Plant Physiol, 1994 Nov, 106(3), 1015 - 1022 Differential Transcript Levels of Genes Associated with Glycolysis and Alcohol Fermentation in Rice Plants (Oryza sativa L.) under Submergence Stress; Umeda M et al.; Expression of genes encoding enzymes involved in specialized metabolic pathways is assumed to be regulated coordinately to maintain homeostasis in plant cells . We analyzed transcript levels of rice (Oryza sativa L.) genes associated with glycolysis and alcohol fermentation under submergence stress . When each transcript was quantified at several times, two types (I and II) of mRNA accumulation were observed in response to submergence stress . Transcripts of type I genes reached a maximum after 24 h of submergence and were reduced by transfer to aerobic conditions or by partial exposure of shoot tips to air . In a submergence-tolerant rice cultivar, transcript amounts of several type I genes, such as glucose phosphate isomerase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase, and enolase, increased significantly compared to an intolerant cultivar after 24 h of submergence . This suggests that the mRNA accumulation of type I genes increases in response to anaerobic stress . mRNA accumulation of type II genes, such as aldolase and pyruvate kinase, reached a maximum after 10 h of submergence . Following transfer to aerobic conditions, their transcript levels were not so rapidly decreased as were type I genes . These results suggest that the mRNA levels of genes engaged in glycolysis and alcohol fermentation may be regulated differentially under submergence stress. Plant Physiol, 1994 Jun, 105(2), 651 - 657 Improved Cytoplasmic pH Regulation, Increased Lactate Efflux, and Reduced Cytoplasmic Lactate Levels Are Biochemical Traits Expressed in Root Tips of Whole Maize Seedlings Acclimated to a Low-Oxygen Environment; Xia JH et al.; We tested the hypothesis (J.-H . Xia and P.H . Saglio {1992} Plant Physiol 100: 40-46) that the enhanced ability of maize (Zea mays) root tips to survive anoxia, elicited by a 4-h exposure to 3% O2 ("acclimation"), is due to less cytoplasmic acidosis early in anoxia . Cytoplasmic pH and fermentation reactions were monitored in excised and intact (attached) maize root tips by simultaneous in vivo 13C- and 31P-NMR spectroscopy . We demonstrate that both excised and intact acclimated root tips have significantly higher cytoplasmic pH values under anoxia . This reduction in cytoplasmic acidosis is greater in intact root tips . Remarkably, cytoplasmic pH does not change when root tips are transferred from 3% O2 to anoxia . The earlier observation of considerable lactate efflux and lowered intracellular lactate in excised, acclimated root tips (ibid.) was extended to intact seedlings . The predominant fermentation end product retained in the cells of acclimated root tips is alanine . We discuss the relationship between cytoplasmic pH and levels of intracellular lactate and alanine in sugar-replete roots, and the role of cytoplasmic pH in determining survival under anoxia. Plant Physiol, 1994 May, 105(1), 61 - 67 Hypoxic Induction of Anoxia Tolerance in Roots of Adh1 Null Zea mays L; Johnson JR et al.; Seedlings of alcohol dehydrogenase 1 null mutants (Adh1-) of Zea mays L., which fail to synthesize alcohol dehydrogenase 1 (ADH1) isozymes, were hypoxically acclimated by 18 h of exposure to an atmosphere of 4% (v/v) O2 in N2 at 25{deg}C . Their ability to tolerate subsequent anoxia by exposure to anaerobic (O2-free) conditions was compared with that of unacclimated seedlings that were transferred immediately from an atmosphere of 40% (v/v) O2 to anaerobic conditions . Only 10% of the root tips of unacclimated seminal roots survived 6 h of anoxia, whereas 70% of the hypoxically acclimated root tips were viable at 24 h . During anoxia, acclimated root tips had enhanced ADH activity compared with unacclimated root tips, through induction of Adh2 . Despite this, enzyme activity was still only about 5% that of acclimated, wild-type root tips and about half that of unacclimated, wild-type root tips . During anoxia, acclimated Adh1- root tips showed a higher rate of anaerobic respiration and ethanol production, greater concentrations of ATP and total adenylates, and a greater adenylate energy charge compared with unacclimated root tips . These results suggest that although enhanced ADH activity may have raised fermentation rates in acclimated Adh1- tissues and thereby contributed to energy metabolism and viability, the high levels of ADH activity inducible in acclimated, wild-type maize root tips appear to be in excess of that required to increase rates of fermentation. Plant Physiol, 1993 Apr, 101(4), 1163 - 1168 Long-Term Anaerobic Metabolism in Root Tissue (Metabolic Products of Pyruvate Metabolism); Good AG et al.; The onset of anaerobiosis in barley root tissue (Hordeum vulgare L . cv Himalaya) results in the following metabolic responses . There are rapid increases in the levels of pyruvate, lactate, and ethanol . Malate and succinate concentrations increase over the first 12 h, after which they return to the levels found in oxygenated root tissue . Alanine concentration increases over the first 12 h, and this is matched by a corresponding decrease in aspartate . The initial stoichiometric decline in aspartate and increase in alanine suggests that the amino group of aspartate is conserved by transaminating pyruvate to alanine . Aspartate catabolism also probably provides the initial source of carbon for reduction to succinate under anoxic conditions . Under long-term anaerobiosis (>24 h), there is no further accumulation of any of the fermentative end products other than ethanol, which also represents the major metabolic end product during long-term anaerobiosis . Although a number of the enzymes involved in fermentative respiration have been found to be induced under anaerobic conditions, neither aspartate amino-transferase nor malate dehydrogenase is induced in barley root tissue . The observations suggest that the long-term adaptations to hypoxic conditions may be quite different than the more well-characterized short-term adaptations. Plant Physiol, 1993 Feb, 101(2), 553 - 560 Evidence for a Large and Sustained Glycolytic Flux to Lactate in Anoxic Roots of Some Members of the Halophytic Genus Limonium; Rivoal J et al.; Soil salinity and anaerobiosis often occur together . This led us to investigate the fermentative metabolism in roots of species from the halophytic genus Limonium (Plumbaginaceae) . Root segments from hypoxically induced plants were incubated for 8 h under strict anoxia in the presence of {U-14C}glucose . In three species (Limonium latifolium, L . nashii, and L . humile), the pattern of 14C-labeled end products was typical of higher plants, with a 14C flux to ethanol higher than that to lactate . However, in four species (L . ramosissimum, L . gougetianum, L perezii, and L . sinuatum), the rate of lactate fermentation was exceptionally high, and in the latter two species the 14C flux to lactate exceeded that to ethanol . These two species secreted most of the lactate produced into the medium . Calculations indicated that the cytoplasm would have been lethally acidified had this secretion not occurred . The effects of factors that might control lactate fermentation or secretion (O2 partial pressure, pH, salt concentration) were studied in two contrasting species: L . sinuatum and L . latifolium . In both species, the lactate:ethanol ratio was higher under hypoxia (0.1-3 kPa O2 partial pressure) than under strict anoxia . In L . sinuatum, this ratio was slightly increased by increasing the pH of the medium from 5.5 to 7.5, but salinity treatment had no effect . The potential contribution of lactate fermentation to the overall carbon and energy metabolism of halophytes is discussed. Plant Physiol, 1993 Feb, 101(2), 407 - 414 Hypoxic and Anoxic Induction of Alcohol Dehydrogenase in Roots and Shoots of Seedlings of Zea mays (Adh Transcripts and Enzyme Activity); Andrews DL et al.; Alcohol dehydrogenase (ADH) is one of a number of enzymes of glycolysis and fermentation known to be synthesized preferentially under low O2 conditions . We examined levels of Adh1 transcripts and of ADH activity in 5-mm root tips, root axes (the remainder of the seminal root), and shoots of maize (Zea mays L . cv TX 5855) seedlings . Seedlings with roots averaging about 60-mm long were transferred from fully aerobic conditions (solutions sparged with 40% {v/v} O2) to anaerobic (O2-free) conditions, or to an intermediate O2 concentration . There was no prior acclimation to low O2 . In root tips, anoxia induced Adh1 transcripts and enzyme activity at 6 h, but this was followed by a rapid decline so that at 12 to 18 h neither were detectable and the root tips were dead . In contrast, higher levels of Adh1 transcripts and enzyme activity were maintained for at least 48 h in root axes and shoots . When induction at 6 h was measured over a wide range of O2 concentrations, a peak in ADH activity occurred in all tissues at 4% (v/v) O2 . Maximum levels of transcripts, however, were in the range of 0 to 4% O2, depending on the tissue . The time course of hypoxic induction (at 4% O2) in root tips showed a peak in transcript levels at 6 h, whereas ADH activity continued to rise throughout the 24-h experiment . These results show that in root tips, ADH induction by anoxia was small and transient relative to induction by hypoxia. Steroids, 2002 Sep, 67(10), 869 - 72 Microbial transformation of hydrocortisone by Acremonium strictum PTCC 5282; Faramarzi MA et al.; The ability of a genus of cephalosporium-like fungus isolated from soil, Acremonium strictum PTCC 5282, for hydrocortisone biotransformation has been investigated . This potential had not been previously examined . The fermentation yielded 11beta,17beta-dihydroxyandrost-4-en-3-one, 11beta,17alpha,20beta,21-tetrahydroxypregn-4-en-3-one and 21-acetoxy-11beta,17alpha,20-trihydroxypregn-4-en-3-one . Each microbial metabolite was purified and characterized using spectroscopic methods. Eur J Biochem, 2002 Sep, 269(18), 4495 - 504 Directed evolution of a glutaryl acylase into an adipyl acylase; Sio CF et al.; Semi-synthetic cephalosporin antibiotics belong to the top 10 of most sold drugs, and are produced from 7-aminodesacetoxycephalosporanic acid (7-ADCA) . Recently new routes have been developed which allow for the production of adipyl-7-ADCA by a novel fermentation process . To complete the biosynthesis of 7-ADCA a highly active adipyl acylase is needed for deacylation of the adipyl derivative . Such an adipyl acylase can be generated from known glutaryl acylases . The glutaryl acylase of Pseudomonas SY-77 was mutated in a first round by exploration mutagenesis . For selection the mutants were grown on an adipyl substrate . The residues that are important to the adipyl acylase activity were identified, and in a second round saturation mutagenesis of this selected stretch of residues yielded variants with a threefold increased catalytic efficiency . The effect of the mutations could be rationalized on hindsight by the 3D structure of the acylase . In conclusion, the substrate specificity of a dicarboxylic acid acylase was shifted towards adipyl-7-ADCA by a two-step directed evolution strategy . Although derivatives of the substrate were used for selection, mutants retained activity on the beta-lactam substrate . The strategy herein described may be generally applicable to all beta-lactam acylases. Pak J Sci, 1968 Jan-Mar, 20(1 and 2), 64 - 7 Contraceptives and other steroid drugs: their production from steroidal sapogenins; Fazli FR; PIP: Sterols, steroidal sapogenins, steroidal alkaloids and alkaloidal amines derived from plant sources provide the starting materials for steroid production . Sarmentogenin (III) a cardiac glycoside, was first used, but the source was limited . Hecogenin (IV), a saponin (Agave sislana), was manufactured to cortisone by the process of Spensley et al . Introduction of an oxygen atom at carbon 11 by microbiological means gave a new series of starting compounds, among them, diosgenin (V) which converts to progesterone, to 11 hydroxyprogesterone by fermentation, cortisone, hydrocortisone and delta compounds . Reviews on the development, physiological and biochemical aspects of oral contraceptives were mentioned . A steroid with activity equivalent to progesterone was made by Ehrenstein in a 12-step synthesis from a cardiac aglycone strophanthidin . Estradiol converted to 19-nor testosterone by Birch and Mukherji provided a breakthrough in production of 19-nor steroids and led to production of 19-nor progesterone (VI) with a higher activity than progesterone . 