Biosci Biotechnol Biochem, 2001 Dec, 65(12), 2755 - 62 Membrane-bound quinoprotein D-arabitol dehydrogenase of Gluconobacter suboxydans IFO 3257: a versatile enzyme for the oxidative fermentation of various ketoses; Adachi O et al.; Solubilization of membrane-bound quinoprotein D-arabitol dehydrogenase (ARDH) was done successfully with the membrane fraction of Gluconobacter suboxydans IFO 3257 . In enzyme solubilization and subsequent enzyme purification steps, special care was taken to purify ARDH as active as it was in the native membrane, after many disappointing trials . Selection of the best detergent, keeping ARDH as the holoenzyme by the addition of PQQ and Ca2+, and of a buffer system involving acetate buffer supplemented with Ca2+, were essential to treat the highly hydrophobic and thus labile enzyme . Purification of the enzyme was done by two steps of column chromatography on DEAE-Toyopearl and CM-Toyopearl in the presence of detergent and Ca2+ . ARDH was homogenous and showed a single sedimentation peak in analytical ultracentrifugation . ARDH was dissociated into two different subunits upon SDS-PAGE with molecular masses of 82 kDa (subunit I) and 14 kDa (subunit II), forming a heterodimeric structure . ARDH was proven to be a quinoprotein by detecting a liberated PQQ from SDS-treated ARDH in HPLC chromatography . More preliminarily, an EDTA-treated membrane fraction lost the enzyme activity and ARDH activity was restored to the original level by the addition of PQQ and Ca2+ . The most predominant unique character of ARDH, the substrate specificity, was highly versatile and many kinds of substrates were oxidized irreversibly by ARDH, not only pentitols but also other polyhydroxy alcohols including D-sorbitol, D-mannitol, glycerol, meso-erythritol, and 2,3-butanediol . ARDH may have its primary function in the oxidative fermentation of ketose production by acetic acid bacteria . ARDH contained no heme component, unlike the type II or type III quinoprotein alcohol dehydrogenase (ADH) and did not react with primary alcohols. Biodegradation, 2001, 12(4), 247 - 57 Protein degradation during anaerobic wastewater treatment: derivation of stoichiometry; Ramsay IR et al.; The stoichiometry of reactions that describe protein degradation in anaerobic treatment systems were investigated . A methodology was developed to describe protein degradation to organic acids using a single reaction step . The reactions for individual amino acid fermentation and their mediating organisms were reviewed . The dominant fermentation pathways were selected based on a number of assumptions . Using the amino acid content of a model protein, it was then possible to determine stoichiometric coefficients for each major organic acid product in the overall degradation of the protein . The theoretical coefficients were then compared to those determined from two experimental runs on a continuously-fed, well-mixed, laboratory-scale anaerobic wastewater treatment system . In general, the coefficients compared well thus validating the use of a single reaction step for the overall catabolic reaction of protein degradation to organic acids . Furthermore, even when the protein concentration in feed or the feed flow rate was doubled, the amino acid fermentation pathways were found to occur predominantly by only one pathway . Although the choice of Stickland reactions over uncoupled degradation provided good comparisons, an electron balance showed that only about 40% of the amino acids could have proceeded coupled to other amino acid reactions . Uncoupled degradation of the remaining amino acids must have relied on the uptake of hydrogen produced from these reactions by hydrogen-consuming methane bacteria. J Biol Chem, 2002 Apr 19, 277(16), 13983 - 8 Epub 2002 Feb 01. Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays; Chambergo FS et al.; Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered . One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina) . Using EST analysis and cDNA microarrays, we find that in T . reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation . Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism . The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation . This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass. J Microbiol Immunol Infect, 2001 Dec, 34(4), 235 - 42 Oxytetracycline production by immobilized Streptomyces rimosus; Yang SS et al.; This study examined whether the production of oxytetracycline by Streptomyces rimosus TM-55 (CCRC 960061) would be improved with calcium alginate immobilization in submerged fermentation compared with free cells . Results showed that in 1-mL culture broth, free cells produced 121 to 124 microg of oxytetracycline, whereas immobilized cells produced 153 to 252 microg . Immobilization of the cells retarded the growth rate of S . rimosus but increased the length of the growth period and improved the oxytetracycline production . The specific oxytetracycline productivity was 33.3 to 34.2 mg in each gram of free dry cells and was 40.2 to 40.7 mg in immobilized dry cells . The optimum immobilization conditions were alginate 2% and bead diameter of 2.13 mm . Oxytetracycline production increased with increasing inoculum density but decreased with increasing bead diameter . Ethylenediaminetetraacetic acid or monovalent ions could react with calcium in the bead or replace it with sodium ion, thereby reducing the strength of the beads. J Nutr, 2002 Feb, 132(2), 238 - 44 Soymilk products affect ethanol absorption and metabolism in rats during acute and chronic ethanol intake; Kano M et al.; In this study we evaluated the effects of soy products on ethanol metabolism during periods of acute and chronic consumption in rats . Gastric ethanol content and blood ethanol and acetaldehyde concentrations were investigated after the oral administration of ethanol (34 mmol/kg) plus soy products such as soymilk (SM) or fermented soymilk (FSM) . The gastric ethanol concentration of the FSM group was greater than that of the control group, whereas portal and aortal blood ethanol concentrations of the FSM group were lower than in controls . The aortal acetaldehyde concentration in the FSM group was lower than that of the control group . The direct effect of isoflavones on liver function was investigated by using hepatocytes isolated from untreated rats . Genistein (5 micromol/L) decreased ethanol (P = 0.045) and tended to decrease acetaldehyde (P = 0.10) concentrations in the culture filtrate . Some variables of ethanol metabolism in the liver were investigated after chronic ethanol exposure for 25 d . Rats consumed a 5% ethanol fluid plus the SM diet, the FSM diet or a control diet . Microsomal ethanol oxidizing activity was significantly lower in the FSM group than the control group . Furthermore, cytosolic glutathione S-transferase activity was higher in the SM and FSM groups than in the control group . Acetaldehyde dehydrogenase activity (low K(m)) in the FSM group (P = 0.15), but not in the SM group (P = 0.31), tended to be greater than in the control group . The amount of thiobarbituric acid reacting substances in the liver of the SM and FSM groups tended to be less than that of the control group (P = 0.18 and 0.10, respectively) . These results demonstrate that soymilk products inhibit ethanol absorption and enhance ethanol metabolism in rats. Appl Environ Microbiol, 2002 Feb, 68(2), 893 - 900 Characterization and description of Anaeromyxobacter dehalogenans gen . nov., sp . nov., an aryl-halorespiring facultative anaerobic myxobacterium; Sanford RA et al.; Five strains were isolated which form a physiologically and phylogenetically coherent group of chlororespiring microorganisms and represent the first taxon in the Myxobacteria capable of anaerobic growth . The strains were enriched and isolated from various soils and sediments based on their ability to grow using acetate as an electron donor and 2-chlorophenol (2-CPh) as an electron acceptor . They are slender gram-negative rods with a bright red pigmentation that exhibit gliding motility and form spore-like structures . These unique chlororespiring myxobacteria also grow with 2,6-dichlorophenol, 2,5-dichlorophenol, 2-bromophenol, nitrate, fumarate, and oxygen as terminal electron acceptors, with optimal growth occurring at low concentrations (<1 mM) of electron acceptor . 2-CPh is reduced by all strains as an electron acceptor in preference to nitrate, which is reduced to ammonium . Acetate, H(2), succinate, pyruvate, formate, and lactate were used as electron donors . None of the strains grew by fermentation . The 16S ribosomal DNA (rDNA) sequences of the five strains form a coherent cluster deeply branching within the family Myxococcaceae within the class Myxobacteria and are mostly closely associated with the Myxococcus subgroup . With the exception of anaerobic growth and lack of a characteristic fruiting body, these strains closely resemble previously characterized myxobacteria and therefore should be considered part of the Myxococcus subgroup . The anaerobic growth and 9.0% difference in 16S rDNA sequence from those of other myxobacterial genera are sufficient to place these strains in a new genus and species designated Anaeromyxobacter dehalogenans . The type strain is 2CP-1 (ATCC BAA-258). Biotechnol Prog, 2002 Jan-Feb, 18(1), 51 - 4 Two-stage culture process for improved production of ganoderic acid by liquid fermentation of higher fungus Ganoderma lucidum; Fang QH et al.; Investigations on the impact of pellet size on the cellular oxygen uptake and accumulation of ganoderic acid (GA) suggested the favorable effect of oxygen limitation on GA formation by the higher fungus Ganoderma lucidum . A two-stage fermentation process was thus proposed for enhanced GA production by combining conventional shake-flask fermentation with static culture . A high cell density of 20.9 g of DW/L (DW = dry cell weight) was achieved through a 4-day shake-flask fermentation followed by a 12-day static culture . A change in the cell morphology and a decrease in the sugar consumption rate were observed during the static culture . The GA production in the new two-stage process was considerably enhanced with its content increased from 1.36 (control) to 3.19 mg/100 mg of DW, which was much higher than previously observed. Biotechnol Prog, 2002 Jan-Feb, 18(1), 29 - 35 Separation characteristics of liquid nematode cultures and the design of recovery operations; Young JM et al.; Production of nematode-based pesticides involves the recovery of a viable nematode life stage known as the infective juvenile (IJ) from fermentation broth . Waste components to be separated from the IJs include non-IJ life stages, dead nematodes, nematode debris, spent media, and the nematode's associated bacteria . This paper reports separation characteristics of liquid cultures and suspensions of the nematodes Phasmarhabditis hermaphrodita, Steinernema feltiae, and Heterorhabditis megidis measured at small scale . Separation characteristics were determined for dead-end filtration, gravity settling and flotation . Results were used to identify large-scale recovery procedures . Separation of culture liquid by dead-end filtration of the crude fermentation broth was not possible due to rapid blinding of filters . However, nematode-water suspensions prepared by gravity settling could be concentrated using this separation method . Settling tests indicated that IJs could be efficiently separated from culture liquid by centrifugation but not by gravity settling . Examination of the effects of nematode concentration indicated an optimum concentration for gravity settling that may entail modest dilution of the fermentation broth . Flocculation of insoluble spent media in suspensions of P . hermaphrodita prevented its separation from nematodes by gravity settling . However, attachment of air bubbles to spent media allowed removal by flotation . Finally, adjustment of continuous phase density using sucrose allowed separation of non-IJ life stages, dead nematodes, and discarded cuticles from the IJs by flotation . The efficiency of this separation decreased with increasing nematode-solute contact time. Biotechnol Prog, 2002 Jan-Feb, 18(1), 6 - 20 Global gene expression differences associated with changes in glycolytic flux and growth rate in Escherichia coli during the fermentation of glucose and xylose; Gonzalez R et al.; The simplicity of the fermentation process (anaerobic with pH, temperature, and agitation control) in ethanologenic Escherichia coli KO11 and LY01 makes this an attractive system to investigate the utility of gene arrays for biotechnology applications . By using this system, gene expression, glycolytic flux, and growth rate have been compared in glucose-grown and xylose-grown cells . Although the initial metabolic steps differ, ethanol yields from both sugars were essentially identical on a weight basis, and little carbon was diverted to biosynthesis . Expression of only 27 genes changed by more than 2-fold in both strains . These included induction of xylose-specific operons (xylE, xylFGHR, and xylAB) regulated by XylR and the cyclic AMP-CRP system and repression of Mlc-regulated genes encoding glucose uptake (ptsHIcrr, ptsG) and mannose uptake (manXYZ) during growth on xylose . However, expression of genes encoding central carbon metabolism and biosynthesis differed by less than 2-fold . Simple statistical methods were used to investigate these more subtle changes . The reproducibility (coefficient of variation of 12%) of expression measurements (mRNA as cDNA) was found to be similar to that typically observed for in vitro measurements of enzyme activities . Using Student's t test, many smaller but significant sugar-dependent changes were identified (p < 0.05 in both strains) . A total of 276 genes were more highly expressed during growth on xylose; 307 genes were more highly expressed with glucose . Slower growth (lower ATP yield) on xylose was accompanied by decreased expression of 62 genes concerned with the biosynthesis of small molecules (amino acids, nucleotides, cofactors, and lipids), transcription, and translation; 5 such genes were expressed at a higher level . In xylose-grown cells, 90 genes associated with the transport, catabolism, and regulation of pathways for alternative carbon sources were expressed at higher levels than in glucose-grown cells, consistent with a relaxation of control by the cyclic AMP-CRP regulatory system . Changes in expression of genes encoding the Embden-Meyerhof-Parnas (EMP) pathway were in excellent agreement with calculated changes in flux for individual metabolites . Flux through all but one step, pyruvate kinase, was predicted to be higher during glucose fermentation . Expression levels (glucose/xylose) were higher in glucose-grown cells for all EMP genes except the isoenzymes encoding pyruvate kinase (pykA and pykF) . Expression of both isoenzymes was generally higher during xylose fermentation but statistically higher in both strains only for pykF encoding the isoenzyme activated by fructose-6-phosphate, a key metabolite connecting pentose metabolism to the EMP pathway . The coordinated changes in expression of genes encoding the EMP pathway suggest the presence of a common regulatory system and that flux control within the EMP pathway may be broadly distributed . In contrast, expression levels for genes encoding the Pentose-Phosphate pathway did not differ significantly between glucose-grown and xylose-grown cells. J Chromatogr A, 2002 Jan 4, 942(1-2), 123 - 31 Direct capture of product from fermentation broth using a cell-repelling ion exchanger; Dainiak MB et al.; A new technique for treating anion exchangers has been proposed allowing direct capture of the fermentation product, shikimic acid directly from the cell-containing fermentation broth . A layer of hydrophilic polymer, poly(acrylic acid) (PAA) has been physically adsorbed on the anion exchanger followed by a covalent cross-linking of PAA . The PAA layer is penetrable for small molecules despite being negatively charged as PAA is, but the polymer layer repels large negatively charged structures like cell debris and cells preventing them from adsorption to the chromatographic matrix . The binding capacity for pure shikimic was about 81 mg/ml adsorbent for both cross-linked PAA-Amberlite and native Amberlite in the fluidized mode of column operation . Binding capacity dropped to 17 and 15 mg per ml adsorbent, respectively, when using filtrated fermentation broth and to about 10 mg/ml adsorbent for cross-linked PAA-Amberlite when using directly the fermentation broth containing cells . Native Amberlite cannot be used for the direct capture of shikimic acid due to the immediate clogging of the column and the collapse of the expanded bed . The cross-linked PAA-Amberlite was used repeatedly for the direct adsorption of shikimic acid from the industrial fermentation broth. J Contam Hydrol, 2001 Dec 15, 53(3-4), 387 - 406 Progression of natural attenuation processes at a crude oil spill site: II . Controls on spatial distribution of microbial populations; Bekins BA et al.; A multidisciplinary study of a crude-oil contaminated aquifer shows that the distribution of microbial physiologic types is strongly controlled by the aquifer properties and crude oil location . The microbial populations of four physiologic types were analyzed together with permeability, pore-water chemistry, nonaqueous oil content, and extractable sediment iron . Microbial data from three vertical profiles through the anaerobic portion of the contaminated aquifer clearly show areas that have progressed from iron-reduction to methanogenesis . These locations contain lower numbers of iron reducers, and increased numbers of fermenters with detectable methanogens . Methanogenic conditions exist both in the area contaminated by nonaqueous oil and also below the oil where high hydrocarbon concentrations correspond to local increases in aquifer permeability . The results indicate that high contaminant flux either from local dissolution or by advective transport plays a key role in determining which areas first become methanogenic . Other factors besides flux that are important include the sediment Fe(II) content and proximity to the water table . In locations near a seasonally oscillating water table, methanogenic conditions exist only below the lowest typical water table elevation . During 20 years since the oil spill occurred, a laterally continuous methanogenic zone has developed along a narrow horizon extending from the source area to 50-60 m downgradient . A companion paper {J . Contam . Hydrol . 53, 369-386} documents how the growth of the methanogenic zone results in expansion of the aquifer volume contaminated with the highest concentrations of benzene, toluene, ethylbenzene, and xylenes. J Contam Hydrol, 2001 Dec 15, 53(3-4), 233 - 67 Processes controlling the distribution and natural attenuation of dissolved phenolic compounds in a deep sandstone aquifer; Thornton SF et al.; Processes controlling the distribution and natural attenuation (NA) of phenol, cresols and xylenols released from a former coal-tar distillation plant in a deep Triassic sandstone aquifer are evaluated from vertical profiles along the plume centerline at 130 and 350 m from the site . Up to four groups of contaminants (phenols, mineral acids, NaOH, NaCl) form discrete and overlapping plumes in the aquifer . Their distribution reflects changing source history with releases of contaminants from different locations . Organic contaminant distribution in the aquifer is determined more by site source history than degradation . Contaminant degradation at total organic carbon (TOC) concentrations up to 6500 mg l(-1) (7500 mg l(-1) total phenolics) is occurring by aerobic respiration NO3-reduction, Mn(IV)-/Fe(III)-reduction, SO4-reduction, methanogenesis and fermentation, with the accumulation of inorganic carbon, organic metabolites (4-hydroxybenzaldehyde, 4-hydroxybenzoic acid), acetate, Mn(II), Fe(II), S(-II), CH4 and H2 in the plume . Aerobic and NO3-reducing processes are restricted to a 2-m-thick plume fringe but Mn(IV)-/Fe(II)-reduction, SO4-reduction, methanogenesis and fermentation occur concomitantly in the plume . Dissolved H2 concentrations in the plume vary from 0.7 to 110 nM and acetate concentrations reach 200 mg l(-1) . The occurrence of a mixed redox system and concomitant terminal electron accepting processes (TEAPs) could be explained with a partial equilibrium model based on the potential in situ free energy (deltaGr) yield for oxidation of H2 by specific TEAPs . Respiratory processes rather than fermentation are rate limiting in determining the distribution of H2 and TEAPs and H2 dynamics in this system . Most (min . 90%) contaminant degradation has occurred by aerobic and NO3-reducing processes at the plume fringe . This potential is determined by the supply of aqueous O2 and NO3 from uncontaminated groundwater, as controlled by transverse mixing, which is limited in this aquifer by low dispersion . Consumption to date of mineral oxides and SO4 is, respectively, <0.15% and 0.4% of the available aquifer capacity, and degradation using these oxidants is <10% . Fermentation is a significant process in contaminant turnover, accounting for 21% of degradation products present in the plume, and indicating that microbial respiration rates are slow in comparison with fermentation . Under present conditions, the potential for degradation in the plume is very low due to inhibitory effects of the contaminant matrix . Degradation products correspond to <22% mass loss over the life of the plume, providing a first-order plume scale half-life >140 years . The phenolic compounds are biodegradable under the range of redox conditions in the aquifer and the aquifer is not oxidant limited, but the plume is likely to be long-lived and to expand . Degradation is likely to increase only after contaminant concentrations are reduced and aqueous oxidant inputs are increased by dispersion of the plume . The results imply that transport processes may exert a greater control on the natural attenuation of this plume than aquifer oxidant availability. Ned Tijdschr Tandheelkd, 1992 Jun, 99(6), 201 - 3 {Caries-- changing patterns}; ten Cate JM; This article briefly describes the pathogenesis of dental caries . It focuses on the relevant chemical processes and histological changes in dental enamel . Recent studies have shown that the mineral balance at the tooth-oral fluid interface are determinants for the occurrence of caries, and more important than the intrinsic tissue properties . During periods of low pH, following bacterial metabolism of fermentable carbohydrates, enamel is demineralized . However, loss of tissue may be compensated by mineral deposition from saliva during subsequent periods at neutral pH ('remineralization') . The mineral balance is positively affected by the presence of low fluoride levels in the oral fluids . Clinically this becomes manifest in the decreasing rates of caries initiation and progression . Also it may affect the appearance of incipient enamel lesions, which in turn could complicate caries diagnosis . The changing patterns of dental changes thus have implications for procedures in preventive and restorative practise. Int J Food Sci Nutr, 2002 Jan, 53(1), 15 - 27 Nutritional and sensory evaluation of tempe-fortified maize-based weaning foods; Egounlety M et al.; Maize-based weaning foods prepared from whole maize meal or dehydrated fermented maize (ogi) flour fortified with soybean, cowpea or groundbean tempe, with or without the addition of melon seed flour were fed to 40 albino weanling rats . The nutrient quality of the weaning foods was monitored by the measurement of protein efficiency ratio (PER), net protein ratio (NPR), true digestibility (TD), biological value (BV) and net protein utilization (NPU) . Amino acid composition and amino acid scores were determined . The weaning foods were also evaluated for sensory properties and acceptability by nursing mothers and their children . Protein efficiency ratio, net protein retention and net protein utilization of the fortified weaning foods were not significantly different (P > 0.05) when compared to casein diet (control) . True digestibility of tempe-fortified maize-based weaning foods averaged 85.4 +/- 3.4% compared to 94.4%, for casein diet . Results from sensory analysis showed that relative to tempe-fortified maize-based weaning foods, most mothers preferred Cerelac, a commercial weaning food, and its porridge because of its vanillic flavour and sweet taste . Despite the low scores recorded for aroma and taste of tempe-fortified maize-based weaning foods, most mothers and their children found porridges prepared from these foods acceptable, with a preference for ogi fortified with cowpea tempe . Tempe-fortified maize-based weaning foods had high protein quality and can support the growth of infants in developing countries especially during the critical weaning period (6-12 months) . They were comparable to Cerelac, a commercial baby food in colour and texture. World J Gastroenterol, 2001 Dec, 7(6), 741 - 51 Management of patients with a short bowel; Nightingale JM; There are two common types of adult patient with a short bowel, those with jejunum in continuity with a functioning colon and those with a jejunostomy . Both groups have potential problems of undernutrition, but this is a greater problem in those without a colon, as they do not derive energy from anaerobic bacterial fermentation of carbohydrate to short chain fatty acids in the colon . Patients with a jejunostomy have major problems of dehydration, sodium and magnesium depletion all due to a large volume of stomal output . Both types of patient have lost at least 60 cm of terminal ileum and so will become deficient of vitamin B(12) . Both groups have a high prevalence of gallstones (45%) resulting from periods of biliary stasis . Patients with a retained colon have a 25% chance of developing calcium oxalate renal stones and they may have problems with D(-) lactic acidosis . The survival of patients with a short bowel, even if they need long-term parenteral nutrition, is good. Membr Cell Biol, 2001, 14(6), 749 - 63 Participation of hyf-encoded hydrogenase 4 in molecular hydrogen release coupled with proton-potassium exchange in Escherichia coli; Bagramyan K et al.; In a previous work (Trchounian et al., Biol . Membrany 16:416-428 (1999) (in Russian)) we reported the interrelations between production of H2 and H+-K+ exchange in fermenting Escherichia coli grown under anaerobic conditions at pH 7.5 . The ion fluxes had stable stoichiometry 2H+/K+ and were N,N'-dicyclohexylcarbodiimide (DCC)-inhibitable at different external pH and K+ activity . In the present study, the H2 production was further studied in fermenting bacteria grown at pH 7.5 or 6.5 . The H2 production was inhibited by DCC and did not occur if bacteria were grown at pH 7.5 in a medium containing formate or upon hypoosmotic stress . The H2 production was not sensitive to osmotic stress when bacteria were grown at pH 6.5 . Formation of H2 and 2H+/K+ exchange were not observed in mutants with deletions of the hyfoperon genes, encoding membrane-associated hydrogenase 4 . K+ influx in these mutants was not sensitive to valinomycin, in contrast to the K+ influx in the parental strain . If grown at pH 6.5, the mutants produced H2 and carried out 2H+/K+ exchange, when subjected to the hyperosmotic stress . The results suggest a participation of hydrogenase 4 in the production of H2 and proton-potassium exchange in fermenting E . coli grown at pH 7.5 . In bacteria grown at pH 6.5 or in a medium containing formate, another membrane-bound hydrogenase, namely hydrogenase 3, may be responsible for the H2 production. Antonie Van