Appl Biochem Biotechnol, 1998 Spring, 70-72, 237 - 48 Production of a novel pyranose 2-oxidase by basidiomycete Trametes multicolor; Leitner C et al.; During a screening for the enzyme pyranose 2-oxidase (P2O) which has a great potential as a biocatalyst for carbohydrate transformations, Trametes multicolor was identified as a promising, not-yet-described producer of this particular enzyme activity . Furthermore, it was found in this screening that the enzyme frequently occurs in basidiomycetes . Intracellular P2O was produced in a growth-associated manner by T . multicolor during growth on various substrates, including mono-, oligo-, and polysaccharides . Highest levels of this enzyme activity were formed when lactose or whey were used as substrates . Peptones from casein and other casein hydrolysates were found to be the most favorable nitrogen sources for the formation of P2O . By applying an appropriate feeding strategy for the substrate lactose, which ensured an elevated concentration of the carbon source during the entire cultivation, levels of P2O activity obtained in laboratory fermentations, as well as the productivity of these bioprocess experiments, could be enhanced more than 2.5-fold. Appl Biochem Biotechnol, 1998 Spring, 70-72, 225 - 35 Use of hemicellulose hydrolysate for beta-glucosidase fermentation; Reczey K et al.; Hydrolysis of cellulose by Trichoderma cellulases often results in a mixture of glucose, cellobiose, and low-mol-wt cellodextrins . Cellobiose is nonfermentable for most yeasts, and therefore it has to be hydrolyzed to glucose by beta-glucosidase prior to ethanol fermentation . In the present study, the beta-glucosidase production of one Penicillium and three Aspergillus strains, which were previously selected out of 24 strains, was investigated on steam pretreated willow . Both steam-pretreated willow and hemicellulose hydrolysate, released during steam explosion of willow, were used as carbon sources . Reference cultivation runs were performed using prehydrolyzed Solka Floc and glucose . The four strains were compared with Trichoderma reesei regarding sugar consumption and beta-glucosidase production . Aspergillus niger and Aspergillus phoenicis proved to be the best enzyme producers on hemicellulose hydrolysate . The maximum beta-glucosidase activity, 4.60 IU/mL, was obtained when A . phoenicis was cultivated on the mixture of hemicellulose hydrolysate and steam-pretreated willow . The maximum yield of enzyme activity, 502 IU/g total carbohydrate, was obtained when Aspergillus foetidus was cultivated on the hemicellulose hydrolysate. Appl Biochem Biotechnol, 1998 Spring, 70-72, 187 - 98 A novel fermentation pathway in an Escherichia coli mutant producing succinic acid, acetic acid, and ethanol; Donnelly MI et al.; Escherichia coli strain NZN111, which is unable to grow fermentatively because of insertional inactivation of the genes encoding pyruvate: formate lyase and the fermentative lactate dehydrogenase, gave rise spontaneously to a chromosomal mutation that restored its ability to ferment glucose . The mutant strain, named AFP111, fermented glucose more slowly than did its wild-type ancestor, strain W1485, and generated a very different spectrum of products . AFP111 produced succinic acid, acetic acid, and ethanol in proportions of approx 2:1:1 . Calculations of carbon and electron balances accounted fully for the observed products; 1 mol of glucose was converted to 1 mol of succinic acid and 0.5 mol each of acetic acid and ethanol . The data support the emergence in E . coli of a novel succinic acid:acetic acid:ethanol fermentation pathway. Appl Biochem Biotechnol, 1998 Spring, 70-72, 137 - 48 Yeast adaptation on softwood prehydrolysate; Keller FA et al.; Several strains and genera of yeast, including Saccharomyces cerevisiae D5A, Pachysolen tannophilus, S . cerevisiae K-1, Brettanomyces custersii, Candida shehatae, and Candida acidothermophilum, are screened for growth on dilute acid-pretreated softwood prehydrolysate . Selected softwood species found in forest underbrush of the western United States, which contain predominantly hexosan hemicellulose, were studied . This phase of the work emphasized debarked Douglas fir . The two best initial isolates were gradually selected for improved growth by adaptation to increasing prehydrolysate concentrations in batch culture, with due consideration of nutrient requirements . Microaerophilic conditions were evaluated to encourage tolerance of pretreatment hydrolysate, as well as ethanol product . Adaptation and simultaneous saccharification and fermentation (SSF) results are used to illustrate improved performance with an adapted strain, compared to the wild type. Crit Rev Food Sci Nutr, 1998 May, 38(4), 259 - 97 Butyric acid from the diet: actions at the level of gene expression; Smith JG et al.; A number of components present in the diet, although nutritionally nonessential, have been discovered to have beneficial effects toward both general health and disease prevention/protection . One such nutrient, butyric acid, can be derived in large quantities from bacterial fementation of dietary fiber in the bowel and is also a component of bovine milk . In gut fermentation, the production of butyric acid defines its delivery point; thus, the synthesis and site of action of butyric acid are in close proximity and have frustrated the investigation of its activities in vivo . Recent research has, however, revealed a number of activities of butyric acid toward isolated cells . In particular, its ability to modify nuclear architecture and induce death by apoptosis in colon cancer cells is arousing great interest . Butyric acid changes the structure of chromatin through its effects on posttranslational modifications, key modifications being acetylation and phosphorylation of the nuclear histones . Butyric acid can also modify the differentiation state of cells, and in the case of cancerous colonic cells overcomes their resistance to normal programmed death . Thus, the activities of this fermentation product of dietary fiber may contribute substantially to the decreased incidence of bowel cancer that has been associated with fiber intake. Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6791 - 6 p21(WAF1) is required for butyrate-mediated growth inhibition of human colon cancer cells; Archer SY et al.; A diet high in fiber is associated with a decreased incidence and growth of colon cancers . Butyrate, a four-carbon short-chain fatty acid product of fiber fermentation within the colon, appears to mediate these salutary effects . We sought to determine the molecular mechanism by which butyrate mediates growth inhibition of colonic cancer cells and thereby to elucidate the molecular link between a high-fiber diet and the arrest of colon carcinogenesis . We show that concomitant with growth arrest, butyrate induces p21 mRNA expression in an immediate-early fashion, through transactivation of a promoter cis-element(s) located within 1.4 kb of the transcriptional start site, independent of p53 binding . Studies using the specific histone hyperacetylating agent, trichostatin A, and histone deacetylase 1 indicate that growth arrest and p21 induction occur through a mechanism involving histone hyperacetylation . We show the critical importance of p21 in butyrate-mediated growth arrest by first confirming that stable overexpression of the p21 gene is able to cause growth arrest in the human colon carcinoma cell line, HT-29 . Furthermore, using p21-deleted HCT116 human colon carcinoma cells, we provide convincing evidence that p21 is required for growth arrest to occur in response to histone hyperacetylation, but not for serum starvation nor postconfluent growth . Thus, p21 appears to be a critical effector of butyrate-induced growth arrest in colonic cancer cells, and may be an important molecular link between a high-fiber diet and the prevention of colon carcinogenesis. Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6739 - 43 The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo; MacAlpine DM et al.; Abf2p is a high mobility group (HMG) protein found in yeast mitochondria that is required for the maintenance of wild-type (rho+) mtDNA in cells grown on fermentable carbon sources, and for efficient recombination of mtDNA markers in crosses . Here, we show by two-dimensional gel electrophoresis that Abf2p promotes or stabilizes Holliday recombination junction intermediates in rho+ mtDNA in vivo but does not influence the high levels of recombination intermediates readily detected in the mtDNA of petite mutants (rho-) . mtDNA recombination junctions are not observed in rho+ mtDNA of wild-type cells but are elevated to detectable levels in cells with a null allele of the MGT1 gene (Deltamgt1), which codes for a mitochondrial cruciform-cutting endonuclease . The level of recombination intermediates in rho+ mtDNA of Deltamgt1 cells is decreased about 10-fold if those cells contain a null allele of the ABF2 gene . Overproduction of Abf2p by >/= 10-fold in wild-type rho+ cells, which leads to mtDNA instability, results in a dramatic increase in mtDNA recombination intermediates . Specific mutations in the two Abf2p HMG boxes required for DNA binding diminishes these responses . We conclude that Abf2p functions in the recombination of rho+ mtDNA. J Biol Chem, 1998 Jun 12, 273(24), 14933 - 41 Isolation and characterization of the gene (CLS1) encoding cardiolipin synthase in Saccharomyces cerevisiae; Chang SC et al.; In eukaryotic cells, cardiolipin (CL) synthase catalyzes the final step in the synthesis of CL from phosphatidylglycerol and CDP-diacylglycerol . CL and its synthesis are localized predominantly to the mitochondrial inner membrane, and CL is generally thought to be an essential component of many mitochondrial processes . By using homology searches for genes potentially encoding phospholipid biosynthetic enzymes, we have cloned the gene (CLS1) encoding CL synthase in Saccharomyces cerevisiae . Overexpression of the CLS1 gene under its endogenous promoter or the inducible GAL1 promoter in yeast and expression of CLS1 in baculovirus-infected insect cells resulted in elevated CL synthase activity . Disruption of the CLS1 gene in a haploid yeast strain resulted in the loss of CL synthase activity, no detectable CL, a 5-fold elevation in phosphatidylglycerol levels, and lack of staining of mitochondria by a dye with high affinity for CL . The cls1::TRP1 null mutant grew on both fermentable and non-fermentable carbon sources but more poorly on the latter . The level and activity of cytochrome c oxidase was normal, and a dye whose accumulation is dependent on membrane proton electrochemical potential effectively stained the mitochondria . These results definitively identify the gene encoding the CL synthase of yeast. J Dairy Sci, 1998 May, 81(5), 1322 - 30 The effect of preservatives based on propionic acid on the fermentation and aerobic stability of corn silage and a total mixed ration; Kung L Jr et al.; For 3 successive yr, whole-plant corn was ensiled in laboratory silos with low percentages of silage preservatives, the primary active ingredient of which was propionic acid . Preservatives were added to forage just prior to ensiling at rates of 0.1 to 0.2% of the fresh forage weight . In all 3 yr, treatments had minor effects on fermentation end products, except that the concentration of propionic acid was greater because of its addition . The mean low and high percentages of preservatives increased aerobic stability of the treated silages by 19 and 57 h, respectively, in Experiment 1 and by 17 and 38 h, respectively, in Experiment 2 . In Experiment 3, aerobic stability was improved by > 90 h by preservatives (0.2% addition) . In a lactation study, a total mixed ration (46% dry matter) was mixed without or with (0.2 or 0.3%) a stabilizer that was designed to prevent spoilage in the feed bunk . The high dose resulted in orts with a lower pH and temperature after 24 h in the feed bunk . However, dry matter intake and milk production were unaffected by treatments . Chemical preservatives based on propionic acid added at low rates did not affect fermentation but were effective in the reduction of heating in corn silage and in a total mixed ration. Alcohol Clin Exp Res, 1998 May, 22(3 Suppl), 170S - 172S Korean status of alcoholics and alcohol-related health problems; Park SC et al.; Recent changes in the socioeconomic status of Korea have caused big differences in alcohol-related social and health problems . The traditional trait of drinking mild fermented beverages with nutritional side dishes and meals has shifted to drinking strongly distilled liquors without any side dishes . Moreover, the alcohol consumption per adult capita of Korea is now 8.1 liters, which parallels the level of other developed countries; it used to be 1.0 liter in 1960 and 7.0 liters in 1980, respectively . But the alcohol consumption per capita of adult males is now 18.4 liters . Consequently, the national incidence of alcohol-related diseases and accidents has rapidly increased . Korean adult males have the highest risk of the incidence of hepatoma . The rate of car accidents caused by drunken driving is about 10-fold higher than in any other developed country. J Dairy Sci, 1998 May, 81(5), 1251 - 61 Trans-octadecenoic acids and milk fat depression in lactating dairy cows; Griinari JM et al.; We examined the role of trans-octadecenoic acids in milk fat depression when low fiber diets were fed . The study consisted of four experimental periods with a 2 x 2 factorial arrangement of treatments to test the effects of dietary fat (saturated vs . unsaturated) and rumen fermentation (high fiber diets vs . low fiber diets) on milk fat depression . Dietary fiber concentration and type of fat had significant effects on milk fat . Effects were most pronounced when unsaturated fat was added to the low fiber diet . When the low fiber diet plus unsaturated fat was fed, milk fat percentage and yield were decreased by 30 and 35%, respectively, compared with the percentage and yield when the high fiber diet plus saturated fat was fed . Alterations in rumen fermentation caused by differences in dietary fiber concentrations had little effect on the amount of trans-octadecenoic acids in milk fat, and the total amount did not correlate with changes in milk fat percentage . Further examination of the isomeric profile of trans-octadecenoic acid revealed substantial differences among the dietary treatments . Although the addition of unsaturated fat resulted in marked increases in the milk fat content of trans-11-octadecenoic acid, regardless of dietary fiber concentration, the low fiber diet plus unsaturated fat increased the content of trans-10-octadecenoic acid . This combination was also associated with a significant decrease in milk fat content and yield . When the low fiber diets were fed, circulating insulin concentrations were elevated, regardless of the type of fat supplement . However, marked milk fat depression occurred only when the low fiber diet was supplemented with unsaturated fat. Appl Microbiol Biotechnol, 1998 Apr, 49(4), 393 - 8 Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var . awamori; Faus I et al.; A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var . awamori . Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var . awamori strain NRRL312 . Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated, and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor . We report the expression and secretion of thaumatin in concentrations of 5-7 mg/l . This recombinant thaumatin is sweet. Appl Microbiol Biotechnol, 1998 Apr, 49(4), 371 - 6 Microbial oxidative metabolism of diclofenac: production of 4'-hydroxydiclofenac using Epiccocum nigrum IMI354292; Webster R et al.; The 4'-hydroxylated metabolite of diclofenac was produced by biocatalysis for probing specific human drug-metabolising enzymes (CYP2C9) . An initial screen of 11 microorganisms was carried out (50 ml scale) to identify the organism best suited to the regioselective conversion of diclofenac to its 4'-hydroxylated metabolite . From this screen, the fungus Epicoccum nigrum IMI354292 was selected as the most suitable microorganism . Scale-up was carried out in a 30-l fermenter to which 2 g diclofenac was added . After 48 h, 50% of the diclofenac had been converted to it 4'-hydroxylated metabolite . The broth was then extracted with ethyl acetate and purified by chromatography and crystallisation . This yielded 0.3 g 4'-hydroxydiclofenac with a purity of at least 99% . The 4'-hydroxydiclofenac produced by E . nigrum was characterised by HPLC, mass spectrometry and NMR. Biosci Biotechnol Biochem, 1998 Apr, 62(4), 710 - 7 Prevention of peroxidative stress in rats fed on a low vitamin E-containing diet by supplementing with a fermented bovine milk whey preparation: effect of lactic acid and beta-lactoglobulin on the antiperoxidative action; Zommara M et al.; We examined the antiperoxidative properties of a fermented bovine milk whey preparation in rats fed on a low vitamin E-containing diet and identified the active principle in the preparation . An exogenous supply of either lactic acid or an amino acid mixture simulated the unfermented whey proteins to prevent red blood cell (RBC) hemolysis and to lower liver thiobarbituric acid reactive substances (TBARS) . The supply of either whey proteins or beta-lactoglobulin resulted in an increase in liver GSH and prevented iron-mediated lipoprotein peroxidation . These protein effects were reproduced in rats orally administered with either GSH or its precursor, gamma-glutamylcysteine . The amount of TBARS formed during in vitro lipoprotein peroxidation were positively correlated with liver TBARS . These results suggest that fermented milk products containing lactic acid and bovine milk whey proteins can ameliorate peroxidative stress in tissues subjected to vitamin E deficiency. J Bacteriol, 1998 May, 180(10), 2689 - 93 Identification of an Na+-dependent malonate transporter of Malonomonas rubra and its dependence on two separate genes; Schaffitzel C et al.; Two membrane proteins encoded by the malonate fermentation gene cluster of Malonomonas rubra, MadL and MadM, have been synthesized in Escherichia coli . MadL and MadM were shown to function together as a malonate transport system, whereas each protein alone was unable to catalyze malonate transport . Malonate transport by MadLM is Na+ dependent, and imposition of a DeltapNa+ markedly enhanced the rate of malonate uptake . The kinetics of malonate uptake into E . coli BL21(DE3) cells synthesizing MadLM at different pH values indicated that Hmalonate- is the transported malonate species . The stimulation of malonate uptake by Na+ ions showed Michaelis-Menten kinetics, and a Km for Na+ of 1.2 mM was determined . These results suggest that MadLM is an electroneutral Na+/Hmalonate- symporter and that it is dependent on two separate genes. J Chromatogr B Biomed Sci Appl, 1998 Apr 10, 707(1-2), 257 - 65 Monoclonal antibody production with on-line harvesting and process monitoring; Zhang J et al.; A semi-automated system has been designed for on-line harvesting and monitoring of monoclonal antibody (mAb) production . {The antibody was directed against the peptide AGPAGTGKTTKDL.} Analytical and purification units were interfaced to the fermenter via a hollow fiber cartridge in which fermentation broth was continuously circulated through the lumen of the hollow fiber system . Permeate from the hollow fiber cartridge was pumped through either an analytical sampling loop or a preparative Protein G column where antibody species were captured . Switching between monitoring and harvesting was achieved by two 3-way toggle valves . Samples from the analytical sampling loop were transported to an analytical Protein G chromatography column for quantitation of all immunoglobulin G species in the fermenter . Data acquisition and processing was performed by the data system of the liquid chromatograph . All valves in the system except the two toggle valves were controlled by the liquid chromatograph . Antibody biosynthesis was monitored for the first 60 h of fermentation . Harvesting was initiated when mAb accumulated in the fermenter . Complete harvesting took approximately 90 h. Aust Dent J, 1998 Apr, 43(2), 105 - 9 Studies on fusobacteria associated with periodontal diseases; Rogers AH; The physiological and metabolic characteristics of representative isolates of the various subspecies of Fusobacterium nucleatum were investigated by growing them in continuous culture in chemically-defined, media . Behaving almost identically, these organisms were found to obtain energy from the fermentation of simple carbohydrates such as glucose or fructose or from the fermentation of certain amino acids, free or in the form of small peptides . The latter can be attacked by aminopeptidase activity which was shown to be essential for the growth of the organism in an environment lacking fermentable carbohydrate and free amino acids but replete with small peptides . This metabolic versatility may explain the presence of F . nucleatum in both supra- and sub-gingival dental plaque and why it is often found together with organisms such as Porphyromonas gingivalis which display powerful endopeptidase activities . Using the technique of allozyme electrophoresis, the current subspeciation of F . nucleatum was shown to be of doubtful validity and evidence, based upon physiological and metabolic properties, for differences in pathogenicity between isolates was not detected . While this organism is a member of various bacterial consortia associated with periodontal diseases, its contribution to the disease process remains unclear. Aust Dent J, 1998 Apr, 43(2), 99 - 104 Purification and characterization of a putative fimbrial protein/receptor of Porphyromonas gingivalis; Dashper SG et al.; Porphyromonas gingivalis has been implicated as a major aetiological agent in certain forms of periodontal disease, P . gingivalis is a Gram-negative, asaccharolytic bacterium that obtains energy from the fermentation of amino acids derived from the hydrolysis of host protein . Virulence factors of this bacterium include the capsule, fimbrial adhesins, cytotoxins and extracellular hydrolytic enzymes . A 43 kDa fimbrillin from P . gingivalis has been isolated and characterized . However, there is evidence that a second type of fimbria exists on the surface of P . gingivalis . A putative P . gingivalis fimbrial protein from a membrane preparation has been isolated and identified . This protein was shown to be reactive with sera from patients harbouring P . gingivalis . A 28 kDa protein fragment was purified by anion exchange, gel filtration and reversed-phase chromatography . N-terminal sequence analysis of the 28 kDa protein fragment revealed homology to the fimbrial precursor protein of Dichelobacter nodosus . A peptide corresponding to the N-terminal 26 amino acyl residues of the 28 kDa protein fragment was synthesized and used to raise antibodies to the protein . Western blot analysis after SDS-PAGE of a P . gingivalis membrane preparation using the antibodies raised to the synthetic peptide detected three proteins of 36, 41 and 67 kDa . When protease inhibitors were not included in the extraction procedure only the 36 and 41 kDa bands were detected . It would appear, therefore, that the intact protein has an M(r) of 67 kDa and that the 28, 36 and 41 kDa bands represent protein fragments produced by endogenous proteolytic activity . Based on sequence homology, the 67 kDa protein is possibly a sub-unit of a second P . gingivalis fimbrial type or a surface receptor. Parasitol Res, 1998 May, 84(5), 354 - 61 Trypanosomatid protozoa in plants of southeastern Spain: characterization by analysis of isoenzymes, kinetoplast DNA, and metabolic behavior; Sanchez-Moreno M et al.; Three flagellates of the family Trypanosomatidae were isolated from mango fruits (Mangifera indica) and from the stems of clover (Trifolium glomeratum) and Amaranth (Amaranthus retroflexus) in southeastern Spain and were adapted to in vitro culture in monophase media . The parasites showed an ultrastructural pattern similar to that of other species of the genus Phytomonas . Mango and clover isolates differed from amaranth isolates in ultrastructural terms . The isolates were characterized by isoenzymatic analysis and by kDNA analysis using five different restriction endonucleases . With eight of the nine enzymatic systems, mango and clover isolates were distinguished from those of amaranth . Nevertheless, with the enzymes malate dehydrogenase and superoxide dismutase, flagellates isolated from clover were differentiated from those isolated from mango . Electrophoretic and restriction-endonuclease analysis of kDNA minicircles showed similar restriction cleavage patterns for the isolates from mango and clover, whereas the patterns of the amaranth isolates differed . The results of the present study confirm that the strains isolated from mango and clover constitute a phylogenetically closely related group of plant trypanosomatids, which is more distantly related to the strain isolated from amaranth . The similarities in the results obtained for isolates from mango and clover foliage, on the one hand, and those obtained from tomato and cherimoya fruits (studied previously), on the other, as well as the geographic proximity of the different plants support the contention that only one strain is involved, albeit one strain that can parasitize different plants . Furthermore, some of the plants appear to act as reservoirs for the parasites . On the other hand, the metabolism studies using {1H}-nuclear magnetic resonance spectroscopy did not reveal that the catabolism of Phytomonas in general follows a pattern common to all the species or isolates . Phytomonas are incapable of completely degrading glucose, excreting a large part of their carbon skeleton into the medium as fermentative metabolites (acetate, ethanol, glycine, glycerol, and succinate). J Nat Prod, 1998 May, 61(5), 648 - 51 A new cytosine glycoside from Streptomyces griseochromogenes produced by the use in vivo of enzyme inhibitors; Zhang Q et al.; The identification of new cytosine glycosides and intermediates in the biosynthetic pathway of the antifungal antibiotic blasticidin S (1) was investigated using in vivo enzyme inhibition . Fermentations of Streptomyces griseochromogenes, the organism that produces 1, supplemented with the arginine analogue argininic acid or the argininosuccinate synthase inhibitor 2-methylaspartic acid were found to produce a new metabolite (7). Microbiologia, 1997 Dec, 13(4), 405 - 11 Trends in wine microbiology; Ramon D; During the last few years many winemakers have started to use pure Saccharomyces cerevisiae strains, frequently isolated from their own geographical regions, to produce wines of more reproductable quality . This microbiological simplification has opened the way for the genetic modification of wine yeast strains . This review concerns the application of molecular techniques in oenology, not only from the point of view of the construction of recombinant strains but also for the study of the population dynamics of wine fermentations. Appl Environ Microbiol, 1998 Jun, 64(6), 2207 - 14 Rapid and reliable identification of food-borne yeasts by Fourier-transform infrared spectroscopy; Kummerle M et al.; Computer-based Fourier-transform infrared spectroscopy (FT-IR) was used to identify food-borne, predominantly fermentative yeasts . Dried yeast suspensions provided the films suitable for FT-IR measurement . Informative windows in the spectrum were selected and combined to achieve optimal results . A reference spectrum library was assembled, based on 332 defined yeast strains from international yeast collections and our own isolates . All strains were identified with conventional methods using physiological and morphological characteristics . In order to assess identification quality, another 722 unknown yeast isolates not included in the reference spectrum library were identified both by classical methods and by comparison of their FT-IR spectra with those of the reference spectrum library . Ninety-seven and one-half percent of these isolates were identified correctly by FT-IR . Easy handling, rapid identification within 24 h when starting from a single colony, and a high differentiation capacity thus render FT-IR technology clearly superior to other routine methods for the identification of yeasts. Appl Environ Microbiol, 1998 Jun, 64(6), 2133 - 40 Effects of pyruvate decarboxylase overproduction on flux distribution at the pyruvate branch point in Saccharomyces cerevisiae; van Hoek P et al.; A multicopy plasmid carrying the PDC1 gene (encoding pyruvate decarboxylase; Pdc) was introduced in Saccharomyces cerevisiae CEN . PK113-5D . The physiology of the resulting prototrophic strain was compared with that of the isogenic prototrophic strain CEN.PK113-7D and an empty-vector reference strain . In glucose-grown shake-flask cultures, the introduction of the PDC1 plasmid caused a threefold increase in the Pdc level . In aerobic glucose-limited chemostat cultures growing at a dilution rate of 0.10 h-1, Pdc levels in the overproducing strain were 14-fold higher than those in the reference strains . Levels of glycolytic enzymes decreased by ca . 