Actinorhodin and undecylprodigiosin production in wild-type and relA mutant strains of Streptomyces coelicolor A3(2) grown in continuous culture

In vitro fertilization and embryo transfer, embryo cryo-preservation, multiple pregnancies, embryo reduction, embryo and gamete donation: each of these questions deserves an articulate ethical and medical discussion. In Italy until this moment we do not have a legislation for the regulation of medically assisted reproduction. One of the most suitable means to monitoring large scale initiatives for medically assisted procreation is through registries that collect standardized information on all treatment cycles performed. In 1992, the Italian national registry on medically assisted procreation was established at the Istituto Superiore di Sanità (ISS, the Italian National Health Institute). The Registry collects data sent voluntarily by centers to the ISS for each cycle of treatment started and for each outcome. The data collected can be used to conduct an adequate statistical-epidemiological analysis and to better evaluate results of specific techniques.     

Localized mutagenesis in Streptomyces coelicolor A3 (2)

The study was conducted to observe in rats the possible modification of ectopic calcification by magnesium-orthophosphate-fluoride combinations, used as additives of diet for reduction of the cariogenicity of the sucrose. In rats, fed low magnesium diets, extra dietary orthophosphate (2%) considerably elevated the calcification of kidneys. Further additions of magnesium and fluoride partially reduced this adverse effect of phosphate. While the calcium content of the aorta in rats, fed low magnesium-high phosphate diet, was considerably elevated, the further addition of magnesium (40 ppm) partially reduced the calcifying effect of phosphate in aorta. Fluoride (15 ppm) together with magnesium (40 ppm) completely reduced it. The appearance of renal calculi caused by a low magnesium diet or by extra phosphate were similar according to light and electron microscopy except for the larger size in the latter case and occasional extratubular calculi found in groups with high phosphate-low magnesium and high phosphate with added magnesium diets.     

The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production

The drrC gene, cloned from the daunorubicin (DNR)- and doxorubicin-producing strain of Streptomyces peucetius ATCC 29050, encodes a 764-amino-acid protein with a strong sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in excision repair of DNA. Expression of drrC was correlated with the timing of DNR production in the growth medium tested and was not dependent on the presence of DNR. Since introduction of drrC into Streptomyces lividans imparted a DNR resistance phenotype, this gene is believed to be a DNR resistance gene. The drrC gene could be disrupted in the non-DNR-producing S. peucetius dnrJ mutant but not in the wild-type strain, and the resulting dnrJ drrC double mutant was significantly more sensitive to DNR in efficiency-of-plating experiments. Expression of drrC in an E. coli uvrA strain conferred significant DNR resistance to this highly DNR-sensitive mutant. However, the DrrC protein did not complement the uvrA mutation to protect the mutant from the lethal effects of UV or mitomycin even though it enhanced the UV resistance of a uvrA+ strain. We speculate that the DrrC protein mediates a novel type of DNR resistance, possibly different from the mechanism of DNR resistance governed by the S. peucetius drrAB genes, which are believed to encode a DNR antiporter.     

Cell division gene ftsQ is required for efficient sporulation but not growth and viability in Streptomyces coelicolor A3(2)

We show that the cell division gene ftsQ of Streptomyces coelicolor A3(2) is dispensable for growth and viability but is needed during development for the efficient conversion of aerial filaments into spores. Combined with our previous demonstration that ftsZ of S. coelicolor is not needed for viability, these findings suggest that cell division has been largely co-opted for development in this filamentous bacterium. This makes S. coelicolor an advantageous system for the study of cell division genes.     

