In-process quality control of wine by planar chromatography versus micro planar chromatography.

High-performance thin-layer chromatography (HPTLC) methods were developed to determine glycerol, gluconic acid, amino acids and sugars for in-process quality control of wine. Twenty wine samples (Pfalz region, Germany) were diluted with methanol and used for quantitative analysis without any further sample preparation. The developed amino acid method provided quantitative and characteristic fingerprints of wine varieties. The amino acid assignments were verified by HPTLC-MS. The developed gluconic acid method was primarily used to control the threshold, indicating a Botrytis cinerea infection of grapes. However, this method also enabled the detection of further organic acids like malic, tartaric and citric acids. A glycerol method was developed for control of the grape must fermentation (spontaneous/regular) and for fraud detection (glycerol adulteration). The HPTLC results of the sugar contents in the wine samples were similar to those of the well-known Luff-Schoorl method. The combined use of these developed HPTLC methods allowed the fermentation control (e.g., alcoholic and malolactic fermentation) and the monitoring of the grapes' overall health status. Without modification, the HPTLC methods for sugar and amino acid analysis could be transferred to circular micro planar chromatography (µ-PLC), showing its potential and benefits in terms of an inexpensive alternative for wineries and distributors.

Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii.

A novel ketoreductase isolated from Zygosaccharomyces rouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance. The 37.8-kDa ketoreductase was purified more than 300-fold to > 95% homogeneity from whole cells with a 30% activity yield. The ketoreductase functions as a monomer with an apparent Km for 3,4-methylenedioxyphenyl acetone of 2.9 mM and a Km for NADPH of 23.5 microM. The enzyme is able to effectively reduce alpha-ketolactones, alpha-ketolactams, and diketones. Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis. The 1.0-kb ketoreductase gene was cloned and sequenced from a Z. rouxii cDNA library using a degenerate primer to the N-terminal sequence of the purified protein. Furthermore, it was expressed in both Escherichia coli and Pichia pastoris and shown to be active. Substrate specificity, lack of a catalytic metal, and extent of protein sequence identity to known reductases suggests that the enzyme falls into the carbonyl reductase enzyme class.

Characterization of an echinocandin B-producing strain blocked for sterigmatocystin biosynthesis reveals a translocation in the stcW gene of the aflatoxin biosynthetic pathway.

Echinocandin B (ECB), a lipopolypeptide used as a starting material for chemical manufacture of the anti-Candida agent LY303366, is produced by fermentation using a strain of Aspergillus nidulans. In addition to ECB, the wild-type strain also produces a significant level of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Characterization of a mutant designated A42355-OC-1 (OC-1), which is blocked in ST biosynthesis, was the result of a chromosomal translocation. The chromosomal regions containing the breakpoints of the translocation were isolated and DNA sequencing and PCR analysis of the chromosomal breakpoints demonstrated the translocation occurred within the stcW gene of the ST biosynthetic pathway, resulting in disruption of the open reading frame for this gene. Biochemical feeding studies indicate the involvement of this gene product in the conversion of averufin to 1-hydroxy versicolorone. This work demonstrates an effective synergy between classical strain improvement methods and molecular genetics.

Enzymatic deacylation of teicoplanin followed by reductive alkylation: synthesis and antibacterial activity of new glycopeptides.

Novel glycopeptides derived from teicoplanin were prepared and evaluated for activity against antibiotic-resistant gram-positive pathogens. Removal of the fatty acid sidechains of teicoplanin was accomplished by enzymatic deacylation. The resulting deacylated teicoplanin was subjected to reductive alkylation resulting in mono- and di-alkylated compounds at the 2 possible primary amines. Deacylated teicoplanin was less active than teicoplanin against enterococci and staphylococci (MIC > or =32 microg/ml). All mono- and di-alkylated products regained some activity, and some had potent activity against both staphylococci and glycopeptide-resistant enterococci. MICs of the most potent di-alkylated compounds ranged from 0.25 approximately 2 microg/ml against glycopeptide-resistant enterococci.

Specific interaction between beta-lactams and soluble penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus: development of a chromogenic assay.