15%, probably due to dilution by the overproduced Pdc protein . In chemostat cultures, the extent of Pdc overproduction decreased with increasing dilution rate . The high degree of overproduction of Pdc at low dilution rates did not affect the biomass yield . The dilution rate at which aerobic fermentation set in decreased from 0.30 h-1 in the reference strains to 0.23 h-1 in the Pdc-overproducing strain . In the latter strain, the specific respiration rate reached a maximum above the dilution rate at which aerobic fermentation first occurred . This result indicates that a limited respiratory capacity was not responsible for the onset of aerobic fermentation in the Pdc-overproducing strain . Rather, the results indicate that Pdc overproduction affected flux distribution at the pyruvate branch point by influencing competition for pyruvate between Pdc and the mitochondrial pyruvate dehydrogenase complex . In respiratory cultures (dilution rate, <0.23 h-1), Pdc overproduction did not affect the maximum glycolytic capacity, as determined in anaerobic glucose-pulse experiments. J Infect Dis, 1998 Jun, 177(6), 1750 - 3 Genotypic and phenotypic changes in the emergence of Escherichia coli O157:H7; Feng P et al.; Escherichia coli O157:H7 is a foodborne pathogen distinguished from typical E . coli by the production of Shiga toxins (Stx) and the inability to ferment sorbitol (SOR) and to express beta-glucuronidase (GUD) activity . An allele-specific probe for the GUD gene (uidA) and multilocus enzyme electrophoresis were used to elucidate stages in the evolutionary emergence of E . coli O157: H7 . A point mutation at +92 in uidA was found only in O157:H7 and its nonmotile relatives, including a SOR+ O157:H clone implicated in outbreaks of hemolytic-uremic syndrome in Germany . The results support a model in which O157:H7 evolved sequentially from an O55:H7 ancestor, first by acquiring the Stx2 gene and then by diverging into two branches; one became GUD- SOR- , resulting in the O157:H7 clone that spread worldwide, and the other lost motility, leading to the O157:H clone that is an increasing public health problem in Europe. Antibiot Khimioter, 1998, 43(4), 7 - 13 {Novel L-glutamate oxidase producing organisms: Streptomyces litmocidini and Streptomyces cremeus}; Dodzin ME et al.; Two novel strains i.e . Streptomyces cremeus 510 and Streptomyces litmocidini 447 producing L-glutamate oxidase were isolated from soil samples and identified . Mathematical design of the experiments made it possible to optimize the fermentation medium composition which provided at least a 5-fold increase of the strain L-glutamate oxidase activity by comparison with the initial activity in the standard medium. Arch Microbiol, 1998 May, 169(5), 387 - 92 Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region; Guillamon JM et al.; In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene . Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species . The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns . This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation. Microb Pathog, 1998 Apr, 24(4), 257 - 62 Human pathogenic Mycoplasma species induced cytokine gene expression in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines; Schaffner E et al.; We addressed the question whether the in vitro interaction of two Epstein-Barr virus (EBV)-genome-positive B cell lines (EB-3 and HilB-gamma) with either Mycoplasma pneumoniae or M . hominis, with the <<AIDS-related>> mycoplasma species (M . fermentans, M . fermentans subsp . incognitus, M . penetrans, M . genitalium) or with mycoplasma species known to be mere commensals of the respiratory tract (M . orale and M . salivarium) would result in expression of mRNAs for IL-2, IL-2R, IL-4 and IL-6 as determined by reverse transcriptase (RT)-PCR after 4 and 24 h of cocultivation . The pattern of cytokine gene expression observed depended on (i) the origin of the transformed cell line, (ii) the pathogenicity of the Mycoplasma species, and (iii) the length of cocultivation . The EBV-immortalized lymphoblastoid cell line HilB-gamma showed mRNA expression for IL-2, IL-2-receptor, IL-4 and IL-6 peaking 24 h after stimulation with M . pneumoniae and all AIDS-related mycoplasma species tested . The Burkitt lymphoma cell line EB-3 showed a distinct and isolated strong II-2/IL-2 R-mRNA expression within 4 h after contact with the pathogenic and all of the AIDS related mycoplasma species . In neither EBV-containing cell line cytokine was gene expression detectable after stimulation with the commensal mycoplasma species, M . orale and M . salivarium, indicating species differences in the ability of mycoplasmas to interact with and stimulate B-cell lines . Our data suggest that some mcyoplasma species may act as immunomodulatory cofactors by eliciting inappropriate cytokine gene expression in B cells latently infected with EBV . Therefore, this cultivation model may prove useful in evaluating the pathogenetic potential of novel isolated mycoplasma species . Biomed Sci Instrum, 1997, 34, 163 - 8 Modeling of recombinant yeast cells: reduction of phase space; Birol G et al.; The mechanism of starch fermentation by recombinant Saccharomyces cerevisiae in batch reactor is studied . Experiments were carried in the presence and absence of oxygen, with different initial starch concentrations . A variety of data concerning biotic and abiotic phases are collected . Nonlinear data analysis techniques are used to determine the block diagram of the system under study . Data analysis and processing reported here, are believed to form a basis in further work in structured modeling of biological systems, recombinant yeast cultures in particular. J Exp Biol, 1998 Apr, 201 ( Pt 8), 1177 - 95 Oxygen sensing and the transcriptional regulation of oxygen-responsive genes in yeast; Kwast KE et al.; The budding yeast Saccharomyces cerevisiae is a facultative aerobe that responds to changes in oxygen availability (and carbon source) by initiating a biochemically complex program that ensures that energy demands are met under two different physiological states: aerobic growth, supported by oxidative and fermentative pathways, and anaerobic growth, supported solely by fermentative processes . This program includes the differential expression of a large number of genes, many of which are involved in the direct utilization of oxygen . Research over the past decade has defined many of the cis-sites and trans-acting factors that control the transcription of these oxygen-responsive genes . However, the manner in which oxygen is sensed and the subsequent steps involved in the transduction of this signal have not been precisely determined . Heme is known to play a pivotal role in the expression of these genes, acting as a positive modulator for the transcription of the aerobic genes and as a negative modulator for the transcription of the hypoxic genes . Consequently, cellular concentrations of heme, whose biosynthesis is oxygen-dependent, are thought to provide a gauge of oxygen availability and dictate which set of genes will be transcribed . But the precise role of heme in oxygen sensing and the transcriptional regulation of oxygen-responsive genes is presently unclear . Here, we provide an overview of the transcriptional regulation of oxygen-responsive genes, address the functional roles that heme and hemoproteins may play in this regulation, and discuss possible mechanisms of oxygen sensing in this simple eukaryotic organism. J Pharm Pharmacol, 1998 Mar, 50(3), 297 - 301 Influence of new fenpropimorph fungicides on the growth and sterol composition in Saccharomyces cerevisiae: relationship between structure and activity; Sajbidor J et al.; The toxicity of fenpropimorph and seven newly synthesized analogues against Saccharomyces cerevisiae has been determined in liquid media . The inhibitory effect of the most efficient derivative is 120 times more than that of standard fenpropimorph . The non-linear relationship between hydrophobicity and toxicity indicates that the binding of the compounds to the receptors does not differ and so the differences in toxicity reflect changes in the rate of metabolism . The presence of inhibitors in the fermentation medium resulted in a reduction in harvested biomass and lipid yield, and changes in sterol composition -- the amount of ergosterol decreased whereas the amounts of lanosterol, dihydroergosterol and squalene increased . The toxicity of the compounds was most influenced by their lipophilicity . Use of this information could lead to development of more potent ergosterol inhibitors. Adv Exp Med Biol, 1998, 434, 285 - 96 Flavor deterioration in yogurt; Harasawa N et al.; Volatile components of flavored yogurt preserved at 5 degrees C in the dark for 0 day, 3 days and 10 days were recovered by simultaneous distillation extraction (SDE) and headspace (HS) procedures . Gas chromatography (GC) and GC-mass spectrometry (GC-MS) analyses of those samples showed remarkable changes in some compounds . Aldehydes which contribute to expressing citrusy notes were reduced to alcohols during fermentation process and storage . As a result, the strength of flavors which expressed well-balanced citrusy notes in yogurt were weakened, and fatty or oily notes mainly caused from alcohols were strengthened reversely . Hydrocarbons were also digested by bacteria during a fermentation process . A small amount of other compounds, such as esters and terpene alcohols changed . Fewer effects of sorption into a package material and chemical reactions, such as hydrolysis esters, hydration or oxidation of hydrocarbons, were observed. Adv Exp Med Biol, 1998, 434, 181 - 8 Antioxidizing potentials of BHA, BHT, TBHQ, tocopherol, and oxygen absorber incorporated in a Ghanaian fermented fish product; Ohshima T et al.; Raw whole fishes (Horse mackerel, Trachurus japonicus) were degutted and separately treated with antioxidants BHA, BHT, TBHQ and tocopherol before fermentation, by completely immersing the samples in 0.1% antioxidant solutions . Fish samples were then salted, fermented and dried to mark the end of processing . A portion of the control samples were packed in an oxygen absorber during storage for 2 months at room temperature to study the effect of oxygen scavenging on lipid oxidation . The moisture content and total lipid decreased with processing and storage . An inductive effect of the fish oil was observed with 2-thiobarbituric acid values and the formation of lysophos-phatidylcholine (LPC) for TBHQ and BHA treated fish during the fermentation process . Free fatty acid formation was detected in all fish samples throughout processing and storage . For samples treated with tocopherol, only *-tocopherol was detected at the end of 2 months storage . Although TBHQ showed the best antioxidative effect during processing, it was the fastest synthetic antioxidant to be depleted . However, relatively high levels of BHA were present in the sample after 2 months storage . Red color patches, suspected to be antioxidant degradative products, were observed around the operculum of TBHQ treated samples after processing. Biochemistry, 1998 Apr 28, 37(17), 6124 - 31 Bacterial expression of a mitochondrial cytochrome c . Trimethylation of lys72 in yeast iso-1-cytochrome c and the alkaline conformational transition; Pollock WB et al.; Saccharomyces cerevisiae iso-1-cytochrome c has been expressed in Escherichia coli by coexpression of the genes encoding the cytochrome (CYC1) and yeast cytochrome c heme lyase (CYC3) . Construction of this expression system involved cloning the two genes in parallel into the vector pUC18 to give the plasmid pBPCYC1(wt)/3 . Transcription was directed by two promoters, Lac and Trc, that were located upstream from CYC1 . Both proteins were expressed in the cytoplasm of E . coli cells harboring the plasmid . Semianaerobic cultures grown in a fermentor produced 15 mg of recombinant iso-1-cytochrome c per liter of culture . Attempts to increase production by addition of IPTG suppressed the number of copies of the CYC1 gene within the population . Wild-type iso-1-cytochrome c expressed with pBPCYC1(wt)/3 in E . coli was compared to the same protein expressed in yeast . At neutral pH, the two proteins exhibit indistinguishable spectroscopic and physical (Tm, Em') characteristics . However, electrospray mass spectrometry revealed that the lysyl residue at position 72 is not trimethylated by E . coli as it is by S . cerevisiae . Interestingly, the pKa of the alkaline transition of the protein expressed in E . coli is approximately 0.6 pKa unit lower than that observed for the cytochrome expressed in yeast (8.5-8.7) . 