P. Nicholas Shaw

Discovery of a New Source of Rifamycin Antibiotics in Marine Sponge Actinobacteria by Phylogenetic Prediction

ABSTRACT Phylogenetic analysis of the ketosynthase (KS) gene sequences of marine sponge-derived Salinispora strains of actinobacteria indicated that the polyketide synthase (PKS) gene sequence most closely related to that of Salinispora was the rifamycin B synthase of Amycolatopsis mediterranei. This result was not expected from taxonomic species tree phylogenetics using 16S rRNA sequences. From the PKS sequence data generated from our sponge-derived Salinispora strains, we predicted that such strains might synthesize rifamycin-like compounds. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis was applied to one sponge-derived Salinispora strain to test the hypothesis of rifamycin synthesis. The analysis reported here demonstrates that this Salinispora isolate does produce compounds of the rifamycin class, including rifamycin B and rifamycin SV. A rifamycin-specific KS primer set was designed, and that primer set increased the number of rifamycin-positive strains detected by PCR screening relative to the number detectable using a conserved KS-specific set. Thus, the Salinispora group of actinobacteria represents a potential new source of rifamycins outside the genus Amycolatopsis and the first recorded source of rifamycins from marine bacteria.

Chromatographic behaviour of positional isomers on porous graphitic carbon

Retention characteristics of six sets of ionizable substituted benzene isomers have been measured on a porous graphitic carbon column using buffered aqueous eluents containing 35% acetonitrile. The elution orders varied with the different types of substituents on the benzene ring and were not readily predictable from the known structures of the isomers. Over the range of mobile phase between pH 2 to 9.4, the retention of the solutes was correlated directly with their degree of ionization, with the ionized form being least retained. An interpretation of these data was suggested based on the solute molecular orientation effect induced by the competing interactions of solute with adsorbent and solvent.

Retention behavior of ionizable isomers in reversed phase liquid chromatography: A comparative study of porous graphitic carbon and octadecyl bonded silica

The retention behaviors of 36 positional isomers of ionizable substituted benzene compounds have been compared on two different packing materials:  porous graphitic carbon (PGC) and octadecyl bonded silica (ODS) using 35% aqueous acetonitrile as the mobile phase. The effect of the mobile phase pH on the solute retention was studied over a range of pH values from pH 2.0 to 7.0. The retention as a function of pH was modeled using equations based on solute ionization. With PGC, the theoretical equations fitted the observed retention data for each class of solute, indicating that the retention mechanism was uniform over the whole pH range. However, with ODS, only the acidic solutes showed agreement with the theoretical model; for the amine-containing compounds, serious deviations from the theory were observed, suggesting that strongly acidic silanols gave added retention at low mobile phase pH. Overall, PGC demonstrated a higher selectivity toward positional isomers than ODS. This was attributed to the greater steric discriminating ability arising from the flat surface of the PGC compared with the more fluid nature of the ODS bonded phase.

Catecholamines derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole: characterization of chemical structure and fluorescence properties

4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was evaluated as a fluorogenic derivatization reagent for the analysis of the catecholamines, dopamine, epinephrine, norepinephrine, and their naturally occurring metabolites, metanephrine and normetanephrine, homovanillic acid, 3,4-dihydroxyphenyl acetic acid. These compounds reacted rapidly with NBD-F under mild conditions to form stable derivatives. The optimal reaction conditions were found to be 12.5 mM borate buffer pH 8.0 in water:acetonitrile (1:1) at 50 degreesC for 5 min. New NBD derivatives of all the catecholamines and metabolites were prepared and purified and were shown by electrospray mass spectrometry to be fully reacted at all available catechol and amine sites, resulting in di- or tri-substituted derivatives. Homovanillic acid and 3,4-dihydroxyphenyl acetic acid reacted with NBD-F but gave non-fluorescent derivatives. The fluorescence excitation wavelength maximum demonstrated a red shift for the derivatives with increasing polarity of the solvent and the fluorescence intensity increased linearly with increasing organic ratio in the solvent-aqueous buffer complex. The presence of electrolyte in the solvent and the electrolyte concentration in the solvent-electrolyte complex had little effect on the fluorescent intensity. The fluorescence quantum yields in acetonitrile were also obtained. The separation behavior of the NBD-catecholamines was determined by high-performance liquid chromatography (HPLC). The studies demonstrated good potential for the application of NBD-F derivatization to the quantitative analysis of catecholamines and related compounds in biological matrices

Separation of pharmaceutical bases from neutral and acidic components by capillary electrochromatography

Capillary electrochromatography (CEC) has proved to be applicable to the separation of neutral and acidic (chromatographed in their ion-suppressed mode) compounds but a major factor holding back its further development has been its perceived inability to analyse basic compounds. In this paper we demonstrate that the addition of a competing base, such as triethylamine (TEA) or triethanolamine (TEOA), to a low pH buffer can achieve excellent CEC analysis of a range of strong pharmaceutical bases. Acceptable peak symmetry and efficiencies of up to 510000 plates m–1 were obtained for bases such as procainamide, nortriptyline and diphenhydramine on CEC Hypersil C18, C8 and phenyl reversed-phase packing materials. The effect of TEA or TEOA concentration and mobile phase pH on the separation behaviour of the bases is discussed. In addition, the paper reports the first example of the simultaneous CEC analysis of acids, bases and neutral compounds by using a mobile phase containing TEOA phosphate at pH 2.5.