Leeuwenhoek, 2001 Sep, 79(3-4), 393 - 9 Influence of killer strains of Saccharomyces cerevisiae on wine fermentation; Perez F et al.; The effect of killer strains of Saccharomyces cerevisiae on the growth of sensitive strains during must fermentation was studied by using a new method to monitor yeast populations . The capability of killer yeast strains to eliminate sensitive strains depends on the initial proportion of killer yeasts, the susceptibility of sensitive strains, and the treatment of the must . In sterile filtered must, an initial proportion of 2-6% of killer yeasts was responsible for protracted fermentation and suppression of isogenic sensitive strains . A more variable initial proportion was needed to get the same effect with non-isogenic strains . The suspended solids that remain in the must after cold-settling decreased killer toxin effect . The addition of bentonite to the must avoided protracted fermentation and the suppression of sensitive strains; however, the addition of yeast dietary nutrients with yeast cell walls did not, although it decreased fermentation lag. Antonie Van Leeuwenhoek, 2001 Sep, 79(3-4), 345 - 52 Yeast population dynamics in spontaneous fermentations: comparison between two different wine-producing areas over a period of three years; Torija MJ et al.; Yeast ecology, biogeography and biodiversity are important and interesting topics of research . The population dynamics of yeasts in several cellars of two Spanish wine-producing regions was analysed for three consecutive years (1996 to 1998) . No yeast starter cultures had been used in these wineries which therefore provided an ideal winemaking environment to investigate the dynamics of grape-related indigenous yeast populations . Non-Saccharomyces yeast species were identified by RFLPs of their rDNA, while Saccharomyces species and strains were identified by RFLPs of their mtDNA . This study confirmed the findings of other reports that non-Saccharomyces species were limited to the early stages of fermentation whilst Saccharomyces dominated towards the end of the alcoholic fermentation . However, significant differences were found with previous studies, such as the survival of non-Saccharomyces species in stages with high alcohol content and a large variability of Saccharomyces strains (a total of 112, all of them identified as Saccharomyces cerevisiae) with no clear predominance of any strain throughout all the fermentation, probably related to the absence of killer phenotype and lack of previous inoculation with commercial strains. Adv Biochem Eng Biotechnol, 2001, 73, 53 - 84 Metabolic engineering of Saccharomyces cerevisiae for xylose utilization; Hahn-Hagerdal B et al.; Metabolic engineering of Saccharomyces cerevisiae for ethanolic fermentation of xylose is summarized with emphasis on progress made during the last decade . Advances in xylose transport, initial xylose metabolism, selection of host strains, transformation and classical breeding techniques applied to industrial polyploid strains as well as modeling of xylose metabolism are discussed . The production and composition of the substrates--lignocellulosic hydrolysates--is briefly summarized . In a future outlook iterative strategies involving the techniques of classical breeding, quantitative physiology, proteomics, DNA micro arrays, and genetic engineering are proposed for the development of efficient xylose-fermenting recombinant strains of S . cerevisiae. Adv Biochem Eng Biotechnol, 2001, 73, 31 - 52 Process development and metabolic engineering for the overproduction of natural and unnatural polyketides; McDaniel R et al.; Polyketide natural products are a rich source of bioactive substances that have found considerable use in human health and agriculture . Their complex structures require that they be produced via fermentation processes . This review describes the strategies and challenges used to develop practical fermentation strains and processes for polyketide production . Classical strain improvement procedures, process development methods, and metabolic engineering approaches are described . The elucidation of molecular mechanisms that underlie polyketide biosynthesis has played an important role in each of these areas over the past few years. Cancer Epidemiol Biomarkers Prev, 2002 Jan, 11(1), 121 - 6 Dietary determinants of plasma enterolactone; Horner NK et al.; Enterolactone is a lignan produced by fermentation of dietary precursors in the human gut . Because lignan precursors are uniquely found in plant foods, plasma enterolactone concentration may serve as a biological marker of plant food consumption . This cross-sectional study examined associations of dietary intake with plasma enterolactone concentration . Weight-stable, 20-40-year-old volunteers (115 women and 78 men in Seattle, Washington) reporting intake of < or =2.5 or > or =4.5 fruit and vegetable servings/day and no antibiotic use for > or =3 months completed a food frequency questionnaire and 3-day food record . Time-resolved fluoroimmunoassay was used to measure plasma enterolactone . Based on diet records, plasma enterolactone was positively correlated with daily vegetable servings (r = 0.17; P < 0.05), fiber (r = 0.36; P < 0.0001), alcohol (r = 0.24; P < 0.001), caffeine (r = 0.21; P < 0.001), and daily botanical group servings {Chenopodiaceae (r = 0.15; P < 0.05), Juglandaceae (r = 0.15; P < 0.05), Leguminosae (r = 0.20; P < 0.001), Pedaliaceae (r = 0.20; P < 0.001), and Vitaceae (r = 0.20; P < 0.001)} . Fat-related variables were not correlated with plasma enterolactone . Based on linear regression models, plasma enterolactone increased by 37.0% (SE = 2.3%) for each 10-g increase in fiber and by 6.6% (SE = 0.2%) for each 50-mg serving of caffeine . Participants consuming 0.5-1 alcoholic drink/day had plasma enterolactone concentrations that were 131.4% (SE = 37.6%) higher than those of nondrinkers . Although plasma enterolactone may be useful as a biological measure of exposure to lignan-containing foods, it may be of limited use as a specific biomarker of fruit and vegetable or plant food intake because coffee, tea, and alcoholic beverages also significantly increase its plasma concentration. Trends Biotechnol, 2002 Feb, 20(2), 72 - 8 Field-assisted extraction of cells, particles and macromolecules; Karumanchi RS et al.; Improved bioseparation techniques are increasingly important for biotechnology because separation is often the limiting factor for the success of biological processes . Manufacturers of new enzymes and pharmaceutical products require improved methods for recovering intact cells and intracellular products . Similarly the isolation, purification and concentration of many biomolecules produced in fermentation processes is extremely important . Often such downstream processing contributes a large portion of the product cost and thus efficient and economical alternative approaches to bioseparation processes are needed to eliminate, reduce or facilitate the handling of solids . Field-assisted separations, which hold immense potential for providing a major improvement in bioseparation in the near future, are considered in this review . Special emphasis is given to multistage methods, which are cost-effective compared with competing technologies . Commercial applications of these methods are detailed, we present suggestions for future work and we analyse the scale-up and economic aspects of these processes. Analyst, 2001 Dec, 126(12), 2149 - 52 Enzymatic reactions for the determination of sugars in food samples using the differential pH technique; Luzzana M et al.; All ATP coupled reactions, when performed at neutral or moderately alkaline pH, produce an acidification of the reaction mixture . The detection of small pH changes--0.1 mpH (1 mpH = 10(-3) pH)--in a constant buffering capacity solution makes it possible to quantify, over a wide concentration range (1-1500 mmol L(-1)), various analytes with very high precision and accuracy . Glucose, fructose, glycerol and gluconic acid can be analysed in less than 1 min with a single step reaction . Wine samples were analysed using the hexokinase reaction for glucose + fructose (sugars undergoing fermentation) and compared against an established method, showing excellent performance over the whole range of concentrations (R = 0.9994) . Increased sensitivity in some applications can be obtained by cycling reactions, e.g . a kinase reaction followed by a phosphatase reaction . in a one step analysis, as required for lactulose assay in milk, a useful indicator of heat treatment damage . A sensitivity well below 0.1 mmol L(-1) in the original milk sample has been demonstrated. Analyst, 2001 Dec, 126(12), 2128 - 33 Enantioseparation of chiral amino acids as the N(O,S)-ethoxycarbonylated diastereomeric esters by achiral dual-capillary column gas chromatography; Lee J et al.; The enantioseparation of 30 racemic amino acids in a single analysis is described for the determination of their absolute configurations . Two-phase extractive ethoxycarbonyl (EOC) reaction with ethyl chloroformate present in the dichloromethane phase was performed to recover amino acids from alkaline aqueous solutions . The resulting N(O,S)-EOC amino acids extracted into an organic solvent after acidification were reacted with a chiral alcohol such as (S)-(+)-3-methylbutan-2-ol, (S)-(+)-butan-2-ol and (S)-(+)-octan-2-ol for gas chromatographic analysis on achiral dual-capillary DB-5 and DB-17 columns of different polarities . Among the chiral reagents examined, (S)-(+)-3-methylbutan-2-ol provided the best diastereomeric structures in resolving all the racemic amino acids into their enantiomeric pairs with high resolution factors (1.2-8.0) . Moreover, the temperature-programmed retention index (I) values measured on the two columns were characteristic of each enantiomer . Hence simple I matching with the reference values was useful in cross-checking for chemical identification and also chiral discrimination . When the present method was applied to a fermented dairy product (Yakult), D-alanine, D-aspartic acid, D-glutamic acid and D-proline were positively detected along with their respective L-forms in addition to glycine. J Nutr Sci Vitaminol (Tokyo), 2001 Oct, 47(5), 335 - 9 Nutritional effects of cyclodextrins on liver and serum lipids and cecal organic acids in rats; Kaewprasert S et al.; The effect of dietary cyclodextrins on liver and serum lipids and cecal organic acid production was investigated . Male Wistar rats were fed a basal diet and a diet containing 5% of alpha-, beta-, or gamma-cyclodextrin . The body weight gain in rats fed the alpha-cyclodextrin diet was not significantly different from rats fed the other three kinds of diets . The feeding of dietary alpha-cyclodextrin increased total lipid and phospholipids in the liver . Beta-cyclodextrin significantly lowered serum total cholesterol and phospholipid levels compared with the basal diet et al . A decrease in serum triacylglycerol levels was also observed in beta-cyclodextrin-fed rats . Dietary alpha-cyclodextrin significantly increased the weight of cecal tissues and contents, and an approximate fourfold increase in acetate, propionate, and total organic acids was noted, indicating the fermentibility of beta-cyclodextrin compared with the basal diet . It seems likely that the suppression of serum cholesterol levels by alpha- and beta-cyclodextrins might be due to the increasing acetate and propionate productions in the cecum . cecal organic acid, cyclodextrin, serum cholesterol, rats J Dairy Sci, 2001 Dec, 84(12), 2760 - 9 Effects of substituting barley grain with corn on ruminal fermentation characteristics, milk yield, and milk composition of Holstein cows; Khorasani GR et al.; The influence of corn or barley, or the equal mixture of both, on digestion characteristics and dairy cow performance was evaluated in metabolic and production experiments . Three rumen-cannulated early-lactation cows were used in a 3 x 3 Latin square design experiment to study the effect on ruminal fermentation characteristics and whole-tract digestion of substituting barley grain with corn . Production responses were determined by the use of 27 early-lactation Holstein cows . Cows in the production study were fed the test diets for 12 wk after a 2-wk covariate period . Results from the metabolic study indicated the effects of grain source on ruminal and total-tract digestion to be minimal . Total ruminal volatile fatty acids and acetate concentrations decreased linearly, butyrate increased linearly, and pH and lactic acid concentration were not affected by increasing levels of corn . Apparent digestibility of DM and organic matter showed a quadratic response with increasing the corn level in the diet, with no dietary effect on neutral detergent fiber, acid detergent fiber, and cellulose digestion . Ruminal fermentation characteristics suggest that substitution of barley grain with corn may alter the site of digestion and the end products of digestion that are absorbed by the animal . Multiparous cows failed to respond to treatment, whereas primiparous animals showed the greater response in milk yield and milk-component yield to diets that contained an equal mixture of corn and barley . These results probably reflect a more optimal synchronization of dietary protein and energy for dairy cows fed the 50:50 barley/corn diet. J Dairy Sci, 2001 Dec, 84(12), 2751 - 9 Effect of type and level of dietary fat on rumen fermentation and performance of dairy cows fed corn silage-based diets; Onetti SG et al.; The objective of this study was to investigate the effects of tallow and choice white grease (CWG) fed at 0, 2, and 4% of the diet dry matter (DM) on rumen fermentation and performance of dairy cows when corn silage is the sole forage source . Fifteen midlactation Holstein cows were used in a replicated 5 x 5 Latin square design with 21-d periods . Treatments were 0% fat (control), 2% tallow, 2% CWG, 4% tallow, and 4% CWG (DM basis) . The forage:concentrate ratio was 50:50, and diets were formulated to contain 18% crude protein and 32% neutral detergent fiber (DM basis) . Cows were allowed ad libitum consumption of diets fed twice daily as total mixed rations . Cows fed supplemental fat had lower DM intake and produced less milk and milk fat than cows fed the control diet . Feeding 4% fat reduced milk production and milk fat yield relative to feeding 2% fat . Treatments had little effect on the concentration of trans-octadecenoic acids in milk fat . Total trans fatty acids were poorly related to changes in milk fat percentage . Ruminal pH and total volatile fatty acids concentration were not affected by supplemental fat . The acetate:propionate ratio, NH3-N, and numbers of protozoa in the rumen were significantly decreased when fat was added to the diets . Source of dietary fat did not affect rumen parameters . There was no treatment effect on in situ corn silage DM and neutral detergent fiber disappearance . Including fat in corn silage-based diets had negative effects on milk production and rumen fermentation regardless of the source or level of supplemental fat. J Dairy Sci, 2001 Dec, 84(12), 2721 - 9 Rumen acid production from dairy feeds . 