Feeding of [5,5-2H(2)]-1-desoxy-D-xylulose and [4,4,6,6,6-2H(5)]-mevalolactone to a geosmin-producing Streptomyces sp. and Fossombronia pusilla

The biosynthesis of the trisnor sesquiterpenoid geosmin (4,8a-dimethyl-octahydro-naphthalen-4a-ol) (1) was investigated by feeding labeled [5,5-2H(2)]-1-desoxy-D-xylulose (11), [4,4,6,6,6-(2)H(5)]-mevalolactone (7) and [2,2-2H(2)]-mevalolactone (9) to Streptomyces sp. JP95 and the liverwort Fossombronia pusilla. The micro-organism produced geosmin via the 1-desoxy-D-xylulose pathway, whereas the liverwort exclusively utilized mevalolactone for terpenoid biosynthesis. Analysis of the labeling pattern in the resulting isotopomers of geosmin (1) by mass spectroscopy (EI/MS) revealed that geosmin is synthesized in both organisms by cyclization of farnesyl diphosphate to a germacradiene-type intermediate 4. Further transformations en route to geosmin (1) involve an oxidative dealkylation of an i-propyl substituent, 1,2-reduction of a resulting conjugated diene, and bicyclization of a germacatriene intermediate 13. The transformations largely resemble the biosynthesis of dehydrogeosmin (2) in cactus flowers but differ with respect to the regioselectivity of the side chain dealkylation and 1,2-reduction     

Regulation of complement activation by C-reactive protein: targeting the complement inhibitory activity of factor H by an interaction with short consensus repeat domains 7 and 8-11.

C-reactive protein (CRP) is a major acute phase protein whose functions are not totally clear. In this study, we examined the interaction of CRP with factor H (FH), a key regulator of the alternative pathway (AP) of complement. Using the surface plasmon resonance technique and a panel of recombinantly expressed FH constructs, we observed that CRP binds to two closely located regions on short consensus repeat (SCR) domains 7 and 8-11 of FH. Also FH-like protein 1 (FHL-1), an alternatively spliced product of the FH gene, bound to CRP with its most C-terminal domain (SCR 7). The binding reactions were calcium-dependent and partially inhibited by heparin. In accordance with the finding that CRP binding sites on FH were distinct from the C3b binding sites, CRP preserved the ability of FH to promote factor I-mediated cleavage of C3b. We propose that the function of CRP is to target functionally active FH and FHL-1 to injured self tissues. Thereby, CRP could restrict excessive complement attack in tissues while allowing a temporarily enhanced AP activity against invading microbes in blood.     

Catabolism of rat growth hormone-releasing factor(1-29) amide in rat serum and liver.

Clinical and veterinary uses of growth hormone-releasing factor [GRF(1- 29)NH2] require the design of analogs that are resistant to proteolysis by serum and liver degrading enzymes. This study investigated rat GRF(1-29)NH2 processing in serum and liver homogenate by means of high pressure liquid chromatography (HPLC). Synthetic rGRF(1-29)NH2 (30 microM) was incubated (0-120 min, 37 degrees C) in serum (49 +/- 8 mg prot./ml). The rGRF(1-29)NH2 (10 microM) was also incubated (0-120 min, 37 degrees C) with liver homogenate (200 +/- 6 micrograms prot./ml). Time course studies of rGRF(1-29)NH2 disappearance showed apparent half-lives of 18 +/- 4 min and 13 +/- 3 min in serum and liver homogenate, respectively. This was accompanied by the appearance of degradation products that were all less hydrophobic than the native peptide. In the serum, two major metabolites were detected and isolated by preparative HPLC. Combined results of amino acid analysis, sequencing, and chromatography with synthetic homologs revealed the presence of rGRF(1-20)OH and (3-20)OH. A small amount of rGRF(12-29)NH2, coeluting with rGRF(3-20)OH, was also found by sequencing. In the liver, rGRF(1-18)OH, (3-18)OH, and (1-10)OH were identified. The peptide bond Ala2-Asp3 (DPP IV cleavage site) was hydrolyzed in both serum and liver. Other tissue-specific cleavage sites were Arg11-Arg12 and Arg20-Lys21 (trypsin-like cleavage site) in the serum, and Tyr10-Arg11 and Tyr18-Ala19 (chymotrypsin-like cleavage site) in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

