The tcmVI region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens encodes the tetracenomycin F1 monooxygenase, tetracenomycin F2 cyclase, and, most likely, a second cyclase.

Certain mutations in the tcmVI region of the Streptomyces glaucescens chromosome affect formation of the D ring of the polyketide antibiotic tetracenomycin C (TCM C). This region lies immediately upstream from the TCM C polyketide synthase genes (tcmKLM), and the nucleotide sequence reveals the presence of three small genes, tcmH, tcmI, and tcmJ. On the basis of the phenotypes of mutants and the effects of these genes, when coupled on a plasmid with the tcmKLMN177 genes (tcmN177 is a 3'-truncated version of tcmN), on the production of TCM intermediates in a TCM- mutant, the tcmH gene encodes the C-5 monooxygenase that converts TCM F1 to TCM D3, the tcmI gene encodes the D-ring cyclase that converts TCM F2 to TCM F1 (mutations in this gene are responsible for the type VI phenotype), and the tcmJ gene most likely encodes the B-ring cyclase that acts in the biosynthesis of TCM F2. Furthermore, it appears that the N-terminal domain of the tcmN gene product (encoded by the tcmN177 gene) acts later in the biosynthesis of TCM F2 than the product of tcmJ, suggesting that the N-terminal domain of the TcmN protein is the C-ring cyclase.     

Sir2 regulates histone H3 lysine 9 methylation and heterochromatin assembly in fission yeast

Hypoacetylated histones are a hallmark of heterochromatin in organisms ranging from yeast to humans. Histone deacetylation is carried out by both NAD(+)-dependent and NAD(+)-independent enzymes. In the budding yeast Saccharomyces cerevisiae, deacetylation of histones in heterochromatic chromosomal domains requires Sir2, a phylogenetically conserved NAD(+)-dependent deacetylase. In the fission yeast Schizosaccharomyces pombe, NAD(+)-independent histone deacetylases are required for the formation of heterochromatin, but the role of Sir2-like deacetylases in this process has not been evaluated. Here, we show that spSir2, the S. pombe Sir2-like protein that is the most closely related to the S. cerevisiae Sir2, is an NAD(+)-dependent deacetylase that efficiently deacetylates histone H3 lysine 9 (K9) and histone H4 lysine 16 (K16) in vitro. In sir2 Delta cells, silencing at the donor mating-type loci, telomeres, and the inner centromeric repeats (imr) is abolished, while silencing at the outer centromeric repeats (otr) and rDNA is weakly reduced. Furthermore, Sir2 is required for hypoacetylation and methylation of H3-K9 and for the association of Swi6 with the above loci in vivo. Our findings suggest that the NAD(+)-dependent deacetylase Sir2 plays an important and conserved role in heterochromatin assembly in eukaryotes.     

Wide-band spectral tuning of heat receptors in the pit organ of the copperhead snake (Crotalinae)

Receptors located in the facial pit organ of certain species of snake signal the presence of prey. Infrared radiation is an effective stimulus suggesting that these receptors may be low-threshold temperature receptors. We recorded from the nerve innervating the pit organ of snakes belonging to the family of Crotalinae while stimulating the receptive area with well-defined optical stimuli. The objective was to determine the sensitivity of these receptors to a wide range (0.400-10.6 micro m) of optical stimuli to determine if a temperature-sensitive or photosensitive protein initiated signal transduction. We found that receptors in the pit organ exhibited a unique broad response to a wide range of electromagnetic radiation ranging from the near UV to the infrared. The spectral tuning of these receptors parallels closely the absorption spectra of water and oxyhemoglobin, the predominant chromophore in tissue. Our results support the hypothesis that these are receptors activated by minute temperature changes induced by direct absorption of optical radiation in the thin pit organ membrane.     

Crystal structure of inositol 1-phosphate synthase from Mycobacterium tuberculosis,a key enzyme in phosphatidylinositol synthesis

Phosphatidylinositol (PI) is essential for Mycobacterium tuberculosis viability and the enzymes involved in the PI biosynthetic pathway are potential antimycobacterial agents for which little structural information is available. The rate-limiting step in the pathway is the production of (L)-myo-inositol 1-phosphate from (D)-glucose 6-phosphate, a complex reaction catalyzed by the enzyme inositol 1-phosphate synthase. We have determined the crystal structure of this enzyme from Mycobacterium tuberculosis (tbINO) at 1.95 A resolution, bound to the cofactor NAD+. The active site is located within a deep cleft at the junction between two domains. The unexpected presence of a zinc ion here suggests a mechanistic difference from the eukaryotic inositol synthases, which are stimulated by monovalent cations, that may be exploitable in developing selective inhibitors of tbINO.     