19-nor-17alpha-ethynyltestosterone (VIII), its acetate derivative, and a related compound (I) account for 80% of consumption of oral contraceptives in the United States . Reviews of nor-steroids by Colton and Kilmstra, and of the chemical developments leading to currently used steroid contraceptives by Djerassi are mentioned . Plant Physiol, 1995 Nov, 109(3), 1069 - 1076 Amylolytic Activities in Cereal Seeds under Aerobic and Anaerobic Conditions; Guglielminetti L et al.; An adequate carbohydrate supply contributes to the survival of seeds under conditions of limited oxygen availability . The amount of soluble, readily fermentable carbohydrates in dry cereal seeds is usually very limited, with starch representing the main storage compound . Starch breakdown during the germination of cereal seeds is the result of the action of hydrolytic enzymes and only through the concerted action of {alpha}-amylase (EC 3.2.1.1), {beta}-amylase (EC 3.2.1.2), debranching enzyme (EC 3.2.1.41), and {alpha}-glucosidase (EC 3.2.1.20) can starch be hydrolyzed completely . We present here data concerning the complete set of starch-degrading enzymes in three cereals, rice (Oryza sativa L.), which is tolerant to anaerobiosis, and wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), which are unable to germinate under anoxia . Among the cereal seeds tested under anoxia, only rice is able to degrade nonboiled, soluble starch, reflecting the ability to degrade the starch granules in vivo . This is explained by the presence of the complete set of enzymes needed to degrade starch completely either as the result of de novo synthesis ({alpha}-amylase, {beta}-amylase) or activation of preexisting, inactive forms of the enzyme (debranching enzyme, {alpha}-glucosidase) . These enzymes are either absent or inactive in wheat and barley seeds kept under anaerobic conditions. Plant Physiol, 1995 Oct, 109(2), 659 - 665 Chill-Induced Changes in the Activity and Abundance of the Vacuolar Proton-Pumping Pyrophosphatase from Mung Bean Hypocotyls; Darley CP et al.; Changes in the properties of extractable vacuolar H+-pumping pyrophosphatase (V-PPase) and vacuolar ATPase activities in chilling-sensitive seedlings of mung bean (Vigna radiata) were investigated . Following chilling at 4{deg}C for 48 h, both hydrolytic and proton-pumping activities of the V-PPase increased 1.5- to 2-fold over controls and remained elevated even after 72 h at low temperatures . Vacuolar ATPase levels did not change significantly throughout the chilling regime . However a large increase in alcohol dehydrogenase activity during chilling suggests a shift toward fermentative metabolism, which can be expected to decrease ATPase activity in situ . Western blotting of vacuolar membrane-enriched fractions from control and treated plants has confirmed that the changes in V-PPase activity are mirrored by increases in the amount of pump protein . Results suggest a specific role for the V-PPase in protecting chill-sensitive plants from the injurious effects of low temperatures via the maintenance of the proton gradient across the vacuolar membrane. Plant Physiol, 1995 Oct, 109(2), 353 - 361 Alternative Oxidase Activity in Tobacco Leaf Mitochondria (Dependence on Tricarboxylic Acid Cycle-Mediated Redox Regulation and Pyruvate Activation); Vanlerberghe GC et al.; Transgenic Nicotiana tabacum (cv Petit Havana SR1) containing high levels of mitochondrial alternative oxidase (AOX) protein due to the introduction of a sense transgene(s) of Aox1, the nuclear gene encoding AOX, were used to investigate mechanisms regulating AOX activity . After purification of leaf mitochondria, a large proportion of the AOX protein was present as the oxidized (covalently associated and less active) dimer . High AOX activity in these mitochondria was dependent on both reduction of the protein by DTT (to the noncovalently associated and more active dimer) and its subsequent activation by certain {alpha}-keto acids, particularly pyruvate . Reduction of AOX to its more active form could also be mediated by intramitochondrial reducing power generated by the oxidation of certain tricarboxylic acid cycle substrates, most notably isocitrate and malate . Our evidence suggests that NADPH may be specifically required for AOX reduction . All of the above regulatory mechanisms applied to AOX in wild-type mitochondria as well . Transgenic leaves lacking AOX due to the introduction of an Aox1 antisense transgene or multiple sense transgenes were used to investigate the potential physiological significance of the AOX-regulatory mechanisms . Under conditions in which respiratory carbon metabolism is restricted by the capacity of mitochondrial electron transport, feed-forward activation of AOX by mitochondrial reducing power and pyruvate may act to prevent redirection of carbon metabolism, such as to fermentative pathways. Biol Rev Camb Philos Soc, 2002 Aug, 77(3), 443 - 53 Carbohydrates: a limit on bacterial diversity within the colon; Rabiu BA et al.; The human large intestine is recognised as a physiologically important organ responsible for the conservation of water and salts . Through its resident bacteria, it is also capable of complex, enzyme catalysed, hydrolytic-digestive functions that have a high biological impact on the host . These microorganisms metabolise dietary components, principally complex carbohydrates that are not hydrolysed or absorbed in the upper gastrointestinal tract, and in this way, sequester energy for the host, through fermentation . This process involves a series of anaerobic, energy-yielding, catabolic reactions which complete digestive processes in the gut, resulting in end products that in turn influence the distribution of microbial species present as well as having some systemic effects . Some of the bacteria are thought to possess important health-promoting activities, especially with respect to their influence on mucosal and systemic immune responses to disease . These bioactivities can be modulated by substrates that support and influence microbial development, growth and survival . For these reasons, it is necessary to review dietary factors that may delimit bacterial diversity, to be able to predict responses and sensitivities to various environmental pressures and manipulations that occur in this area of human microbiology. Appl Microbiol Biotechnol, 2002 Sep, 59(6), 721 - 6 Epub 2002 Jul 19. Isolation and characterization of a Trichodermastrain capable of fermenting cellulose to ethanol; Stevenson DM et al.; The direct fermentation of cellulosic biomass to ethanol has long been a desired goal . To this end, we screened the environment for fungal strains capable of this conversion when grown on minimal medium . One strain, identified as a member of the genus Trichoderma and designated strain A10, was isolated from cow dung and initially produced about 0.4 g ethanol l(-1) . This strain cannot grow on any substrate under anaerobic conditions, but can ferment microcrystalline cellulose or several sugars to ethanol . Ethanol accumulation was eventually increased, by selection and the use of a vented fermentation flask, to 2 g l(-1) when the fermentation was carried out in submerged culture in minimal medium . The highest levels of ethanol, >5.0 g l(-1), were obtained by the fermentation of glucose . Little ethanol was produced by the fermentation of xylose, although other fermentation products such as succinate and acetate were observed . Strain A10 was also found to utilize (aerobically) a wide range of carbon sources . In addition, auxotrophic mutants were generated and used to demonstrate parasexuality by complementation between auxotrophs and between morphological mutants . The ability of this strain to use a wide variety of carbohydrates (including crystalline cellulose) combined with its minimal nutrient requirements and the availability of a genetic system suggests that the strain merits further investigation of its ability to convert biomass to ethanol. Plant Physiol, 2002 Sep, 130(1), 256 - 64 Biochemical characterization of the Arabidopsis protein kinase SOS2 that functions in salt tolerance; Gong D et al.; The Arabidopsis Salt Overly Sensitive 2 (SOS2) gene encodes a serine/threonine (Thr) protein kinase that has been shown to be a critical component of the salt stress signaling pathway . SOS2 contains a sucrose-non-fermenting protein kinase 1/AMP-activated protein kinase-like N-terminal catalytic domain with an activation loop and a unique C-terminal regulatory domain with an FISL motif that binds to the calcium sensor Salt Overly Sensitive 3 . In this study, we examined some of the biochemical properties of the SOS2 in vitro . To determine its biochemical properties, we expressed and isolated a number of active and inactive SOS2 mutants as glutathione S-transferase fusion proteins in Escherichia coli . Three constitutively active mutants, SOS2T168D, SOS2T168D Delta F, and SOS2T168D Delta 308, were obtained previously, which contain either the Thr-168 to aspartic acid (Asp) mutation in the activation loop or combine the activation loop mutation with removal of the FISL motif or the entire regulatory domain . These active mutants exhibited a preference for Mn(2+) relative to Mg(2+) and could not use GTP as phosphate donor for either substrate phosphorylation or autophosphorylation . The three enzymes had similar peptide substrate specificity and catalytic efficiency . Salt overly sensitive 3 had little effect on the activity of the activation loop mutant SOS2T168D, either in the presence or absence of calcium . The active mutant SOS2T168D Delta 308 could not transphosphorylate an inactive protein (SOS2K40N), which indicates an intramolecular reaction mechanism of SOS2 autophosphorylation . Interestingly, SOS2 could be activated not only by the Thr-168 to Asp mutation but also by a serine-156 or tyrosine-175 to Asp mutation within the activation loop . Our results provide insights into the regulation and biochemical properties of SOS2 and the SOS2 subfamily of protein kinases. Plant Physiol, 1997 Mar, 113(3), 925 - 932 Dynamics of Acetaldehyde Production during Anoxia and Post-Anoxia in Red Bell Pepper Studied by Photoacoustic Techniques; Zuckermann H et al.; Acetaldehyde (AA), ethanol, and CO2 production in red bell pepper (Capsicum annum L.) fruit has been measured in a continuous flow system as the fruit was switched between 20% O2 and anaerobic conditions . Minimum gas phase concentrations of 0.5 nL L-1, 10 nL L-1, and 1 mL L-1, respectively, can be detected employing a laser-based photoacoustic technique . This technique allows monitoring of low production rates and transient features in real time . At the start of anaerobic treatment respiration decreases by 60% within 0.5 h, whereas AA and ethanol production is delayed by 1 to 3 h . This suggests a direct slow-down of the tricarboxylic acid cycle and a delayed onset of alcoholic fermentation . Reexposure of the fruit to oxygen results in a 2- to 10-fold upsurge in AA production . A short anoxic period leads to a sharp transient peak lasting about 40 min, whereas after numerous and longer anoxic periods, post-anoxic AA production stays high for several hours . High sensitivity of the fruit tissue to oxygen is further evidenced by a sharp decrease in post-anoxic AA production upon an early return to anaerobic conditions . Ethanol oxidation by the "peroxidatic" action of catalase is proposed to account for the immediate post-anoxic AA upsurge. Plant Physiol, 1997 Feb, 113(2), 657 - 661 Molecular Genetic Evidence of the Ability of Alternative Oxidase to Support Respiratory Carbon Metabolism; Vanlerberghe GC et al.; With the cytochrome pathway inhibited, AOX was able to support considerable growth of cultured tobacco (Nicotiana tabacum cv Petit Havana SR1) cells but the efficiency of carbon utilization decreased dramatically . Antisense cells with decreased AOX protein did not grow, whereas sense cells with elevated AOX protein had higher growth and respiration rates than the wild type . In antisense cells a large accumulation of pyruvate resulted in aerobic ethanolic fermentation. Int J Food Microbiol, 2002 Sep 15, 78(1-2), 119 - 31 Commercial bacterial starter cultures for fermented foods of the future; Hansen EB; Starter cultures for fermented foods are today developed mainly by design rather than by screening . The design principles are based on knowledge of bacterial metabolism and physiology as well as on the interaction with the food product . In the genomics era, we will obtain a wealth of data making design on a rational basis even simpler . The design tools available are food grade tools for genetic, metabolic and protein engineering and an increased use of laboratory automation and high throughput screening methods . The large body of new data will influence the future patterns of regulation . It is currently difficult to predict in what direction the future regulatory requirements will influence innovation in the food industry . It can either become a promoting force for the practical use of biotechnology to make better and safer products, or it can be limiting the use of starter cultures to a few strains with official approval . Successful cultures based on modern technology is expected to be launched in the areas of: probiotics, bioprotection, general improvement of yield and performance for the existing culture market and probably the introduction of cultures for fermenting other food products . A scientific basis for dramatic innovations that could transform the culture industry is currently being established. J Nutr, 2002 Sep, 132(9), 2638 - 43 A poorly fermented gel from psyllium seed husk increases excreta moisture and bile acid excretion in rats; Marlett JA et al.; Psyllium seed husk (PSH) increases stool output and lowers blood cholesterol levels in humans . PSH and three fractions isolated from it were meal-fed to colectomized rats and fermented in vitro to test the hypothesis that viscous, gel-forming fraction B was responsible for these physiological actions . Control rats were fed 50 g/kg cellulose . The concentration of each PSH fraction in the test meals was equivalent to its concentration in PSH . Yields of the fractions were: A, 171; B, 575; and C, 129 g/kg of PSH . The wet weight and moisture content of ileal excreta (IE) from rats fed test meals containing PSH or fraction B were greater than those measured in excreta from rats fed meals containing cellulose or the other two PSH fractions . Total bile acids in IE did not differ between rats fed PSH or fraction B and were greater in these groups than in the other groups . Fraction A was not fermented