1 . Effects on feed intake and milk production of dairy cows offered grass or corn silages; Dewhurst RJ et al.; Earlier studies developed a new approach to feed evaluation, measuring the net acid load that develops during rumen fermentation . Two concentrates were formulated to be isoenergetic and isonitrogenous, with extremes of rumen acid load . A third treatment comprised a 50:50 mixture of these concentrates . These concentrates were evaluated along with ryegrass silage and corn silage . The feeds were evaluated in a continuous culture system adapted to deliver and record the quantities of acid or alkali needed to maintain a constant pH (6.2 to 6.3) . This study confirmed the anticipated ranking of concentrates for rumen acid load, as well as the highly acidogenic nature of corn silage . The concentrates were formulated to balance corn silage and were offered to early-lactation Holstein-Friesian cows at 50% of dry matter intake, with either ryegrass silage or corn silage . Feed intake was lower for animals offered corn silage-based diets (17.4 vs . 22.2 kg of dry matter/d) . Increasing concentrate acid load led to a large decline in dry matter intake for corn silage, although not for grass silage . Feed intake effects were reflected in significant effects on yield of milk (31.0, 29.9, and 26.9 kg/d for low-, medium-, and high-acid load concentrates, respectively) and milk solids . Milk protein concentration was unaffected by concentrate type with corn silage diets but tended to be higher when high acid load concentrates were fed with grass silage . This may reflect the effect of the high starch concentrate rectifying a shortage of glucogenic precursors or microbial protein with the grass silage-based diet. Toxicol Sci, 2002 Feb, 65(2), 288 - 98 Spinosad insecticide: subchronic and chronic toxicity and lack of carcinogenicity in Fischer 344 rats; Yano BL et al.; Spinosad is an insecticide derived from a naturally occurring bacterium via fermentation . The toxicity of spinosad was characterized in subchronic and chronic toxicity/oncogenicity studies conducted according to standard toxicology regulatory guidelines . Subchronic toxicity was evaluated in groups of 10 Fischer 344 rats/sex given feed containing 0, 0.05, 0.1, 0.2, or 0.4% spinosad (Study 1) or 0, 0.003, 0.006, 0.012, or 0.06% spinosad (Study 2) for 13 weeks . Lower body weights and increased mortality occurred in rats given 0.4% spinosad . Microscopic effects were observed in the adrenal glands, liver, lymphoid cells, reproductive tissues, kidney, thyroid, stomach, lung, and skeletal muscle of rats given > or = 0.05% spinosad, and consisted primarily of vacuolation of cells; however, degenerative, regenerative, and/or inflammatory changes were also noted in some tissues . Vacuolation within a number of tissues was ultrastructurally characterized by an increase in size and number of lysosomes that contained extensive membranous whorls consistent with phospholipidosis . The no observed effect level (NOEL) in the 13-week studies was 0.012% (24 mg/kg/day) spinosad . Chronic toxicity and oncogenicity were evaluated in groups of 60 Fischer 344 rats/sex given feed containing 0, 0.005, 0.02, 0.05, or 0.1% spinosad for up to 2 years . Rats given 0.1% spinosad for 1 year had microscopic effects similar to those observed in the subchronic studies . Vacuolation and inflammation of the thyroid gland also occurred in rats given 0.05% spinosad for 1 year . Excessive mortality occurred in rats from the oncogenicity study given 0.1% spinosad by 21 months, and surviving rats were euthanized because the maximum tolerated dose had been exceeded . Rats given 0.05% spinosad for 2 years had vacuolation and/or inflammation involving the thyroid, lymphoid tissue, and lung . Rats given 0.05% spinosad had similar numbers of neoplasms as control rats, indicating that spinosad was not carcinogenic at dose levels up to 0.05% . The NOEL at 2 years was 0.005% (2.4 mg/kg/day) spinosad. Toxicol Sci, 2002 Feb, 65(2), 276 - 87 Spinosad insecticide: subchronic and chronic toxicity and lack of carcinogenicity in CD-1 mice; Stebbins KE et al.; The potential toxicologic and oncogenic effects of spinosad, a natural fermentation product with insecticidal properties, were investigated . The 13-week toxicity study consisted of groups of 10 CD-1 mice/sex provided diets containing 0, 0.005, 0.015, 0.045, or 0.12% spinosad (Study 1) . The 0.12% group was terminated on Test Day 44 due to mortality and overt clinical signs of toxicity . An 18-month chronic oncogenicity study consisted of groups of 50 CD-1 mice/sex provided diets containing 0, 0.0025, 0.008, or 0.036% spinosad (Study 2) . Two interim groups of 10 mice/sex/group were terminated after 3 and 12 months . Females given 0.036% were terminated on Day 455 due to markedly lower body weights and feed consumption, as well as excessive mortality . Because of the early termination of the female high-dose group, additional groups of 10 male and female mice (12-month interim necrospy) and 50 male and female mice (18-month necropsy) were provided diets containing 0, 0.0008, or 0.024% spinosad (Study 3) to fully assess potential chronic toxicity and oncogenicity . Standard toxicologic parameters were evaluated consistent with existing regulatory guidelines . The primary effect in the 13-week and 18-month studies was intracellular vacuolation of histiocytic and epithelial cells in numerous tissues and organs at doses of > or = 0.015% . The histological vacuolation corresponded to ultrastructural lysosomal lamellar inclusion bodies . This alteration was consistent with phospholipidosis, a condition that results from accumulation of polar lipids in lysosomes . Lesions with no apparent direct relation to vacuolation were hyperplasia of the glandular mucosa of the stomach, skeletal muscle myopathy, bone marrow necrosis, and anemia with associated splenic hematopoiesis . The incidence of tumors in mice given spinosad was not increased relative to controls at any dose level . The no observed effect level for the 13-week study was 0.005% (6 mg/kg/day) spinosad, and for the chronic toxicity/oncogenicity study was 0.008% (11 mg/kg/day) spinosad for male and female CD-1 mice. Food Addit Contam, 2002 Jan, 19(1), 55 - 61 Mycotoxins in South African traditionally brewed beers; Odhav B et al.; Traditionally brewed alcoholic beverages are regularly consumed by most ethnic black South Africans . Maize and barley, both of which are used for producing locally brewed alcoholic beer, are frequently contaminated by mycotoxin-producing moulds . The study was undertaken to investigate whether these toxins are present in raw grains and the traditional beers imbibed by the local black African population . It was established that the raw ingredients (sorghum, sorghum malt grains, maize grits), commercially produced traditional beers (Utshwala and Utshwala special) and home-brewed beers (Umqombotha, Isiqatha, Imfulamfula) were contaminated by bacteria and fungi (both yeasts and moulds) . The contaminating moulds were isolated and identified . The contaminated samples were analysed for aflatoxins B1, B2, G1 and G2, zearalenone, citrinin, deoxynivalenol, and ochratoxin A using a multi-mycotoxin thin-layer chromatography screening method and the toxins were quantified by high-performance liquid chromatography . Grain samples were infected by . Aspergillus flavus, A . alliaceus, A . clavatus, Penicillium spp., Rhizopus spp . and Mucor spp . Sorghum malt grain samples contained the toxin zear alenone . No mycotoxin-producing fungi were present in the fermented beers but two of six commercial beer samples contained aflatoxins (200 and 400 microg l(-1) and 45% (13 of 29) of the home-brewed beers had zear alenone (range 2.6-426 microg l(-1) and/or ochratoxin A (3-2340 microg l(-1). J Anim Sci, 2001 Dec, 79(12), 2949 - 56 In vitro study of the biochemical origin and production limits of odorous compounds in cattle feedlots; Miller DN et al.; Livestock odors are closely correlated to airborne concentrations of volatile organic compounds (VOC), which are a complex mixture of carbon-, sulfur-, and nitrogen-containing compounds produced primarily during the incomplete anaerobic fermentation of animal manure by microorganisms . Volatile fatty acids, alcohols, and aromatic ring compounds comprise a substantial fraction of VOC, yet very little is known about their biochemical origin and environmental factors controlling their production . The anaerobic production of fermentation products and consumption of substrates (CP, starch, and nonstarch carbohydrate) were analyzed in slurries of fresh (< 24 h) and aged (> 1 d) cattle manure over several weeks . Ethanol, acetate, propionate, butyrate, lactate, and H2 were the major products of fermentation . Aged cattle manure produced twice the concentration of VFA during incubation produced by the fresh manure (P < 0.001) . Aromatic compounds (phenols, indoles, and benzoates) remained unchanged in both manures . Production of VFA from fresh manure was inhibited when the pH fell below 4.5 . It is likely that the presence of calcareous soil, which has a high buffering capacity, and lactate-consuming microorganisms minimized acidification in the aged manure slurries . Low starch content limited VFA production in the aged manure . Starch was the likely biochemical source for fermentation products in both manures based on the strong negative correlations between fermentation product and starch content (r = -0.944 and -0.773) and ratio of fermentation products produced to starch consumed (r = 0.64 and 0.72) for fresh and aged manure, respectively . Nonstarch carbohydrate served an indeterminate role in the production of fermentation products . Nonstarch carbohydrate decreased by 4.7 and 23.4 g/L in the fresh and aged manure, respectively, whereas the starch content decreased by 18.6 and 22.4 g/L in the fresh and aged manure, respectively . The concentration of CP did not change, which suggests a balance between protein consumption and new bacterial biomass production . We conclude that the types of substrates in cattle manure and the feedlot soils where they are deposited are significant factors in the production of odors. Mol Genet Genomics, 2002 Jan, 266(5), 838 - 47 Epub 2001 Nov 07. Co-ordinate regulation of lactate metabolism genes in yeast: the role of the lactate permease gene JEN1; Lodi T et al.; In the yeast Saccharomyces cerevisiae, the first step in lactate metabolism is its transport across the plasma membrane, a proton symport process mediated by the product of the gene JEN1 . Under aerobic conditions, the expression of JEN1 is regulated by the carbon source: the gene is repressed by glucose and induced by non-fermentable substrates . JEN1 expression is also controlled by oxygen availability, but is unaffected by the absence of haem biosynthesis . JEN1 is negatively regulated by the repressors Mig1p and Mig2p, and requires Cat8p for full derepression . In this report we demonstrate that, in addition to these regulators, the Hap2/3/4/5 complex interacts specifically with a CAAT-box element in the JEN1 promoter, and acts to derepress JEN1 expression . We also provide evidence for transcriptional stimulation of JEN1 by the protein kinase Snf1p . Data are presented which provide a better understanding of the molecular mechanisms implicated in the co-regulation of genes involved in the metabolism of lactate. J Food Prot, 2002 Jan, 65(1), 12 - 7 A conductance method for the identification of Escherichia coli O157:H7 using bacteriophage AR1; Chang TC et al.; The feasibility of using a specific phage (AR1) in conjunction with a conductance method for the identification of Escherichia coli O157:H7 was evaluated . The multiplication of strains of E . coli O157:H7 was inhibited by AR1; therefore, a time point (detection time, DT) at which an accelerating change in conductance in the culture broth was not obtained . Bacterial strains were subcultured on sorbitol-MacConkey agar and incubated at 35 degrees C for 24 h, and the ability of the bacteria to ferment sorbitol was recorded . An aliquot of 0.5 ml of the bacterial suspension (10(7) CFU/ml) and 0.5 ml of the phage suspension (10(8) PFU/ml) were added to the conductance tube of a Malthus analyzer containing 5 ml of culture broth . The tubes were incubated at 35 degrees C, and conductance changes in the tubes were continuously monitored at 6-min intervals for 24 h by the instrument . A positive reaction was defined as an E . coli strain that could not utilize sorbitol and caused no conductance change (i.e., no DT) within an incubation period of 24 h . Of the 41 strains of E . coli O157:H7 tested, all produced positive reactions . When a total of 155 strains of non-O157:H7 E . coli were tested, 14 did not have a DT within 24 h . However, among these 14 strains, 13 were sorbitol fermenters, and the remaining one was a nonfermenter . Therefore, by definition, only one strain produced a false-positive reaction . The sensitivity and specificity of the present method were 100% (41 of 41) and 99.4% (154 of 155), respectively . The present method incorporating conductimetric measurement and phage AR1 for the identification of E . coli O157:H7 was simple and capable of automation. Int J Cancer, 2002 Feb 10, 97(5), 567 - 73 Modulation of HMG-N2 binding to chromatin by butyrate-induced acetylation in human colon adenocarcinoma cells; Luhrs H et al.; Butyrate, a short chain fatty acid (SCFA), is generated by anaerobic fermentation of undigested carbohydrates within the colon . Butyrate enhances acetylation of core histones, a process directly linked to the formation of active chromatin and gene expression . However, additional chromatin components also contribute to the formation of transcriptionally active chromatin . The high mobility group protein N2 (HMG-N2), a nonhistone protein, is involved in chromatin structure modulation . We examined the effects of butyrate on HMG-N2 expression, hyperacetylation and chromatin binding . HT29 human adenocarcinoma cells were incubated with butyrate . Levels of HMG-N2 mRNA and of total or acetylated HMG-N2 protein were analyzed . Protein dynamics were investigated with transfected cells expressing HMG-N2-EGFP fusion proteins . Treatment of HT29 cells with butyrate led to significant hyperacetylation of HMG-N2 . Levels of HMG-N2 protein remained unchanged . Northern blot analysis revealed a significant reduction in HMG-N2 mRNA levels after treatment with butyrate . Analysis of HMG-N2-EGFP transfected HT29 cells demonstrated that butyrate treatment changes the binding properties of HMG-N2-EGFP to chromatin . In addition, butyrate treatment resulted in solubilization of endogenous acetylated HMG-N2 into the supernatant of permeabilized cells . We demonstrate that butyrate treatment is associated with hyperacetylation of HMG-N2 protein in HT29 cells . The modulation of this nonhistone chromatin protein resulted in altered binding properties to chromatin . This may represent an additional step in changing chromatin structure and composition with subsequent consequences for transcription and gene expression . Modulation of nonhistone chromatin proteins, like the ubiquitous HMG-N2 proteins, may be partly responsible for the wide range of butyrate-associated effects . Biotechnol Bioeng, 2002 Mar 20, 77(6), 632 - 40 Monitoring the aroma production during wine-must fermentation with an electronic nose; Pinheiro C et al.; This work discusses the feasibility of using the electronic nose for the on-line and real-time monitoring of the production of a complex aroma profile during a bioconversion process . As a case study, the formation of the muscatel aroma during the wine-must fermentation was selected . During wine-must fermentation, aroma compounds responsible for the organoleptic character are produced in the ppm range, while simultaneously one of the main metabolic products, ethanol, is produced in much higher quantities (up to 10% wt) . Because the sensors of the electronic nose array are cross-selective to different volatile compounds, it was investigated in detail how far the electronic nose was able to evaluate the aroma profile along the fermentation . This article discusses and evaluates subsequently the integration of a membrane separation process-organophilic pervaporation-for selectively enriching aroma compounds relative to ethanol, to improve sample discrimination . Biotechnol Bioeng, 2002 Mar 20, 77(6), 601 - 13 Estimation of hyphal tensile strength in production-scale Aspergillus oryzae fungal fermentations; Li ZJ et al.; Fragmentation of filamentous fungal hyphae depends on two phenomena: hydrodynamic stresses, which lead to hyphal breakage, and hyphal tensile strength, which resists breakage . The goal of this study was to use turbulent hydrodynamic theory to develop a correlation that allows experimental data of morphology and hydrodynamics to be used to estimate relative (pseudo) tensile strength (sigma(pseudo)) of filamentous fungi . Fed-batch fermentations were conducted with a recombinant strain of Aspergillus oryzae in 80 m(3) fermentors, and measurements were made of both morphological (equivalent hyphal length, L) and hydrodynamic variables (specific power input, epsilon; kinematic viscosity, v) . We found that v increased over 100-fold during these fermentations and, hence, Kolmogorov microscale (lambda) also changed significantly with time . In the impeller discharge zone, where hyphal fragmentation is thought to actually take place, lambda was calculated to be 700-3500 microm, which is large compared to the size of typical fungal hyphae (100-300 microm) . This result implies that eddies in the viscous subrange are responsible for fragmentation . Applying turbulent theory for this subrange, it was possible to calculate sigma(pseudo)from morphological and hydrodynamic measurements . Pseudo tensile strength was not constant but increased to a maximum during the first half and then decreased during the second half of each fermentation, presumably due to differences in physiological state . When a literature correlation for hyphal fragmentation rate (k(frag)) was modified by adding a term to account for viscosity and tensile strength, the result was better qualitative agreement with morphological data . Taken together, these results imply hyphal tensile strength can change significantly over the course of large-scale, fed-batch fungal fermentations and that existing fragmentation and morphology models may be improved if they accounted for variations in hyphal tensile strength with time . Nature, 2002 Jan 24, 415(6870), 454 - 6 Anaerobic microbial metabolism can proceed close to thermodynamic limits; Jackson BE et al.; Many fermentative bacteria obtain energy for growth by reactions in which the change in free energy (DeltaG') is less than that needed to synthesize ATP . These bacteria couple substrate metabolism directly to ATP synthesis, however, by classical phosphoryl transfer reactions . An explanation for the energy economy of these organisms is that biological systems conserve energy in discrete amounts, with a minimum, biochemically convertible energy value of about -20 kJ mol-1 (refs 1, 2, 3) . This concept predicts that anaerobic substrate decay ceases before the minimum free energy value is reached, and several studies support this prediction . Here we show that metabolism by syntrophic associations, in which the degradation of a substrate by one species is thermodynamically possible only through removal of the end product by another species, can occur at values close to thermodynamic equilibrium (DeltaG' approximately 0 kJ mol-1) . The free energy remaining when substrate metabolism halts is not constant; it depends on the terminal electron-accepting reaction and the amount of energy required for substrate activation . Syntrophic associations metabolize near thermodynamic equilibrium, indicating that bacteria operate extremely efficient catabolic systems. Health Care Anal, 2001, 9(3), 299 - 319 Historical and philosophical reflections on patient autonomy; Tauber AI; Contemporary American medical ethics was born during a period of social ferment, a key theme of which was the espousal of individual rights . Driven by complex cultural forces united in the effort to protect individuality and self-determined choices, an extrapolation from case law to rights of patients was accomplished under the philosophical auspices of 'autonomy.' Autonomy has a complex history; arising in the modern period as the idea of self-governance, it received its most ambitious philosophical elaboration in Kant's moral philosophy . In examining the Kantian construction, it is evident that neither his universal moral imperative nor his rigorous application of self-legislated ethical action can sustain our own notions of moral agency in a pragmatic, pluralistic society . But the Kantian position is useful in highlighting that self-governance is not equivalent to 'autonomy,' and this distinction defines the limits of autonomy in the clinical setting . A critique of Engelhardt's idea of 'principle of permission' is used to illustrate autonomy's eclipse as a governing principle for medical ethics. J Agric Food Chem, 2002 Jan 30, 50(3), 648 - 52 Time course study of substrate utilization by Aspergillus flavus in medium simulating corn (Zea mays) kernels; Mellon JE et al.; Utilization of the three major corn reserve materials, starch, triglycerides (refined corn oil), and zein (storage protein), by Aspergillus flavus was monitored in vitro over a 7-day fermentation . Medium composition in which proportions of reserve materials initially approximated proportions in mature corn kernels changed little over the first 18 h . Subsequently, hydrolysis of both starch and triglycerides occurred simultaneously, with peak concentrations of glucose and free fatty acids on day 2 of the fermentation period . Fatty acid concentrations dropped relatively rapidly after day 2 but increased again after day 6 . Aflatoxin B(1) production increased after 36 h, with a peak at day 4 . Aflatoxin B(1) production paralleled fungal biomass production during the exponential growth phase . A . flavus did not appear to preferentially utilize any of the released fatty acids . A number of fungus-specific metabolites were detected, including arabitol, erythritol, mannitol, trehalose, and kojic acid . Mannitol exceeded the other metabolites in concentration, and the timing of mannitol production closely paralleled that of aflatoxin B(1) . Kojic acid concentrations peaked at day 6 . In contrast to previously described selective use of simple carbohydrates by A . flavus, less discrimination was displayed when faced with utilization of complex substrates such as starch or triglycerides. J Agric Food Chem, 2002 Jan 30, 50(3), 565 - 70 Total phenol, catechin, and caffeine contents of teas commonly consumed in the United kingdom; Khokhar S et al.; Levels of total phenol, catechins, and caffeine in teas commonly consumed in the United Kingdom have been determined using reversed phase high-performance liquid chromatography . Tea bags or tea leaves were purchased from local supermarkets and extracted in boiling water for 5 min . The resulting data showed considerable variability in both total phenols {80.5-134.9 mg/g of dry matter (DM) in black teas and 87-106.2 mg/g of DM in green teas} and catechins (5.6-47.5, 51.5-84.3, and 8.5-13.9 mg/g of DM in black, green, and fruit teas, respectively); this was most probably a result of differing agronomic conditions, leaf age, and storage during and after transport, as well as the degree of fermentation . Caffeine contents of black teas (22-28 mg/g of DM) were significantly higher than in less fermented green teas (11-20 mg/g of DM) . The relative concentration of the five major tea catechins ranked EGCG > ECG > EC > EGC > C . The estimated U.K . dietary intakes of total tea catechins, calculated on the basis of an average tea consumption of three cups of tea (200 mL cup, 1% tea leaves w/v), were 61.5, 92.7, and 405.5 mg/day from fruit teas, black teas, and green teas, respectively . The coefficients of variation were 19.4, 88.6, and 17.3%, respectively, indicating the wide variation in these intakes . The calculated caffeine intake ranged between 92 and 146 mg/day . In addition, many individuals will consume much larger quantities of tea, of various strengths (as determined by the brewing conditions employed) . This broad spread of U.K . daily intakes further emphasizes the need for additional research to relate intake and effect in various population groups. J Agric Food Chem, 2002 Jan 30, 50(3), 549 - 54 Aroma-active components in fermented bamboo shoots; Fu SG et al.; Bamboo shoots (Phyllostachys pubescens) were fermented and prepared in a traditional Taiwanese manner . Static and dynamic headspace extractions of volatile compounds were conducted by solid phase microextraction (SPME) and by cryogenic focusing purge and trap, respectively . Volatile analysis was conducted with gas chromatography and mass spectrometry . Gas chromatography-olfactometry (GCO) was conducted utilizing the Osme time-intensity method . Of 70 volatile compounds detected, 29 possessed aroma activity, and the most odor active included p-cresol (barn-like), 2-heptanol (mushroom), acetic acid (vinegar), and 1-octen-3-ol (mushroom) . SPME extracted 66 compounds, purge and trap extracted 14 compounds, and 12 compounds were common to both methods . The Osme GCO technique coupled with SPME is an effective tool for the extraction and evaluation of aroma-active headspace volatiles. Biochim Biophys Acta, 2002 Jan 17, 1553(1-2), 39 - 56 C4-dicarboxylate carriers and sensors in bacteria; Janausch IG et al.; Bacteria contain secondary carriers for the uptake, exchange or efflux of C4-dicarboxylates . In aerobic bacteria, dicarboxylate transport (Dct)A carriers catalyze uptake of C4-dicarboxylates in a H(+)- or Na(+)-C4-dicarboxylate symport . Carriers of the dicarboxylate uptake (Dcu)AB family are used for electroneutral fumarate:succinate antiport which is required in anaerobic fumarate respiration . The DcuC carriers apparently function in succinate efflux during fermentation . The tripartite ATP-independent periplasmic (TRAP) transporter carriers are secondary uptake carriers requiring a periplasmic solute binding protein . For heterologous exchange of C4-dicarboxylates with other carboxylic acids (such as citrate:succinate by CitT) further types of carriers are used . The different families of C4-dicarboxylate carriers, the biochemistry of the transport reactions, and their metabolic functions are described . Many bacteria contain membraneous C4-dicarboxylate sensors which control the synthesis of enzymes for C4-dicarboxylate metabolism . The C4-dicarboxylate sensors DcuS, DctB, and DctS are histidine protein kinases and belong to different families of two-component systems . They contain periplasmic domains presumably involved in C4-dicarboxylate sensing . In DcuS the periplasmic domain seems to be essential for direct interaction with the C4-dicarboxylates . In signal perception by DctB, interaction of the C4-dicarboxylates with DctB and the DctA carrier plays an important role. Biochem J, 2002 Feb 1, 361(Pt 3), 597 - 604 Phosphorylation of a novel zinc-finger-like protein, ZPR9, by murine protein serine/threonine kinase 38 (MPK38); Seong HA et al.; We have identified previously a new murine protein serine/threonine kinase, MPK38, closely related to the sucrose-non-fermenting protein kinase family {Gil, Yang, Lee, Choi and Ha (1997) Gene 195, 295-301} . Using the C-terminal half of the putative human counterpart of MPK38, HPK38, as a bait in a yeast two-hybrid screen of a human HeLa cDNA library, it was discovered that the zinc-finger-motif-containing protein, termed zinc-finger-like protein 9 (ZPR9), bound both HPK38 and MPK38 . In a co-expression assay, ZPR9 associated with MPK38 in vivo, and we showed that the ZPR9 is also phosphorylated by MPK38 . In addition, ZPR9 physically interacts with itself in mammalian cells . The ZPR9 cDNA hybridized with a mRNA species of approx . 