Cell physiology and antibiotic production of Streptomyces coelicolor grown on solid medium

Summary In order to examine the physiology ofStreptomyces coelicolor when growing on solid media, we have employed a membrane overlay technique and used a new approach to extract substrate and product compounds from the agar. Comparisons made with liquid grown cultures indicate a change from non-growth associated productivity of actinorhodin in liquid culture, to growth associated production on agar plates. In contrast, the temporal control of methylenomycin production was virtually identical under both culture conditions. Considerable extracellular protein production was observed during growth on agar.     

Development and maturation of surghum seeds on detached panicles grown in vitro

Summary A procedure for culturing detached panicles of sorghum, Sorghum bicolor (L.) Moench, was developed to achieve flowering, fertilization, and subsequent seed development and maturation in vitro. Sixteen sorghum genotypes (five high and eleven low in tannin) were tested for their ability to develop normally in culture. Panicles collected one to two days before the initiation of anthesis were cultured in flasks containing liquid medium. Contamination and medium darkening were the major obstacles encountered. Up to 55% of the panicles cultured reached physiological maturity in vitro. The frequency of seed set ranged from 30 to 97% depending upon genotype and medium. Seed and glume color were normal. Seed produced in vitro resembled those grown in vivo and germinated well, but were smaller than normal (100 kernel weight reached 50 to 70% of the control). Grain polyphenols were synthesized in the cultured panicles. Seed of high tannin genotypes produced in vitro were lower in total phenols and tannins and higher in flavan-4-ols and the 3-deoxyanthocyanidin pigments than control seed. This technique can be used for harvesting late-maturing stocks and for various sorghum studies.     

Intracellular aminopeptidases inStreptomyces lividans 66

Summary We have investigated the aminopeptidase activities present inStreptomyces lividans strains. The majority of these activities proved to be intracellular with multiple active species. Two aminopeptidase P genes were identified to be responsible for the ability to hydrolyze amino terminal peptide bonds adjacent to proline residues. Two other broad spectrum aminopeptidases were found to display homology at both the DNA and protein levels. One showed significant homology to PepN proteins, particularly around the putative zinc-binding residues which are important for catalysis. The second broad spectrum activity was not analyzed in detail but showed a different spectrum of substrate specificity to that of PepN.     

Differential molecular responses to abscisic acid and osmotic stress in viviparous maize embryos

Substantial quantities of mRNA encoding the abundant Em polypeptide accumulate, in planta, in developing embryos of maize (Zea mays L.). By contrast, accumulation of Em mRNA is only barely detectable in embryos with the vp-5/vp-5 genotype [an abscisic acid (ABA)-deficient viviparous phenotype]. Em mRNA is not detectable within viviparous embryos of the vp-1/vp-1 genotype that are non-responsive to ABA. Culture of immature wild-type and vp-5/vp-5 embryos in the presence of exogenous ABA or of an osmotically active agent prevents precocious germination and results in expression of the Em genes. When vp-1/vp-1 embryos are cultured under similar conditions, only the application of osmotic stress prevents precocious germination. However, Em mRNA does not accumulate either in ABA-treated or stressed, arrested embryos, indicating a requirement for ABA perception through a VP-1-mediated mechanism for Em gene expression. Nevertheless, vp-1/vp-1 embryos do show both ABA and stress responses at the molecular level. Treatment with ABA causes the accumulation of mRNA encoding a polypeptide of approx. 30 kDa, whilst osmotic stress induces the accumulation both of a 30-kDa polypeptide and a set of approx. 20-kDa polypeptides. This indicates the existence of discrete, parallel ABA and stress response pathways in developing maize embryos.Abbreviations ABA abscisic acid - cDNA copy-DNA - DAP days after pollination - kDa kilodaltons - MS Murashige and Skoog medium - LEA late embryogenesis abundant - NEpHGE non-equilibrium pH gradient gel electrophoresis - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

The synthesis of oligoribonucleotides containing O6-methylguanosine: the role of conserved guanosine residues in hammerhead ribozyme cleavage.