We investigated the enzymatic acylation of penicillin-binding protein 2a (PBP 2a) from methicillin-resistant Staphylococcus aureus by beta-lactams. Using a purified, soluble form of the protein (PBP 2a'), we observed beta-lactam-induced in vitro precipitation following first-order kinetics with respect to protein concentration. We used electrospray mass ionization spectrometry to show that the protein precipitate predominantly contained PBP 2a', with the beta-lactam bound to it in a 1:1 molar ratio. Using nitrocefin, a chromogenic beta-lactam, we confirmed the correlation between PBP 2a' precipitation and its beta-lactam-dependent enzymatic acylation by monitoring the absorbance associated with the precipitate. Finally, dissolving the precipitate in urea, we developed a simple in vitro chromogenic assay to monitor beta-lactam-dependent enzymatic acylation of PBP 2a'. This assay represents a significant improvement over the traditional radioactive penicillin-binding assay.

Perfluorocarbon-associated gas exchange in normal and acid-injured large sheep.

OBJECTIVES: We hypothesized that a) perfluorocarbon-associated gas exchange could be accomplished in normal large sheep; b) the determinants of gas exchange would be similar during perfluorocarbon-associated gas exchange and conventional gas ventilation; c)in large animals with lung injury, perfluorocarbon-associated gas exchange could be used to enhance gas exchange without adverse effects on hemodynamics; and d) the large animal with lung injury could be supported with an FIO2 of <1.0 during perfluorocarbon-associated gas exchange. DESIGN: Prospective, observational animal study and prospective randomized, controlled animal study. SETTING: An animal laboratory in a university setting. SUBJECTS: Thirty adult ewes. MEASUREMENT AND MAIN RESULTS: Five normal ewes (61.0 +/- 4.0 kg) underwent perfluorocarbon-associated gas exchange to ascertain the effects of tidal volume, end-inspiratory pressure, and positive end-expiratory pressure (PEEP) on oxygenation. Respiratory rate, tidal volume, and minute ventilation were studied to determine their effects on CO2 clearance. Sheep, weighing 58.9 +/- 8.3 kg, had lung injury induced by instilling 2 mL/kg of 0.05 Normal hydrochloric acid into the trachea. Five minutes after injury, PEEP was increased to 10 cm H2O. Ten minutes after injury, sheep with Pao2 values of <100 torr (<13.3 kPa) were randomized to continue gas ventilation (control, n=9) or to institute perfluorocarbon-associated gas exchange (n=9) by instilling 1.6 L of unoxygenated perflubron into the trachea and resuming gas ventilation. Blood gas and hemodynamic measurements were obtained throughout the 4-hr study. Both tidal volume and end-inspiratory pressure influenced oxygenation in normal sheep during perfluorocarbon-associated gas exchange. Minute ventilation determined CO2 clearance during perfluorocarbon-associated gas exchange in normal sheep. After acid aspiration lung injury, perfluorocarbon-associated gas exchange increased PaO2 and reduced intrapulmonary shunt fraction. Hypoxia and intrapulmonary shunting were unabated after injury in control animals. Hemodynamics were not influenced by the institution of perfluorocarbon-associated gas exchange. CONCLUSIONS: Tidal volume and end-inspiratory pressure directly influence oxygenation during perfluorocarbon-associated gas exchange in large animals. Minute ventilation influences clearance of CO2. In adult sheep with acid aspiration lung injury, perfluorocarbon-associated gas exchange at an FIO2 of <1.0 supports oxygenation and improves intrapulmonary shunting, without adverse hemodynamic effects, when compared with conventional gas ventilation.

Capillary electrophoretic analysis of serine hydroxymethyltransferase in Escherichia coli fermentation broth.

Serine hydroxymethyltransferase (SHMT) expressed in Escherichia coli was analyzed in fermentation broth through the use of capillary electrophoresis (CE), a method which provided advantages over the traditional techniques of slab gel electrophoresis and chromatography. In addition, via CE the difficult resolution and quantitation of SHMT holoenzyme and apoenzyme were achieved. Using this method, a pyridoxal-5'-phosphate (PLP) cofactor/SHMT dimer molar ratio of 0.65 was estimated to be present in holoenzyme in the absence of excess PLP. This determination correlated well with results obtained by other techniques, including electrospray ionization mass spectrometry (ESI-MS). CE and ESI-MS analyses both provided evidence for significant differences between the folded conformations of SHMT holoenzyme and apoenzyme.

Determination of ammonia, ethanol and acetic acid in solution using membrane introduction mass spectrometry.