1H NMR spectroscopy of the bacterially expressed cytochrome collected at high pH revealed the presence of a third alkaline conformer that is not observed in the corresponding spectrum of the cytochrome expressed in yeast . These observations suggest that Lys72 can serve as an axial ligand to the heme iron of alkaline iso-1-ferricytochrome c if it is not modified posttranscriptionally to trimethyllysine. J Biol Chem, 1998 Apr 17, 273(16), 9829 - 36 The PEL1 gene (renamed PGS1) encodes the phosphatidylglycero-phosphate synthase of Saccharomyces cerevisiae; Chang SC et al.; Phosphatidylglycerophosphate (PG-P) synthase catalyzes the synthesis of PG-P from CDP-diacylglycerol and sn-glycerol 3-phosphate and functions as the committed and rate-limiting step in the biosynthesis of cardiolipin (CL) . In eukaryotic cells, CL is found predominantly in the inner mitochondrial membrane and is generally thought to be an essential component of many mitochondrial functions . We have determined that the PEL1 gene (now renamed PGS1), previously proposed to encode a second phosphatidylserine synthase of yeast (Janitor, M., Jarosch, E., Schweyen, R . J., and Subik, J . (1995) Yeast 13, 1223-1231), in fact encodes a PG-P synthase of Saccharomyces cerevisiae . Overexpression of the PGS1 gene product under the inducible GAL1 promoter resulted in a 14-fold increase in in vitro PG-P synthase activity . Disruption of the PGS1 gene in a haploid strain of yeast did not lead to a loss of viability but did result in a dependence on a fermentable carbon source for growth, a temperature sensitivity for growth, and a petite lethal phenotype . The pgs1 null mutant exhibited no detectable in vitro PG-P synthase activity and no detectable CL or phosphatidylglycerol (PG); significant CL synthase activity was still present . The growth arrest phenotype and lack of PG-P synthase activity of a pgsA null allele of Escherichia coli was corrected by an N-terminal truncated derivative of the yeast PG-P synthase . These results unequivocally demonstrate that the PGS1 gene encodes the major PG-P synthase of yeast and that neither PG nor CL are absolutely essential for cell viability but may be important for normal mitochondrial function. J Dairy Sci, 1998 Apr, 81(4), 1036 - 45 Influence of nonenzymatically browned soybeans on ruminal fermentation and lactational performance of dairy cows; Abel-Caines SF et al.; Four ruminally fistulated Holstein cows were assigned to one of four total mixed rations (TMR) in a 4 x 4 Latin square with 3-wk periods to investigate the effects of added lipid from nonenzymatically browned soybeans or soybean oil on ruminal metabolism and milk fatty acids . All rations contained 50% forage and 1) 4% added lipid from soybean oil, 2) 4 or 6% added lipid from nonenzymatically browned soybeans, or 3) no added lipid (control) . The TMR that contained nonenzymatically browned soybeans increased milk C18:2 by 35% compared with the TMR that contained soybean oil . A lactation trial was conducted in which 60 Holstein cows were assigned to one of five TMR from wk 3 to 18 of lactation . The TMR contained 1) 4.5% added lipid from soybean oil, 2) 1.5% added lipid from nonenzymatically browned soybeans and 3% from soybean oil, 3) 3% added lipid from nonenzymatically browned soybeans and 1.5% from soybean oil, 4) 4.5% added lipid from nonenzymatically browned soybeans, and 5) 4.5% added lipid from Ca salts of long-chain fatty acids . Dry matter intake was reduced by 11% for cows fed the TMR containing soybean oil only compared with that for cows fed the other TMR . Milk C18:2 and C18:3 were increased as the inclusion of nonenzymatically browned soybeans in the TMR increased . All percentages of the nonenzymatically browned soybeans fed to cows resulted in fat-corrected milk yields that were similar to those of cows fed the TMR that contained Ca salts of fatty acids. J Dairy Sci, 1998 Apr, 81(4), 1028 - 35 Influence of maturity of grass silage and flaked corn starch on the production and metabolism of volatile fatty acids in dairy cows; De Visser H et al.; An experiment employing a Latin square design was used to quantify the effects of two stages of maturity of grass silage (early cut and late cut) and three concentrations of flaked corn starch (0, 2, and 4 kg) on the molar proportion of rumen volatile fatty acids (VFA), the production of rumen VFA, and the net fluxes of VFA in the splanchnic tissue of cows . The molar proportions of VFA in rumen fluid were similar for cows fed both silages . When the silage diets were supplemented with starch, the proportion of propionic acid increased for cows fed diets containing early cut grass silage, but no effects were found for cows fed diets containing late cut grass silage . Estimated gastrointestinal production of acetate, propionate, butyrate, and branched-chain fatty acids plus valerate was related to intake of metabolizable energy and organic matter fermented into VFA . The portal release of acetate was approximately 14% lower than the estimated production of acetate by cows fed diets containing early cut grass silage, but cows fed diets containing late cut grass silage showed a variable difference between estimated production and portal release (31, 24, and 15%, respectively) as starch supplementation increased . The portal release of butyrate plus beta-hydroxybutyrate and the release of branched-chain fatty acids plus valerate were approximately 70 and 25%, respectively, of the estimated gastrointestinal production . Propionate production was similar to the portal release of propionate . Net flux measurements in splanchnic tissue in combination with gastrointestinal digestion and kinetics provide information that increases the knowledge of pathways and metabolism and quantifies the availability of individual nutrients for milk production in dairy cows. J Antibiot (Tokyo), 1997 Nov, 50(11), 893 - 9 Cymbimicin A and B, two novel cyclophilin-binding structures isolated from actinomycetes; Fehr T et al.; Two novel metabolites, cymbimicins A and B, were isolated from the culture broth of a strain of Micromonospora sp . by screening for cyclophilin binding metabolites from actinomycete strains . Cymbimicin A binds to cyclophilin A with a high affinity six fold lower than to that of cyclosporin A . The binding affinity of cymbimicin B is about 100 times lower . The taxonomy of the producing strain, fermentation, isolation, physical and biological properties and structure elucidation are described. J Nutr Sci Vitaminol (Tokyo), 1998 Feb, 44(1), 103 - 12 Effect of indigestible saccharides on B lymphocyte response of intestinal mucosa and cecal fermentation in rats; Kudoh K et al.; The effects of water-soluble and -insoluble indigestible saccharides (IDS) on immune responses of the intestinal tract were studied . Male 4-week-old Sprague Dawley rats were fed for three weeks on diets containing several kinds of IDS at 5% . The results revealed that the proportion of kappa-light chain and IgA-presenting lymphocytes in small intestinal and cecal mucosa differed in increased number depending on the type of IDS . The response of colonic mucosa was not pronounced . The amounts of short-chain fatty acid (SCFA) and lactic acid in the cecal contents of the other test groups except the celfur group tended to be higher than those in the cellulose group, particularly in the lactulose group where many acids showed significant increases . The correlation between the proportion of kappa-light chain and IgA-presenting lymphocytes in the cecal mucosa and lactic acid in the cecal contents was significant, but that between the proportion of both lymphocytes and SCFA was not . Based on the above, we concluded that the oral administration of IDS induces the proliferation of kappa-light chain and IgA-producing B lymphocytes in small intestinal and cecal mucosa, but the degree of response differs depending on the type of IDS . It is thus suggested that IDS are involved in the intestinal immune system of rats. J Antibiot (Tokyo), 1998 Mar, 51(3), 323 - 7 Micacocidin A, B and C, novel antimycoplasma agents from Pseudomonas sp . I . Taxonomy, fermentation, isolation, physico-chemical properties and biological activities; Kobayashi S et al.; A Gram-negative bacterium was found to produce a new zinc-containing antibiotics, micacocidin A and related compounds containing Cu or Fe, micacocidin B and C, respectively . These antibiotics were isolated by column chromatography on silica gel, and then separated by preparative TLC and HPLC . These new antibiotics exhibited an excellent activity against Mycoplasma species . The producing bacterium was characterized and ascribed to the genus Pseudomonas. J Antibiot (Tokyo), 1998 Mar, 51(3), 303 - 16 Cell wall active antifungal compounds produced by the marine fungus Hypoxylon oceanicum LL-15G256 . II . Isolation and structure determination; Schlingmann G et al.; Fermentations of the marine fungus Hypoxylon oceanicum (LL-15G256) were found to have potent antifungal activity . Isolation and purification of the antifungal agents provided two classes of compounds, macrocyclic polylactones and the lipodepsipeptides 15G256 gamma (1), 15G256 delta (2) and 15G256 epsilon (3) . The isolation and structure elucidation of the lipodepsipeptides, all containing D-glutamate, L-serine, and the rare amino acid beta-ketotryptohan, are described in this paper. J Antibiot (Tokyo), 1998 Mar, 51(3), 296 - 302 Cell wall active antifungal compounds produced by the marine fungus hypoxylon oceanicum LL-15G256 . I . Taxonomy and fermentation; Abbanat D et al.; The cell wall targeted antifungal activity of Hypoxylon oceanicum LL-15G256 extracts resulted from the production of novel lipodepsipeptides and previously reported macrocyclic polylactones . In an optimized medium, titers of the lipodepsipeptide and the polylactones reached approximately 200-400 mg/liter and 25-50 mg/liter, respectively . The optimum fermentation temperature for production of 15G256 gamma was 28 degrees C . Seawater appeared to have an inhibitory effect on metabolite accumulation at lower fermentation temperatures. J Antibiot (Tokyo), 1998 Mar, 51(3), 253 - 60 Epostatin, new inhibitor of dipeptidyl peptidase II, produced by Streptomyces sp . MJ995-OF5 . I . Taxonomy of producing strain, fermentation, isolation, physico-chemical properties and biological properties; Akiyama T et al.; A new inhibitor of dipeptidyl peptidase II (DPP-II, EC 3.4.14.2), designated as epostatin, was discovered in the fermentation broth of a strain isolated in our institute . The strain has been identified as Streptomyces sp . MJ995-OF5 on the basis of taxonomic studies . Epostatin was obtained as a yellow powder after seqential purification by chromatography on Diaion HP-20, n-butanol extraction, Sephadex LH-20 column chromatography and centrifugal partition chromatography (CPC) . Epostatin inhibited DPP-II competitively in a dose dependent manner . The compound was slightly inhibitory against other dipeptidyl peptidases. Protein Expr Purif, 1998 Apr, 12(3), 331 - 9 Dog zona pellucida glycoprotein-3 (ZP3): expression in Escherichia coli and immunological characterization; Santhanam R et al.; An internal fragment (978 bp) corresponding to the dog zona pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by polymerase chain reaction from a full-length cDNA clone . The amplified SacI and PstI restricted fragment was cloned in-frame downstream of the T5 promoter under lac operator control for expression in the pQE-30 vector . Recombinant DZP3 (rec-DZP3) was expressed as a polyhistidine fusion protein in Escherichia coli . Optimum expression of rec-DZP3 was observed at 1.0 mM isopropyl-beta-D-thiogalactopyronoside . Immunoblots with a murine monoclonal antibody, MA-451 (raised against porcine ZP3beta-a homologue of DZP3 and cross-reactive with dog zona pellucida), revealed a major band of 42 kDa . Localization studies revealed that the recombinant protein was present only in an insoluble intracellular fraction . Further optimization studies revealed that the level of expression of rec-DZP3 was significantly higher in Luria broth medium containing glycerol rather than glucose and maximum expression was observed when cultures were induced during the mid-log phase of growth . Batch fermentation with glycerol as the carbon source yielded 30 mg/L of rec-DZP3 compared to 4 mg/L from a shake flask culture . Immunization of two male rabbits with Ni-NTA-purified rec-DZP3 and two female dogs with the rec-DZP3 conjugated to diphtheria toxoid generated high antibody titers against rec-DZP3 as determined by enzyme-linked immunosorbent assay . Rabbit immune serum reacted with porcine ZP3beta but failed to react with porcine ZP3alpha in a Western blot . Moreover, antisera when tested by indirect immunofluorescence on dog ovarian sections showed positive fluorescence with zona pellucida . The availability of rec-DZP3 will help in evaluating its efficacy for fertility regulation in stray dogs . J Nutr, 1998 Apr, 128(4), 688 - 93 Fermentation products of sugar-beet fiber by cecal bacteria lower plasma cholesterol concentration in rats; Hara H et al.; Plasma cholesterol