during 3 d of incubation; fraction B was poorly fermented, with approximately 30% of the constituent sugars disappearing; and fraction C was rapidly and nearly completely fermented . These results indicate that the gel-forming fraction we isolated from PSH is the physiologically active component of the husks. Trends Biotechnol, 2002 Oct, 20(10), 426 - 32 Meeting the consumer challenge through genetically customized wine-yeast strains; Pretorius IS et al.; Wine producers are facing intensifying competition brought about by a widening gap between wine production and wine consumption, a shift of consumer preferences away from basic commodity wine to top quality wine, and economic globalization . Consequently, they are calling for a total revolution in the 'magical' world of wine . The process of transforming the wine industry from a production- to a market-orientated industry results in an increasing dependence on, amongst others, biotechnological innovation . Market-orientated wine-yeast strains are currently being developed for the cost-competitive production of wine with minimized resource inputs, improved quality and low environmental impact . The emphasis is on the development of Saccharomyces cerevisiae strains with improved fermentation, processing and biopreservation abilities, and capacities for an increase in the wholesomeness and sensory quality of wine. Anal Bioanal Chem, 2002 Sep, 374(1), 167 - 72 Epub 2002 Aug 10. Determination of yeast assimilable nitrogen content in wine fermentations by sequential injection analysis with spectrophotometric detetection; Muik B et al.; An automated method for measuring the primary amino acid concentration in wine fermentations by sequential injection analysis with spectrophotometric detection was developed . Isoindole-derivatives from the primary amino acid were formed by reaction with o-phthaldialdehyde and N-acetyl- L-cysteine and measured at 334 nm with respect to a baseline point at 700 nm to compensate the observed Schlieren effect . As the reaction kinetic was strongly matrix dependent the analytical readout at the final reaction equilibrium has been evaluated . Therefore four parallel reaction coils were included in the flow system to be capable of processing four samples simultaneously . Using isoleucine as the representative primary amino acid in wine fermentations a linear calibration curve from 2 to 10 mM isoleucine, corresponding to 28 to 140 mg nitrogen/L (N/L) was obtained . The coefficient of variation of the method was 1.5% at a throughput of 12 samples per hour . The developed method was successfully used to monitor two wine fermentations during alcoholic fermentation . The results were in agreement with an external reference method based on high performance liquid chromatography . A mean-t-test showed no significant differences between the two methods at a confidence level of 95%. J Dairy Sci, 2002 Aug, 85(8), 1976 - 87 Whole linted cottonseed as a forage substitute fed with ground or steam-flaked corn: digestibility and performance; Harvatine DI et al.; Six ruminally and duodenally cannulated Holstein cows were used in a 6 x 6 Latin square design . The objective was to evaluate any potential interactions in site of nutrient digestion when neutral detergent fiber (NDF) from cottonseed was incrementally substituted for forage NDF (FNDF) from alfalfa silage and when starch availability was varied by feeding ground (G) or steam-flaked (SF) corn . Iso-NDF diets were forage control with G corn (21% FNDF), 5% whole cottonseed (WCS) with G or SF corn (18% FNDF), 10% WCS with G or SF corn (15% FNDF), and 15% WCS with G corn (12% FNDF) . Ruminal or total tract digestibilities of organic matter (OM) or nonstructural carbohydrate (NSC) were unaffected, but efficiency of microbial protein synthesis decreased as WCS increased . Ruminal NDF digestibility was not affected despite a linear decrease in pH, but postruminal NDF digestibility decreased with increasing WCS . Ruminal digestibilities of OM and NSC were higher for SF than G corn but did not affect efficiency of microbial N synthesis . Dry matter intake increased quadratically with increasing level of WCS but decreased when SF replaced G corn . Milk yield did not differ across treatments . Milk fat percentage was affected quadratically and milk protein increased linearly with increasing WCS . Milk fat percentage decreased but milk protein was not affected when SF replaced G corn . Lack of an interaction between corn source and level of WCS substitution suggests that WCS was equally effective in maintaining ruminal fermentation and digestibility in diets varying in ruminal starch availability. J Dairy Sci, 2002 Aug, 85(8), 1969 - 75 The effect of delayed ensiling and application of a propionic acid-based additive on the fermentation of barley silage; Mills JA et al.; Prolonged exposure to air can adversely affect the silage fermentation process . To investigate a possible method to overcome this problem, we determined if a buffered propionic acid-based additive, applied to chopped, whole-plant barley exposed to air before ensiling, would affect the subsequent fermentation . Wilted forage was chopped and treated with nothing, or with 0.1% (wt/wt wet forage) of a buffered propionic acid-based additive and ensiled immediately in quadruplicate 20-L laboratory silos . Portions of the chopped forage, untreated and treated, were left in loose piles in a barn for 24 h before ensiling . Another portion of the untreated silage exposed to air for 24 h was also treated with 0.1% of the additive just before ensiling . Prolonged exposure to air before ensiling increased the numbers of yeasts on forages by more than 1,000-fold . The concentrations of water-soluble carbohydrates decreased by more than 50%; the ammonia-N concentrations increased 40%, and pH increased by more than 1 unit as a result of exposure to air . These changes were less in forage that was treated with the additive at chopping . After 60 d, silages of forages that were exposed to air before ensiling had a higher pH, higher concentrations of ammonia-N and butyric acid, and lower concentrations of lactic and acetic acids than silages of forage that had been ensiled immediately after harvest . In situ DM digestibility was lowest in untreated silages that had been exposed to air before ensiling . In contrast, treatment with the additive, applied before or after exposure to air, prevented the reduction in in vitro digestion. J Dairy Sci, 2002 Aug, 85(8), 1947 - 57 Effects of forage particle size and grain fermentability in midlactation cows . II . Ruminal pH and chewing activity; Krause KM et al.; Our study investigated the effects of, and interactions between, level of dietary ruminally fermentable carbohydrate (RFC) and forage particle size on rumen pH and chewing activity for dairy cows fed one level of dietary NDF . Also, correlations between intake, production, chewing, and ruminal pH parameters were investigated . Eight cows (61 days in milk) were assigned to four treatments in a double 4 x 4 Latin square . Treatments were arranged in a 2 x 2 factorial design; finely chopped alfalfa silage (FS) and coarse alfalfa silage (CS) were combined with concentrates based on either dry, cracked-shelled corn (DC; low RFC) or ground, high-moisture corn (HMC; high RFC) . Diets were fed ad libitum as a total mixed rations with a concentrate:forage ratio of 60:40 . Diets averaged 18.7% crude protein, 24.0% neutral detergent fiber, 18.3% , acid detergent fiber and 27.4% starch on a DM basis . Mean particle size of the four diets were 6.3, 2.8, 6.0, and 3.0 mm for DCCS, DCFS, HMCCS, and HMCFS, respectively . Decreasing forage particle size decreased ruminal pH from 6.02 to 5.81, and increasing level of RFC decreased pH from 5.99 to 5.85 . Minimum daily ruminal pH decreased from 5.66 to 5.47 when level of RFC was increased, and decreased from 5.65 to 5.48 when forage particle size decreased . Time below pH 5.8 per day increased from 7.4 h to 10.8 h when level of RFC increased, and increased from 6.4 h to 11.8 h when forage particle size was decreased . Area below 5.8 showed the same relationship with RFC and forage particle size . Also, forage particle size affected the postprandial pH pattern . Cows spent more time eating when fed CS compared with FS (274 vs . 237 min/d), and time spent eating decreased when level of RFC was increased (271 vs . 241 min/d) . Decreasing forage particle size decreased time spent ruminating (485 vs . 320 min/d), rumination periods (15.3 vs . 11.7), and duration of rumination periods (29 vs . 26 min) . Increasing level of RFC increased time spent ruminating per kg NDF intake (68.5 vs . 79.5 min/kg) . Milk fat percentage was correlated to mean ruminal pH (r = 0.41), time spent below pH 5.8 (r = -0.55), and area below 5.8 (r = -0.57), but not to intake or chewing variables . DMI of particles retained on a screen equivalent in size to the top screen of the Penn State particle separator was the intake parameter explaining most of the variation in mean ruminal pH (r = 0.27) and was correlated to time spent ruminating (r = 0.61) and chewing (r = 0.61). Microbiology, 2002 Sep, 148(Pt 9), 2783 - 8 Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization; Hamacher T et al.; For an economically feasible production of ethanol from plant biomass by microbial cells, the fermentation of xylose is important . As xylose uptake might be a limiting step for xylose fermentation by recombinant xylose-utilizing Saccharomyces cerevisiae cells a study of xylose uptake was performed . After deletion of all of the 18 hexose-transporter genes, the ability of the cells to take up and to grow on xylose was lost . Reintroduction of individual hexose-transporter genes in this strain revealed that at intermediate xylose concentrations the yeast high- and intermediate-affinity transporters Hxt4, Hxt5, Hxt7 and Gal2 are important xylose-transporting proteins . Several heterologous monosaccharide transporters from bacteria and plant cells did not confer sufficient uptake activity to restore growth on xylose . Overexpression of the xylose-transporting proteins in a xylose-utilizing PUA yeast strain did not result in faster growth on xylose under aerobic conditions nor did it enhance the xylose fermentation rate under anaerobic conditions . The results of this study suggest that xylose uptake does not determine the xylose flux under the conditions and in the yeast strains investigated. Wien Klin Wochenschr, 2002 May 15, 114(8-9), 289 - 300 Short-chain fatty acids: bacterial mediators of a balanced host-microbial relationship in the human gut; Saemann MD et al.; The luminal compartment of the gastrointestinal tract is colonized by a large and highly complex microflora providing not only nutritional advances, but also representing a potential immunological challenge for the host . Under physiological conditions, the immune cells of the colonic mucosa do not defeat the microflora . In contrast, in cases of inflammatory bowel disease (IBD), the intestinal microflora appears to be the target of immune reactivity as demonstrated in various genetic studies and animal models of mucosal inflammation . The mechanisms responsible for the maintenance of this immunological unresponsiveness in the mucosal compartment are still largely enigmatic though recent studies indicate that luminal components might control this peculiar state . The bacterial fermentation product n-butyrate has been identified as such as critical molecule . Apart from its essential nutritional function for colonocytes, an anti-inflammatory activity of this short-chain fatty acid (SCFA) has been recognized in vitro and in vivo . Regarding its molecular mode of action, an interference with transcription factors critical for the production of pro-inflammatory cytokines has been found . This overview discusses the physiological functions of this bacterial metabolite and its emerging role as a potent regulator of mucosal homeostasis . Special emphasis is laid on potential therapeutic implications of SCFA in the treatment of several forms of colitis. Environ Technol, 2002 Aug, 23(8), 863 - 75 Fermentation of municipal primary sludge: effect of SRT and solids concentration on volatile fatty acid production; Bouzas A et al.; Laboratory bench-scale experiments were conducted to investigate the performance of primary sludge fermentation for volatile fatty acids production . Primary sludges from two major wastewater treatment plants located in Valencia (Pinedo and Carraixet) were used . Experiments were performed at solids retention times between 4 and 10 days, and total volatile solids concentrations between 0.6% and 2.8% . Operation at two temperatures (20 degrees C and 30 degrees C) was also checked . Results indicated the importance of feed sludge characteristics on volatile fatty acids yields, being approximately double for the Carraixet wastewater treatment plant sludge than for the Pinedo plant . In both cases, higher volatile fatty acids yields were observed at higher total volatile solids concentrations . Solids retention times above 6 days scarcely improve volatile fatty acids yields, while experiments conducted at 4 days of solids retention times show an important decrease in volatile fatty acids yields . On raising temperature an increase in volatile fatty acids yields was observed, mainly due to an improvement in the hydrolysis of particulate organic matter. Electrophoresis, 2002 Jul, 23(14), 2216 - 22 Comparison of lysis methods and preparation protocols for one- and two-dimensional electrophoresis of Aspergillus oryzae intracellular proteins; Nandakumar MP et al.; Filamentous fungal fermentations are used