Differential expression of superoxide dismutases containing Ni and Fe/Zn in Streptomyces coelicolor

Streptomyces coelicolor contains two distinct superoxide dismutase (SOD) activities detected on native PAGE. The level of each changed differently depending on growth media and scarcely responded to paraquat, a superoxide-generating agent. The total SOD activity doubled in late exponential phase compared with that in mid-exponential phase and less than double upon treatment with plumbagin, another superoxide-generating agent. The two SODs from S. coelicolor ATCC 10147 (Müller) strain were purified to near homogeneity. SOD1, a tetramer of 13.4-kDa subunits, was found to be a novel type of SOD containing 0.74 mol nickel/mol subunit as determined by atomic absorption spectroscopy. SOD2, a tetramer of 22.2-kDa subunits, was found to contain 0.36 mol iron and 0.26 mol zinc/mol subunit. The N-terminal amino acid sequences of both SODs were determined. SOD2 is similar to manganese-containing superoxide dismutases (MnSODs) and iron-containing superoxide dismutases (FeSODs) from other organisms, whereas SOD1 is less similar to known SODs but still contains a few conserved amino acids. The effects of metals and chelating agents on the expression of these two SODs were examined. The presence of nickel at micromolar concentrations in growth media induced the expression of SOD1 (nickel-containing superoxide dismutase; NiSOD), whereas the expression of SOD2 (iron/zinc-containing superoxide dismutase; FeZnSOD) was repressed. The changes in SOD activities were positively correlated with the amount of each enzyme as determined by immunoblotting, suggesting that metals do not modulate the activity per se but the amount of each protein.     

The cell wall-anchored Streptomyces reticuli avicel-binding protein (AbpS) and its gene

Streptomyces reticuli produces a 35-kDa cellulose-binding protein (AbpS) which interacts strongly with crystalline forms of cellulose (Avicel, bacterial microcrystalline cellulose, and tunicin cellulose); other polysaccharides are recognized on weakly (chitin and Valonia cellulose) or not at all (xylan, starch, and agar). The protein could be purified to homogeneity due to its affinity to Avicel. After we sequenced internal peptides, the corresponding gene was identified by reverse genetics. In vivo labelling experiments with fluorescein isothiocyanate (FITC), FITC-labelled secondary antibodies, or proteinase K treatment revealed that the anchored AbpS protrudes from the surfaces of the hyphae. When we investigated the hydrophobicity of the deduced AbpS, one putative transmembrane segment was predicted at the C terminus. By analysis of the secondary structure, a large centrally located alpha-helix which has weak homology to the tropomyosin protein family was found. Physiological studies showed that AbpS is synthesized during the late logarithmic phase, independently of the carbon source.     

Analysis of a Streptomyces coelicolor A3(2) locus containing the nucleoside diphosphate kinase (ndk) and folylpolyglutamate synthetase (folC) genes

Conjugates of oligodeoxyribonucleotide phosphorothioate (ODN-PS) with folic acid, retinoic acid, arachidonic acid, and methoxypoly(ethylene glycol)propionic acid have been synthesized. The procedure involved the initial solid-phase preparation of 5'-amino-functionalized ODN-PS using N-pent-4-enoyl-derived (PNT) nucleoside phosphoramidites followed by conjugation of the oligonucleotide either to the ligand acids, using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide as a coupling reagent, or to their corresponding succinimidyl derivatives. Subsequent exposure of the support to aqueous ammonium hydroxide (28%, 2 h, 55 degrees C) resulted in the release of the fully deprotected ODN conjugates, which were purified by reversed-phase HPLC or by preparative polyacrylamide gel electrophoresis. The identity of the oligonucleotide conjugates was confirmed by MALDI-TOF mass spectral analysis.     

Cloning, purification, and properties of a phosphotyrosine protein phosphatase from Streptomyces coelicolor A3(2)