In silico characterization of the functional and structural modules of the hemagglutinin protein from the swine-origin influenza virus A (H1N1)-2009

The 2009 swine-origin influenza virus (S-OIV,H1N1 subtype) has developed into a new pandemic influenza as announced by the World Health Organization.In order to uncover clues about the determinants for virulence and pathogenicity of the virus,we characterized the functional modules of the surface glycoprotein hemagglutinin (HA),the most important protein in molecular epidemiology and pathogenesis of influenza viruses.We analyzed receptor binding sites,basic patch,neutralization antibody epitopes and T cell epitopes in the HA protein of the current S-OIV according to the corresponding functional and structural modules previously characterized in other H1 HA molecules or HA molecules of other subtypes.We compared their differences and similarities systematically.Based on the amino acids defined as the functional and structural modules,the HA protein of 2009 S-OIV should specifically bind to the human 2,6-receptor.The D225G/E mutation in HA,which is found in some isolates,may confer dual binding specificity to the 2,3and 2,6-receptor based on previously reported work.This HA variant contains two basic patches,one of which results in increased basicity,suggesting enhanced membrane fusion function.The 2009 S-OIV HA also has an extra glycosylation site at position 276.Four of the five antibody neutralization epitopes identified in A/RP/8/34(H1N1) were exposed,but the other was hidden by a glycosylation site.The previously identified cytotoxic T cell epitopes in various HA molecules were summarized and their corresponding sequences in 2009 S-OIV HA were defined.These results are critical for understanding the pathogenicity of the virus and host immune response against the virus.     

Synthesis and 1H NMR spectroscopic characterization of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]]

The reaction of trans-[Pt(NH3)2Cl2] with the sodium salt of [d(ApGpGpCpCpT)]2 in aqueous solution at 37 degrees C was monitored by reversed-phase high-performance liquid chromatography and UV spectroscopy. Two intermediates, most likely monofunctional adducts, were observed, which subsequently formed one predominant single-stranded product, as well as several polymeric species proposed to be interstrand cross-linked products. The single-stranded adduct was structurally characterized by 1H NMR spectroscopy. From the pH dependence of the chemical shifts, two-dimensional homonuclear chemical shift correlation (COSY) spectroscopy, and one- and two-dimensional nuclear Overhauser effect (NOESY) experiments, the platinum(II) moiety was found to be coordinated to the N7 positions of adenine(1) and guanine(3), with the intervening guanine(2) base destacked from its neighboring residues. This intrastrand 1,3 adduct induces changes in the backbone torsion angles and causes the deoxyribose ring of adenine(1) to switch from a C2'-endo to a predominantly C3'-endo conformation. The other deoxyribose rings retain B DNA type conformations. The structure of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]] differs from those previously reported for cis-DDP 1,2- and 1,3-intrastrand oligonucleotide adducts but is consistent with the structures of trans-DDP 1,3-intrastrand adducts of two previously reported trinucleotides.     

Influence of sarcosine or N-methylalanine in position 7 on the antagonistic properties of [1-deaminopenicillamine]- and [1-(beta-mercapto-beta, beta-cyclopentylmethylene-propionic acid)]-vasopressin

Substituting sarcosine or N-methylalanine for proline in the inhibitory vasopressin analogs dPAVP and d(CH2)5AVP had the following effects: 1) milk ejection and antidiuretic activities were severely depressed, 2) pressor antagonism was maintained but weakened somewhat, and 3) antagonism in the uterus in vitro was maintained, but no consistent pattern was seen.     

Purification and characterization of two distinct forms of rat adrenal cytochrome P450(11) beta: functional and structural aspects