concentration is reduced by feeding some dietary fibers but the mechanism is not fully understood . We examined whether cecal fermentation products are involved in lowering plasma cholesterol by feeding rats a highly fermentable sugar-beet fiber (SBF) in four separate experiments . These were designed to investigate the effects on plasma cholesterol of oral ingestion of fermentation products on plasma cholesterol, the effects of the products in comparison with that of a short-chain fatty acid (SCFA) mixture, effects of individual SCFA and effects of alteration of energy and nitrogen ratio in the diet by the addition of the SCFA mixture . Cecal contents of rats were cultured with SBF by using a jar fermenter under anaerobic conditions, and the supernatant from the culture medium, containing fermentation products of SBF, was collected and freeze-dried before feeding to rats . Yield of fermentation products as dry weight from the fiber was 80-90% . In rats fed a diet containing fermentation products (80 g/kg diet), plasma cholesterol concentrations were lower than in rats of the fiber-free group 3, 7 and 14 d after feeding the test diet . Major SCFA in the fermentation products were sodium salts of acetic, propionic and butyric acids . Plasma cholesterol concentration in rats fed the diet containing a mixture of equal amounts of the three SCFA salts (66 g/kg diet) as the fermentation products diet was also lower than that in the fiber-free group and was not different from those in rats fed SBF (100 g/kg diet) and the fermentation products . In rats fed an acetate-containing diet but not in rats fed diets without acetate, plasma cholesterol was significantly lower than in the fiber-free group . In conclusion, absorption of SCFA from cecal fermentation products lowers plasma cholesterol . Acetate, and not propionate, may be responsible for lowering plasma cholesterol concentration. Yeast, 1998 Feb, 14(3), 255 - 69 During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1; Ernandes JR et al.; In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (= GGS1 = FDP1 = BYP1 = CIF1 = GLC6 = TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo . We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells . However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation . The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels . In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1 delta mutant and the wild-type strain . Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase . We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose . Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total D-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular) . The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, +/- 10 mM in a hxk1 delta hxk2 delta glk1 delta and 2-3 mM in a tps1 delta strain . In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced . Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption . Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1 . However, it mimics it transiently during the initiation of fermentation . Afterwards, the Tps1-dependent control system is apparently able to restrict properly up to 50-fold higher hexokinase activity. Biotechnol Prog, 1998 Mar, 14(2), 318 - 25 Correlation of fermentation yield with yeast extract composition as characterized by near-infrared spectroscopy Kasprow RP, Lange AJ, Kirwan DJ. Complex, ill-defined mixtures of natural origin are often used as nutrients in the production of biological products through microbial fermentation . Product yields are affected by variation in these natural products . It was desired to examine near-infrared spectroscopy as a rapid screening tool for qualifying raw material lots . Specifically, the characterization of yeast extract was investigated . The model system consisted of a Merck & Co., Inc., microbial fermentation process . Cell mass and specific product yields are dependent upon variations in the yeast extract used in the medium . Partial least-squares regression on the second-derivative spectral absorbances of various yeast extracts in the ranges 1150-1380, 1554-1826, and 2100-2300 nm resulted in the development of models with multiple correlation coefficients of 0.99 for cell mass yields and 0.96 for specific product yields in large-scale fermentations . These models could also be used to predict cell mass yields in 15 L batch fermentations and specific product yields in 2-L shake flasks. Biotechnol Prog, 1998 Mar, 14(2), 248 - 58 Computational fluid dynamics model for predicting flow of viscous fluids in a large fermentor with hydrofoil flow impellers and internal cooling coils Kelly WJ, Humphrey AE. Considerable debate has occurred over the use of hydrofoil impellers in large-scale fermentors to improve mixing and mass transfer in highly viscous non-Newtonian systems . Using a computational fluid dynamics software package (Fluent, version 4.30) extensive calculations were performed to study the effect of impeller speed (70-130 rpm), broth rheology (value of power law flow behavior index from 0.2 to 0.6), and distance between the cooling coil bank and the fermentor wall (6-18 in.) on flow near the perimeter of a large (75-m3) fermentor equipped with A315 impellers . A quadratic model utilizing the data was developed in an attempt to correlate the effect of A315 impeller speed, power law flow behavior index, and distance between the cooling coil bank and the fermentor wall on the average axial velocity in the coil bank-wall region . The results suggest that there is a potential for slow or stagnant flow in the coil bank-wall region which could result in poor oxygen and heat transfer for highly viscous fermentations . The results also indicate that there is the potential for slow or stagnant flow in the region between the top impeller and the gas headspace when flow through the coil bank-wall region is slow . Finally, a simple guideline was developed to allow fermentor design engineers to predict the degree of flow behind a bank of helical cooling coils in a large fermentor with hydrofoil flow impellers. Biotechnol Prog, 1998 Mar, 14(2), 233 - 40 Influence of morphology on product formation in aspergillus awamori during submerged fermentations Johansen CL, Coolen L, Hunik JH. The relationship between fungal morphology and heterologous protein production was examined for an Aspergillus awamori strain during a series of fermentations with a batch phase followed by a fed-batch phase . Agitation rate and inoculation concentration were used as controlled variables to generate different fungal morphologies in 20-dm3 stirred tank reactors . Morphology was quantitatively characterized using Image Analysis . The different agitation rates and inoculum concentrations had large effects on the development in hyphal length and number of tips during the fermentations . A reduced inoculum concentration resulted in a more branched mycelium . The different agitation rates affected the morphology after 30 h of fermentation significantly but did not affect the start time of fragmentation . A 3-fold increase in hyphal length increased the apparent viscosity by a factor of 7 . The observed morphological differences had only a limited effect on product formation, suggesting that the structural features such as hyphal length and number of tips are of less importance for product formation . The primary effect of morphology on product formation is due to viscosity. J Clin Microbiol, 1998 May, 36(5), 1371 - 7 Genotypic and phenotypic analysis of Mycoplasma fermentans strains isolated from different host tissues; Campo L et al.; A correlation was found between the expression of a specific Mycoplasma fermentans surface antigen (Pra, proteinase-resistant antigen) and the site of isolation of the organism from the infected host . Strains which expressed Pra were most frequently associated with cells of bone marrow origin, and strains which lacked expression of Pra were most commonly isolated from the respiratory tract, genital tract, and arthritic joints, i.e., epithelial cell surfaces . Pra was previously shown to be resistant to degradation by proteinases and was hypothesized to play a protective role at the organism surface and perhaps to influence which host tissue site was colonized by the organism . The methods used for this phenotyping scheme required isolation and growth of the mycoplasma in quantities sufficient for immunoblot analysis using monoclonal antibodies . We wanted to determine a more rapid and less cumbersome technique to supplement this method for determining the Pra phenotype directly in clinical specimens . Here we describe PCR studies to investigate the movement of a previously identified M . fermentans insertion sequence (IS)-like element . These data showed a correlation between a specific IS genotype and the Pra+ phenotype . Production of a 160-bp product using a single set of IS-based primers was associated with expression of Pra . The genomic IS location resulting in the 160-bp product was determined by using Southern blot analysis and was found to be a stable insertion site characteristic of genotype I strains . Additional analyses of sequences within and flanking the IS insertion sites revealed another pair of PCR primer sites which resulted in the consistent production of a 450-bp amplicon . The stability of this site was dependent on the absence of the IS-like element between the primer sites . The production of this 450-bp amplicon correlated with the Pra mutant phenotype and was characteristic of genotype II strains . The data showed that the sequence within the IS may be unstable and that reliable genotyping sequences are more easily found in the stable genomic sites which flank the IS element. J Clin Microbiol, 1998 May, 36(5), 1226 - 31 Genotypic characterization of seven strains of Mycoplasma fermentans isolated from synovial fluids of patients with arthritis; Schaeverbeke T et al.; We performed a genotypic characterization of seven strains of Mycoplasma fermentans which have been isolated from the synovial fluid of patients with rheumatoid arthritis (n = 2), spondyloarthropathy (n = 1), and unclassified arthritis (n = 4) . We compared them to three reference strains (strains PG18 and K7 and incognitus strain) and to a clinical isolate from the urethra of a patient with nongonococcal urethritis . The characterization methods included electrophoresis of native DNA, arbitrarily primed PCR, and restriction fragment length polymorphism analysis following conventional and pulsed-field gel electrophoresis . Southern blot analysis with a probe internal to an insertion sequence was performed with the restriction products produced by the last two techniques . No extrachromosomal DNA sequences were detected . The M . fermentans strains identified by these methods did not present a unique profile, but they could be separated into two main categories: four articular isolates were genetically related to PG18 and the three other isolates, the urethral isolate, and the incognitus strain were related to K7 . We also looked for the presence of the bacteriophage MAV1 (associated with the arthritogenic property of Mycoplasma arthritidis in rodents) in the M . fermentans strains . MAV1 DNA was not detected in either the clinical isolates or the reference strains of M . fermentans. J Biol Chem, 1998 Apr 3, 273(14), 8308 - 16 Evolution of an Escherichia coli protein with increased resistance to oxidative stress; Lu Z et al.; L-1,2-Propanediol:NAD+ 1-oxidoreductase of Escherichia coli is encoded by the fucO gene, a member of the regulon specifying dissimilation of L-fucose . The enzyme normally functions during fermentative growth to regenerate NAD from NADH by reducing the metabolic intermediate L-lactaldehyde to propanediol which is excreted . During aerobic growth L-lactaldehyde is converted to L-lactate and thence to the central metabolite pyruvate . The wasteful excretion of propanediol is minimized by oxidative inactivation of the oxidoreductase, an Fe2+-dependent enzyme which is subject to metal-catalyzed oxidation (MCO) . Mutants acquiring the ability to grow aerobically on propanediol as sole carbon and energy source can be readily selected . These mutants express the fucO gene constitutively, as a result of an IS5 insertion in the promoter region . In this study we show that continued selection for aerobic growth on propanediol resulted in mutations in the oxidoreductase conferring increased resistance to MCO . In two independent mutants, the resistance of the protein was respectively conferred by an Ile7 --> Leu and a Leu8 --> Val substitution near the NAD-binding consensus amino acid sequence . A site-directed mutant protein with both substitutions showed an MCO resistance greater than either mutant protein with a single amino acid change. J Immunol, 1998 Feb 1, 160(3), 1330 - 9 Activation of mitogen-activated protein kinase pathways by Mycoplasma fermentans membrane lipoproteins in murine macrophages: involvement in cytokine synthesis; Rawadi G et al.; Stimulation of monocytes and resident macrophages by mycoplasmas induces production of numerous cytokines . We have previously reported that membrane lipoproteins derived from Mycoplasma fermentans are responsible for the induction of proinflammatory cytokines by monocytic cells and that triggering protein tyrosine kinase activation is an essential requirement for this biologic effect . In the present study, we have investigated the effect of M . fermentans-derived membrane lipoproteins (LAMPf) on mitogen-activated protein kinase (MAPK) cascades in the murine macrophage cell line RAW 264.7 and have analyzed the contribution of these pathways to the cytokine induction mediated by this agent . Treatment of murine macrophages with LAMPf resulted in significant activation of MAPK family members extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), and p38 . Unlike LPS, these effects were demonstrated to be independent of the presence of serum . The activation of MAPKs paralleled the tyrosine kinase activation and peaked at 30 min after stimulation . The specific p38 inhibitor SB203580 abrogated the mycoplasma-induced IL-6, IL-1beta, and TNF-alpha synthesis . The selective MAPK/extracellular signal-regulated kinase 1 (MEK-1) inhibitor PD-98059 blocked both IL-1beta and TNF-alpha but not IL-6 production by RAW 264.7 cells in response to LAMPf . Additionally, transfection of murine macrophages with a JNK dominant negative mutant significantly reduced only IL-6 production . These data underscore the role of MAPKs as signal transduction molecules controlling the expression of cytokines upon mycoplasma stimulation. Lett Appl Microbiol, 1998 Mar, 26(3), 199 - 204 Dynabeads plus 3 M Petrifilm HEC versus Vitek Immunodiagnostic Assay System for detection of E . coli O157 in minced meat; Grif K et al.; The potentially low infective dose of Escherichia coli O157 makes it necessary to be able to detect low numbers in food, and the lack of sensitivity of direct plating has led to the development of various enrichment and detection methods . Until now, the most selective procedure for detection of E . coli O157 isolates was the immunomagnetic separation (IMS) method . The number of sorbitol non-fermenting micro-organisms other than E . coli O157 that adhere non-specifically to the magnetic beads hampers the application of IMS . The use of IMS in conjunction with 3 M Petrifilm-HEC yielded EHEC O157 in 21 of 165 samples of minced meat (12.7%) . Without advance application of IMS, Petrifilm plates often yield confluent growth and colonies too numerous to count . The Vitek Immunodiagnostic Assay System (VIDAS-ECO) showed good sensitivity when testing artificially contaminated beef samples, but only four of 21 naturally contaminated samples were recognized . The addition of 3 M Petrifilm to IMS resulted in less growth of contaminants and eliminated much of the need to test presumed colonies for confirmation . The combination of IMS and 3 M Petrifilm-HEC is a fast and efficient screening procedure for E . coli O157 in minced meat. J Paediatr Child Health, 1998 Feb, 34(1), 79 - 82 Bowel dysfunction in cystic fibrosis: importance of breath testing; Lewindon PJ et al.; OBJECTIVE: To investigate the prevalence of carbohydrate malabsorption and bacterial overgrowth in children with cystic fibrosis (CF) and abnormal stool pattern referred for breath hydrogen testing . METHODS: Results from 89 tests using lactose, sucrose and lactulose in 54 children with CF were compared with 5430 tests on children with non-CF-related stool abnormalities . RESULTS: Children with CF were more frequently unable to ferment lactulose to hydrogen (39% vs . 20%, P<0.03); they had significantly longer oro-caecal transit times (mean 99 vs . 68 min, P<0.0003); they had a higher incidence of bacterial overgrowth (32% vs . 7%, P<0.003) and sucrose malabsorption (47% vs . 14.5%, P<0.004); but they had no increase in lactose malabsorption (40% vs . 31%) . Children with bacterial overgrowth in both groups had longer transit times (CF 123 min, non-CF 108 min) compared to the non-CF children without overgrowth (68 min) and reference normal children (69 min) . CONCLUSIONS: Bacterial overgrowth and carbohydrate malabsorption, particularly of sucrose, should be considered when assessing children with CF and abnormal stool patterns. Protein Sci, 1998 Apr, 7(4), 994 - 1005 Mutagenesis of histidine 26 demonstrates the importance of loop-loop and loop-protein interactions for the function of iso-1-cytochrome c; Fetrow JS et al.; In yeast iso-1-cytochrome c, the side chain of histidine 26 (His26) attaches omega loop A to the main body of the protein by forming a hydrogen bond to the backbone atom carbonyl of glutamic acid 44 . The His26 side chain also forms a stabilizing intra-loop interaction through a hydrogen bond to the backbone amide of asparagine 31 . To investigate the importance of loop-protein attachment and intra-loop interactions to the structure and function of this protein, a series of site-directed and random-directed mutations were produced at His26 . Yeast strains expressing these variant proteins were analyzed for their ability to grow on non-fermentable carbon sources and for their intracellular production of cytochrome c . While the data show that mutations at His26 lead to slightly decreased intracellular amounts of cytochrome c, the level of cytochrome c function is decreased more . The data suggest that cytochrome c reductase binding is affected more than cytochrome c oxidase or lactate dehydrogenase binding . We propose that mutations at this residue increase loop mobility, which, in turn, decreases the protein's ability to bind redox partners. Mol Cell Biol, 1998 May, 18(5), 2940 - 8 Carbon source-dependent phosphorylation of hexokinase PII and its role in the glucose-signaling response in yeast; Randez-Gil F et al.; The HXK2 gene is required for a variety of regulatory effects leading to an adaptation for fermentative metabolism in Saccharomyces cerevisiae . However, the molecular basis of the specific role of Hxk2p in these effects is still unclear . One important feature in order to understand the physiological function of hexokinase PH is that it is a phosphoprotein, since protein phosphorylation is essential in most metabolic signal transductions in eukaryotic cells . Here we show that Hxk2p exists in vivo in a dimeric-monomeric equilibrium which is affected by phosphorylation . Only the monomeric form appears phosphorylated, whereas the dimer does not . The reversible phosphorylation of Hxk2p is carbon source dependent, being more extensive on poor carbon sources such as galactose, raffinose, and ethanol . In vivo dephosphorylation of Hxk2p is promoted after addition of glucose . This effect is absent in glucose repression mutants cat80/grr1, hex2/reg1, and cid1/glc7 . Treatment of a glucose crude extract from cid1-226 (glc7-T152K) mutant cells with lambda-phosphatase drastically reduces the presence of phosphoprotein, suggesting that CID1/GLC7 phosphatase together with its regulatory HEX2/REG1 subunit are involved in the dephosphorylation of the Hxk2p monomer . An HXK2 mutation encoding a serine-to-alanine change at position 15 {HXK2 (S15A)} was to clarify the in vivo function of the phosphorylation of hexokinase PII . In this mutant, where the Hxk2 protein is unable to undergo phosphorylation, the cells could not provide glucose repression of invertase . Glucose induction of HXT gene expression is also affected in cells expressing the mutated enzyme . Although we cannot rule out a defect in the metabolic state of the cell as the origin of these phenomena, our results suggest that the phosphorylation of hexokinase is essential in vivo for glucose signal transduction. Neurochem Res, 1998 Apr, 23(4), 455 - 61 Manda, a fermented natural food, suppresses lipid peroxidation in the senescent rat brain; Kawai M et al.; The level of lipid peroxidation reflects the degree of free radical-induced oxidative damage in brain tissue of the elderly . We examined the effects of Manda, a product prepared by yeast fermentation of several fruits and black sugar, on lipid peroxidation in the senescent rat brain as model of aging . Senescent rats were provided with a diet containing 50 g/100 g Manda for 8 days, supplemented on day 8 with an intragastric administration of Manda (6.0 g/kg body wt.) twice daily . The hydroxyl radical scavenging activity was generated by the FeSO4-H2O2 system and analyzed by electron spin resonance spectrometry . Using this method, the addition of Manda (2.88 mg/ml) to brain homogenates of adult rats (0.06 mg/ml) had an additive inhibitory effect on lipid peroxidation compared with control adult rats not treated with Manda . Incubation of brain homogenates with Manda for 2 h and 3 h, significantly inhibited the increase in lipid peroxides (malondialdehydes and 4-hydroxyalkenals) levels in aged rats due to auto-oxidation . In addition, oral administration of Manda significantly suppressed the age-related increase in lipid peroxidation in the hippocampus and striatum, although such change was not observed in the cerebral cortex . Although Manda contains trace level of alpha-tocopherol, the level of alpha-tocopherol in Manda did no correlate with its antioxidant effect . Our results suggest that Manda protects against age-dependent oxidative neuronal damage caused by oxidative stress and that this protective effect may be due, in part, to its scavenging activity against free radicals. J Dairy Sci, 1998 Mar, 81(3), 765 - 76 Protein preservation and ruminal degradation of ensiled forage treated with heat, formic acid, ammonia, or microbial inoculant; Polan CE et al.; The objectives of this study were to determine whether treatment of forage with heat would reduce proteolysis during subsequent fermentation . In Experiment 1, direct-cut barley forage and alfalfa were untreated, microwaved, or steamed and then ensiled in laboratory silos as wilted forages . Silages of microwaved or steamed forage showed a marked increase in N bound to neutral detergent fiber and in the recovery of protein; however, alfalfa silages also had high pH values and concentrations of butyric acid . In Experiment 2, steam heating was compared with formic acid and NH3 treatments for the prevention of proteolysis in alfalfa silages . Silage of steamed alfalfa had a greater amount of N bound to neutral detergent fiber and greater recovery of protein than did control silage or silages of forage treated with formic acid or NH3 . Silage of steamed forage had lower pH values than did silages of wilted, direct-cut, or control forage . Microbial innoculant added to steamed forage increased the recovery of protein . Silage of steamed forage had less aerobic stability than did silage of direct-cut forage . Ruminal degradability of crude protein (CP) and organic matter of silage from both experiments was evaluated . Degradability of CP was 8 to 26 percentage units lower in silages of microwaved or steamed forage in Experiment 1 than in silage of unheated forage because of slower degradation rates, but all had similar undegraded CP after incubation for 72 h . In Experiment 2, wilting, steam, formic acid, and NH3 treatments affected CP degradability similarly, but CP degradability was decreased when compared with silage of direct-cut forage without treatment. J Chromatogr A, 1998 Mar 6, 798(1-2), 109 - 16 Improved ion chromatography-integrated pulsed amperometric detection method for the evaluation of biogenic amines in food of vegetable or animal origin and in fermented foods; Draisci R et al.; An improved method for the simultaneous determination of underivatized biogenic amines, cadaverine, putrescine, spermidine, histamine, tyramine and some amino acids precursors, histidine and tyrosine, in food products, based on ion-exchange chromatography (IC) with integrated pulsed amperometric detection (IPAD) has been developed . The method was successfully used for the analysis of biogenic amines and amino acids in food both of vegetable (kiwi, Actinidia chinensis) and animal origin, (fish, pilchard), as well as in fermented foods, such as cheese (Emmenthal) and dry sausages (salami) . The method was also successfully used to study the changes in biogenic amines during the ripening of dry fermented sausages (salami) . The analytes were extracted from foods with perchloric acid and the extracts were purified by liquid-liquid partition using n-hexane . Determination of biogenic amines was performed through cation-exchange chromatography with isocratic elution and IPAD . The detection limits for the analytes under investigation were found to range from 1.25 to 2.50 ng, at a signal-to-noise ratio of 3:1 . Average recoveries ranged from 85.5 to 97.4% and R.S.D . values ranged from 3.4 to 8.8 . The proposed method offers a number of advantages over our previous IPAD method, such as the application to a larger number of analytes and matrices, a simpler extraction procedure and clean-up, isocratic elution using low acid and base concentrations, an improved chromatographic separation and a lower detection limit. J Chromatogr A, 1998 Mar 6, 798(1-2), 83 - 90 Rapid reversed-phase high-performance liquid chromatography method for quantitation, at high pH, of the recombinant apolipoprotein A-IMilano in Escherichia coli fermentation broth; Mulugeta E et al.; An automated reversed-phase high-performance liquid chromatography method for quantitative determination of recombinant apolipoprotein A-IMilano (r-Apo A-IM) in E . coli fermentation broth has been developed and evaluated . The use of a unique matrix (Poros IIR/H) makes it possible to achieve rapid separation and good resolution at high pH . The r-Apo A-IM-containing fraction is well separated from other proteins allowing a reliable quantification . The automation and high sample throughput of this method makes it very useful for routine determination of r-Apo A-IM in fermentation broth and in eluates from the various purfication steps . With suitable modifications and adaptions this method is likely to be useful for similar rapid analytical determination of recombinant proteins in complex solutions. Int J Syst Bacteriol, 1998 Jan, 48 Pt 1, 305 - 9 Phylogenetic position of rare human mycoplasmas, Mycoplasma faucium, M . buccale, M . primatum and M . spermatophilum, based on 16S rRNA gene sequences; Rawadi G et al.; The nucleotide sequence of the 16S rRNA genes of four rare human mycoplasma species, Mycoplasma faucium, M . buccale, M . primatum and M . spermatophilum, were partially sequenced and compared to published rRNA genes of mycoplasmas to determine