Disruption of NaS1 sulfate transport function in mice leads to enhanced acetaminophen-induced hepatotoxicity.

Sulfate is required for detoxification of xenobiotics such as acetaminophen (APAP), a leading cause of liver failure in humans. The NaS1 sulfate transporter maintains blood sulfate levels sufficiently high for sulfonation reactions to work effectively for drug detoxification. In the present study, we identified two loss‐of‐function polymorphisms in the human NaS1 gene and showed the Nas1‐null mouse to be hypersensitive to APAP hepatotoxicity. APAP treatment led to increased liver damage and decreased hepatic glutathione levels in the hyposulfatemic Nas1‐null mice compared with that in normosulfatemic wild‐type mice. Analysis of urinary APAP metabolites revealed a significantly lower ratio of APAP‐sulfate to APAP‐glucuronide in the Nas1‐null mice. These results suggest hyposulfatemia increases sensitivity to APAP‐induced hepatotoxicity by decreasing the sulfonation capacity to metabolize APAP. In conclusion, the results of this study highlight the importance of plasma sulfate level as a key modulator of acetaminophen metabolism and suggest that individuals with reduced NaS1 sulfate transporter function would be more sensitive to hepatotoxic agents. (HEPATOLOGY 2006;43: 1241–1247.)

Effects of the mango components mangiferin and quercetin and the putative mangiferin metabolite norathyriol on the transactivation of peroxisome proliferator-activated receptor isoforms.

Mangos are a source of bioactive compounds with potential health-promoting activity. This study evaluated the abilities of the mango components quercetin and mangiferin and the aglycone derivative of mangiferin, norathyriol, to modulate the transactivation of peroxisome proliferator-activated receptor isoforms (PPARs). PPARs are transcription factors important in many human diseases. Through the use of a gene reporter assay it was shown that quercetin inhibited the activation of all three isoforms of PPARs (PPARgamma IC(50) = 56.3 microM; PPARalpha IC(50) = 59.6 microM; PPARbeta IC(50) = 76.9 microM) as did norathyriol (PPARgamma IC(50) = 153.5 microM; PPARalpha IC(50) = 92.8 microM; PPARbeta IC(50) = 102.4 microM), whereas mangiferin did not inhibit the transactivation of any isoform. These findings suggest that mango components and metabolites may alter transcription and could contribute to positive health benefits via this or similar mechanisms.

Mango extracts and the mango component mangiferin promote endothelial cell migration.

This study tested the hypothesis that mango extracts contain bioactive molecules capable of modulating endothelial cell migration, an essential step in the formation of new blood vessels or angiogenesis. The formation of new blood vessels is an important therapeutic target for diseases such as limb ischemia, coronary infarction or stroke. We examined the effect of mango peel and flesh extracts as well as the individual polyphenolic molecules, mangiferin and quercetin, on bovine aortic cell migration using a modified Boyden chamber assay. Our results show that mangiferin, and extracts rich in mangiferin, increase endothelial cell migration. The dose-effect relationship for various extracts further suggests that this action of mangiferin is modulated by other components present in the extracts. The promigratory effect of mango extracts or mangiferin was unrelated to an effect on cell proliferation, and did not involve a change in the production of matrix metalloprotease-2 or -9 by the endothelial cells. Taken together, these results suggest that mangiferin present in mango extracts may have health promoting effects in diseases related to the impaired formation of new blood vessels.

High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man

High-performance liquid chromatographic assay procedures have been developed for naproxen, ibuprofen and diclofenac in human plasma and synovial fluid samples. A single liquid-liquid extraction procedure was used to isolate each compound from acidified biological matrix prior to the quantitative analysis. A Spherisorb ODS column (12.5 cm x 4.6 mm I.D.) was used for all the chromatography. Naproxen was eluted with a mobile phase of methanol-Sörensens buffer at pH 7 (37:63, v/v). Ibuprofen and diclofenac were eluted using mobile phases of methanol-water at pH 3.3 (65:35, v/v and 63:37, v/v, respectively). Diphenylacetic acid was used as the internal standard for the assay of naproxen and flurbiprofen was used in the analysis of ibuprofen and diclofenac. Inter- and intra-day coefficients of variation were less than 7%. The assays were used in clinical studies of the three drugs in osteo- and rheumatoid arthritis patients.

An improved extraction method for the HPLC determination of morphine and its metabolites in plasma

A new, simple and rapid extraction procedure coupled with a combined coulometric-fluorescence HPLC assay is described for the simultaneous determination of morphine (M) and morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), and normorphine (NM) in plasma. The effect of concentration and pH of selected ion-pairing agents on the extraction of these compounds from plasma by solid-phase extraction was investigated. The extraction procedure was optimized in terms of recovery, reproducibility and lack of interference from endogenous materials. The optimized method uses tetrabutylammonium hydrogen sulphate (TBAHS) at pH 10 followed by separation on a single C18 solid-phase extraction cartridge. For routine analysis the procedure provides high and reproducible recoveries over a concentration range of 1.0-1000 ng ml-1 for morphine, M6G and normorphine and 20-1000 ng ml-1 for M3G. The method was used successfully to analyse plasma samples from a pharmacokinetic study in which sheep had received an intravenous dose of 0.015 mg kg-1 of M6G.