The synthesis is described of oligoribonucleotides containing the modified nucleoside O6-methylguanosine. Solid-phase oligoribonucleotide assembly was carried out by use of 2'-silyl-protected nucleoside phosphoramidites, a new O6-methylguanosine-containing synthon and a mild deprotection method. The O6-methylguanosine-modified oligonucleotides were used in the study of the role of conserved residues G5, G8 and G12 in hammerhead ribozyme cleavage. Hammerheads thus substituted at any of these positions showed an approximately 75-fold reduction in kcat whereas Km was unaffected. Hammerheads with modifications at G5 or G8 showed a significant reduction in magnesium binding affinity whereas modification at G12 had no effect. The results show that the three conserved G residues play crucial but different role sin hammerhead cleavage.     

A histidine binding protein ofEscherichia coli: a component of cystine binding protein ofEscherichia coli

Summary Commercially obtained cystine binding protein (CBP), an osmotic shock protein ofEscherichia coli, was studied in an effort to determine its binding characteristics. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis of commercially obtained CBP showed three protein bands. N-terminal amino acid microsequencing and subsequent computer search revealed that the sequence of one of these proteins (25-kDa) was nearly identical to histidine binding protein (HisJ) ofSalmonella typhimurium. Purification of CBP by HPLC yielded four protein peaks, of which one bound histidine exclusively. Binding was maximal at pH 5.0 to 6.0, at 4°C, did not require calcium or magnesium ions and was not inhibited by reduction of CBP disulfide bonds. Amino acids other than histidine or cystine did not bind to CBP. These data show that commercially available CBP is not a homogenous protein; it contains a histidine as well as a cystine binding component.     

Costs of minimally invasive laser surgery compared with transurethral electrocautery resection of the prostate.

We reviewed hospital charges for patients undergoing uncomplicated endoscopic surgical resection for symptomatic bladder outlet obstruction due to benign prostatic hyperplasia over a 1-year period at a single institution. Of 115 patients, 67 underwent transurethral electrocautery resection of the prostate, and 48 underwent endoscopic neodymium:yttrium-aluminum-garnet laser ablation of the prostate under direct vision. Analysis showed a cost differential between these 2 surgical treatments in excess of $2,000, favoring laser prostatectomy (P < .0001) over transurethral electrocautery resection. The single greatest difference between the treatments was the ability to manage all patients receiving laser treatment as outpatients, whereas the mean and median hospital stay after transurethral electrocautery resection was 3.0 days. Taking additional cost variables into account and decreasing the cost of laser delivery systems would further increase this cost differential in favor of laser therapy. The diminished postoperative morbidity associated with laser treatment also promises lower total costs over the long term.     

The effect of elevated levels of thyroxine on the aerobic capacity of locomotor muscles of the tufted duck,Aythya fuligula

Following extended periods of relative inactivity, or prior to migration, birds are able to increase the aerobic capacity of their locomotory muscles. Thyroid hormones may influence this process. A preliminary study was undertaken to assess the ability of elevated levels of thyroxine to increase the aerobic capacity of the locomotory and cardiac muscles of adult tufted ducks. Administration of thyroxine in the food for 8 weeks had little effect on body mass or on the masses of the pectoralis, semitendinosus and iliofibularis muscles, although there were increases in resting oxygen consumption and in the mass of the cardiac ventricles. The maximum activity of the aerobic enzyme, citrate synthase, was significantly greater in the left ventricle, liver, and iliofibularis muscles (P<0.005) of treated birds. However, while there was clearly no difference in activity in the semimembranosus leg muscle, that of the pectoralis was not quite significant (P=0.078). It is concluded that addition of supra-physiological levels of exogenous thyroxine may induce a differential increase in the maximum activity of citrate synthase in the locomotor muscles of the tufted duck, which is correlated with the fibre type composition of these muscles. These results are consistent with those found in studies on rats, with slow oxidative fibres being the most sensitive, and fast glycolytic fibres the least sensitive, to thyroxine treatment.Abbreviations BM body mass - CS citrate synthase - CYTOX cytochrome c oxidase - FG last glycolytic - FOG fast oxydative glycolytic - VO₂ oxygen consumption - SO slow oxidative - T₄ thyroxine - T₃ triiodothyronine