We have investigated by restriction fragment analysis genomic abnormalities involving the c-myc gene in DNA isolated from adenomas and hepatocellular carcinomas (HCCs). Adenomas and HCCs were induced by the "resistant hepatocyte" protocol in diethylnitrosamine-initiated male F344 rats. Southern-blot analysis of EcoRI-restricted DNA from normal liver, early and late adenomas, 12 weeks (EAs) and 30 weeks (LAs) after initiation, and HCCs, showed 2 bands of 18 and 3.2 kb hybridizing with c-myc, in all tissues. c-myc amplification occurred in almost all HCCs, and in the majority of EAs and LAs. These results were confirmed by dilution analysis. c-myc amplification was also seen in adenomas and HCCs by Southern analysis with HindIII-restricted DNA, and in HCCs by differential PCR. c-myc mRNA increase occurred in all adenomas and HCCs, but it was higher in the lesions showing gene amplification. Moreover, a 13-kb DNA extraband, hybridizing with c-myc, was found in the HindIII-restricted DNA from HCCs, but not in normal liver and adenomas, and a 7.1-kb extra band was present in EcoRI-digested DNA from one LA. EcoRI-restricted DNA from some adenomas exhibited a decrease in intensity of the 18-kb fragment, and an increase in intensity of the 3.2-kb fragment. No alteration in banding pattern occurred in the beta-actin gene in adenomas. These results provide evidence of amplification and some other rearrangements involving the c-myc gene, in pre-malignant and malignant liver lesions, induced by the RH protocol, and suggest a role of c-myc rearrangement in the progression of adenomas to malignancy.     

The zntA gene of Escherichia coli encodes a Zn(II)-translocating P-type ATPase

The first Zn(II)-translocating P-type ATPase has been identified as the product of o732, a potential gene identified in the sequencing of the Escherichia coli genome. This gene, termed zntA, was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain exhibited hypersensitivity to zinc and cadmium salts but not salts of other metals, suggesting a role in zinc homeostasis in E. coli. Everted membrane vesicles from a wild-type strain accumulated 65Zn(II) and 109Cd(II) by using ATP as an energy source. Transport was sensitive to vanadate, an inhibitor of P-type ATPases. Membrane vesicles from the zntA::kan strain did not accumulate those metal ions. Both the sensitive phenotype and transport defect of the mutant were complemented by expression of zntA on a plasmid.     

Saccharomyces cerevisiae Msh2p and Msh6p ATPase activities are both required during mismatch repair

In the Saccharomyces cerevisiae Msh2p-Msh6p complex, mutations that were predicted to disrupt ATP binding, ATP hydrolysis, or both activities in each subunit were created. Mutations in either subunit resulted in a mismatch repair defect, and overexpression of either mutant subunit in a wild-type strain resulted in a dominant negative phenotype. Msh2p-Msh6p complexes bearing one or both mutant subunits were analyzed for binding to DNA containing base pair mismatches. None of the mutant complexes displayed a significant defect in mismatch binding; however, unlike wild-type protein, all mutant combinations continued to display mismatch binding specificity in the presence of ATP and did not display ATP-dependent conformational changes as measured by limited trypsin protease digestion. Both wild-type complex and complexes defective in the Msh2p ATPase displayed ATPase activities that were modulated by mismatch and homoduplex DNA substrates. Complexes defective in the Msh6p ATPase, however, displayed weak ATPase activities that were unaffected by the presence of DNA substrate. The results from these studies suggest that the Msh2p and Msh6p subunits of the Msh2p-Msh6p complex play important and coordinated roles in postmismatch recognition steps that involve ATP hydrolysis. Furthermore, our data support a model whereby Msh6p uses its ATP binding or hydrolysis activity to coordinate mismatch binding with additional mismatch repair components.     

The PEL1 gene (renamed PGS1) encodes the phosphatidylglycero-phosphate synthase of Saccharomyces cerevisiae