1.7 kb in Northern-blot analysis . The ZPR9 transcript was detected in all tissues examined, including lung, kidney, spleen,liver and brain . Co-expression of ZPR9 with MPK38 caused the accumulation of ZPR9 in the nucleus . These findings suggest a potentially important role for ZPR9 in MPK38-mediated signal transduction, and that ZPR9 is a physiological substrate of MPK38 in vivo. Bioresour Technol, 2002 Feb, 81(3), 261 - 3 Solid-state production of biopulp by Phanerochaete chrysosporium using steam-exploded wheat straw as substrate; Chen H et al.; A novel material for biopulp-making, steam-exploded wheat straw (SEWS), was studied . During the steam explosion process, the hemicellulose was partly degraded and became water-soluble sugar as the carbon resource of the chosen microbe growth, and compared with non-SEWS, the degradation amount of cellulose decreased and the degradation amount of lignin increased for the fermented steam-exploded wheat straw (FSEWS) cultured with Phanerochaete chrysosporium ME-446 . Under the optimum conditions of solid-state ferrmentation (SSF), the degradation amount of lignin reached 60% on the 5th day and the fermented straw residue could be used directly as the material for pulp making. J Exp Biol, 1979 Oct, 82, 303 - 20 Metabolic sources of heat and power in tuna muscles . II . Enzyme and metabolite profiles; Guppy M et al.; Tuna appear able to maintain their muscles at 5-10 degrees C above ambient by balancing heat produced in situ and conserved by a counter-current heat exchanger with heat lost to the sea . Metabolite profiles under three different activity states (rest, burst swimming, and steady state swimming during feeding frenzies at sea) were used to identify which metabolic processes in white and red muscles could account for observed excess temperatures . During burst swimming, transient changes in metabolite levels indicate that the metabolism of both red and white muscle contributes to powering burst swimming; red muscle work is sustained mainly by oxidative metabolism while white muscle work depends upon an intense anaerobic glycolysis . The rate of metabolism in red muscle is easily high enough to account for the measured (10 degrees C) increase in temperature at this time . However, in white muscle, anaerobic glycolysis can account for only about a 2 degrees C maximum rise in temperature . The highest sustained swimming speeds and the highest muscle temperatures in skipjack are found during feeding frenzies at sea . As in burst swimming, during steady-state swimming red muscle temperatures can be accounted for by oxidative metabolism . In the case of white muscle, the lactate measurements indicate that anaerobic glycolysis could only lead to a 0.3 degree C temperature rise . However, if the fraction of utilized glycogen that is not fermented (about 60%) is assumed to be fully oxidized, enough heat is generated to raise white muscle temperatures by over 10 degrees C . The observed excess temperature at this time is about 8-10 degrees C, showing that anarerobic carbohydrate metabolism in white muscle is probably the major heat source during feeding frenzies . These interpretations are fully consistent with enzyme profiles of red and white muscles in tuna . They do not, however, explain why tuna have warm muscles . The latter problem is briefly discussed. Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 594 - 7 {The effects of different glucose feeding modes on hEGF production in an excretory recombinant Escherichia coli K12 system}; Xu ZN et al.; The effects of different glucose feeding modes on hEGF expression were evaluated in an excretory recombinant E . coli K12 system . The results showed that, compared with batch cultivation, the plasmid stability and density of plasmid-retaining cells were improved by all three glucose feeding modes (intermittent, pH-stat and constant-rate) . It was shown that hEGF yields were improved up to 25.5% and 28.1% by intermittent or pH-stat glucose feeding respectively . Especially, up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min . The effects of further combined feeding with other medium components (ampicillin, nitrogen sources, and inorganic salts) and inducer on hEGF yield were also examined in the bench-top fermentor. Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 584 - 6 {Purification of vancomycin by using magnetic affinity technique}; Guo LA et al.; A new kind of magnetic affinity microspheres (MAMS), whose ligand is D-Ala-D-Ala, was prepared using agarose as matrix . By using this new MAMS vancomycin was purified directly from crude fermentation liquor with only one step . The purity and the mass recovery of vancomycin measured by reverse-phase HPLC were 97% and 87%, respectively . The characteristic of this method was simpler, faster, cheaper and more effective than that of currently used ones. Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 579 - 83 {Factors of laccase producing and fermentation conditions by a new white-rot fungus AH28-2}; Xiao YZ et al.; White-rot fungus AH28-2, a newly isolated strain, produced effectively laccase by induction when grown on a synthetic medium . Aromatic compounds of low molecular weight had an inducing influence on laccase production and its isoenzyme compositions . The using of o-toluidine or syringic acid had the best inducing effect . Cu2+ concentration in medium had distinguished effect on laccase production . Enzyme activity was notably increased by Cu2+ and reached the maximum when Cu2+ final concentration was 5 mumol/L . Mn2+ inhibited the synthesis of laccase . Carbon and nitrogen limitation were not beneficial to laccase synthesis, while high nutrient organic medium was beneficial to the growth of cell and the synthesis of laccase . Using cellobiose as the sole carbon source, the highest level enzyme activity reached 82,923 . 7 u/L under the condition of optimum fermentation with ABTS as substrate . This enzyme activity was 2.9-fold higher compared to the reported data on international references in recent years. Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 575 - 8 {Studies on lignocellulolytic enzymes production and biomass degradation of Pleurotus sp2 and Trametes gallica in wheat straw cultures}; Xie J et al.; Pleurotus sp2 and Trametes gallica were selected in this assay because of their high activities of lignocellulolytic enzymes and the enzyme peaks appeared at the early stage of liquid state fermentation . Solid state fermentation was also investigated for their abilities and behaviors of enzyme-production . The capabilities and characteristics of the two strains in degrading biomass were studied . When Pleurotus sp2 was incubated in wheat straw powder containing the liquid medium of low-nitrogen, no-carbon and high inorganic salt, the activities of MnP and Lac reached the peaks on the tenth day, but the activities of hemicellulases reached the peak on the 40th day . Pleurotus sp2 caused 17.6% of biomass loss . When T . gallica was incubated in wheat straw powder containing the liquid medium of hlig-nitrogen, or low-nitrogen, no-carbon and high inorganic salt, the activities of MnP reached the peaks on the tenth day, the lac and hemicelluloses on the 40th day, and the lignin peroxidases reached the peaks on the 50th day, and it caused more than 64% of biomass loss . Among them the hemicellulose was degraded by 71.96%, and the cellulose 66.21% . T . gallica was very capable of degrading lignin of wheat straw and caused 34.37% loss during 20 days, 46 . 71% loss during 30 days and 70.14% loss during 60 days . It was interesting that T . gallica degraded lignin preferentially with respect to cellulose, which was very beneficial to biopulping of paper industry. Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 570 - 4 {The bottleneck steps limiting maturation of penicillin G acylase in Escherichia coli}; Xu JN et al.; We have identified the bottleneck steps limiting maturation of penicillin G acylase (PAC) through comparison of the maturation performance for various PAC-expression systems (Pac, Tac, T7, Vgb + T7) with different efficiencies of proteolysis, subunit folding and assembly . The maturation of PAC could be limited by various steps, such as translocation, periplasmic proteolysis, subunit folding and assembly depending on the host/vector systems . In BL21(pPA6) cells, maturation of PAC were limited by proteolysis and folding steps; the efficiency of proteolysis was 57.2%; the subunit folding and assembly capacity was 0.72 . In BL21(pKKpacSP) cells, the stability and folding of alpha subunit was bottleneck steps . In T7 and dissolved-oxygen regulation expression systems, PAC proprecursor could be maturated efficiently . Results also indicate that the folding of alpha peptide plays a key role in folding of precursor for PAC in E . coli . Developing proper host/vector systems and fermentation technology with superior abilities on subunit folding and assembly of precursor for PAC could be plausible for enhancing production of PAC . In this study, pac could be expressed (transcribed, translated and maturated) efficiently under the control of T7 promoter. Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 557 - 60 {The RAPD analysis of haploid strain of thermotolearnt yeast}; Zhu XF et al.; The haploid strains HZ line from the thermololeant strain HU-TY-1 of Saccharomyces cerevisiae have been obtained by sporulation, and the analysis of growth and fermentation test was performed . The strains HZ-21 and HZ-84 were used as the analysis of random amplified polymerphic DNA (RAPD) . The result showed that there were some polymorphic DNA fragments of genomic DNA among haploid, the diploid parent strain HU-TY-1 and the original strain LK, some of them may be correlation with thermotolerant property. Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 485 - 90 {Progress on the biosynthesis of medium-chain-length polyhydroxyalkanoates by microorganisms}; Yan Q et al.; Polyhydroxylkanoates(PHAs) are a class of polyesters produced as reserve materials by a large number of microorganisms under metabolic stress . The most fascinating feature of PHAs is its degradability, and which is supposed to take place of the traditional plastics made from petroleum in the future . PHAs are divided into two classes: short-chain-length PHAs(scl-PHAs) and medium-chain-length PHAs . mcl-PHAs is more welcome owing to its more wide crystallinity and higher extension to break than scl-PHAs, especially when some kind of new functional groups were incorporated into the side chain of the polyester . Since Psedumonas oleovorans is the most typical microorganism to produce mcl-PHAs, here the author summarized how P . oleovorans synthesize the mcl-PHAs and the production of mcl-PHAs by fermentation and give some of the idea about the future research of this field. Arch Microbiol, 2001 Dec, 177(1), 107 - 12 Epub 2001 Oct 30. A genetic screen for suppressors of Escherichia coli Tat signal peptide mutations establishes a critical role for the second arginine within the twin-arginine motif; Buchanan G et al.; The Escherichia coli Tat protein export pathway transports folded proteins synthesized with N-terminal twin-arginine signal peptides . Twin-arginine signal sequences contain a conserved SRRxFLK "twin-arginine" amino acid sequence motif which is required for protein export by the Tat pathway . The E . coli trimethylamine N-oxide reductase (TorA) is a Tat-dependent periplasmic molybdoenzyme that facilitates anaerobic respiration with trimethylamine N-oxide as terminal electron acceptor . Here, we describe mutant strains constructed with modified TorA twin-arginine signal peptides . Substitution of the second arginine residue of the TorA signal peptide twin-arginine motif with either lysine or aspartate, or the simultaneous substitution of both arginines with lysine residues, completely abolished export . In each case, the now cytoplasmically localised TorA retained full enzymatic activity with the artificial electron donor benzyl viologen . However, the mutant strains were incapable of anaerobic growth with trimethylamine N-oxide and the non-fermentable carbon-source glycerol . The growth phenotype of the mutant strains was exploited in a genetic screen with the aim of identifying second-site suppressor mutations that allowed export of the modified TorA precursors. Int Dent J, 2001, 