNac1-3Gal1-4Glc-Cer, lactotriaosylceramide - iLc5 iLcOse5Cer, GlcNAc1-3nLcOse4Cer - nLc6 nLcOse6Cer, Gal1-4iLcOse5Cer - SA^–Gal^–₁AGP asialo-agalacto1-acid glycoprotein - TLC thin layer chromatography     

Selenium,zinc, and thyroid hormones in healthy subjects

Iodothyronine 5′ deiodinase, which is mainly responsible for peripheral T₃ production, has recently been demonstrated to be a selenium (Se)-containing enzyme. The structure of nuclear thyroid hormone receptors contains Zinc (Zn) ions, crucial for the functional properties of the protein. In the elderly, reduced peripheral conversion of T₄ to T₃ with a lower T₃/T₄ ratio and overt hypothyroidism are frequently observed. We measured serum Se and RBC GSH-Px (as indices of Se status), circulating and RBC Zinc (as indices of Zn status), thyroid hormones and TSH in 109 healthy euthyroid subjects (52 women, 57 men), carefully selected to avoid abnormally low thyroid hormone levels induced by acute or chronic diseases or calorie restriction. The subjects were subdivided into three age groups. To avoid under- or malnutrition conditions, dietary records were obtained for a sample of 24 subjects, randomly selected and representative of the whole population for age and sex. Low T₃/T₄ ratios and reduced Se and RBC GSH-Px activity were observed only in the older group. A highly significant linear correlation between the T₃/T₄ ratio and indices of Se status was observed in the older group of subjects (r=0.54;p<0.002, for Se;r=0.50;p<0.002, for RBC GSH-Px). Indices of Zn status did not correlate with thyroid hormones, but RBC Zn was decreased in older as compared with younger subjects. We concluded that reduced peripheral T₄ conversion is related to impaired Se status in the elderly.     

Bioavailability of selenium accumulated by selenite-reducing bacteria

The bioavailability of selenium (Se) was determined in bacterial strains that reduce selenite to red elemental Se (Se^o). A laboratory strain ofBacillus subtilis and a bacterial rod isolated from soil in the vicinity of the Kesterson Reservoir, San Joaquin Valley, CA, (Microbacterium arborescens) were cultured in the presence of 1 mM sodium selenite (Na₂SeO₃). After harvest, the washed, lyophilizedB. subtilis andM. arborescens samples contained 2.62 and 4.23% total Se, respectively, which was shown to consist, within error, entirely of Se^o. These preparations were fed to chicks as supplements to a low-Se, vitamin E-free diet. Three experiments showed that the Se in both bacteria had bioavailabilities of approx 2% that of selenite. A fourth experiment revealed that gray Se^o had a bioavailability of 2% of selenite, but that the bioavailability of red Se^o depended on the way it was prepared (by reduction of selenite). When glutathione was the reductant, bioavailability resembled that of gray Se^o and bacterial Se; when ascorbate was the reductant, bioavailability was twice that level (3–4%). These findings suggest that aerobic bacteria such asB. subtilis andM. arborescens may be useful for the bioremediation of Se-contaminated sites, i.e., by converting selenite to a form of Se with very low bioavailability.     

Expression of a moderately anionic peroxidase is induced by aluminum treatment in tobacco cells: Possible involvement of peroxidase isozymes in aluminum ion stress

To clarify the mechanism of aluminum (Al) toxicity and Al tolerance, we isolated a new clone (pAL201) from a tobacco cDNA library. Northern blot hybridization analysis indicated that the expression of pAL201 is induced by Al treatment and phosphate (P₁) starvation. The complete cDNA sequence suggested that this clone encodes a moderately anionic peroxidase (EC 1.11.1.7). Analysis by isoelectric focussing indicated that a moderately anionic peroxidase (approximately pI 6.7) and two cationic peroxidases (pI 9.2 and 9.7) in the soluble fraction are activated by Al treatment and P₁ starvation, while two moderately anionic isozymes are repressed by these stresses. We suppose that Al ion stress can control the activity of some peroxidase isozymes, one of which is probably induced by enhanced gene expression of pAL201. There is a possibility that some of these isozymes have some functions in Al ion stress.     

Fibre length and gibberellins A1 and A20 are decreased in Eucalyptus globules by acylcyclohexanedione injected into the stem

Trinexapacethyl (TriEt), an acylcyclohexanedionetype inhibitor of gibberellin (GA) biosynthesis, was applied to 3-year-old Eucalyptus globules saplings by localised injection near the base of each stem. The objective was to alter cambial region GA levels and to study the effects on secondary xylem fibre development. Seven weeks later wood samples, with bark and cambial region intact, were removed 10 and 30 cm above the point of injection. Fusiform cambial cell dimensions were compared with those of fibre-tracheids in the most recently formed 100 um of secondary xylem. Increasing TriEt applications from 5 to 5 000 mg active ingredient significantly reduced average fibre length, and to a lesser extent average fusiform cambial cell length. Also reduced was the number of cells in the cambial zone and the number of differentiating fibres with primary walls. However, no trends were evident for changes in fibre diameter, the proportion of vessel elements or the ratio of cambial ray cells to fusiform cambial cells. Two gibberellins (GA₁ and GA₂₀), indole-3-acetic acid (IAA) and abscisic acid (ABA) were quantified in cambial region tissues by gas chromatographymass spectrometry using stable isotope labelled internal standards. Increasing TriEt application reduced both GA₁ and GA₂₀ levels. Effects on IAA and ABA were not significant, although their levels tended to be lower at the highest TriEt application rate. The elongation of secondary xylem fibres was positively correlated with higher levels of endogenous GA₁ (r_s= 0.74, P < 0.01) and GA₂₀ (r_s= 0.72, P < 0.01). These results support a causal role for GA₁ in cambial cell division. They are also consistent with the hypothesis that the elongation of differentiating secondary xylem fibres in woody an–giosperms is dependent on GA₁ levels in the cambial region.     

Molecular genetics of sulfate assimilation in plants

The sulfate assimilation pathway is the primary route by which higher plants obtain the sulfur necessary for growth. Sulfur is involved in a myriad of processes of central importance in metabolism. In the past few years much has been learned about this pathway and its regulation through analysis'of the genes encoding the enzymes and proteins that make up the sulfate assimilation pathway. The recent molecular genetic analysis builds on the biochemical and physiological groundwork of past studies. Further, gene analysis has provided the opportunity to compare directly the evolution of sulfate assimilation in plants and other organisms.,