to produce billions of dollars of biochemical and pharmaceutical products annually, yet are plagued by a number of poorly understood problems that would benefit from proteomic analysis . Unfortunately, few publications are available which describe extraction of filamentous fungal proteins for two-dimensional electrophoresis . The goal here was to develop protocols for extraction of fungal proteins, from both wild-type and a recombinant strain of the industrially important filamentous fungi Aspergillus oryzae, to be used for both one- and two-dimensional electrophoresis (1-DE and 2-DE) . Because fungal cell walls are exceptionally resistant to fragmentation, four lysis protocols were tested: (i) boiling in strong alkali solution, (ii) boiling in Sodium dodecyl surfate (SDS), (iii) chemical lysis in Y-PER(R) reagent, and (iv) mechanical lysis via rapid agitation with glass beads in a Mini-BeadBeater(R) . For both 1-DE and 2-DE, rapid agitation with glass beads was found to be the most efficient extraction method, yielding both mini- and large-format gels with little streaking or spot tailing, and proteins comprising a broad range of molecular weights and pI values. Biotechnol Bioeng, 2002 Sep 20, 79(6), 694 - 700 Supercritical fluid extraction of a lignocellulosic hydrolysate of spruce for detoxification and to facilitate analysis of inhibitors; Persson P et al.; This work describes a novel approach to detoxify lignocellulosic hydrolysates and facilitate the analysis of inhibitory compounds, namely supercritical fluid extraction (SFE) . The efficiency of the fermentation of lignocellulosic dilute-acid hydrolysates depends upon the composition of the hydrolysate and the organism used . Furthermore, it has been shown that inhibitors in the hydrolysate reduce the fermentation yield . This knowledge has given rise to the need to identify and remove the inhibiting compounds . Sample clean-up or work-up steps, to provide a clean and concentrated sample for the analytical system, facilitate the characterization of inhibitors, or indeed any compound in the hydrolysates . Removal of inhibitors was performed with countercurrent flow supercritical fluid extraction of liquid hydrolysates . Three different groups of inhibitors (furan derivatives, phenolic compounds, and aliphatic acids) and sugars were subsequently analyzed in the hydrolysate, extracted hydrolysate, and extract . The effect of the SFE treatment was examined with respect to fermentability with Saccharomyces cerevisiae . Not only did the extraction provide a clean and concentrated sample (extract) for analysis, but also a hydrolysate with increased fermentability as well as lower concentrations of inhibitors such as phenolics and furan derivatives . Biotechnol Bioeng, 2002 Sep 20, 79(6), 674 - 81 Rapid sampling for analysis of in vivo kinetics using the BioScope: a system for continuous-pulse experiments; Visser D et al.; In this article we present a novel device, the BioScope, which allows elucidation of in vivo kinetics of microbial metabolism via perturbation experiments . The perturbations are carried out according to the continuous-flow method . The BioScope consists of oxygen permeable silicon tubing, connected to the fermentor, through which the broth flows at constant velocity . The tubing has a special geometry (serpentine channel) to ensure plug flow . After leaving the fermentor, the broth is mixed with a small flow of perturbing agent . This represents the start of the perturbation . The broth is sampled at different locations along the tubing, corresponding to different incubation times . The maximal incubation time is 69 s; the minimally possible time interval between the samples is 3-4 s . Compared to conventional approaches, in which the perturbation is carried out in the fermentor, the BioScope offers a number of advantages . (1) A large number of different perturbation experiments can be carried out on the same day, because the physiological state of the fermentor is not perturbed . (2) In vivo kinetics during fed-batch experiments and in large-scale reactors can be investigated . (3) All metabolites of interest can be measured using samples obtained in a single experiment, because the volume of the samples is unlimited . (4) The amount of perturbing agent spent is minimal, because only a small volume of broth is perturbed . (5) The system is completely automated . Several system properties, including plug-flow characteristics, mixing, oxygen and carbon dioxide transfer rates, the quenching time, and the reproducibility have been explored, with satisfactory results . Responses of several glycolytic intermediates in Saccharomyces cerevisiae to a glucose pulse, measured using a conventional approach are compared to results obtained with the BioScope . The agreement between the results demonstrates that the BioScope is indeed a promising device for studying in vivo kinetics . Biotechnol Bioeng, 2002 Sep 20, 79(6), 664 - 73 Mass transfer effects on the reaction rate for heterogeneously distributed immobilized yeast cells; Gutenwik J et al.; Here we examine the efficiency of different immobilized cell gradients applied to immobilized Saccharomyces cerevisiae fermenting glucose to ethanol . We developed a simulation model to fully study the competing effects of mass transfer hindrance and kinetics . It is based on a diffusion-reaction model and can be used to analyze the different cell concentration profiles inside an immobilized gel bead, in terms of effectiveness factors, productivity, and mass flux . The internal diffusion coefficient, which varies with the local cell concentration, as well as the external mass transfer, is taken into account when describing the efficiency . Although the diffusion hindrance is greater at higher cell concentrations, high cell concentration is still advantageous in the present case because the increase in reaction rate outweighs the diffusion hindrance . Thus, high cell concentrations contribute to increased productivity . The influence of the cell concentration gradient on the efficiency of the beads is negligible . Within the range of cell profiles studied it has been established that the location of the cells within the bead is of lesser importance . However, a steep cell gradient increases the importance of the external mass transfer . Biotechnol Bioeng, 2002 Sep 20, 79(6), 653 - 63 Water and glucose gradients in the substrate measured with NMR imaging during solid-state fermentation with Aspergillus oryzae; Nagel FJ et al.; Gradients inside substrate particles cannot be prevented in solid-state fermentation . These gradients can have a strong effect on the physiology of the microorganisms but have hitherto received little attention in experimental studies . We report gradients in moisture and glucose content during cultivation of Aspergillus oryzae on membrane-covered wheat-dough slices that were calculated from (1)H-NMR images . We found that moisture gradients in the solid substrate remain small when evaporation is minimized . This is corroborated by predictions of a diffusion model . In contrast, strong glucose gradients developed . Glucose concentrations just below the fungal mat remained low due to high glucose uptake rates, but deeper in the matrix glucose accumulated to very high levels . Integration of the glucose profile gave an average concentration close to the measured average content . On the basis of published data, we expect that the glucose levels in the matrix cause a strong decrease in water activity . The results demonstrate that NMR can play an important role in quantitative analysis of water and glucose gradients at the particle level during solid-state fermentation, which is needed to improve our understanding of the response of fungi to this nonconventional fermentation environment . Biotechnol Bioeng, 2002 Sep 20, 79(6), 628 - 40 Use of frozen bagged seed inoculum for secondary metabolite and bioconversion processes at the pilot scale; Junker B et al.; Frozen bagged seed inoculum was prepared, thawed and tested for seven cultures . Thawing techniques were developed and other key influences on thawing rate were quantified; seed bag thawing without a water bath rarely required more than 4 to 5 h and was as short as 0.5 to 1 h for lower fill volume bags . Testing included growth of bagged seed as a function of bag fill volume (0.5, 1.0, 2.0, and 3.5 L), comparison of culture age at time of bagging, growth of bagged versus laboratory-prepared seed, productivity of production cultures derived from bagged versus laboratory-prepared seed, growth of bagged seed as a function of volume percent glycerol added at time of bagging, and growth of bagged seed as a function of frozen storage time and temperature . For each culture tested, conditions were developed such that seed tanks inoculated with bagged seed showed only minimal delay in attaining the target oxygen uptake rate (OUR) relative to seed tanks inoculated with laboratory-prepared inoculum . Although the bag fill volume did influence culture growth in some cases, bag fill volumes required were reasonable (typically 2.0 to 3.5 L) compared with laboratory seed inoculum volumes of 2.0 L . In the most remarkable example, frozen bagged seed was prepared from a second-stage seed-tank cultivation of Glarca lozoyensis, then thawed and inoculated into first-stage seed medium . It grew to the desired OUR in a similar timeframe as laboratory-prepared inoculum inoculated into first-stage seed medium . Thus, the frozen bagged seed replaced an existing laboratory inoculum preparation period of 7 days without an appreciable delay in either of the two subsequent seed-tank growth stages . Furthermore, productivities were found to be comparable for bagged-seed-derived and laboratory-seed-derived production cultivations for four different fermentation processes . Biotechnol Bioeng, 2002 Sep 30, 79(7), 804 - 15 Pattern analysis techniques to process fermentation curves: application to discrimination of enological alcoholic fermentations; Roger JM et al.; In fermentation processes, kinetic curves are generally aimed at control purposes . However, these curves could also contain information about inherent features of the product (such as origin, quality, etc.) . This article presents several pattern analysis techniques used to classify fermentation curves . An application to alcoholic fermentation is presented as an illustration: it aims at retrieving the origin of a must from its fermentation curve . The fermentation kinetics of five vineyard musts, harvested over 9 years on the same parcels, were recorded . From these curves two sets of variables were generated: The first (p(1)) gathers all the kinetic curve points . The second (p(2)) contains a restrained number of variables, generated by the expert knowledge of the enologist . The set p(2) was processed by two very different techniques: a linear one (factorial discriminant analysis) and a nonlinear one (artificial neural networks) . The set p(1) was processed by a new chemometric technique, the discriminant partial least-squares regression . For all the sets and the techniques used the selection of variables was studied . The interest in the latter is largely demonstrated both by theoretical and practical discussions . The discrimination results (up to 94% of good predictions) enhance the interest of the on-line measurements and their use in such pattern analysis tools . Biotechnol Bioeng, 2002 Sep 30, 79(7), 713 - 23 Overexpression of an archaeal protein in yeast: secretion bottleneck at the ER; Smith JD et al.; Archaeal enzymes have great potential for industrial use; however, expressing them in their natural hosts has proven challenging . Growth conditions for many archaea are beyond typical fermentation capabilities, and to compound the problem, archaea generally achieve much lower biomass yields than Escherichia coli or Saccharomyces cerevisiae . To determine whether a eukaryotic host, S . cerevisiae, would be a suitable alternative for archaeal protein production, we examined the expression of the tetrameric beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus . We engineered the beta-glucosidase to facilitate secretion into the culture medium and have demonstrated the beta-glucosidase's secretion and activity . We determined the dependence of beta-glucosidase secretion on gene copy number and obtained a transformant capable of secreting approximately 10 mg/L in batch culture . All transformants retained large intracellular fractions of beta-glucosidase, indicative of an intracellular bottleneck . Cell fractionation by sucrose density centrifugation and immunofluorescence identified the endoplasmic reticulum as the secretion bottleneck . Preliminary evidence indicates that the cause of this bottleneck is misfolding of the monomeric beta-glucosidase, rather than tetrameric association . Expression at moderately elevated temperatures (between 30 and 40 degrees C) improved beta-glucosidase yields, suggesting that higher temperature expression may improve folding and secretion yields . Biotechnol Bioeng, 2002 Oct 20, 80(2), 139 - 43 Recovery and separation of cell lysate proteins using hydrogels guided by aqueous two-phase extraction principles; Putka CS et al.; The addition of poly(ethylene glycol) and salts to clarified cell lysates of Thiosphaera pantotropha increases sorption of microbial proteins into dextran hydrogels, consistent with the thermodynamics of aqueous two-phase extraction . Addition of 12 wt% PEG-10,000 to the lysate increased total sorption of protein by the dextran gel from 5.2 mg/g dextran to 37 mg/g; addition of either 0.1 M potassium iodide or tetrabutylammonium fluoride along with PEG to the lysate increased protein sorption to more than 63 mg/g, a 12-fold increase . SDS-PAGE demonstrated that the type of salt added controls which proteins are absorbed by the gel . Previously demonstrated only with model solutions, these results suggest another approach to recovery and separation strategies for proteins produced by fermentation . Microbiol Mol Biol Rev, 2002 Sep, 66(3), 506 - 77, table of contents Microbial cellulose utilization: fundamentals and biotechnology; Lynd LR et al.; Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature . The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures . Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics . A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations . Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step . Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance . A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts. Biochem Biophys Res Commun, 2002 Sep 6, 296(5), 1148 - 51 The antioxidant cocktail effective microorganism X (EM-X) inhibits oxidant-induced interleukin-8 release and the peroxidation of phospholipids in vitro; Deiana M et al.; The antioxidant beverage EM-X is derived from the ferment of unpolished rice, papaya, and sea-weeds with effective microorganisms . Oxidative stress enhances the expression of proinflammatory genes, causing the release of the chemokine interleukin-8 (IL-8), which mediates a multitude of inflammatory events . Human alveolar epithelial cells (A549) were treated with H(2)O(2) (100 microM) or TNF-alpha (10ng/ml) alone or with the addition of EM-X (100 microl/ml), incubated for 20h, and the release of IL-8, measured using ELISA . EM-X inhibited the release of IL-8 at the transcriptional level in A549 cells . EM-X also decreased the iron/ascorbate dependent peroxidation of ox-brain phospholipids in a concentration dependent manner . A TEAC value of 0.10+/-0.05mM was obtained for EM-X, indicating antioxidant potential . We suggest that the anti-inflammatory and antioxidant properties of EM-X are dependent on the flavonoid contents of the beverage. J Agric Food Chem, 2002 Sep 11, 50(19), 5378 - 85 Quantitation of odor-active compounds in rye flour and rye sourdough using stable isotope dilution assays; Kirchhoff E et al.; Application of the aroma extract dilution analysis on a flavor distillate prepared from freshly ground rye flour (type 1150) revealed 1-octen-3-one (mushroom-like), methional (cooked potato), and (E)-2-nonenal (fatty, green) with the highest flavor dilution (FD) factors among the 26 odor-active volatiles identified . Quantitative measurements performed by stable isotope dilution assays and a comparison to the odor thresholds of selected odorants in starch suggested methional, (E)-2-nonenal, and hexanal as contributors to the flour aroma, because their concentrations exceeded their odor thresholds by factors >100 . Application of the same approach on a rye sourdough prepared from the same batch of flour revealed 3-methylbutanal, vanillin, 3-methylbutanoic acid, methional, (E,E)-2,4-decadienal, 2,3-butanedione, and acetic acid as important odorants; their concentrations exceeded their odor thresholds in water and starch by factors >100 . A comparison of the concentrations of 20 odorants in rye flour and the sourdough made therefrom indicated that flour, besides the fermentation process, is an important source of aroma compounds in dough . However, 3-methylbutanol, acetic acid, and 2,3-butanedione were much increased during fermentation, whereas (E,E)-2,4-decadienal and 2-methylbutanal were decreased . Similar results were obtained for five different flours and sourdoughs, respectively, although the amounts of some odorants in the flour and the sourdough differed significantly within batches. J Agric Food Chem, 2002 Sep 11, 50(19), 5318 - 25 Effect of different forms of alkali treatment on specific fermentation inhibitors and on the fermentability of lignocellulose hydrolysates for production of fuel ethanol; Persson P et al.; Treatment with alkali, particularly overliming, has been widely used as a method for the detoxification of lignocellulose hydrolysates prior to ethanolic fermentation . However, the mechanisms behind the detoxification effect and the influence of the choice of cation have not been well understood . In this study, a dilute acid hydrolysate of spruce and an inhibitor cocktail consisting of six known inhibitors were used to investigate different alkali detoxification methods . The various treatments included the addition of calcium hydroxide, sodium hydroxide, potassium hydroxide, and ammonia to pH 10.0 and subsequent adjustment of the pH to 5.5 with either sulfuric or hydrochloric acid as well as treatment with the corresponding amounts of calcium, sodium, and potassium as sulfate or chloride salts at pH 5.5 . An RP-HPLC method was developed for the separation of 18 different inhibitors in the hydrolysate, including furaldehydes and phenolics . Detection and quantification were carried out by means of UV, DAD, and ESI-MS in negative mode . Treatment of the spruce hydrolysate with alkali resulted in up to approximately 40% decrease in the concentration of furaldehydes . The effects on the aromatic compounds were complex . Furthermore, SFE was performed on the precipitate formed during alkali treatment to evaluate the inhibitor content of the precipitate, and the following RP-HPLC analysis implied that potential inhibitors were removed mainly through conversion rather than through filtration of precipitate . Parallel experiments in which sulfuric acid or hydrochloric acid was used for acidification to pH 5.5 after alkali treatment indicated that the choice of anion did not affect the removal of inhibitors . Detoxification with calcium hydroxide and ammonia resulted in better fermentability using Saccharomyces cerevisiae than detoxification with sodium hydroxide . The results from the experiments with the inhibitor cocktail indicated that the positive effects of alkali treatment are difficult to explain by removal of the inhibitors only and that possible stimulatory effects on the fermenting organism warrant further attention. J Dairy Sci, 2002 Jul, 85(7), 1767 - 76 Efficacy of carbohydrate sources for milk production by cows fed diets based on alfalfa silage; Broderick GA et al.; The effectiveness of three carbohydrate sources, high-moisture ear corn (HMEC), cracked shelled corn (CSC), and a 50:50 mixture of HMEC plus dried citrus pulp (DCP), fed with or without supplemental rumen-undegraded protein as expeller soybean meal (ESBM), was assessed in 48 multiparous dairy cows . All diets contained (dry mater {DM} basis) 50% alfalfa silage, 10% ryegrass silage, 28% NDF, and one of six concentrates: A) 38% HMEC; B) 38% CSC; C) 19% DCP plus 19% HMEC; D) 27% HMEC plus 12% ESBM; E) 27% CSC plus 12% ESBM; or F) 13% DCP, 13% HMEC, and 12% ESBM . Diets A, B, and C averaged 19% crude protein, of which 53% was nonprotein nitrogen (NPN), and diets D, E, and F averaged 22% crude protein, of which 40% was NPN . Cows were fed a high-energy covariate diet for 2 wk, blocked into eight groups of six, based on covariate protein yield, then randomly assigned to diets that were fed for 12 wk . Feeding ESBM increased DM intake, yields of milk, fat-corrected milk, fat, protein, SNF, and milk and blood urea concentration and decreased weight loss . There were no production differences between HMEC and CSC . However, DM intake, yields of milk, fat-corrected milk, fat, protein, lactose, SNF, and milk SNF content all were lower on the diets containing DCP versus HMEC and CSC . A 6 x 6 Latin square trial conducted at the same time with six ruminally cannulated cows showed similar effects of diet on DM intake and milk production . Ruminal ammonia was elevated by ESBM but not ruminal total amino acids and branched-chain volatile fatty acids . Ruminal propionate was highest on HMEC diets and lowest on DCP diets; acetate, butyrate and acetate-to-propionate ratio were lowest on HMEC diets and highest on DCP diets . These results indicated that, compared to HMEC and CSC, feeding the pectin-rich carbohydrate source DCP altered ruminal fermentation but depressed intake and milk production in lactating cows. Water Sci Technol, 2002, 45(12), 167 - 74 Application of membrane-coupled anaerobic volatile fatty acids fermentor for dissolved organics recovery from coagulated raw sludge; Kim JO et al.; To investigate the treatment performance of membrane-coupled anaerobic volatile fatty acids fermentor system, the effects of operational parameters for volatile fatty acids production were evaluated through experiments and a mathematical model . The volatile fatty acids recovery ratio was largely affected by the change of hydraulic retention time, reaching its maximum value at 12 hrs . Over the range of hydraulic retention time 8 to 96 hrs, the volatile fatty acids recovery ratio decreased with the increase of hydraulic retention time above 12 hrs, while the ratio of mineralization and gasification increased . Hydraulic retention time and membrane filtration ratio should be maintained less than 1 day and above 0.9, respectively, to attain over 40% of organic materials recovery ratio at 10 days of solids retention time . When the hydrolysis rate constant was 0.01 hr-1, the organic loading rate should be maintained at above 1.0 (kgC/m3/day) to attain over 45% of volatile fatty acids recovery ratio . Based on experimental and simulated results, membrane-coupled anaerobic volatile fatty acids fermentor system was thought to be effective for dissolved organics recovery from coagulated sewage sludge. Appl Environ Microbiol, 2002 Sep, 68(9), 4534 - 8 Fumarate-mediated inhibition of erythrose reductase, a key enzyme for erythritol production by Torula corallina; Lee JK et al.; Torula corallina, a strain presently being used for the industrial production of erythritol, has the highest erythritol yield ever reported for an erythritol-producing microorganism . The increased production of erythritol by Torula corallina with trace elements such as Cu(2+) has been thoroughly reported, but the mechanism by which Cu(2+) increases the production of erythritol has not been studied . This study demonstrated that supplemental Cu(2+) enhanced the production of erythritol, while it significantly decreased the production of a major by-product that accumulates during erythritol fermentation, which was identified as fumarate by instrumental analyses . Erythrose reductase, a key enzyme that converts erythrose to erythritol in T . corallina, was purified to homogeneity by chromatographic methods, including ion-exchange and affinity chromatography . In vitro, purified erythrose reductase was significantly inhibited noncompetitively by increasing the fumarate concentration . In contrast, the enzyme activity remained almost constant regardless of Cu(2+) concentration . This suggests that supplemental Cu(2+) reduced the production of fumarate, a strong inhibitor of erythrose reductase, which led to less inhibition of erythrose reductase and a high yield of erythritol . This is the first report that suggests catabolite repression by a tricarboxylic acid cycle intermediate in T . corallina. Appl Environ Microbiol, 2002 Sep, 68(9), 4448 - 56 Metabolic control analysis of glycerol synthesis in Saccharomyces cerevisiae; Cronwright GR et al.; Glycerol, a major by-product of ethanol fermentation by Saccharomyces cerevisiae, is of significant importance to the wine, beer, and ethanol production industries . To gain a clearer understanding of and to quantify the extent to which parameters of the pathway affect glycerol flux in S . cerevisiae, a kinetic model of the glycerol synthesis pathway has been constructed . Kinetic parameters were collected from published values . Maximal enzyme activities and intracellular effector concentrations were determined experimentally . The model was validated by comparing experimental results on the rate of glycerol production to the rate calculated by the model . Values calculated by the model agreed well with those measured in independent experiments . The model also mimics the changes in the rate of glycerol synthesis at different phases of growth . Metabolic control analysis values calculated by the model indicate that the NAD(+)-dependent glycerol 3-phosphate dehydrogenase-catalyzed reaction has a flux control coefficient (C(J)v1) of approximately 0.85 and exercises the majority of the control of flux through the pathway . Response coefficients of parameter metabolites indicate that flux through the pathway is most responsive to dihydroxyacetone phosphate concentration (R(J)DHAP= 0.48 to 0.69), followed by ATP concentration (R(J)ATP = -0.21 to -0.50) . Interestingly, the pathway responds weakly to NADH concentration (R(J)NADH = 0.03 to 0.08) . The model indicates that the best strategy to increase flux through the pathway is not to increase enzyme activity, substrate concentration, or coenzyme concentration alone but to increase all of these parameters in conjunction with each other. Ann Allergy Asthma Immunol, 2002 Aug, 89(2), 197 - 202 Glucoamylase: another fungal enzyme associated with baker's asthma; Quirce S et al.; BACKGROUND: Aspergillus-derived enzymes are widely used as dough additives in the baking industry . These enzymes may give rise to immunoglobulin (Ig)E-mediated sensitization and occupational asthma . Glucoamylase (or amyloglucosidase) is an important industrial enzyme obtained from Aspergillus niger and used to provide fermentable sugars for yeast to improve loaf volume and texture . OBJECTIVE: The aim of our study was to investigate the potential allergenic role of