Anti-Helicobacter pylori activities of six Iranian plants

BACKGROUND: Helicobacter pylori is the major worldwide cause of bacterial gastrointestinal infections in adults and children. Antibiotic therapy and a combination of two or three drugs have been widely used to eradicate these infections. However, development of drug resistance in bacteria calls for new sources of drugs, and plants seem to be a logical source of new antibacterial compounds. METHODS: The anti-H. pylori activities of six native Iranian plants (Glycyrrhiza aspera, Juglans regia, Ligustrum vulgare, Thymus kotschyanus, Trachyspermum copticum and Xanthium brasilicum) and seven antibiotics were determined against 70 clinical isolates from children using the disk susceptibility assay. Minimum inhibitory concentrations were also measured for the biologically active extracts. One extract with the best anti-H. pylori activity was fractionated by silica gel and thin layer chromatography and the active compounds were identified by hydrogen nuclear magnetic resonance ((1)HNMR) spectroscopy. RESULTS: All plant extracts showed anti-H. pylori activity by the disk sensitivity method, but the most active extracts were those from X. brasilicum and T. copticum. In fact, the anti-H. pylori activities of the two extracts were superior to the disk antibiotic susceptibility profile. Minimum inhibitory concentrations were within the range of 31.25-250 micro g/ml. Fractionation and chemical identification of the extract from X. brasilicum showed the presence of two substances, a flavonoid and a xanthanolide. CONCLUSIONS: Due to the rise in antibiotic resistance, new sources of anti-H. pylori drugs are needed. The use of medicinal plants and/or their chemical components may have potential benefit in eradicating such problems.     

A high-throughput assay to identify compounds that can induce dimerization of the erythropoietin receptor

Catalytic chromatography exploits both specific biological affinity and catalytic specificity to selectively purify enzymes. Two different applications are presented. Purification of EcoRI restriction endonuclease to apparent homogeneity was accomplished in a single step with significantly greater yield and purification than was obtained with affinity chromatography. An attempt to purify the multiple DNA polymerase activities of Escherichia coli was also developed. Five well-resolved peaks of DNA polymerase activity were fractionated. In this new chromatographic mode, the enzyme binds immobilized substrate coupled to a column in the absence of some required cofactor. When the missing cofactor is added, the enzyme converts substrate to product and selectively elutes from the column.     

The selective orexin 1 receptor antagonist SB-334867-A impairs acquisition and consolidation but not retrieval of spatial memory in Morris water maze

The novel neuropeptides orexin-A and orexin-B derive from a common 130-amino acid precursor molecule (prepro-orexin), are mainly localized to neurons within and around the lateral hypothalamus, and exhibit high affinity to the closely related G-Protein-coupled receptors orexin 1 and 2 receptor (OX1R, OX2R). Orexinergic neurons send their axons to the hippocampal formation (CA1, CA2 and dentate gyrus), which expresses OX1Rs. Recent studies have shown that central administration of orexin-A and orexin-B have effects on learning and memory but literature concerning the role of orexinergic system in cognition remains controversial. More recently, antagonists have been described. The most potent and selective is SB-334867-A, which has an affinity of 40 nM at OX1R which is at least 50-fold selective over OX2R. It is likely that the intracerebroventricular (i.c.v.) administration may block OX1Rs in many brain regions. Previously we have shown that intra-CA1 injection of SB-334867-A impairs acquisition, consolidation and retrieval of spatial memory in MWM task. In the present study, the effect of pre-training, post-training and pre-probe of trial intra-DG (dentate gyrus) administration of SB-334867-A (1.5, 3, 6 microg/0.5 microl) on acquisition, consolidation and retrieval in a single-day testing version of MWM (Morris water maze) task was examined. Our results show impaired acquisition and consolidation of MWM task for SB-334867-A as compared with the control group. However, SB-334867-A had no effect on retrieval in spatial memory. Also, this antagonist had no effect on escape latency of a non-spatial visual discrimination task. Therefore, it seems that endogenous orexin-A and orexin-B, through DG OX1Rs, play an important role in spatial learning and memory in the rat.     

Analysis of the nucleotide sequence of the Streptomyces glaucescens tcmI genes provides key information about the enzymology of polyketide antibiotic biosynthesis.