Methods have been developed to allow applications of membrane introduction mass spectrometry (MIMS) to monitor solution phase components of fermentation broths using electron ionization. The solutions are transported by flow injection analysis (FIA) through a direct insertion membrane probe, fitted with a silicone membrane in the sheet configuration. Analytes of interest pass through the membrane and are ionized by electron implant ionization. The compounds monitored are ammonia, acetic acid, and ethanol, with ammonia being detected as the monochloramine derivative which is generated at pH 10 upon addition of hypochlorite. Quantitation is achieved using external standard solutions. The dynamic range for the quantification of ammonia is 2-8000 ppm, and for ethanol and acetic acid 10-1000 ppm. This method provides rapid detection of analytes of interest, on-line monitoring capabilities, and the advantage of electron ionization. The introduction of samples into the mass spectrometer is achieved readily and automatically, the response time is a few seconds, and there are no memory effects.

Conformer selection of protein ions by ion mobility in a triple quadrupole mass spectrometer.

Electrospray mass spectra of multiply charged protein molecules show two distinct charge state distributions proposed to correspond to a more highly charged, open conformational form and a lower charged, folded form. Elastic collisions carried out in the radiofrequency-only collision cell of a triple quadrupole mass spectrometer have dramatic effects on the appearance of the mass spectra. The different cross sectional areas of the conformers allow preferential selection of one charge state distribution over the other on the basis of ion mobility. Preferential selection is dependent on the nature and pressure of the target gas as well as the nature of the protein. In the case of positively charged horse heart apomyoglobin (MW 16,951 da), a high charge state distribution centered around (M + 20H)(20+) predominates at low target gas pressures and a second distribution centered around (M + 10H)(10+) predominates at high target gas pressures. Bimodal distributions are observed at intermediate pressures and, remarkably, charge states between the two distributions are not effectively populated under most of the conditions examined. Hard sphere collision calculations show large differences in collision frequencies and in the corresponding kinetic energy losses for the two conformational states and they demonstrate that the observed charge state selectivity can be explained through elastic collisions.

Metabolism of des(64,65)-human proinsulin in the rat. Evidence for the proteolytic processing to insulin.

The metabolism of des(64,65)-human proinsulin was examined in rats after subcutaneous administration. Profiles of circulating insulin-like immunoreactivity in rat plasma 25 min after subcutaneous administration were evaluated by anion exchange fast protein liquid chromatography and reversed-phase high-performance liquid chromatography. Both techniques indicated the presence of circulating immunoreactivity having retention characteristics of human insulin. This metabolite peak comprised 5-10% of circulating immunoreactivity; the remainder had retention characteristics of des(64,65)-human proinsulin. The peaks of immunoreactive material were isolated and their structure determined using reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry. The major circulating component co-eluted with des(64,65)-human proinsulin and had an identical mass spectrum. Two circulating metabolites were identified. These metabolites co-eluted by reversed-phase high-performance liquid chromatography with human insulin and diarginyl(B31,32)-human insulin and had mass spectra identical to the standard compounds. The data indicate proteolytic processing of des(64,65)-human proinsulin involves an initial tryptic cleavage at the carboxy side of ArgB32, with the formation of human insulin by the subsequent action of a carboxypeptidase to remove the ArgB31-ArgB32 dipeptide from diarginyl(B31,32)-human insulin. The results suggest that some of the pharmacological activity of des(64,65)-human proinsulin may be mediated in part by circulating insulin-like metabolites.

Characterization of the interaction between human alpha-thrombin and methyl 3-(2-methyl-1-oxopropoxy)[1]benzothieno[3,2-b]furan-2-carboxylate (LY806303) using electrospray mass spectrometry and tandem mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) have been used for the first time to study the interaction of human alpha-thrombin with methyl 3-(2-methyl-1-oxopropoxy)[1]benzothieno[3,2-b]furan-2-carbox ylate (LY806303; 1), a potent and selective inhibitor whose mechanism of action was never fully defined. Using ESI-MS, it is shown that inhibitor 1 covalently modifies human alpha-thrombin as evidenced by a shift in the molecular weight of the native protein by 72 Da, which is consistent with isobutyrylation (C4H7O; 71 Da) of the enzyme at a single site. Tryptic digestion of the modified protein and tandem mass spectral analysis of isolated peptide fragments indicate that compound 1 acylates Ser-205 of the heavy chain of alpha-thrombin. Ser-205, along with His-43 and Asp-99 make up the catalytic triad within the active site of thrombin.

Proteolysis of human growth hormone by rat thyroid gland in vitro: application of electrospray mass spectrometry and N-terminal sequencing to elucidate a metabolic pathway.