glucoamylase in baker's asthma . METHODS: We report four subjects with work-related allergic respiratory symptoms who were exposed to glucoamylase and other starch-cleaving enzymes used as baking additives . The causative role of glucoamylase in work-related asthma was investigated by immunologic tests and specific inhalation challenges (SIC) . Glucoamylase allergenic components were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting . RESULTS: Skin prick tests to glucoamylase (10 mg/mL) gave a positive response in all patients . Further, a positive skin prick test to alpha-amylase was obtained in the four patients and to hemicellulase in two of them . SIC to glucoamylase elicited isolated early asthmatic responses in the three patients tested, and SIC to alpha-amylase elicited early asthmatic responses in two patients and a dual asthmatic response in another patient . Immunoblotting with glucoamylase showed several IgE-binding bands with molecular masses between 33 and 96 kD . IgE-inhibition assays showed scarce to moderate allergenic cross-reactivity between glucoamylase and alpha-amylase . CONCLUSIONS: These bakers had developed IgE-mediated occupational asthma to glucoamylase and alpha-amylase . Fungal glucoamylase is widely used as a baking additive and this enzyme may give rise to allergic respiratory reactions among exposed workers. Life Sci Space Res, 1969, 7, 102 - 9 Intestinal hydrogen and methane of men fed space diet; Calloway DH et al.; Intestinal bacteria form two gases, hydrogen (H2) and methane (CH4), that could constitute a fire hazard in a closed chamber . So H2 and CH4 pass from the anus but these gases are also transported by the blood to the lungs and removed to the atmosphere . Several factors affect gas formation: 1) amount and kind of fermentable substrate; 2) abundance, types, and location of microflora; and 3) psychic and somatic conditions that affect the gut . We evaluated the first factor by studying men fed different diets and have also recorded influences of uncontrollable factors . One group of 6 men ate Gemini-type diet (S) and another received a bland formula (F), for 42 days . Breath and rectal gases were analyzed during the first and final weeks . Flatus gases varied widely within dietary groups but much more gas was generated with diet S than with F . In the first 12-hour collection, subjects fed S passed 3 to 209 ml (ATAP) of rectal H2 (avg 52) and 24 to 156 ml (avg 69) from the lungs (assuming normal pulmonary ventilation) . With F, these values were 0 to 3 ml (avg 1) and 6 to 36 ml (avg 20) . Subjects were calmer during the second test . Gas production was lower with S than initially; F values were unchanged . Methane differed idiosyncratically, presumably due to differences in flora . Computed from 12-hour values, maximum potential daily H2 and CH4 are per man: for S, 730 ml and 382 ml; for F, 80 and 222 ml . Volumes would be larger at reduced spacecraft and suit pressures. J Antibiot (Tokyo), 2002 Jun, 55(6), 565 - 70 Arylomycins A and B, new biaryl-bridged lipopeptide antibiotics produced by Streptomyces sp . Tü 6075 . I . Taxonomy, fermentation, isolation and biological activities; Schimana J et al.; New lipopeptide antibiotics, colourless arylomycins A series and yellow arylomycins B series were detected in the culture filtrate and mycelium extracts of Streptomyces sp . Tu 6075 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening . Arylomycins are a family of lipohexapeptide antibiotics, which represent the first examples of biaryl-bridged lipopeptides . They show antibiotic activities against Gram-positive bacteria. J Infect Dis, 2002 Aug 15, 186(4), 493 - 500 Epub 2002 Aug 02. Clinical course and the role of shiga toxin-producing Escherichia coli infection in the hemolytic-uremic syndrome in pediatric patients, 1997-2000, in Germany and Austria: a prospective study; Gerber A et al.; Hemolytic-uremic syndrome (HUS) is mainly associated with foodborne infections by Shiga toxin-producing Escherichia coli (STEC) . From January 1997 through December 2000, 394 children with HUS were evaluated in a prospective multicenter surveillance study in Germany and Austria (incidences, 0.7/100,000 and 0.4/100,000 children <15 years old, respectively) . Blood leukocytosis was associated with increased detection of STEC in stool cultures (P<.01) and a more severe disease course . Risk of death was associated with cerebral involvement (P<.01) . Most strikingly, non-O157:H7 STEC were detected in 43% of stool cultures of patients with HUS: O26 was detected in 15%, sorbitol-fermenting O157:H(-) in 10%, O145 in 9%, O103 in 3%, and O111 in 43% . Patients with O157:H7 serotypes required dialysis for a longer time and had bloody diarrhea detected more frequently, compared with patients with non-O157:H7 serotypes (P<.05) . This large study in children with HUS underlines the rising importance of non-O157:H7 serotypes, and, despite increased public awareness, the number of patients remained unchanged. J Nat Prod, 2002 Aug, 65(8), 1091 - 5 Durhamycin A, a potent inhibitor of HIV Tat transactivation; Jayasuriya H et al.; Tat is a small HIV protein essential for both viral replication and the progression of HIV disease . In our efforts to discover Tat inhibitors from natural product screening of microbial fermentation extracts, we discovered durhamycin A (1) as a potent inhibitor (IC(50) = 4.8 nM) of Tat transactivation . Detailed NMR and MS/MS studies were utilized to elucidate the structure of 1 as a new member of the aureolic acid family of antibiotics . It consists of tetrasaccharide and disaccharide moieties attached to the aglycone, which is hitherto unknown in the aureolic acid family . Three other novel analogues, durhamycin B (2), compound (3), and the aglycone (4), were also discovered or chemically prepared that were less potent than durhamycin A. Curr Microbiol, 2002 Oct, 45(4), 281 - 6 Regulation of Escherichia coli formate hydrogenlyase activity by formate at alkaline pH; Mnatsakanyan N et al.; Escherichia coli possesses two hydrogenases, Hyd-3 and Hyd-4 . These, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenlyases, FHL-1 and FHL-2, both catalyzing the decomposition of formate to H(2) and CO(2) during fermentative growth . FHL-1 is the major pathway at acidic pH whereas FHL-2 is proposed for slightly alkaline pH . In this study, regulation of activity of these pathways by formate has been investigated . In cells grown under fermentative conditions on glucose in the presence of 30 mM formate at pH 7.5, intracellular pH was decreased to 7.1, the activity of Fdh-H raised 3.5-fold, and the production of H(2) became mostly Hyd-3 dependent . These results suggest that at alkaline pH formate increases an activity of Fdh-H and of Hyd-3 both but not of Hyd-4. J Agric Food Chem, 2002 Aug 28, 50(18), 5236 - 40 Characterization of resistant starch type III from banana (Musa acuminata); Lehmann U et al.; Banana starch (Musa acuminata var . Nandigobe) was evaluated for its use in generating resistant starch (RS) type III . Structural, physicochemical, and biological properties of these products were analyzed . The investigated process includes debranching of the native starch and retrogradation under different storage temperatures and starch concentrations . After enzymatic debranching, a high amount of low-molecular-weight polymers with a degree of polymerization between 10 and 35 glucose units beside a higher molecular weight fraction were found . The resulting products comprised RS contents of about 50% . After heat-moisture treatment, the RS yield increased up to 84% . Peak temperatures of about 145 degrees C found in DSC measurements pointed to a high thermal stability of the RS products . In vitro fermentations of the RS products, carried out with intestinal microflora of healthy humans, resulted in a molar ratio of acetate:propionate:butyrate of about 49:17:34 . The established method allowed the production of a high-quality RS with prebiotic properties for health preventing applications. Water Sci Technol, 2002, 45(10), 163 - 8 Influence of pulsation on start-up of UASB reactors; Franco A et al.; The aim of this work is to study the influence of pulsation on the start-up of lab-scale UASB reactors . Pulsation was produced by an Elastic Membrane Pulsator (EMP) . The application of this device in previous works improved the performance of continuous fixed-bed fermentors and reduced the formation of preferential pathways, the retention of gas metabolites within the bed and the resistance to mass transfer . These characteristics seem to be suitable for feeding UASB reactors . In this work, the influence of pulsation frequency was studied in two pulsed UASB reactors operated in parallel with a non-pulsed one . One of them (P1) operated at high frequencies (periods of 50 and 200 s between each pulsation) and the other (P2) at low frequencies (periods of 3600 and 900 s between each pulsation) . An important improvement of the removal efficiency for pulsed reactors with respect to the non-pulsed one was obtained . The structure of the biomass was observed at the end of the process by scanning electron microscopy . In general, granulation of biomass was improved when operating in pulsing form. Biochemistry, 2002 Aug 27, 41(34), 10603 - 7 In vitro evolution of amphioxus insulin-like peptide to mammalian insulin; Guo ZY et al.; By site-directed mutagenesis, six insulin residues related to the insulin-receptor interaction were grafted, partially or fully, onto the corresponding position of a recombinant amphioxus insulin-like peptide (ILP) that contained the A- and B-domains of the deduced amphioxus ILP . After fermentation, purification, and enzymatic cleavage, six insulin-like double-chain ILP analogues were obtained: {A2Ile}ILP, {B12Val, B16Tyr}ILP, {B25Phe}ILP, {A2Ile, B12Val, B16Tyr, B25Phe}ILP (four-mutated ILP), {A2Ile, B12Val, B16Tyr, B24Phe, B25Phe}ILP (five-mutated ILP), and {A2Ile, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr}ILP (six-mutated ILP) . Circular dichroism analysis showed that such replacement did not significantly affect their secondary and tertiary structure compared with that of the wild-type ILP . The insulin-receptor-binding activity of the four-, five-, and six-mutated ILP was 0.14%, 11%, and 11% of native insulin, respectively; the other three ILP analogues acquired none of the detectable insulin-receptor-binding potency . The growth-promoting activities of the five- and six-mutated ILP were both about 50% of native insulin, while that of the wild-type ILP was not detectable . By structure-function-based mutagenesis, the completely inactive amphioxus ILP was converted into a molecule with moderate mammalian insulin activity . These results indicated the following: first, the grafted as well as those inborn insulin-receptor-binding related residues can form an insulin-receptor-binding patch on the ILP analogues; second, the ILP can be used as a scaffold molecule to investigate the role of the insulin residues; third, the natural evolution of amphioxus ILP to mammalian insulin is a possible process and can be mimicked in the laboratory. Yeast, 2002 Jun 15, 19(8), 713 - 26 The hexose transporters of Saccharomyces cerevisiae play different roles during enological fermentation; Luyten K et al.; We investigated the role of hexose transporters in a Saccharomyces cerevisiae strain derived from an industrial wine strain by carrying out a functional analysis of HXT genes 1-7 under enological conditions . A strain in which the sugar carrier genes HXT1-HXT7 were deleted was constructed and the HXT genes were expressed individually or in combination to evaluate their role under wine alcoholic fermentation conditions . No growth or fermentation was observed in winemaking conditions for the hxt1-7 delta strain . The low-affinity carriers Hxt1 and Hxt3 were the only carriers giving complete fermentation of sugars when expressed alone, indicating that these carriers play a predominant role in wine fermentation . However, these two carriers have different functions . The Hxt3 transporter is thought to play a major role, as it was the only carrier that gave an almost normal fermentation profile when produced alone . The hxt1 carrier was much less effective during the stationary phase and its role is thought to be restricted to the beginning of fermentation . The high-affinity carriers Hxt2, Hxt6 and/or Hxt7 were also required for normal fermentation . These high-affinity transporters have different functions: hxt2 is involved in growth initiation, whereas Hxt6 and/or Hxt7 are required at the end of alcoholic fermentation . This work shows that the successful alcoholic fermentation of wine involves at least four or five hexose carriers, playing different roles at various stages in the fermentation cycle. Infect Immun, 2002 Sep, 70(9), 4925 - 35 Identification and functional mapping of the Mycoplasma fermentans P29 adhesin; Leigh SA et al.; Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes . Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved . We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M . fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not . Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop . A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity . Together these results suggest that the direct interaction of M . fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein . Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype . Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species . This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29). Protein Expr Purif, 2002 Aug, 25(3), 379 - 88 cDNA cloning, high-level expression, purification, and characterization of an avian Cu,Zn superoxide dismutase from Peking duck; Liu W et al.; As a special species of avian, Peking duck is often used as a model for exploring effective factors against cardio-cerebrovascular diseases, and therefore investigations of antioxidant enzymes including superoxide dismutase are intriguing . By using 3(')-RACE with a gene-specific primer, a cDNA encoding duck Cu,Zn SOD was amplified from the total RNA extracted from Peking duck liver . Three free cysteine residues are found in the deduced amino acid sequence of duck SOD, among which Cys153 at the carbonyl-terminal is a distinctive feature . Production with a high yield of recombinant duck Cu,Zn SOD was achieved in Escherichia coli after the reconstituted expression vector pET-3a-dSOD was transformed into the bacterial strain BL21(DE3)pLysS . After two steps of anion exchange chromatography, a great quantity of the purified enzyme (100mg/L fermented culture) with an enzymatic activity comparable to that of native duck and bovine SOD was finally obtained . Duck SOD is a homodimer with 153 residues for each subunit . The molecular mass of the recombinant enzyme is 15,540.0Da measured by mass spectrum, which well coincides with the estimated size of the sequence but significantly differs from that of the native counterpart . Five charge isomers were observed on isoelectricfocusing (IEF) . The most interesting observation is that the thermal stability of duck SOD is much lower than that of the bovine enzyme as revealed by irreversible heat inactivation at 70 degrees C . These properties are discussed in relation to the distinctive free Cys residues in duck Cu,Zn SOD. J Food Prot, 2002 Aug, 65(8), 1297 - 303 Microbial quality of water supply to an urban community in Trinidad; Agard L et al.; A microbiological study was conducted to determine the quality of the water supply to an urban community in San Fernando proper in south Trinidad using total coliforms and thermotolerant coliforms as indicators of water pollution . The membrane filter technique was used to detect total coliforms and thermotolerant coliforms on endo agar and MFc agar, respectively . The residual chlorine levels in water from the reservoir, from standpipes along the distribution line, and from households were determined with a commercial test kit . Of a total of 104 drinking water samples obtained from households, 84 (80.8%), 56 (53.8%), and 70 (67.3%) tested positive for total coliforms, thermotolerant coliforms, and Escherichia coli, respectively . The difference was statistically significant (P < 0.05, chi2) . Of the 81 water samples collected from the Water and Sewerage Authority (WASA) main supply to households, 38 (46.9%), 13 (16.0%), and 27 (33.3%) were contaminated by total coliforms, thermotolerant coliforms, and E . coli, respectively, and the difference was statistically significant (P < 0.05, chi2) . Eight (20.5%) of 39 water samples from standpipes along the distribution line tested positive for total coliforms, compared with 4 (10.3%) samples testing positive for thermotolerant coliforms . All five samples of treated water obtained from the reservoir tested negative for coliforms . There was a significant difference (P = 0.004) in the mean residual chlorine levels in water from the reservoir, water from standpipes, and water from households . Similarly, as the level of residual chlorine decreased, there was a statistically significant (P = 0.004) increase in the prevalence of total coliforms in water from 0.0% (treated reservoir water) to 15.2% (standpipe) to 53.5% (household mains) to 80.0% (household drinking water) . There was also a statistically significant difference (P < 0.001, chi2) in the prevalence of total coliforms in drinking water and in water from the WASA main supply to households . Of the 105 E . coli strains tested, 7 (6.7%), 16 (15.2%), and 22 (21.0%) were mucoid, hemolytic, and non-sorbitol fermenters, respectively . It was concluded that the high degree of contamination of drinking water in households poses a health hazard to consumers. Lett Appl Microbiol, 2002, 35(3), 195 - 202 Production of red pigment by submerged culture of Paecilomyces sinclairii; Cho YJ et al.; AIMS: From a survey of submerged culture of edible mushrooms, a high pigment-producing fungus Paecilomyces sinclairii was selected and its optimal culture conditions investigated . METHODS AND RESULTS: The optimal culture conditions for pigment production were as follows: inoculum age, 3 d; temperature, 25 degrees C; initial pH, 6.0; carbon source, 1.5% (w/v) soluble starch; nitrogen source, 1.5% (w/v) meat peptone . Although addition of 10 mmol l(-1) CaCl2 to the culture medium slightly increased pigment production, most of the bio-elements examined had no notable or detrimental effect on pigment production . CONCLUSIONS: Under the optimal conditions obtained in the flask culture tested, a ninefold increase in pigment production (4.4 g l(-1)) was achieved using a 5(-l) batch fermenter . Paecilomyces sinclairii secreted water-soluble red pigment into the culture medium . The pigment colour was strongly dependent on the pH of the solution: red at pH 3-4, violet at pH 5-9 and pink at pH 10-12 . SIGNIFICANCE AND IMPACT OF THE STUDY: The high concentration of pigment (4.4 g l(-1)) produced by P . sinclairii demonstrates the possibility of commercial production of pigment by this strain, considering its relatively high production yield and light stability. Biochemistry, 2002 Aug 20, 41(33), 10462 - 71 Enzymes involved in fatty acid and polyketide biosynthesis in Streptomyces glaucescens: role of FabH and FabD and their acyl carrier protein specificity; Florova G et al.; Malonyl acyl carrier protein (ACP) is used as an extender unit in each of the elongation steps catalyzed by the type II dissociated fatty acid synthase (FAS) and polyketide synthase (PKS) of Streptomyces glaucescens . Initiation of straight-chain fatty acid biosynthesis by the type II FAS involves a direct condensation of acetyl-CoA with this malonyl-ACP to generate a 3-ketobutyryl-ACP product and is catalyzed by FabH . In vitro experiments with a reconstituted type II PKS system in the absence of FabH have previously shown that the acetyl-ACP (generated by decarboxylation of malonyl-ACP), not acetyl-CoA, is used to initiate tetracenomycin C (TCM C) biosynthesis . We have shown that sgFabH activity is present in S . glaucescens fermentations during TCM C production, suggesting that it could contribute to initiation of TCM C biosynthesis in vivo . Isotope incorporation studies with {CD3}acetate and {13CD3}acetate demonstrated significant intact retention of three deuteriums into the starter unit of palmitate and complete washout of deuterium label into the starter unit of TCM C . These observations provide evidence that acetyl-CoA is not used directly as a starter unit for TCM C biosynthesis in vivo and argue against an involvement of FabH in this process . Consistent with this conclusion, assays of the purified recombinant sgFabH with acetyl-CoA demonstrated activity using malonyl-ACP generated from either FabC (the S . glaucescens FAS ACP) (k(cat) 42.2 min(-1), K(m) 4.5 +/- 0.3 microM) or AcpP (the E . coli FAS ACP) (k(cat) 7.5 min(-1), K(m) 6.3 +/- 0.3 microM) but not TcmM (the S . glaucescens PKS ACP) . In contrast, the sgFabD which catalyzes conversion of malonyl-CoA to malonyl-ACP for fatty acid biosynthesis was shown to be active with TcmM (k(cat) 150 min(-1), K(m) 12.2 +/- 1.2 microM), AcpP (k(cat) 141 min(-1), K(m) 13.2 +/- 1.6 microM), and FabC (k(cat) 560 min(-1), K(m) 12.7 +/- 2.6 microM) . This enzyme was shown to be present during TCM C production and could play a role in generating malonyl-ACP for both processes . Previous demonstrations that the purified PKS ACPs catalyze self-malonylation and that a FabD activity is not required for polyketide biosynthesis are shown to be an artifact of the expression and purification protocols . The relaxed ACP specificity of FabD and the lack of a clear alternative are consistent with a role of FabD in providing malonyl-ACP precursors for PKS as well as FAS processes . In contrast, the ACP specificity of FabH, isotope labeling studies, and a demonstrated alternative mechanism for initiation of the PKS process provide unequivocal evidence that FabH is involved only in the FAS process. J Environ Manage, 2002 May, 65(1), 25 - 38 Wastewater management in a cane molasses distillery involving bioresource recovery; Nandy T et al.; Waste management involving bioresource recovery in a cane molasses-based distillery engaged in the manufacture of rectified spirit (alcohol) is described . The spentwash generated from the distillation of fermenter wash is highly acidic (pH 4.0-4.3) with high rates of biochemical and chemical oxygen demand (BOD: 52-58, COD: 92-100 kg/m3) and suspended solids (2.0-2.5 kg/m3) . Biogas is recovered from high strength raw spentwash through the full-scale application of a biomethanation system as pretreatment option, comprising anaerobic fixed film reactors . This, combined with subsequent concentration through multiple effect evaporators (MEE), and utilization of concentrated effluent for biocomposting of pressmud (another by-product of the industry) for production of biomanure contributes to the elimination of effluent discharges. Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 501 - 8 Epub 2002 Jun 22. Genetic and physiological analysis of branched-chain alcohols and isoamyl acetate production in Saccharomyces cerevisiae; Yoshimoto H et al.; Branched-chain alcohols, such as isoamyl alcohol and isobutanol, and isoamyl acetate are important flavor components of yeast-fermented alcoholic beverages . Analysis of a null mutant of the BAT2 gene encoding cytosolic branched-chain amino acid aminotransferase, and a transformant with multi-copy plasmids containing the BAT2 gene showed that the BAT2 gene product plays an important role in the production of branched-chain alcohols and isoamyl acetate . Fermentation tests using the bat2 null mutant transformed with multi-copy plasmids carrying the ATF1 gene, which encodes alcohol acetyltransferase, indicated that modified expression of BAT2 and ATF1 genes could significantly alter the proportion of branched-chain alcohols and isoamyl acetate synthesized . Furthermore, fermentation tests using different ratios of nitrogen source and RNA blot analyses demonstrated that transcription of L-leucine biosynthetic ( LEU) and BAT genes is co-regulated by nitrogen source, that production of isoamyl alcohol depends on this transcription, and that ATF transcription increased with increased concentrations of nitrogen source . Our data suggest that changes in isoamyl alcohol production by nitrogen source are due to transcriptional co-regulation of LEU and BAT genes, and that production of isoamyl acetate is dependent on isoamyl alcohol production and ATF transcription. Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 443 - 8 Epub 2002 Jun 27. The influence of bark on the fermentation of Douglas-fir whitewood pre-hydrolysates; Robinson J et al.; Douglas-fir ( Pseudotsuga menziesii) whitewood was supplemented with increasing concentrations of bark (0-30%) and was pretreated using SO(2)-catalysed steam explosion . The presence of bark in the feedstock resulted in the decreased recovery of total sugars, furfural and 5-hydroxymethylfurfural in the resultant pre-hydrolysate . No detrimental impact on monomer sugar recovery was observed . The concentration of lipophilic extractives present in the pre-hydrolysate increased with increasing bark loading, to a maximum of 0.43 g x l(-1) . The water-soluble pre-hydrolysates were fermented by Saccharomyces cerevisiae to determine the impact of bark on sugar consumption and ethanol production . Despite the inclusion of bark, fermentation of all pre-hydrolysates resulted in the complete consumption of hexose sugars within 48 h . Ethanol yields were greater than 0.43 g x g(-1) for all pre-hydrolysates regardless of bark content, indicating that, up to a content of 30%, bark had a negligible impact on the fermentation of the pre-hydrolysates to ethanol. Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 436 - 42 Epub 2002 Jul 03. Fermentation performance and intracellular metabolite patterns in laboratory and industrial xylose-fermenting Saccharomyces cerevisiae; Zaldivar J et al.; Heterologous genes for xylose utilization were introduced into an industrial Saccharomyces cerevisiae, strain A, with the aim of producing fuel ethanol from lignocellulosic feedstocks . Two transformants, A4 and A6, were evaluated by comparing the performance in 4-l anaerobic batch cultivations to both the parent strain and a laboratory xylose-utilizing strain: S . cerevisiae TMB 3001 . During growth in a minimal medium containing a mixture of glucose and xylose (50 g/l each), glucose was preferentially consumed . During the first growth phase on glucose, the specific growth rates were 0.26, 0.32, 0.27 and 0.30 h(-1) for strains TMB 3001, A (parental strain), A4, and A6, respectively . The specific ethanol productivities were 0.04, 0.13, 0.04 and 0.03 g/g.per hour, for TMB 3001, A, A4 and A6, respectively . The specific xylose consumption rates were 0.06, 0.21 and 0.14 g/g.per hour, respectively for strains TMB 3001, A4 and A6 . Xylose consumption resulted mainly in the formation of xylitol, with biomass and ethanol being minor products . The metabolite profile of intermediates in the pentose phosphate pathway and key glycolytic intermediates were determined during growth on glucose and xylose, respectively . The metabolite pattern differed depending on whether glucose or xylose was utilized . The levels of intracellular metabolites were higher in the industrial strains than in the laboratory strain during growth on xylose. Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 409 - 18 Epub 2002 Jul 03. Microbial alkaline pectinases and their industrial applications: a review; Hoondal GS et al.; The biotechnological potential of pectinolytic enzymes from microorganisms has drawn a great deal of attention from various researchers worldwide as likely biological catalysts in a variety of industrial processes . Alkaline pectinases are among the most important industrial enzymes and are of great significance in the current biotechnological arena with wide-ranging applications in textile processing, degumming of plant bast fibers, treatment of pectic wastewaters, paper making, and coffee and tea fermentations . The present review features the potential applications and uses of microbial alkaline pectinases, the nature of pectin, and the vast range of pectinolytic enzymes that function to mineralize pectic substances present in the environment . It also emphasizes the environmentally friendly applications of microbial alkaline pectinases thereby revealing their underestimated potential . The review intends to explore the potential of these enzymes and to encourage new alkaline pectinase-based industrial technology. Cytotherapy, 2001, 3(3), 233 - 42 Manufacture and quality control of CAMPATH-1 antibodies for clinical trials; Phillips J et al.; BACKGROUND: CAMPATH-1 Abs have been used for T-cell depletion in stem-cell transplantation since the early 1980s . During that time there has been substantial progress in manufacturing techniques and quality control procedures . This article summarizes the methods used to produce the Abs for clinical use and describes results of quality control tests on representative batches . METHODS: Rat hybridoma and recombinant CHO cells were cultured in hollow-fiber fermentors . Antibodies were purified from the culture supernatant by fractionation with ammonium sulphate, or by column chromatography . Additional steps were added to assure the removal of DNA and viruses . A range of analytical methods was used to characterize the antibodies . Samples were stored frozen at -70 degrees C and re-analyzed many years later to assess the long-term stability . RESULTS: Hollow-fiber fermentors provided a simple and reliable means for antibody production, with yields between 3-10 mg/h and a convenient concentration for further processing (0.6-2.0 mg/mL) . All of the CAMPATH-1 Abs (rat IgM, rat IgG2b and human IgG1) could be purified by affinity chromatography on Protein A, but the low pH required for elution caused unacceptable aggregation of the IgM . CAMPATH-1H contained approx . 20% dimeric IgG, which could be removed by size exclusion chromatography . Antibodies were stable for at least 6 years at -70 degrees C, but there was unacceptable aggregation of CAMPATH-1M in one batch stored for 9 years . DISCUSSION: Pilot-scale production of MAbs for clinical studies is feasible in a small academic center, but regulatory requirements now demand that great attention is paid to all aspects of manufacturing and quality assurance . Although the underlying principles of cell culture and protein chemistry remain the same, the level of documentation, validation and quality control has increased greatly over the last 20 years. Curr Pharm Des, 2002, 8(19), 1707 - 12 Epothilones: a novel class of non-taxane microtubule-stabilizing agents; Altaha R et al.; The epothilones are a novel class of non-taxane microtubule-stabilizing agents obtained from the fermentation of the cellulose degrading myxobacteria, Sorangium cellulosum . Preclinical studies have shown that the epothilones are more potent than the taxanes and active in some taxane-resistant models . Similar to paclitaxel and other taxanes, the epothilones block cells in mitosis, resulting in cell death . The chief components of the fermentation process are epothilones A and B, with epothilones C and D found in smaller amounts . Trace amounts of other epothilones have also been detected . Pre-clinical studies have shown that epothilone B is the most active form, exhibiting significantly higher antitumor activity than paclitaxel and docetaxel . Several phase I and phase II clinical trials are ongoing with epothilone B and BMS 247550, an epothilone B analog . Preliminary reports indicate these agents are active against human cancers in heavily pre-treated patients . The epothilones appear to be well tolerated, with a side effect profile that is similar to that reported with the taxanes . This article will review some basic aspects of epothilone chemistry and biology, and pre-clinical and preliminary clinical experience with epothilone B and its analog, BMS 247550. Mycopathologia, 2002, 154(3), 127 - 38 Effect of liquid culture media on morphology, growth, propagule production, and pathogenic activity of the Hyphomycete, Metarhizium flavoviride; Fargues J et al.; Two isolates of Metarhizium spp . were studied for propagule production, because of their pathogenic activity towards locusts and grasshoppers (Mf189 = M . flavoviride (or M . anisopliae var . acridum) strain IMI 330189, and Mf324 = M . flavoviride strain ARSEF324) . Both isolates were grown in seven different liquid media, which have been developed for mass production of various Hyphomycetes, considered as candidates for microbial control of noxious insects . Shake-flask experiments were carried out at 28 degress C in the dark . Production was quantified for 72 h and the effects of the tested media were evaluated on propagule concentration, morphology and pathogenicity . Based on preliminary experiments, all tested media were supplemented with 0.4% Tween 80 to avoid the formation of pellets and to produce unicellular propagules . Submerged propagule yields were higher with Mf189 than with Mf324 in all seven media . While high concentrations of propagules (1.4 to 2.4 x 10(8) propagules ml(-1) for MF189 and 1.4 to 8.3 x 10(7) propagules ml(-1) for Mf324) were produced in four media (Adamek, Catroux, Jackson, and Jenkins-Prior media), production of propagules was lower in the three other media (Goral, Kondryatiev, and Paris media) . Both isolates produced oblong blastospore-like propagules, except in Kondryatiev medium in which they provided ovoid propagules . In this case, Mf189 submerged propagules looked like aerial conidia, but scanning observations did not demonstrate a typical conidiogenesis via phialides . In Kondryatiev medium, Mf324 submerged propagules were significantly smaller than aerial conidia . Infection potential of submerged propagules was assayed on Schistocerca gregaria . Second-instar larvae fed for 48 h on fresh wheat previously contaminated by a spraying suspension of each inoculum titrated at 10(7) propagules ml(-1) . All seven media produced submerged propagules that were highly infectious for S . gregaria larvae . Shake flask culture assays permitted us to select three low-costmedia, Adamek, Jenkins-Prior, and Catroux for improving scale-up of liquid fermentation focused on mass-production of Metarhizium propagules for mycoinsecticides devoted to locust control. Eur J Gastroenterol Hepatol, 2002 Jul, 14(7), 753 - 6 Evaluation of hydrogen excretion after lactulose administration as a screening test for causes of irritable bowel syndrome; Sen S et al.; OBJECTIVE : To determine whether it is possible to separate cases of irritable bowel syndrome associated with excess total hydrogen production (as a surrogate of colonic fermentation; these patients may be offered an exclusion diet as treatment) from other causes of irritable bowel syndrome by determining the amount of hydrogen excreted on patients' breath after oral administration of lactulose . DESIGN : Comparison of 24-hour hydrogen excretion and breath hydrogen following lactulose in untreated patients fulfilling the Rome criteria for irritable bowel syndrome, normal controls and irritable bowel syndrome patients who had previously failed to improve on an exclusion diet . METHODS : Colonic fermentation was measured by indirect calorimetry over 24 h . Immediately after calorimetry, the patients who were fasting received 20 g lactulose; end-expiratory breath samples were then collected every 30 min for 3 h . Hydrogen concentrations were determined by an electro-chemical cell . RESULTS : The total 24-hour excretion of hydrogen was significantly greater in the irritable bowel syndrome group (median 333.7 ml/24 h, interquartile range 234.7-445.67) compared to the normal volunteers (median 203.1 ml/24 h, interquartile range 131.4-256; P = 0.002) or the failed-diet group (median 204.5 ml/24 h, interquartile range 111.35-289.13; P = 0.015) . No difference was detected in breath excretion of hydrogen following lactulose in any group . CONCLUSION : Total hydrogen production over 24 h is increased in some patients with irritable bowel syndrome who may respond to exclusion diets . However, this sub-group of patients cannot be identified by measuring breath-hydrogen excretion after lactulose. Anticancer Res, 2002 May-Jun, 22(3), 1569 - 74 Diverse biological activities of fermented pine seed shell extract; Mihara S et al.; Intraperitoneal administration of fermented pine seed shell extract (PSSE) (up to 2 g/kg) induced no apparent acute toxicity to mice . Pretreatment of mice with PSSE protected them from the lethality of Escherichia coli infection . PSSE showed a very weak cytotoxic activity against both normal and tumor cells and no anti-HIV activity, but stimulated the mouse macrophage-like Raw 264.7 cells to produce nitric oxide (NO) and citrulline . ESR spectroscopy showed that PSSE produced no detectable radicals, but effectively scavenged O2- (generated by the hypoxanthine-xanthine oxidase reaction), hydroxyl radical (generated by the Fenton reaction) and NO (generated by NOC-7) . Comparison of PSSE with other natural products, such as polyphenols and vitamins, further confirmed the close association between radical intensity and radical scavenging activity, suggesting the bimodal action of natural products . Although the biological activities of PSSE were relatively lower than those of other natural products, the present study suggests the possible medicinal efficacy of PSSE. J Agric Food Chem, 2002 Aug 14, 50(17), 4895 - 9 Content of biogenic amines in a Chardonnay wine obtained through spontaneous and inoculated fermentations; Torrea D et al.; This paper describes the content of biogenic amines in wines obtained from a Chardonnay must inoculated with different strains of Saccharomyces cerevisiae and in a wine fermented with the indigenous yeasts (control wine) . The concentrations of nonvolatile amines and phenethylamine in the wines from the inoculated must were superior to those of the control wine . This was probably due to the fact that consumption of the precursor amino acids of these amines, during fermentation, was also greater in the inoculated samples than in the control sample . Furthermore, from the results obtained it may be said that, at least to some extent, the nonvolatile amines were formed by yeasts during fermentation . The concentrations of dimethylamine, ethylamine, and pyrrolidine (volatile amines) were different for the different wines, although they did not reach concentrations sufficiently high to have any effect on the aroma. FEMS Microbiol Rev, 2002 Aug, 26(3), 285 - 309 Enzymology and bioenergetics of respiratory nitrite ammonification; Simon J; Nitrite is widely used by bacteria as an electron acceptor under anaerobic conditions . In respiratory nitrite ammonification an electrochemical proton potential across the membrane is generated by electron transport from a non-fermentable substrate like formate or H(2) to nitrite . The corresponding electron transport chain minimally comprises formate dehydrogenase or hydrogenase, a respiratory quinone and cytochrome c nitrite reductase . The catalytic subunit of the latter enzyme (NrfA) catalyzes nitrite reduction to ammonia without liberating intermediate products . This review focuses on recent progress that has been made in understanding the enzymology and bioenergetics of respiratory nitrite ammonification . High-resolution structures of NrfA proteins from different bacteria have been determined, and many nrf operons sequenced, leading to the prediction of electron transfer pathways from the quinone pool to NrfA . Furthermore, the coupled electron transport chain from formate to nitrite of Wolinella succinogenes has been reconstituted by incorporating the purified enzymes into liposomes . The NrfH protein of W . succinogenes, a tetraheme c-type cytochrome of the NapC/NirT family, forms a stable complex with NrfA in the membrane and serves in passing electrons from menaquinol to NrfA . Proteins similar to NrfH are predicted by open reading frames of several bacterial nrf gene clusters . In gamma-proteobacteria, however, NrfH is thought to be replaced by the nrfBCD gene products . The active site heme c group of NrfA proteins from different bacteria is covalently bound via the cysteine residues of a unique CXXCK motif . The lysine residue of this motif serves as an axial ligand to the heme iron thus replacing the conventional histidine residue . The attachment of the lysine-ligated heme group requires specialized proteins in W . succinogenes and Escherichia coli that are encoded by accessory nrf genes . The proteins predicted by these genes are unrelated in the two bacteria but similar to proteins of the respective conventional cytochrome c biogenesis systems. J Altern Complement Med, 2002 Jun, 8(3), 315 - 23 A