their position in the Mollicutes phylogenetic tree . Nucleotide sequence motif and overall similarities allowed positioning of these mycoplasmas in the hominis phylogenetic group, as defined by Weisburg et al . {Weisburg, W . G., Tully, J . G., Rose, D . L . & 9 other authors (1989) . J Bacteriol 171, 6455-6467} . Furthermore, these mycoplasmas could be clustered into two different subdivisions of the hominis group: (i) M . faucium and M . buccale were found to be included in the M . fermentans subdivision, and (ii) M . primatum and M . spermatophilum were included in the M . hominis one . Variable regions of the 16S rRNA genes were used to determine specific PCR primers to detect and identify M . faucium. Physiol Rev, 1998 Apr, 78(2), 547 - 81 The free radical theory of aging matures; Beckman KB et al.; The free radical theory of aging, conceived in 1956, has turned 40 and is rapidly attracting the interest of the mainstream of biological research . From its origins in radiation biology, through a decade or so of dormancy and two decades of steady phenomenological research, it has attracted an increasing number of scientists from an expanding circle of fields . During the past decade, several lines of evidence have convinced a number of scientists that oxidants play an important role in aging . (For the sake of simplicity, we use the term oxidant to refer to all "reactive oxygen species," including O2-., H2O2, and .OH, even though the former often acts as a reductant and produces oxidants indirectly.) The pace and scope of research in the last few years have been particularly impressive and diverse . The only disadvantage of the current intellectual ferment is the difficulty in digesting the literature . Therefore, we have systematically reviewed the status of the free radical theory, by categorizing the literature in terms of the various types of experiments that have been performed . These include phenomenological measurements of age-associated oxidative stress, interspecies comparisons, dietary restriction, the manipulation of metabolic activity and oxygen tension, treatment with dietary and pharmacological antioxidants, in vitro senescence, classical and population genetics, molecular genetics, transgenic organisms, the study of human diseases of aging, epidemiological studies, and the ongoing elucidation of the role of active oxygen in biology. Physiol Rev, 1998 Apr, 78(2), 393 - 427 Contributions of microbes in vertebrate gastrointestinal tract to production and conservation of nutrients; Stevens CE et al.; The vertebrate gastrointestinal tract is populated by bacteria and, in some species, protozoa and fungi that can convert dietary and endogenous substrates into absorbable nutrients . Because of a neutral pH and longer digesta retention time, the largest bacterial populations are found in the hindgut or large intestine of mammals, birds, reptiles, and adult amphibians and in the foregut of a few mammals and at least one species of bird . Bacteria ferment carbohydrates into short-chain fatty acids (SCFA), convert dietary and endogenous nitrogenous compounds into ammonia and microbial protein, and synthesize B vitamins . Absorption of SCFA provides energy for the gut epithelial cells and plays an important role in the absorption of Na and water . Ammonia absorption aids in the conservation of nitrogen and water . A larger gut capacity and longer digesta retention time provide herbivores with additional SCFA for maintenance energy and foregut-fermenting and copoprophagic hindgut-fermenting species with access to microbially synthesized protein and B vitamins . Protozoa and fungi also contribute nutrients to the host . This review discusses the contributions of gut microorganisms common to all vertebrates, the numerous digestive strategies that allow herbivores to maximize these contributions, and the effects of low-fiber diets and discontinuous feeding schedules on these microbial digestive processes. Biochem J, 1998 May 1, 331 ( Pt 3), 877 - 83 Metabolic control analysis of the bc1 complex of Saccharomyces cerevisiae: effect on cytochrome c oxidase, respiration and growth rate; Boumans H et al.; A number of strains varying in steady-state level of assembled bc1 complex were used to test the conclusions from inhibitor titration experiments with isolated mitochondria that, in cells of Saccharomyces cerevisiae grown on non-fermentable carbon sources, the control coefficient of the bc1 complex on the mitochondrial respiratory capacity equals 1 and the respiratory chain consists of supermolecular respiratory units {Boumans, Grivell and Berden (1998) J . Biol . Chem . 273, 4872-4877} . In addition, the control coefficient of mitochondrial respiration on the growth rate was determined . It was found that a reduced level of bc1 complex is accompanied by an almost parallel decrease in steady-state level of cytochrome c oxidase . Since the linear relationship between level of active bc1 complex and respiratory capacity still holds, it is concluded that cytochrome c oxidase has disappeared from respiratory units that are already deficient in the bc1 complex and that the cytochrome c oxidase in a respiratory unit is destabilized when the bc1 complex is deficient . The control coefficient of the bc1 complex, and thus of mitochondrial electron-transfer capacity, on respiration of intact cells (without uncoupler added) is 0.20 . Addition of uncoupler results in an increase in the coefficient to 0.36 . Thus changing the respiratory state changes the distribution of control, increasing the control coefficient of electron-transfer activity as the respiratory state goes towards State 3u . Rates of growth of the strains on different carbon sources were determined and subsequently fitted to calculate control coefficients of the bc1 complex (and therefore of the respiratory capacity) on growth . Little variation was found between lactate-, ethanol- and glycerol-containing media, control coefficients being around 0.18 at pH 5 . At pH 7 the control coefficient increased to 0.57, indicative of a higher dependence of the cell on ATP derived from oxidative phosphorylation . During growth on glucose-containing medium, the bc1 complex has no control on the growth rate, as indicated by the fact that all strains, including a respiratory-deficient strain, grow as fast as the wild-type . However, the presence of respiratory capacity in the wild-type does result in a higher growth yield compared with the respiratory-deficient strain, indicating that, in contrast with what is generally assumed, in S . cerevisiae the 'Pasteur effect' is not restricted to special experimental conditions. Medicine (Baltimore), 1998 Mar, 77(2), 73 - 82 D-lactic acidosis . A review of clinical presentation, biochemical features, and pathophysiologic mechanisms; Uribarri J et al.; This report describes a case of d-lactic acidosis observed by the authors and then reviews all case reports of d-lactic acidosis in the literature in order to define its clinical and biochemical features and pathogenetic mechanisms . The report also reviews the literature on metabolism of d-lactic acid in humans . The clinical presentation of d-lactic acidosis is characterized by episodes of encephalopathy and metabolic acidosis . The diagnosis should be considered in a patient who presents with metabolic acidosis and high serum anion gap, normal lactate level, negative Acetest, short bowel syndrome or other forms of malabsorption, and characteristic neurologic findings . Development of the syndrome requires the following conditions 1) carbohydrate malabsorption with increased delivery of nutrients to the colon, 2) colonic bacterial flora of a type that produces d-lactic acid, 3) ingestion of large amounts of carbohydrate, 4) diminished colonic motility, allowing time for nutrients in the colon to undergo bacterial fermentation, and 5) impaired d-lactate metabolism . In contrast to the initial assumption that d-lactic acid is not metabolized by humans, analysis of published data shows a substantial rate of metabolism of d-lactate by normal humans . Estimates based on these data suggest that impaired metabolism of d-lactate is almost a prerequisite for the development of the syndrome. Appl Biochem Biotechnol, 1998 Mar, 69(3), 177 - 89 Saccharification and alcohol fermentation in starch solution of steam-exploded potato; Kobayashi F et al.; Steam explosion pretreatment of potato for the efficient production of alcohol was experimentally studied . The amount of water-soluble starch increased with the increase of steam pressure, but the amounts of methanol-soluble material and Klason lignin remained insignificant, regardless of steam pressure . The potatoes exploded at high pressure were hydrolyzed into a low molecular liquid starch, and then easily converted into ethanol by simultaneous saccharification and fermentation using mixed microorganisms: an amylolytic microorganism, Aspergillus awamori, and a fermentation microorganism, Saccharomyces cerevisiae . The maximal ethanol concentration was 4.2 g/L in a batch culture at 15 g/L starch concentration, and 3.6 g/L in a continuous culture fed the same starch concentration . In the fed-batch culture, the maximal ethanol concentration increased more than twofold, compared to the batch culture. J Pediatr Gastroenterol Nutr, 1998 Apr, 26(4), 432 - 6 Butyrate and glucose metabolism in isolated colonocytes in the developing rat colon; Krishnan S et al.; BACKGROUND: The newborn colon is devoid of microflora, in that bacterial colonization is established after birth . Short chain fatty acids, products of bacterial fermentation, are the major energy source for colonocytes . Because it is not known whether colonocytes in the newborn can metabolize butyrate, this was examined in newborn and infant rat colon . METHODS: Isolated colonocytes from rats of different perinatal ages were incubated with 14C-labeled butyrate or glucose in vitro . Complete oxidation was estimated by the production of 14C-labeled carbon dioxide, whereas intermediate metabolites were measured enzymatically . RESULTS: Oxidation of butyrate (in micromoles per hour per milligram of protein) was highest in newborns (5.83+/-1.76), declining to 1.32+/-0.28 at day 10 and to 0.34+/-0.04 in adult rats . Glucose oxidation was also highest at birth (0.39+/-0.23), with a minor increase at approximately day 20 (weaning period) before decreasing to adult levels (0.05+/-0) . Butyrate oxidation was substantially higher than was glucose oxidation in all age groups . Production of metabolic intermediates paralleled substrate oxidation . Acetoacetate production was 4.35+/-2.68, 2.07+/-1.29, and 0.27+/-0.09 nmol/hr per milligram of protein in newborns, at postnatal day 10, and in adults, respectively . The corresponding values for beta-hydroxybutyrate were 3.62+/-3.35, 0.2+/-0.07, and 0.09+/-0.03 nmol/hr per milligram of protein; and L-lactate production was 0.54+/-0.52, 0.06+/-0.04, and 0.02+/-0 micromol/hr per milligram of protein respectively . CONCLUSIONS: Neonatal rat colon epithelial cells resemble adult colonocytes in their preference for butyrate as a metabolic substrate, indicating a constitutive expression of this property. Scand J Gastroenterol, 1998 Mar, 33(3), 242 - 6 The ability of enteric diarrhoeal pathogens to ferment starch to short-chain fatty acids in vitro; Krishnan S et al.; BACKGROUND: Short-chain fatty acids (SCFA), produced in the normal colon by bacterial fermentation, are decreased in acute diarrhoea . This may have deleterious effects on epithelial function in the colon . METHODS: The ability of several diarrhoeal pathogens to produce SCFA when incubated with starch in vitro was studied . Isolated pathogens were incubated for 24 h with either no added substrate, glucose, or starch under anaerobic conditions, and SCFA were quantitated by gas-liquid chromatography . RESULTS: Unlike the normal colonic flora, the pathogens produced acetate but not propionate or butyrate . D-Lactate was also produced by all the pathogens studied . When the pathogens were incubated in anaerobic medium containing starch, significantly greater amounts of acetate and significantly lesser amounts of lactate were produced . CONCLUSIONS: The inability of enteric pathogens to produce butyrate may impair epithelial cell function, whereas production of D-lactate may enhance mucosal damage in diarrhoeal disease . The presence of luminal starch may be helpful in shifting the fermentation profile to a more favourable pattern. J Antibiot (Tokyo), 1998 Feb, 51(2), 170 - 7 Directed biosynthesis of peptaibol antibiotics in two Trichoderma strains . I . Fermentation and isolation; Leclerc G et al.; Peptaibols are linear alpha-aminoisobutyric acid-containing peptide antibiotics originating from soil fungi mainly of the genus Trichoderma and biosynthesized in complex mixtures of closely related analogues by a polyenzymatic pathway . Addition of amino acids such as alpha-aminoisobutyric acid (Aib), glutamic acid or arginine, to the fermentation medium of two Trichoderma strains, T . harzianum and T . longibrachiatum, has been shown to result in the simplification of the natural peptaibol mixtures, leading in each case to the almost exclusive biosynthesis of a single peptide . Surprisingly, the obtained peptides are Aib-enriched, whether the added amino acid is Aib, Glu or Arg . By adding Aib to the fermentation medium of T . harzianum, two new Aib-rich peptaibols were isolated . Moreover, adding glutamic acid to the culture medium of T . longibrachiatum, which produces both neutral and acidic 20-residue peptaibols with either glutamine or glutamic acid at position 18, increases the production of the acidic peptides . However, arginine which is a positively charged amino acid generally absent from peptaibol sequences, is not incorporated in