Phosphatidylglycerophosphate (PG-P) synthase catalyzes the synthesis of PG-P from CDP-diacylglycerol and sn-glycerol 3-phosphate and functions as the committed and rate-limiting step in the biosynthesis of cardiolipin (CL). In eukaryotic cells, CL is found predominantly in the inner mitochondrial membrane and is generally thought to be an essential component of many mitochondrial functions. We have determined that the PEL1 gene (now renamed PGS1), previously proposed to encode a second phosphatidylserine synthase of yeast (Janitor, M., Jarosch, E., Schweyen, R. J., and Subik, J. (1995) Yeast 13, 1223-1231), in fact encodes a PG-P synthase of Saccharomyces cerevisiae. Overexpression of the PGS1 gene product under the inducible GAL1 promoter resulted in a 14-fold increase in in vitro PG-P synthase activity. Disruption of the PGS1 gene in a haploid strain of yeast did not lead to a loss of viability but did result in a dependence on a fermentable carbon source for growth, a temperature sensitivity for growth, and a petite lethal phenotype. The pgs1 null mutant exhibited no detectable in vitro PG-P synthase activity and no detectable CL or phosphatidylglycerol (PG); significant CL synthase activity was still present. The growth arrest phenotype and lack of PG-P synthase activity of a pgsA null allele of Escherichia coli was corrected by an N-terminal truncated derivative of the yeast PG-P synthase. These results unequivocally demonstrate that the PGS1 gene encodes the major PG-P synthase of yeast and that neither PG nor CL are absolutely essential for cell viability but may be important for normal mitochondrial function.     

Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor

This report reviews published information on the clinical pharmacokinetics of antitumour agents in patients with liver dysfunction, associated with primary liver disease or liver metastases. Information was available for anthracyclines and their related compounds, antimetabolites, cyclophosphamide, vinca alkaloids, taxanes and epipodophyllotoxins. Changes in the pharmacokinetic profile or metabolism in patients with mild or severe hepatobiliary dysfunction are described and the relationships between serum levels, parameters employed for measuring hepatic function and toxic or therapeutic effects are examined. Current knowledge of the pharmacokinetics of antineoplastic agents in liver disease is far from complete, mostly obtained in small numbers of non-homogeneous patients often presenting only moderate liver dysfunction, and empirical guidelines for dose assessment are still largely applied in clinical practice. Because of the complex pathophysiological mechanisms of liver insufficiency in cancer patients, there is still doubt whether endogenous markers are useful. Although caution in treating cancer patients with liver insufficiency is compulsory, for most compounds there seems no need to recommend dose reductions for moderate impairment. However, for the tubulin acting agents, vincristine, vinblastine and possibly for paclitaxel and docetaxel, there is strong evidence that dose adjustment is mandatory in order to avoid excessive neutropenia and neurotoxicity.     

Evidence that the extracytoplasmic function sigma factor sigmaE is required for normal cell wall structure in Streptomyces coelicolor A3(2)

The sigE gene of Streptomyces coelicolor A3(2) encodes an RNA polymerase sigma factor belonging to the extracytoplasmic function (ECF) subfamily. Constructed sigE deletion and disruption mutants were more sensitive than the parent to muramidases such as hen egg white lysozyme and to the CwlA amidase from Bacillus subtilis. This correlated with an altered muropeptide profile, as determined by reverse-phase high-performance liquid chromatography analysis of lytic digests of purified peptidoglycan. The sigE mutants required high levels of magnesium for normal growth and sporulation, overproducing the antibiotic actinorhodin and forming crenellated colonies in its absence. Together, these data suggest that sigE is required for normal cell wall structure. The role of sigmaE was further investigated by analyzing the expression of hrdD, which is partially sigE dependent. The hrdD gene, which encodes the sigmaHrdD subunit of RNA polymerase, is transcribed from two promoters, hrdDp1 and hrdDp2, both similar to promoters recognized by other ECF sigma factors. The activities of hrdDp1 and hrdDp2 were reduced 20- and 3-fold, respectively, in sigE mutants, although only hrdDp1 was recognized by EsigmaE in vitro. Growth on media deficient in magnesium caused the induction of both hrdD promoters in a sigE-dependent manner.     

The YGR194c (XKS1) gene encodes the xylulokinase from the budding yeast Saccharomyces cerevisiae

To test the hypothesis that the major irrational evaluative beliefs postulated by Rational Emotive Behavior Therapy are related to marital conflict, 15 married couples participated in a thought-listing procedure. During this procedure, three idiosyncratic scenes portraying marital conflict and three control scenes free of conflict were identified for and presented to each member of the dyad. Analysis indicated that the conflict-portraying scenes were associated with significantly more irrational evaluative beliefs and significantly fewer rational cognitions than the control scenes.