Key information about the biosynthesis of polyketide metabolites has been uncovered by sequence analysis of the tetracenomycin C polyketide synthase genes (tcml) from Streptomyces glaucescens GLA.0. The sequence data revealed the presence of three complete open reading frames (ORFs). ORF1 and ORF2 appear to be translationally coupled and would encode proteins containing 426 and 405 amino acids, respectively. The two deduced proteins are homologous to known beta-ketoacyl synthases. ORF3 begins 70 nucleotides after the stop codon of ORF2 and would code for an 83 amino acid protein with a strong resemblance to known bacterial, animal and plant acyl-carrier proteins (ACP). The presence of an ACP gene within the tcm gene cluster suggests that different ACPs are used in fatty acid and polyketide biosynthesis in Streptomyces. We conclude from these data and earlier information that polyketide biosynthesis in S. glaucescens, and most likely in other bacteria, involves a multienzyme complex consisting of at least five types of enzymes: acylCoA transferases that load the acyl and 2-carboxyacyl precursors onto the ACP; a beta-ketoacyl synthase that, along with the acylated ACP, forms the poly-beta-ketoacyl intermediates; a poly-beta-ketone cyclase that forms carbocyclic structures from the latter intermediates; a beta-ketoacyl oxidoreductase that forms beta-hydroxyacyl intermediates or reduces ketone groups in fully formed polyketides; and a thioesterase that releases the assembled polyketide from the enzyme.     

Unique temperature-activated neurons from pit viper thermosensors

Rattlesnakes, copperheads, and other pit vipers have highly sensitive heat detectors known as pit organs, which are used to sense and strike at prey. However, it is not currently known how temperature change triggers cellular and molecular events that activate neurons supplying the pit organ. We dissociated and cultured neurons from the trigeminal ganglia (TG) innervating the pit organs of the Western Diamondback rattlesnake (Crotalus atrox) and the copperhead (Agkistrodon contortix) to investigate electrophysiological responses to thermal stimuli. Whole cell voltage-clamp recordings indicated that 75% of the TG neurons from C. atrox and 74% of the TG neurons from A. contortix showed a unique temperature-activated inward current (I_T). We also found an I_T-like current in 15% of TG neurons from the common garter snake, a species that does not have a specialized heat-sensing organ. A steep rise in the current-temperature relationship of I_T started just below 18°C, and cooling temperature-responsive TG neurons from 20°C resulted in an outward current, suggesting that I_T is on at relatively low temperatures. Ion substitution and Ca²⁺ imaging experiments indicated that I_T is primarily a monovalent cation current. I_T was not sensitive to capsaicin or amiloride, suggesting that the current did not show similar pharmacology to other mammalian heat-sensitive membrane proteins. Our findings indicate that a novel temperature-sensitive conductance with unique ion permeability and low-temperature threshold is expressed in TG neurons and may be involved in highly sensitive heat detection in snakes. snake; thermosensory; trigeminal; ion conductance     

ANGPTL4 silencing via antisense oligonucleotides reduces plasma triglycerides and glucose in mice without causing lymphadenopathy

Angiopoietin-like 4 (ANGPTL4) is an important regulator of plasma triglyceride (TG) levels and an attractive pharmacological target for lowering plasma lipids and reducing cardiovascular risk. Here, we aimed to study the efficacy and safety of silencing ANGPTL4 in the livers of mice using hepatocyte-targeting GalNAc-conjugated antisense oligonucleotides (ASOs). Compared with injections with negative control ASO, four injections of two different doses of ANGPTL4 ASO over 2 weeks markedly downregulated ANGPTL4 levels in liver and adipose tissue, which was associated with significantly higher adipose LPL activity and lower plasma TGs in fed and fasted mice, as well as lower plasma glucose levels in fed mice. In separate experiments, injection of two different doses of ANGPTL4 ASO over 20 weeks of high-fat feeding reduced hepatic and adipose ANGPTL4 levels but did not trigger mesenteric lymphadenopathy, an acute phase response, chylous ascites, or any other pathological phenotypes. Compared with mice injected with negative control ASO, mice injected with ANGPTL4 ASO showed reduced food intake, reduced weight gain, and improved glucose tolerance. In addition, they exhibited lower plasma TGs, total cholesterol, LDL-C, glucose, serum amyloid A, and liver TG levels. By contrast, no significant difference in plasma alanine aminotransferase activity was observed. Overall, these data suggest that ASOs targeting ANGPTL4 effectively reduce plasma TG levels in mice without raising major safety concerns.