51(6 Suppl 1), 408 - 12 Dental considerations for dietary counselling; Duggal MS et al.; A decline in the prevalence of dental caries over three decades has occurred without a significant change in the consumption of fermentable carbohydrates, indicating that good dental health is achievable with the presence of cariogenic factors in the diet . Since, in many countries 80% of the caries is present in only 20% of the population, 'targeted intervention' would seem a better preventive option, stressing the judicious use of fluoride, plaque control, fissure sealants and a sensible diet . Dietary modification is notoriously difficult to achieve, being incumbent upon the subject's willingness to effect a change in behaviour . Many texts refer to the frequency of consumption of carbohydrates as being all-important, recommending a reduction in the frequency . However, recent evidence suggests it is the frequency of toothbrushing with a fluoride containing dentifrice which is of fundamental importance in promoting remineralisation of enamel . Dietary advice should be formulated which is both realistic and positive . Trying to dissuade children from consuming products, which they perceive to be tasty and pleasurable is counter productive and more emphasis should be given to tooth brushing using a fluoride toothpaste . Also, a fundamental shift away from the idea of 'good foods versus bad foods' is required and more emphasis laid on good diets as opposed to bad diets . Children should be able to enjoy foods traditionally considered 'bad' from a dental viewpoint, as long as they brush their teeth with a fluoride containing dentifrice and have a sensible approach to their consumption. Acta Microbiol Immunol Hung, 2001, 48(3-4), 429 - 40 The biochemistry of citric acid accumulation by Aspergillus niger; Karaffa L et al.; Fungi, in particular Aspergilli, are well known for their potential to overproduce a variety of organic acids . These microorganisms have an intrinsic ability to accumulate these substances and it is generally believed that this provides the fungi with an ecological advantage, since they grow rather well at pH 3 to 5, while some species even tolerate pH values as low as 1.5 . Organic acid production can be stimulated and in a number of cases conditions have been found that result in almost quantitative conversion of carbon substrate into acid . This is exploited in large-scale production of a number of organic acids like citric-, gluconic- and itaconic acid . Both in production volume as well as in knowledge available, citrate is by far the major organic acid . Citric acid (2-hydroxy-propane-1,2,3-tricarboxylic acid) is a true bulk product with an estimated global production of over 900 thousand tons in the year 2000 . Till the beginning of the 20th century, it was exclusively extracted from lemons . Since the global market was dominated by an Italian cartel, other means of production were sought . Chemical synthesis was possible, but not suitable due to expensive raw materials and a complicated process with low yield . The discovery of citrate accumulation by Aspergillus niger led to a rapid development of a fermentation process, which only a decade later accounted for a large part of the global production . The application of citric acid is based on three of its properties: (1) acidity and buffer capacity, (2) taste and flavour, and (3) chelation of metal ions . Because of its three acid groups with pKa values of 3.1, 4.7 and 6.4, citrate is able to produce a very low pH in solution, but is also useful as a buffer over a broad range of pH values (2 to 7) . Citric acid has a pleasant acid taste which leaves little aftertaste . It sometimes enhances flavour, but is also able to mask sweetness, such as the aspartame taste in diet beverages . Chelation of metal ions is a very important property that has led to applications such as antioxidant and preservative . Moreover, it is a "natural" substance and fully biodegradable. Acta Microbiol Immunol Hung, 2001, 48(3-4), 393 - 411 The glutathione metabolism of the beta-lactam producer filamentous fungus Penicillium chrysogenum; Pocsi I et al.; Glutathione (gamma-L-glutamyl-L-cysteinyl-glycine; GSH) shares structural similarities with the beta-lactam biosynthetic intermediate ACV-tripeptide (delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine) . Not surprisingly, GSH has been reported to inhibit the beta-lactam biosynthetic machinery quite effectively and, hence, strategies to decrease the intracellular GSH concentrations without influencing negatively the physiological status of idiophasic mycelia would attract industrial interests . Here we present a detailed map of the GSH metabolic network of P . chrysogenum and show a promising way to keep the GSH pool selectively down under penicillin producing conditions . This procedure includes a well-controlled and transient lowering of pH at the beginning of the production phase, and it relies on the GSH-dependent detoxification of the protonophore penicillin side-chain precursors phenoxyacetic acid (POA) and phenylacetic acid (PA) . Encouraging preliminary fed-batch fermentation experiments have been performed to test this technological proposal . Interestingly, the mechanism of the activation of POA and PA to the appropriate CoA derivatives has remained yet to be answered but the involvement of GSH seems to be rather unlikely in this case . Our data also challenge the hypothesis that the formation of different kinds of penicillins would be an alternative to GSH-dependent detoxification processes in P . chrysogenum. J Bacteriol, 2002 Feb, 184(3), 672 - 8 Novel pathway for alcoholic fermentation of delta-gluconolactone in the yeast Saccharomyces bulderi; van Dijken JP et al.; Under anaerobic conditions, the yeast Saccharomyces bulderi rapidly ferments delta-gluconolactone to ethanol and carbon dioxide . We propose that a novel pathway for delta-gluconolactone fermentation operates in this yeast . In this pathway, delta-gluconolactone is first reduced to glucose via an NADPH-dependent glucose dehydrogenase (EC 1.1.1.47) . After phosphorylation, half of the glucose is metabolized via the pentose phosphate pathway, yielding the NADPH required for the glucose-dehydrogenase reaction . The remaining half of the glucose is dissimilated via glycolysis . Involvement of this novel pathway in delta-gluconolactone fermentation in S . bulderi is supported by several experimental observations . (i) Fermentation of delta-gluconolactone and gluconate occurred only at low pH values, at which a substantial fraction of the substrate is present as delta-gluconolactone . Unlike gluconate, the latter compound is a substrate for glucose dehydrogenase . (ii) High activities of an NADP(+)-dependent glucose dehydrogenase were detected in cell extracts of anaerobic, delta-gluconolactone-grown cultures, but activity of this enzyme was not detected in glucose-grown cells . Gluconate kinase activity in cell extracts was negligible . (iii) During anaerobic growth on delta-gluconolactone, CO(2) production exceeded ethanol production by 35%, indicating that pyruvate decarboxylation was not the sole source of CO(2) . (iv) Levels of the pentose phosphate pathway enzymes were 10-fold higher in delta-gluconolactone-grown anaerobic cultures than in glucose-grown cultures, consistent with the proposed involvement of this pathway as a primary dissimilatory route in delta-gluconolactone metabolism. Int J Food Microbiol, 2001 Dec 30, 71(2-3), 169 - 76 Survival and characterization of Escherichia coli strains in a typical Mexican acid-fermented food; Sainz T et al.; In this study, the presence and pathogenic characteristics of Escherichia coli strains in pozol, an acid-fermented maize beverage consumed in South-eastern Mexico, were determined . Seventy-three E . coli strains were isolated at early and late times (6 and 48 h) during the pozol fermentation process, when pH values of the doughs were 6.7-4.7 (6 h) and 4.7-3.7 (48 h) . Serotypes that belong to diarrheagenic E . coli serogroups O18, O88, O8, O11, O20, O173 were identified . HEp-2 cell adherence in vitro assays showed localized, diffuse and aggregative adherence patterns among some of these strains . A DNA colony hybridization analysis with different probes showed the presence of virulence genes related to diarrheal pathogenesis . Thirty-three percent of the E . coli strains were tetracycline-resistant and 95% had a 20 kb plasmid . The presence and survival of potentially pathogenic E . coli in acid-fermented pozol suggest that such foods may be a potential source of foodborne outbreaks. Reprod Nutr Dev, 2001 Jul-Aug, 41(4), 297 - 308 Variation of proteolytic activity in ewe during in vivo digestion of two pea-based diets differing by their fermentability characteristics; Sales-Duval M et al.; In order to study the effects of a small difference in starch and nitrogen availability on proteolysis, two different diets were supplied to four ewes fitted with rumen fistulae . They differed in the ratio of fermentable nitrogen over fermentable energy . with 144 g of fermentable nitrogen (FN) per kg of fermentable energy (FE) for diet I and 126 g FN x kg(-1) FE for diet II . The diets were constituted of 700 g hay grass, 200 g ground pea and either 100 g ground wheat (diet I) or 100 g corn starch (diet II) . After two weeks of an adapting period to the diets, rumen content was sampled after feeding over time . The rate of disappearance of soluble proteins was 2.5 times higher with diet II and ammonia concentrations were significantly lower (from -28 to -43%) with diet II . Total proteolytic activity, by considering all the bacterial compartments, was significantly higher with diet II (+40 EU/mL x h(-1)): changes in the total proteolytic activity in the particulate and the liquid phases of the rumen could explain the difference observed between the two diets . Moreover, with diet II, exopeptidase activities increased more in the liquid phase, especially leucine aminopeptidase and Dipeptidyl peptidase I (DPP-I), and the diversity of endopeptidase activities increased in the particulate phase . These two facts could account for the higher total proteolytic activity in the rumen content with diet II. Biotechnol Bioeng, 2002 Mar 5, 77(5), 589 - 98 Production of teicoplanin by Actinoplanes teichomyceticus in continuous fermentation; Vara AG et al.; Production of the potent antibiotic teicoplanin by Actinoplanes teichomyceticus was studied in batch and in chemostat cultures . It is found that the producing strain deactivates to a non-producing strain named NP-12 . This strain is used to find the growth kinetics of the A . teichomyceticus without interference from the product teicoplanin . In batch experiments with NP-12 grown on glucose at different initial concentrations and with different added amounts of teicoplanin, the strong inhibitory effect of teicoplanin was determined . These results obtained on NP-12 were validated in a series of chemostat experiments with the processing strain . All experiments in batch and in chemostat cultures were well represented by Monod kinetics with respect to the carbon and energy source (glucose) and with a substantial inhibitory effect of teicoplanin . Further experiments were made with the producing strain in a continuous reactor coupled to a microfilter that delivers a cell-free permeate . It was found that the derived kinetics almost exactly simulated the behavior of the cell recirculation reactor in addition to when the cell concentration in the reactor was more than four times higher than in the chemostat . For industrial production of teicoplanin, a continuous reactor with cell recirculation and working with a low effluent glucose concentration was by far the best mode of operation . Finally, the deactivation of the producing strain to NP-12 was modeled by a two-step deactivation mechanism . Deactivation was independent of dilution rate but dependent on the inoculum preparation and on the previous history of the inoculum . J Biol Chem, 2002 Mar 29, 277(13), 10973 - 81 Epub 2002 Jan 11. Uptake of exogenous coenzyme Q and transport to mitochondria is required for bc1 complex stability in yeast coq mutants; Santos-Ocana C et al.; Coenzyme Q (Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells but also is present in other cellular membranes where it acts as an antioxidant . Because Q synthesis machinery in Saccharomyces cerevisiae is located in the mitochondria, the intracellular distribution of Q indicates the existence of intracellular Q transport . In this study, the uptake of exogenous Q(6) by yeast and its transport from the plasma membrane to mitochondria was assessed in both wild-type and in Q-less coq7 mutants derived from four distinct laboratory yeast strains . Q(6) supplementation of medium containing ethanol, a non-fermentable carbon source, rescued growth in only two of the four coq7 mutant strains . Following culture in medium containing dextrose, the added Q(6) was detected in the plasma membrane of each of four coq7 mutants tested . This detection of Q(6) in the plasma membrane was corroborated by measuring ascorbate stabilization activity, as catalyzed by NADH-ascorbate free radical reductase, a transmembrane redox activity that provides a functional assay of plasma membrane Q(6) . These assays indicate that each of the four coq7 mutant strains assimilate exogenous Q(6) into the plasma membrane . The two coq7 mutant strains rescued by Q(6) supplementation for growth on ethanol contained mitochondrial Q(6) levels similar to wild type . However, the content of Q(6) in mitochondria from the non-rescued strains was only 35 and 8%, respectively, of that present in the corresponding wild-type parental strains . In yeast strains rescued by exogenous Q(6), succinate-cytochrome c reductase activity was partially restored, whereas non-rescued strains contained very low levels of activity . There was a strong