It is generally accepted that the last three steps of aldosterone biosynthesis are catalyzed by a single enzyme, i.e., cytochrome P450(11) beta (P450XIB). We have previously reported that rat adrenal mitochondria may be capable of producing two forms of P450(11) beta which differ in molecular weight (49 and 51 kDa). In the present study we describe the purification, the enzymatic activities, and some structural properties of these two proteins. Using zona fasciculata mitochondria, the 51-kDa protein was purified to electrophoretic homogeneity by means of octyl-Sepharose chromatography. In a reconstituted system the protein catalyzed 18- and 11 beta-hydroxylation of deoxycorticosterone, but exhibited no 18-hydroxylation or 18-hydroxydehydrogenation of corticosterone. The 49-kDa protein was isolated from zona glomerulosa mitochondria of rats kept on a low-sodium, high-potassium regimen. Using octyl-Sepharose chromatography, it could be separated from the 51-kDa protein. A reconstituted eluate fraction, containing the 49-kDa protein, converted deoxycorticosterone not only to 18-OH-deoxycorticosterone and corticosterone, but also to 18-OH-corticosterone and aldosterone. These findings indicate that the rat adrenal cortex is capable of producing two distinct forms of active cytochrome P450(11) beta. A structural relationship of the 49- and 51-kDa proteins was indicated by experiments involving limited proteolysis. Thus, digestion with alpha-chymotrypsin and V8-protease yielded very similar peptide maps for both proteins. During potassium repletion of potassium-deficient rats, the disappearance of the active 51-kDa protein coincided with the appearance of the 49-kDa protein. These results are suggestive of a post-translational processing mechanism converting the 51-kDa protein into the smaller 49-kDa form. However, the 49-kDa protein might also be encoded by a distinct gene, regulated separately depending on the physiological conditions.     

Synthesis and 1H NMR studies of 3-(D-erythro-glycerol-1-yl)-1H-pyrazolo[3,4-b]quinoxaline and its 7-chloro and 7-methyl analogues

3-(D-erythro-Glycerol-1-yl)-1H-pyrazolo[3,4-b]quinoxaline and its 7-chloro and 7-methyl analogues (11 and 12) were prepared from the corresponding quinoxalines. The 7-substituted analogues 11 and 12 were obtained as the preponderant isomers, and the 6-substituted analogues as the minor isomers. The structure and position of the substituent were determined by 1H NMR studies. The effect of substitution on the chemical shift of other protons is discussed.     

Demonstration of two exchangeable non-catalytic and two cooperative catalytic sites in isolated bovine heart mitochondrial F1, using the photoaffinity labels [2-3H]8-azido-ATP and [2-3H]8-azido-ADP

The photoreactive nucleotides [2-3H]8-azido-ATP and [2-3H]8-azido-ADP could be used to label the nucleotide binding sites on isolated mitochondrial F1-ATPase to a maximum of 4 mol of nucleotide per mol F1, also when the F1 was depleted of tightly bound nucleotides. At a photolabel concentration of 300-1000 microM, label was found on both alpha and beta subunits in a typically 1:3 ratio, independent of the total amount bound. Under these conditions the covalent binding of two nucleotides is needed for full inactivation (Wagenvoord, R.J., Van der Kraan, I. and Kemp, A. (1977) Biochim. Biophys. Acta 460, 17-24). At lower concentrations of [2-3H]8-azido-ATP (20 microM), it was found that covalent binding of only 1 mol of nucleotide per mole F1 was required for complete inactivation to take place indicating catalytic site cooperativity in the mechanism of ATP hydrolysis. Under those conditions, radioactivity was only found on the beta subunits, which would indicate that the catalytic site is located on a beta subunit and that a second site is located on the alpha/beta interface. It is found that four out of the six nucleotide binding sites are exchangeable and can be labelled with 8-azido-AT(D)P, i.e., two catalytic sites and two non-catalytic sites.     

Synthesis, complete 1H assignments and conformations of the self-complementary hexadeoxyribonucleotide [d(CpGpApTpCpG)]2 and its fragments by high field NMR.

The two deoxyribonucleotides [d(CpGpApTpCpG)]2 and [d(CpGpCpG)]2 were synthesized by the phosphotriester method. Their duplex form under the conditions of the 1H-nmr experiments was proven by end 32P labeling with T4 polynucleotide kinase followed by butt end joining employing the absolute specificity of T4 ligase for double stranded DNA and analysis using gel electrophoresis and autoradiography. Complete nmr assignment of the 1H chemical shifts and coupling constants was achieved. The assignments were secured using sequential decoupling, NOE difference measurements, and two-dimensional COSY and SECSY experiments. Spectrum simulation confirmed the experimental values of chemical shifts and coupling constants. The techniques for the assignment outlined together with 31P and 2-D heteronuclear shift correlation permit an approach to a systematic analysis of more complex single-strand and duplex oligodeoxyribonucleotides.     