trichorzins when added to the fermentation medium of T . harzianum. J Antibiot (Tokyo), 1998 Feb, 51(2), 107 - 11 TMC-49A, a novel transcriptional up-regulator of low density lipoprotein receptor, produced by Streptomyces sp . AS1345; Koguchi Y et al.; Microbial metabolites were screened for a transcriptional up-regulator of low density lipoprotein (LDL) receptor by a reporter assay . TMC-49A was discovered as an up-regulator obtained from the fermentation broth of Streptomyces sp . AS1345 . The structure of TMC-49A was elucidated to be butyl N-phenethylcarbamate by spectroscopic analyses . This compound enhanced the synthesis of LDL receptor in human hepatoma HepG2 cells as assessed by a receptor binding assay . Taxonomy of the producing strain is also described. FEMS Immunol Med Microbiol, 1997 Dec, 19(4), 275 - 83 Mycoplasma salivarium induces interleukin-6 and interleukin-8 in human gingival fibroblasts; Shibata K et al.; Analysis by an enzyme-linked immunosorbent assay for cytokines indicated that whole cells, intracellular materials and cell membranes of Mycoplasma salivarium induced interleukin-6 and interleukin-8 in a human gingival fibroblast cell line, Gin-1 cells . This was confirmed by reverse transcription-polymerase chain reaction analysis of mRNAs of these cytokines . Studies with inhibitors of second-messenger pathway indicated that a protein kinase C-dependent pathway was involved in the expression of the activity of the cell membranes . In addition, whole cells of other mycoplasmas (M . hominis, M . arthritidis, M . arginini, M . fermentans, M . penetrans, M . pirum and M . pneumoniae) tested for comparative purposes were also shown to possess the activity . Thus, this study demonstrated that mycoplasmas possess the activity to induce interleukin-6 and interleukin-8 in human fibroblasts. Biochem Biophys Res Commun, 1998 Mar 27, 244(3), 903 - 7 Protective role of black tea against oxidative damage of human red blood cells; Halder J et al.; The purpose of our study was to explore the possible scavenging property of black tea and catechins, the major flavonols of tea-leaf, against damage by oxidative stress . For this purpose, human red blood cell (rbc) was taken as the model and the oxidative damage was induced by a variety of inducers, e.g . phenylhydrazine (PHX), Cu(2+)-ascorbic acid, and xanthine/xanthine oxidase systems . Lipid peroxidation of pure erythrocyte membrane and of whole red blood cell could be completely prevented by black tea extract . Similarly, black tea provided total protection against degradation of membrane proteins . Finally, membrane fluidity studies as monitored by the fluorescent probe 1,6 diphenyl hexa 1,3,5-triene (DPH) showed considerable disorganization of its architecture that could be restored back to normal on addition of black tea or free catechins . Black tea extract in comparison to free catechins seemed to be a better protecting agent against various types of oxidative stress . Apparently, conversion of catechins to partially polymerized products such as theaflavin or thearubigin during 'fermentation' process for making black tea has no deleterious effect on its scavenging properties. J Anim Sci, 1998 Mar, 76(3), 888 - 95 Ensiling effects of the ethanol fractionation of forages using gas production; Doane PH et al.; We studied the use of gas curve subtraction to distinguish between two fractions soluble in neutral detergent solution . Samples of unfractionated (whole) forage, residue insoluble in 90% ethanol, and isolated NDF were fermented in vitro, and gas production was monitored . The gas volume associated with the ethanol solubles (A fraction) was determined as the difference between the gas from the whole forage and from the ethanol residue . The gas yield associated with the fraction insoluble in 90% ethanol but soluble in neutral detergent solution (B fraction) was determined by subtracting the isolated NDF gas curve from the corresponding ethanol residue curve . This experiment included three forages (alfalfa, bromegrass, and orchardgrass) harvested at two maturities and preserved by freeze-drying or ensiling to form a 3 x 2 x 2 factorial arrangement . Ensiling reduced the rate of gas formation from the A fraction by approximately 30% (P < .01) . Ensiling increased (P < .05) the size of the A fraction (2 to 10% of DM) but did not change the volume of gas produced (P > .05) . The gas yield from the B1 fraction was reduced 40% (P < .05) by ensiling with no significant change in rate . Curve subtraction of gas production profiles may be used to obtain rate estimates for multiple neutral detergent soluble pools . The separation of the neutral detergent solubles into two pools clarified the effects caused by ensiling . Changes due to ensiling on the rate of gas produced were associated with the A fraction, and the effects on final gas volume were associated with the B1 fraction. J Hypertens, 1998 Feb, 16(2), 175 - 80 Alcohol intake and blood pressure: the importance of time elapsed since last drink; Moreira LB et al.; BACKGROUND: A positive association of chronic exposure to alcoholic beverages with blood pressure and the prevalence of hypertension has been described in epidemiological surveys, but the influence of time elapsed since last ingestion in this setting was not demonstrated . DESIGN: A cross-sectional, population-based survey . METHODS: In total 1089 adults from Porto Alegre, randomly selected from a population-based, multi-stage probability sample, were interviewed at home . The average daily alcohol intake of each subject was calculated taking into account the concentration of ethanol in the beverages (distilled or fermented beverages), and the time elapsed between the last ingestion of ethanol and the moment of blood pressure determination . Standardized sitting blood pressure and anthropometric parameters were collected . The magnitude and shape of the associations were analyzed considering blood pressure as a continuous variable and the prevalence of arbitrarily defined hypertension . Simple and multiple linear regression models, including models to identify nonlinear associations, with quadratic and cubic terms of the amount of alcohol consumed, were employed . Blood pressure means were compared by analysis of variance and analysis of covariance . The association between hypertension and exposure to ethanol was analyzed through logistic regression models, controlling for various potential confounders . RESULTS: Positive nonlinear associations of the amount of alcohol consumed with blood pressure and the prevalence of hypertension (> or = 160/95 mmHg) were found, independent of age, years of education, smoking, and use of oral contraceptive and antihypertensive drugs . The consumption of 30 g/day ethanol was associated with increases of 1.5 and 2.3 mmHg in diastolic and systolic blood pressures, respectively, for men, and 2.1 and 3.2 mmHg, respectively, for women . The prevalence of hypertension was higher among those ingesting more than 30 g/day (odds ratio = 2.9, P < 0.01) . The time elapsed between the last ingestion and blood pressure measurement was independently associated with the prevalence of hypertension . Men with last consumption of alcohol 13-23 h prior to measurement had odds of being hypertensive 2.6 (confidence interval 1.3-5.0) greater than did subjects who had consumed alcoholic beverages 24 h and more before the blood pressure determination . For men, systolic and diastolic blood pressures were lower during the first 3 h after ingestion and increased afterward . Frequency of consumption and type of beverage consumed were not independently associated with level of blood pressure . CONCLUSION: A time-dependent association between alcohol consumption and effects on blood pressure, demonstrated in experimental studies, was found for free-living individuals selected at random. J Dairy Sci, 1998 Feb, 81(2), 462 - 70 Effect of soybean hulls, soy lecithin, and soapstock mixtures on ruminal fermentation and milk composition in dairy cows; Abel-Caines SF et al.; Two experiments were conducted to determine effects of soybean hulls, soy lecithin, and soapstock mixtures on ruminal fermentation, milk composition, and ruminal microbial populations . In Experiment 1, 20 Holstein dairy cows were assigned to one of five total mixed rations (TMR) in replicated 5 x 5 Latin squares to measure the effect of TMR on intake and milk composition . Four ruminally fistulated cows were used in a 4 x 5 Youden square to measure the effect of TMR on ruminal fermentation . The TMR consisted of 40 or 50% alfalfa and corn silages, 13% soybean hulls, and 47 or 37% of a concentrate containing either 2.25% soybean oil; 2.25% lipid from a mixture of soybean hulls, soy lecithin, and soapstock; or no added lipid . The ratios of soy lecithin to soapstock evaluated were 1:1, 2.5:1, and 4:1 (dry basis) . The TMR containing soy lecithin and soapstock at ratios of 1:1 and 2.5:1 resulted in higher milk C18:2 than did the soybean oil TMR . Rate of ruminal NDF digestion of soybean hulls was reduced for the soybean oil TMR . In Experiment 2, a semi-continuous culture system was used to determine effects of soybean oil or an optimal mixture of soy lecithin and soapstock (1:1, wt/wt) on fermentation of soybean hulls and microbial populations . The TMR containing the lipid mixture increased the ratio of acetate to propionate compared with the TMR containing soybean oil and resulted in higher total protozoal counts than did the control TMR . The TMR containing mixtures of soy lecithin and soapstock at ratios of 1:1 and 2.5:1 elevated milk C18:2, and no negative effect on NDF digestion was detected, indicating some degree of ruminal protection. J Antibiot (Tokyo), 1998 Jan, 51(1), 50 - 7 Cloning and heterologous expression of genes from the kinamycin biosynthetic pathway of Streptomyces murayamaensis; Gould SJ et al.; The genes for most of the biosynthesis of the kinamycin antibiotics have been cloned and heterologously expressed . Genomic DNA of Streptomyces murayamaensis was partially digested with MboI and a library of approximately 40 kb fragments in E . coli XL1-BlueMR was prepared using the cosmid vector pOJ446 . Hybridization with the actI probe from the actinorhodin polyketide synthase genes identified two clusters of polyketide genes . After transferal of these clusters to S . lividans ZX7, expression of one cluster was established by HPLC with photodiode array detection . Peaks were identified from the kin cluster for dehydrorabelomycin, kinobscurinone, and stealthin C, which are known intermediates in kinamycin biosynthesis . Two shunt metabolites, kinafluorenone and seongomycin were also identified . The structure of the latter was determined from a quantity obtained from large-scale fermentation of one of the clones. J Antibiot (Tokyo), 1998 Jan, 51(1), 14 - 20 Novel lactone compounds from Mortierella verticillata that induce the human low density lipoprotein receptor gene: fermentation, isolation, structural elucidation and biological activities; Dekker KA et al.; Among methods of controlling hypercholesterolemia and hyperlipidemia is the direct stimulation of hepatic low density lipoprotein (LDL) receptors . Two novel lactone compounds, CJ-12,950 and CJ-13,357, containing and unusual oxime moiety, were isolated from a zygomycete Mortierella verticillata . These lactones are potent inducers of the LDL receptor gene in vitro, that enhanced LDL receptor expression in human hepatocytes 2-fold at 100 nM. Infect Immun, 1998 Apr, 66(4), 1467 - 72 Molecular analysis of Shiga toxigenic Escherichia coli O111:H- proteins which react with sera from patients with hemolytic-uremic syndrome; Voss E et al.; Western blot analysis was used to assess the reactivity of convalescent-phase sera from patients who were associated with an outbreak of hemolytic-uremic syndrome (HUS) caused by fermented sausage contaminated with Shiga toxin-producing Escherichia coli (STEC) . The predominant STEC isolated from HUS patients belonged to serotype O111:H-, and reactivity to O111:H- whole-cell lysates, treated or untreated with proteinase K, was examined . As expected, all five serum samples demonstrated a marked anti-lipopolysaccharide response, but several protein bands were also immunoreactive, particularly one with an apparent size of 94 kDa . One convalescent-phase serum sample was subsequently used to screen an O111:H- cosmid bank and 2 of 900 cosmid clones were found to be positive, both of which contained a similar DNA insert . Western blot analysis of one of these clones identified three major immunoreactive protein bands of approximately 94, 70, and 50 kDa . An immune response to the three proteins was detectable with all five convalescent-phase serum samples but not with healthy human serum . Immunoreactive 94- and 50-kDa species were produced by a deletion derivative of the cosmid containing a 7-kb STEC DNA insert . Sequence analysis of this region indicated that it is part of the locus for enterocyte effacement, including the eaeA gene which encodes intimin . The deduced amino acid sequence of the O111:H- intimin was 88.6% identical to intimin from O157:H7 STEC, and the most divergent region was the 200 residues at the carboxyl terminus, which were only 75% identical . Such variation may be antigenically significant as serum from a HUS patient infected only with the O111:H- STEC reacted with intimin from an enteropathogenic E . coli O111 strain, as well as several other eaeA-positive STEC isolates, but not with an eaeA-positive STEC belonging to serotype O157:H- . Sera from two of the other HUS patients also failed to react with intimin from this latter strain . However, intimin from O157:H- STEC did react with serum from a patient infected with both O111:H- and O157:H- STEC. Crit Rev Food Sci Nutr, 19