The present studies were designed to provide structural characterization of peptide metabolites of biosynthetic human growth hormone (hGH) formed by rat thyroid gland proteases in vitro. Electrospray ionization mass/spectrometry (ESI-MS) and N-terminal sequencing were used to characterize the peptide metabolites. The predominant enzyme in the thyroid gland preparations was a chymotrypsin-like serine protease which was biochemically similar to rat mast cell protease-I. Metabolic intermediates were formed by cleavage of hGH exclusively at Tyr/Phe/Leu-Xaa bonds. After a 5- or 45-min incubation of hGH with thyroid gland S9 pellet fraction, the majority of metabolites formed were two-chain variants of hGH having masses ranging from 16,002 to 22,143 Da. These metabolites were formed as a result of proteolysis in the large disulfide loop region of hGH in combination with processing at Tyr42-Ser43. Based upon the time-related appearance and structural characterization of these intermediates, a sequence of metabolic events is proposed. The initial event appears to be cleavage by the chymotrypsin-like protease between Tyr143-Ser144 to form a two-chain hGH. This intermediate is then cleaved between Tyr42-Ser43, liberating the N-terminal peptide, Phe1-Tyr42. Two other metabolites were generated as a result of the deletion of the peptides Lys140-Tyr143 and Ser144-Phe146 from the large loop region. The identification of similar metabolites truncated by a single amino acid at the carboxyl terminus indicated the action of a carboxypeptidase on these metabolic products. After a 4.5-hr incubation, the protease isolated from the S9 pellet fraction degraded hGH to > 20 small peptides, having masses < or = 2300 Da.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermospray liquid chromatography-mass spectrometry with a quadrupole ion trap mass spectrometer.

A theromospray ion source using corona discharge ionization was interfaced to a quadrupole ion trap mass spectrometer via a multi-element lens system. Ions were injected into the trap periodically where they were stabilized by collisions with helium bath gas. Mass spectra were recorded on the trapped ions using the mass-selective instability scan mode. Data are shown for a peptide and a nucleoside and the effects of some experimental variables on the spectra are explored.

Pulsed gas introduction into quadrupole ion traps.

Two different Paul-type quadrupole ion traps were equipped with pulsed-valve gas inlets. The duration of a gas pulse inside the trap is variable, and pulses as short as 50 ms (FWHH) have been measured, allowing the use of several gas pulses during one experiment. The benefits of pulsed valves are outlined and demonstrated for chemical ionization experiments and for the use of selective ion-molecule reactions in structure determination of ions and neutral molecules.

Progressive necrotizing surgical infections--a unified approach.

Four types of progressive necrotizing surgical infections have been described, based on the type of infecting organism, the type and depth of soft tissue penetration and clinical findings, and the type of surgical therapy recommended, e.g., necrotizing fasciitis, clostridial cellulitis. A mortality rate of up to 50% continues to be reported. An experience with 20 such cases indicates that there is a considerable overlap in clinical-physical findings and bacteriology such that classification schemes are confusing and lead to treatment delays and the use of inappropriate therapy. The infections all seem to be variations of the same disease process, a spreading, necrotizing infection. Of the 20 cases, four were treated with antibiotics and delayed (1 to 3 days) excision of necrotic tissue with 75% deaths; four cases received antibiotics and multiple surgical incisions with 100% deaths. The other 12 cases were treated with a unified approach of resuscitation, antibiotics (penicillin, clindamycin, tobramycin), immediate surgical excision (3 to 4 hours) of all necrotic tissue, aggressive nutritional support, and early skin coverage, with an 8.3% mortality. We conclude that there seems to be no need to classify necrotizing infections into different types. Recognizing them as the same disease process and treating them with a unified approach resulted in a significant reduction in mortality.

Microtechniques for the gas chromatographic determination of barbiturates in small blood samples.

A method for the determination of therapeutic levels of barbituric acids in 25 microliter of whole blood is described. After extraction and controlled concentration of the extract to a volume of 5 microliter, the barbituric acids are N,N'-dimethylated using a microrefluxer. Of the total extract 20-100% is injected into the gas chromatograph. Low blanks, recoveries of 70--80% and peak ratios that are comparable to those in calibration experiments are obtained provided the detailed working instructions are followed strictly. In addition, barbiturates were determined (1 ng in 25 microliter blood) using column-switching devices and nitrogen-sensitive detection.