Synthesis and biological evaluation of 1-(benzenesulfonamido)-2-[5-(N-hydroxypyridin-2(1H)-one)]acetylene regioisomers: a novel class of 5-lipoxygenase inhibitors

A hitherto unknown class of linear acetylene regioisomers were designed such that a SO₂NH₂ group was located at the ortho-, meta-, or para-position of the acetylene C-1 phenyl ring, and a N-hydroxypyridin-2(1H)-one moiety was attached via its C-5 position to the C-2 position on an acetylene template (scaffold). All three regioisomers inhibited 5-lipoxygenase (5-LOX), where the relative potency order was 2-SO₂NH₂ (IC₅₀ = 10 μM) >3-SO₂NH₂ (IC₅₀ = 15 μM) >4-SO₂NH₂ (IC₅₀ = 68 μM) relative to the reference drug nordihydroguaiaretic acid (NDGA; IC₅₀ = 35 μM). The 2-SO₂NH₂ regioisomer (ED₅₀ = 86.0 mg/kg po) exhibited excellent oral anti-inflammatory (AI) activity that was more potent than aspirin (ED₅₀ = 128.9 mg/kg) and marginally less potent than ibuprofen (ED₅₀ = 67.4 mg/kg). The N-hydroxypyridin-2(1H)one moiety provides a novel pharmacophore for the design of cyclic hydroxamic mimetics capable of chelating 5-LOX iron for exploitation in the design of 5-LOX inhibitory AI drugs.     

Structural and functional characterization of galactooligosaccharides in Nostoc commune: beta-D-galactofuranosyl-(1-->6)-[beta-D-galactofuranosyl-(1-->6)]2-beta-D-1,4-anhydrogalactitol and beta-(1-->6)-galactofuranosylated homologues

A new class of galactooligosaccharides has been identified from the terrestrial cyanobacterium Nostoc commune by MS and NMR techniques. These consist of beta-D-galactofuranosyl-(1-->6)-[beta-D-galactofuranosyl-(1-->6)]n-beta-d-1,4-anhydrogalactitols with n ranging from 2 to 8, corresponding to compounds designated 1 through 7. In total these saccharides amounted to approximately 0.35% of the dry thallus of N. commune, while in several other cyanobacteria they were not detected. Possibly they play some role in protection from damage by heat and desiccation as suggested by experiments with heterologous systems. For example, phosphoglucomutase (EC 2.7.5.1) from rabbit muscle was protected against heat inactivation by these oligosaccharides, and alpha-amylase (EC 3.2.1.1) from porcine pancreas by the oligosaccharides 6 and 7. The homologues of lower molecular mass, however, enhanced heat sensitivity of alpha-amylase. The viability of Escherichia coli was completely abolished by desiccation, whereas in the presence of 4 survival rates were approximately 50% of controls not subjected to desiccation. The newly identified saccharides are compared with known galactofuranose-based oligo- and polysaccharides and possible biological functions of them are discussed.     

Synthesis and antitumor activity of cyclopentadienyltitanium substituted polyoxotungstate [CoW11O39(CpTi)]7- (Cp=eta5-C5H5)

A novel polyoxotungstate [CoW(11)O(39)(CpTi)](7-) (Cp=eta(5)-C(5)H(5)) has been prepared. This complex exhibits the highest antitumor activity in vitro among the cyclopentadienyltitanium substituted polyoxometalates investigated and has a remarkable inhibitory action on three types of human cancer cells, SSMC-7721, HL-60 and HLC, in vivo.     

7-[3-(4-[2,3-Dimethylphenyl]piperazinyl)propoxy]-2(1H)-quinolinone (OPC-4392), a presynaptic dopamine autoreceptor agonist and postsynaptic D2 receptor antagonist

