Biosynthesis of rubradirin as an ansamycin antibiotic from Streptomyces achromogenes var. rubradiris NRRL3061

The four overlapping cosmids from the rubradirin producer, Streptomyces achromogenes var rubradiris NRRL 3061, have 58 ORFs within a 105.6 kb fragment. These ORFs harbored essential genes responsible for the formation and attachment of four distinct moieties, along with the genes associated with regulatory, resistance, and transport functions. The PKS (rubA) and glycosyltransferase (rubG2) genes were disrupted in order to demonstrate a complete elimination of rubradirin production. The rubradirin biosynthetic pathway was proposed based on the putative functions of the gene products, the functional identification of sugar genes, and the mutant strains. The GeneBank accession number for the sequence reported in this paper is AJ871581.     

The new generation dihydropyridine type calcium blockers, bearing 4-phenyl oxypropanolamine, display alpha-/beta-adrenoceptor antagonist and long-acting antihypertensive activities

A new series of dihydropyridine derivatives, bearing oxypropanolamine moiety on phenyl ring at the 4-position of the dihydropyridine base, were prepared. Oxypropanolamine was synthesized by replacing the phenolic OH of vanillin or other compounds, having a phenyl aldehyde group, with epichlorohydrin, followed by cleavaging the obtained epoxide compounds with tert-butylamine, n-butylamine or 2-methoxy-1-oxyethylamino benzene (guaiacoxyethylamine), respectively. Obtained various oxypropanolamine compounds, still remaining a phenyl aldehyde moiety, were then performed by Hantzsch condensation reaction with methylacetoacetate or ethylacetoacetate, respectively, to give our new series of dihydropyridine linked with the 4-phenyl ring. These compounds were evaluated for inotropic, chronotropic, and aorta contractility that associated with calcium channel and adrenoceptor antagonist activities. Dihydropyridine derivatives that with oxypropanolamine side chain on their 4-phenyl ring associated alpha-/beta-adrenoceptor blocking activities created a new family of calcium entry and the third generation beta-adrenoceptor blockers. Optimizing this research to obtain more potent alpha-/beta-adrenoceptor blocking and long-acting antihypertensive oxypropanolamine on the 4-phenyl ring of dihydropyridine series compounds was thus accomplished and classified as third generation dihydropyridine type calcium channel blockers, in comparison with previous short-acting type nifedipine and long-acting type amlodipine. We concluded that compounds 1a, 1b and 1g showed not only markedly high calcium-antagonistic activity but also the highest antihypertensive effect; compounds 1b, 1c, 1f, 1g, 1i and 1j induced sustained antihypertensive effects are major and attributed to their calcium entry and alpha-adrenoceptor blocking activities in the blood vessel due to their introduction of 2-methoxy, 1-oxyethylamino benzene moiety in the side chain on the 4-phenyl ring of dihydropyridine. Bradycardiac effects of all the compounds 1a-1j resulted from calcium entry and beta-adrenoceptor blocking, which attenuate the sympathetic activation-associated reflex tachycardia in the heart. We selected compound 1b as candidate compound for further pharmacological and pre-clinical evaluation studies.     

Binding potency of peptide fragments of type 1 collagen cross-linked N-telopeptide measured by an enzyme-linked immunosorbant assay

Osteoporosis represents a major healthcare problem affecting elderly person. Urinary level of the crosslinked N-telopeptide of type I collagen is a sensitive marker of bone resorption. Ten overlapping peptides covering the N-telopeptide of alpha-2 type I collagen were synthesized, purified, and assayed for their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibody by using a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). Peptides 1, 2, and 3, containing the N-terminal sequence of N-telopeptide, showed higher binding potency than peptides 4-10, suggesting that these peptides may contain binding sites for anti-NTX antibodies, and can serve as the lead for further preparation of their antibodies in order to develop novel bioassays for monitoring the bone loss in humans.     

The extracellular calcium-sensing receptor is required for cholecystokinin secretion in response to L-phenylalanine in acutely isolated intestinal I cells

The extracellular calcium-sensing receptor (CaSR) has recently been recognized as an L-amino acid sensor and has been implicated in mediating cholecystokinin (CCK) secretion in response to aromatic amino acids. We investigated whether direct detection of L-phenylalanine (L-Phe) by CaSR results in CCK secretion in the native I cell. Fluorescence-activated cell sorting of duodenal I cells from CCK-enhanced green fluorescent protein (eGFP) transgenic mice demonstrated CaSR gene expression. Immunostaining of fixed and fresh duodenal tissue sections confirmed CaSR protein expression. Intracellular calcium fluxes were CaSR dependent, stereoselective for L-Phe over D-Phe, and responsive to type II calcimimetic cinacalcet in CCK-eGFP cells. Additionally, CCK secretion by an isolated I cell population was increased by 30 and 62% in response to L-Phe in the presence of physiological (1.26 mM) and superphysiological (2.5 mM) extracellular calcium concentrations, respectively. While the deletion of CaSR from CCK-eGFP cells did not affect basal CCK secretion, the effect of L-Phe or cinacalcet on intracellular calcium flux was lost. In fact, both secretagogues, as well as superphysiological Ca(2+), evoked an unexpected 20-30% decrease in CCK secretion compared with basal secretion in CaSR(-/-) CCK-eGFP cells. CCK secretion in response to KCl or tryptone was unaffected by the absence of CaSR. The present data suggest that CaSR is required for hormone secretion in the specific response to L-Phe by the native I cell, and that a receptor-mediated mechanism may inhibit hormone secretion in the absence of a fully functional CaSR.     

Nonenzymatic chromophore attachment in biliproteins: conformational control by the detergent Triton X-100

While chromophore attachment to alpha-subunits of cyanobacterial biliproteins has been studied in some detail, little is known about this process in beta-subunits. The ones of phycoerythrocyanin and C-phycocyanin each carry two phycocyanobilin (PCB) chromophores covalently attached to cysteins beta84 and beta155. The differential nonenzymatic reconstitution of PCB to the apoproteins, PecA, PecB, CpcA and CpcB, as well as to mutant proteins of the beta-subunits lacking either one of the two binding cysteins, was studied using overexpression of the respective genes. PCB adds selectively to Cys-84 of CpcA, CpcB, PecA, and PecB, but the bound chromophore has a nonnative configuration, and in the case of CpcA, is partly oxidized to mesobiliverdin (MBV). The oxidation is independent of thiols but can be suppressed by ascorbate. The addition to Cys-beta84 is suppressed in the presence of detergents like Triton X-100, in favor of an addition to Cys-beta155 yielding the correctly bound chromophore. Triton X-100 also inhibits oxidation of the chromophore during addition to CpcA. The effect of Triton X-100 was studied on the isolated components of the reconstitution system. Absorption, fluorescence and circular dichroism spectra indicate a major conformational change of the chromophore upon addition of the detergent, which probably controls the site selectivity of the addition reaction, and inhibits the oxidation of PCB to MBV.