correlation between mitochondrial Q(6) content, succinate-cytochrome c reductase activity, and steady state levels of the cytochrome c(1) polypeptide . These studies show that transport of extracellular Q(6) to the mitochondria operates in yeast but is strain-dependent . When Q biosynthesis is disrupted in yeast strains with defects in the intracellular transport of exogenous Q, the bc(1) complex is unstable . These results indicate that delivery of exogenous Q(6) to mitochondria is required fore activity and stability of the bc(1) complex in yeast coq mutants. Life Sci, 2001 Nov 30, 70(2), 219 - 27 Fermented soybean-derived Touchi-extract with anti-diabetic effect via alpha-glucosidase inhibitory action in a long-term administration study with KKAy mice; Fujita H et al.; A water-soluble Touchi-extract (TE) exerts a potent inhibitory activity against rat intestinal alpha-glucosidase in foodstuffs, and elicited anti-glycemic effects in rats and humans with single-bolus oral administration . In the present study, genetically modified diabetic model KKAy mice were used to examine the long-term effects of TE . Eight-week-old male KKAy mice were fed with CE-2 chow containing 0.08 and 0.4% of TE for 60 days . In the latter group, fasting blood glucose levels decreased (6.68 +/- 0.41 mmol/L) significantly (p<0.05) after a 60-day ingestion period compared with controls (8.75 +/- 0.54 mmol/L) . Moreover, postprandial blood glucose levels were also significantly reduced (16.79 +/- 2.28 mmol/L; p<0.01) after ingesting TE for only 30 days compared with controls (28.49 +/- 0.59 mmol/L) . On oral TE treatment for 60 days, postprandial increases in the blood glucose level after oral loading of sucrose (2 g/kg) at 30 (p<0.05) and 60 (p<0.01) min were significantly depressed compared with controls . Indexes for serum lipids; viz., total cholesterol (p<0.05) and triglyceride (p<0.01) levels significantly decreased after TE ingestion . Indexes for hepatic functions, such as glutamic-oxaloacetic transaminase (p<0.01), glutamic-pyruvic transaminase and gama-glutamyl transpeptide levels, were similarly suppressed . Organ weights of the heart, kidney, jejunum, liver and spleen increased in control KKAy mice due to hyperinsulinemia . Interestingly, the respective organ weights decreased (p<0.05, 0.01) and the jejunum length was reduced (p<0.05) significantly in the TE-treated groups . All in all, TE demonstrated an anti-hyperglycemic effect and may have potential use in the management of non-insulin-dependent diabetic mellitus. Bioseparation, 2001, 10(1-3), 87 - 97 Direct product sequestration of a recombinant cutinase from batch fermentations of Saccharomyces cerevisiae; Calado CR et al.; The recovery of cutinase of Fusarium solani pisi produced by the yeast Saccharomyces cerevisiae was studied in a fluidised bed adsorption system directly integrated with a productive fermenter (so-called direct product sequestration; DPS) . The relative efficiency of this system was compared with the one of a conventional purification process by discrete sequences of fermentation, broth clarification, ultrafiltration and fixed bed anion exchange chromatography . By direct product sequestration of the extracellular heterologous cutinase it was possible, through only one unit operation: (i) to perform broth clarification, (ii) to obtain a high cutinase concentration factor, and (iii) to recover cutinase with a specific activity that equalled that obtained with the conventional purification process . It was also possible (iv) to substantially reduce the total process time, (v) to improve the overall yield, and (vi) to increase cutinase productivity . Furthermore, the procedure outlined is suitable for large scale bioprocess exploitation. Biotechnol Bioeng, 2002 Feb 15, 77(4), 420 - 9 Hybrid model building methodology using unsupervised fuzzy clustering and supervised neural networks; Ronen M et al.; This paper suggests a model building methodology for dealing with new processes . The methodology, called Hybrid Fuzzy Neural Networks (HFNN), combines unsupervised fuzzy clustering and supervised neural networks in order to create simple and flexible models . Fuzzy clustering was used to define relevant domains on the input space . Then, sets of multilayer perceptrons (MLP) were trained (one for each domain) to map input-output relations, creating, in the process, a set of specified sub-models . The estimated output of the model was obtained by fusing the different sub-model outputs weighted by their predicted possibilities . On-line reinforcement learning enabled improvement of the model . The determination of the optimal number of clusters is fundamental to the success of the HFNN approach . The effectiveness of several validity measures was compared to the generalization capability of the model and information criteria . The validity measures were tested with fermentation simulations and real fermentations of a yeast-like fungus, Aureobasidium pullulans . The results outline the criteria limitations . The learning capability of the HFNN was tested with the fermentation data . The results underline the advantages of HFNN over a single neural network . Pol J Pharmacol, 2001 May-Jun, 53(3), 201 - 14 Drug addiction . Part I . Psychoactive substances in the past and presence; Vetulani J; Substances capable of changing the functions of the central nervous system are widely distributed in plant kingdom, and many of them were discovered by ancient food-gatherers at the dawn of humanity . In the Old World only a few substances producing euphoria or altered states of consciousness and having habit-forming properties are still widely used . They are the products of poppy (opium, morphine), hemp (hashish, marijuana), and of fermentation of various organic materials alkohol . This list has recently been joined by the psilocybin-containing mushrooms . The addiction-forming compounds originated in the New World and widely spread are tobacco (nicotine) and cocaine . In the 19th and 20th, century the development of medicinal chemistry resulted in several synthetic compounds, originally proposed as therapeutics, such as barbiturates, benzodiazepines and amphetamines . Due to legal problems, to avoid production of the substances already prohibited, many designer drugs were manufactured . In addition, several compounds were synthesized as recreational drugs . Also some compounds that were not regarded as drugs, such as aromatic hydrocarbons and other cleansing agents, as well as steroids were found to have properties of dangerous, habit-forming agents . The attitude of society and the pattern of use of psychoactive substances have changed with time, particularly in the last decades . The active principles are now more addictive because of concentration, purification, chemical modifications and the way of ingestion, which now favors most rapid transport to the central nervous system . The substance abuse approaches the level of global epidemics, and the recent usage of drugs of addition is also reviewed. Public Health Nutr, 2001 Oct, 4(5), 943 - 52 Dietary soy isoflavone intake in older Japanese American women; Rice MM et al.; OBJECTIVE: In a sample of older Japanese American women, we aimed to: (1) describe the most commonly consumed soy foods, (2) estimate dietary soy isoflavone intake, (3) describe characteristics associated with dietary soy isoflavone intake, and (4) compare our estimates with previously published estimates in other Japanese samples . DESIGN: A 14-item soy food-frequency questionnaire was administered to older Japanese American women and responses were converted to quantitative estimates of soy isoflavones (genistein plus daidzein) . Multiple regression was used to examine characteristics associated with dietary soy isoflavone intake, including self-reported lifestyle and cultural factors and dietary intake of various foods ascertained from a semi-quantitative food-frequency questionnaire . To compare our estimates with other samples, a review of the literature was conducted . SETTING/SUBJECTS: Data are from 274 women aged 65+ years, recruited from a longitudinal cohort study of Japanese Americans in King County, Washington State . RESULTS: The soy foods most commonly consumed were tofu (soybean curd), miso (fermented soybean paste) and aburaage (fried thin soybean curd) . The mean intake of dietary soy isoflavones was 10.2 (standard deviation (SD), 12.4) mg day(-1), approximately a quarter to a half that of previously published estimates in Japanese samples . Dietary soy isoflavone intake was positively associated with speaking Japanese, the consumption of traditional Japanese dishes (kamaboko, manju and mochi), low-fat/non-fat milk and yellow/red vegetables, vitamin E supplement use, and walking several blocks each day . Dietary soy isoflavone intake was negatively associated with the consumption of butter . CONCLUSIONS: The estimated dietary soy isoflavone intake in Japanese American women living in King County, Washington State was about a quarter to a half that of women living in Japan . Dietary soy isoflavone intake was associated with speaking Japanese and healthy lifestyle and dietary habits. Appl Biochem Biotechnol, 2001 Oct-Dec, 96(1-3), 71 - 82 Semi-solid-state fermentation of Eicchornia crassipes biomass as lignocellulosic biopolymer for cellulase and 3-glucosidase production by cocultivation of Aspergillus niger RK3 and Trichoderma reesei MTCC164; Kumar R et al.; An aquatic weed biomass, Eicchornia crassipes, present in abundance and leading to a threatening level of water pollution was used as substrate for cellulase and beta-glucosidase production using wild-type strain Aspergillus niger RK3 that was isolated from decomposing substrate . Alkali treatment of the biomass (10%) resulted in a 60-66% increase in endoglucanase, exoglucanase, and beta-glucosidase production by the A . niger RK3 strain in semi-solid-state fermentation . Similarly, the alkali-treated biomass led to a 45-54% increase in endo- and exoglucanase and a higher (98%) increase in beta-glucosidase production by Trichoderma reesei MTCC164 under similar conditions . However, the cocultivation of A . niger RK3 and T . reesei MTCC164 at a ratio of 3:1 showed a 20-24% increase in endo- and exoglucanase activities and about a 13% increase in the beta-glucosidase activity over the maximum enzymatic activities observed under single culture conditions . Multistep physical (ultraviolet) and chemical (N-methyl-N'-nitrosoguanidine, sodium azide, colchicine) mutagenesis of the A . niger RK3 strain resulted in a highly cellulolytic mutant, UNSC-442, having an increase of 136, 138, and 96% in endoglucanase, exoglucanase, and beta-glucosidase, activity, respectively . The cocultivation of mutant UNSC-442 along with T . reesei MTCC164 (at a ratio of 3:1) showed a further 10-11% increase in endo- and exoglucanase activities and a 29% increase in beta-glucosidase activity in semi-solid-state fermentation. Eur J Clin Microbiol Infect Dis, 2001 Nov, 20(11), 765 - 9 Bacteremia due to Leptotrichia trevisanii sp . nov; Tee W et al.; A thin, filamentous, non-motile, aerotolerant, anaerobic, gram-negative bacterium was isolated from the blood of a 46-year-old man who was diagnosed as having acute myeloid leukemia . The organism had a positive catalase reaction but was negative in indole and oxidase tests . A commercially available system failed to identify the bacterium, but 16S rRNA gene sequencing showed it to be most closely related (97% similarity) to a recently isolated Leptotrichia sp . The DNA base composition was 29.7% mol G+C, and the organism produced lactate as the sole end-product of glucose fermentation . These data indicate the isolate is a new species of Leptotrichia for which the name Leptotrichia trevisanii sp . nov . is proposed. EMBO J, 2002 Jan 15, 21(1-2), 135 - 44 Acute glucose starvation activates the nuclear localization signal of a stress-specific yeast transcription factor; Gorner W et al.; In yeast, environmental conditions control the transcription factor Msn2, the nuclear accumulation and function of which serve as a sensitive indicator of nutrient availablity and environmental stress load . We show here that the nuclear localization signal (NLS) of Msn2 is a direct target of cAMP-dependent protein kinase (cAPK) . Genetic analysis suggests that Msn2-NLS function is inhibited by phosphorylation and activated by dephosphorylation . Msn2-NLS function is unaffected by many stress conditions that normally induce nuclear accumulation of full-length Msn2 . The Msn2-NLS phosphorylation status is, however, highly sensitive to carbohydrate fluctuations during fermentative growth . Dephosphorylation occurs in >2 min after glucose withdrawal but the effect is reversed rapidly by refeeding with glucose . This response to glucose depletion is due to changes in cAPK activity rather than an increase in protein phosphatase activity . Surprisingly, the classical glucose-sensing systems are not connected to this rapid response system . Our results further imply that generic stress signals do not cause short-term depressions in cAPK activity . They operate on Msn2 by affecting an Msn5-dependent nuclear export and/or retention mechanism. EMBO J, 2002 Jan 15, 21(1-2), 43 - 52 Shy1p is necessary for full expression of mitochondrial COX1 in the yeast model of Leigh's syndrome; Barrientos A et al.; SHY1 codes for a mitochondrial protein required for full expression of cytochrome oxidase (COX) in Saccharomyces cerevisiae . Mutations in the homologous human gene (SURF1) have been reported to cause Leigh's syndrome, a neurological disease associated with COX deficiency . The function of Shy1p/Surf1p is poorly understood . Here we have characterized revertants of shy1 null mutants carrying extragenic nuclear suppressor mutations . The steady-state levels of COX in the revertants is increased by a factor of 4-5, accounting for their ability to r