7-[3-(4-[2,3-dimethylphenyl]piperazinyl)propoxy]-2(1H)-quinolinone (OPC-4392), was synthesized in our laboratories and compared with apomorphine, 3-(3-hydroxyphenyl)-N-n-propylpiperidine (3-PPP) and dopamine antagonists in a series of tests designed to characterize dopamine receptor activation and inhibition. The assertion that OPC-4392 acts as an agonist at presynaptic dopamine autoreceptors is supported by the following behavioral and biochemical observations: OPC-4392, 3-PPP and apomorphine inhibited the reserpine-induced increase in DOPA accumulation in the forebrain of mice and in the frontal cortex, limbic forebrain and striatum of rats. In addition, the gamma-butyrolactone (GBL)-induced increase in DOPA accumulation in the mouse forebrain was also inhibited by OPC-4392, 3-PPP and apomorphine. Haloperidol antagonized the inhibitory effect of OPC-4392 in both instances. The inhibitory effect of OPC-4392 on GBL-induced DOPA accumulation lasted for at least 8 hours after oral administration to mice, while that of 3-PPP and apomorphine disappeared in 4 hours after subcutaneous injection. OPC-4392 failed to increase spontaneous motor activity in reserpinized mice, enhance spontaneous ipsilateral rotation in rats with unilateral striatal kainic acid (KA) lesions, induce contralateral rotation in rats with unilateral striatal 6-hydroxydopamine (6-OHDA) lesions and inhibit 14C-acetylcholine (Ach) release stimulated by 20 mM KCl in rat striatal slices. In addition, OPC-4392 appears to block postsynaptic D2 receptors since OPC-4392, as well as dopamine antagonists, was able to inhibit stereotyped behavior and climbing behavior induced by apomorphine in mice, displace the 3H-spiroperidol binding to rat synaptosomal membranes in vitro and reverse the inhibitory effect of apomorphine on Ach release in rat striatal slices. These results suggest that OPC-4392 acts as a dopamine agonist at presynaptic autoreceptors related to dopamine synthesis and acts as dopamine antagonist at postsynaptic D2 receptors.     

Synthesis of new 1-[2-Azido-2-(2,4-dichlorophenyl)ethyl]-1H/-imidazoles and in vitro evaluation of their antifungal activity

New 1-[2-azido-2-(2,4-dichlorophenyl)ethyl]-1H/-imidazole were synthesized by nucleophilic substitution of various tertiary alcohols with azide anion in presence of boron trifluoride-diethyl etherate. Their antifungal activity was evaluated against Candida albicans, Candida glabrata, Aspergillus fumigatus and an azole-resistant petite mutant of C. glabrata. Preliminary SAR results are discussed.     

alpha-DNA. I. Synthesis, characterization by high field 1H-NMR, and base-pairing properties of the unnatural hexadeoxyribonucleotide alpha-[d(CpCpTpTpCpC)] with its complement beta-[d(GpGpApApGpG)].

The novel deoxyribonucleotide alpha-[d(CpCpTpTpCpC)] and its complement beta-[d(GpGpApApGpG)] were synthesized by the phosphotriester method. 1H-NMR-NOE examination of the alpha-hexamer revealed that the cytosine and thymine bases appear to adopt anti conformations in this strand. In addition the deoxyribose of the thymidine moieties may adopt average conformations approximating to C3'-endo while the cytidine furanose groups are close to C2'-endo conformations. Both hyperchromicity in thermal melting and detection of base paired imino protons in 1H-NMR studies in H2O provide evidence for the annealing of alpha-d[CCTTCC] with its complement beta-d[GGAAGG] in potassium phosphate buffer pH 7.1 containing 10 mM magnesium chloride. Under these conditions thermal melting begins at 38 degrees C and its complete at approximately 45 degrees C. NOE experiments do not permit a decision on the polarity of annealing (predicted to be parallel) for this particular pair of sequences.     

Synthesis of new 1-[2-Azido-2-(2,4-dichlorophenyl)ethyl]-1H/-imidazoles and in vitro evaluation of their antifungal activity

New 1-[2-azido-2-(2,4-dichlorophenyl)ethyl]-1H/-imidazole were synthesized by nucleophilic substitution of various tertiary alcohols with azide anion in presence of boron trifluoride-diethyl etherate. Their antifungal activity was evaluated against Candida albicans, Candida glabrata, Aspergillus fumigatus and an azole-resistant petite mutant of C. glabrata. Preliminary SAR results are discussed.     

Utilization by the isolated perfused rat liver of N-acetyl-D-[1-14C]galactosamine and N-[3H]acetyl-D-galactosamine for the biosynthesis of glycoproteins.

The isolated perfused rat liver system has been used to monitor the utilization of N-[3H]acetyl-D-galactosamine and N-acetyl-D-[1-14C]galactosamine for the biosynthesis of radiolabelled glycoproteins, which are subsequently secreted into the plasma. Both radiolabels appear in a number of different glycoproteins, predominantly as sialic acid and N-acetylglucosamine. The ratio of labelled sialic acid to labelled N-acetylglucosamine varies for different glycoproteins, but the bulk of N-acetyl-D-galactosamine is incorporated without deacetylation.