Clin Sci (Lond), 1997 Aug, 93(2), 109 - 12 Fermentable dietary fibre, intestinal microflora and plasma hormones in the rat; Ghatei MA et al.; 1 . Conventional and germ-free rats were fed a fibre-free elemental diet with or without the addition of fermentable dietary fibres . We have previously reported that fibre was associated with greatly increased epithelial cell proliferation, but only in the conventional group, implying that it is the breakdown of fibre by the colonic microflora that is the main determinant of mucosal proliferation in the hind gut . The relationship of these changes to various plasma hormones implicated in intestinal growth control are described in this paper . 2 . The most dramatic finding was that plasma levels of enteroglucagon and peptide YY were greatly increased in the germ-free groups . The response of these rats to fibre differed in that fibre decreased levels of enteroglucagon and peptide YY in the germ-free animals, but increased them in the conventional rats . Gastrin and insulin levels were significantly lowered in the fibre-supplemented groups, but were not affected by the microflora . 3 . These results corroborate our previous findings that the effects of fibre and its fermentation are dynamically complex, and demonstrate that, like proliferation, direct effects and indirect fermentation-derived effects on plasma hormones also coexist. Appl Microbiol Biotechnol, 1997 Aug, 48(2), 141 - 8 Production of D-ribose by fermentation; De Wulf P et al.; The production of D-ribose by fermentation has received much attention lately, possibly because of the use of this pentose to synthesize antiviral and anticancer drugs . This review briefly outlines the methods that have been used to synthesize D-ribose since it was identified in yeast RNA, and focuses in particular on the latest developments in D-ribose fermentation, which have led to D-ribose yields that exceed 90 g/1 . Furthermore, the various transketolase-deficient D-ribose-producing mutants that are used, and the biochemical and genetic rationales applied to select them or to enhance their D-ribose productivities, are dealt with . Attention is also drawn to the unusual pleiotropic characteristics of the mutant strains, as well as to the industrial and academic applications of D-ribose. Glycoconj J, 1997 Aug, 14(5), 669 - 76 Cellulose depolymerization to glucose and other water soluble polysaccharides by shear deformation and high pressure treatment; Kokorevics A et al.; The simultaneous action of shear deformation and high pressure (SDHP) creates changes in the structure of wood and its main components (cellulose, hemicelluloses, lignin) . The formation of water and alkali soluble polysaccharides under SDHP action, proceeds in seconds in the solid state, without the use of any reagents and solvents . Therefore, SDHP seems to be a technologically safe method and friendly to the environment . The amorphization of cellulose crystallites and depolymerization of cellulose chains were observed under a wide range of pressures (1-6 GPa), both for cellulose samples and the cellulose part of wood . Similar depolymerization occurs in the hemicellulose part of wood . The decomposition of polysaccharides under SDHP causes the formation of the water soluble part, whose content increases with pressure and the applied shear deformation . A maximum solubility of 40% and 55% was registered at 6 GPa following treatment of cellulose and birch wood samples . A higher output in the case of wood can be explained by a specific role of lignin under SDHP, which acts as a 'grinding stone' during cellulose and hemicelluloses destruction . As shown by high-performance size exclusion chromatography, the water soluble fraction obtained from cellulose contained glucose (2.6%), cellobiose (9.6%), cellotriose (16.6%) and other higher water soluble oligomers (71%) . Almost complete dissolution (98%) of the treated cellulose sample can be achieved by extraction with 10% NaOH solution . The SDHP treated birch wood was subjected to submerged fermentation (with Trichoderma viride), and a 13% output of proteins was obtained . In this case, the water soluble part played the role of the so called 'start sugars'. Antonie Van Leeuwenhoek, 1997 Aug, 72(2), 101 - 9 Formation of formate and hydrogen, and flux of reducing equivalents and carbon in Ruminococcus flavefaciens FD-1; Shi Y et al.; A pathway for conversion of the metabolic intermediate phosphoenolpyruvate (PEP) and the formation of acetate, succinate, formate, and H2 in the anaerobic cellulolytic bacterium Ruminococcus flavefaciens FD-1 was constructed on the basis of enzyme activities detected in extracts of cells grown in cellulose- or cellobiose-limited continuous culture . PEP was converted to acetate and CO2 (via pyruvate kinase, pyruvate dehydrogenase, and acetate kinase) or carboxylated to form succinate (via PEP carboxykinase, malate dehydrogenase, fumarase, and fumarate reductase) . Lactate was not formed even during rapid growth (batch culture, mu = 0.35/h) . H2 was formed by a hydrogenase rather than by cleavage of formate, and 13C-NMR and 14C-exchange reaction data indicated that formate was produced by CO2 reduction, not by a cleavage of pyruvate . The distribution of PEP into the acetate and succinate pathways was not affected by changing extracellular pH and growth rates within the normal growth range . However, increasing growth rate from 0.017/h to 0.244/h resulted in a shift toward formate production, presumably at the expense of H2 . This shift suggested that reducing equivalents could be balanced through formate or H2 production without affecting the yields of the major carbon-containing fermentation endproducts. Arch Microbiol, 1997 Oct, 168(4), 297 - 301 Thiosulfate as a metabolic product: the bacterial fermentation of taurine; Denger K et al.; Thiosulfate (S2O32-) is a natural product that is widely utilized in natural ecosystems as an electron sink or as an electron donor . However, the major biological source(s) of this thiosulfate is unknown . We present the first report that taurine (2-aminoethanesulfonate), the major mammalian solute, is subject to fermentation . This bacterial fermentation was found to be catalyzed by a new isolate, strain GKNTAU, a strictly anaerobic, gram-positive, motile rod that formed subterminal spores . Thiosulfate was a quantitative fermentation product . The other fermentation products were ammonia and acetate, and all could be formed by cell-free extracts. Arch Microbiol, 1997 Oct, 168(4), 290 - 6 Regulatory O2 tensions for the synthesis of fermentation products in Escherichia coli and relation to aerobic respiration; Becker S et al.; In an oxystat, the synthesis of the fermentation products formate, acetate, ethanol, lactate, and succinate of Escherichia coli was studied as a function of the O2 tension (pO2) in the medium . The pO2 values that gave rise to half-maximal synthesis of the products (pO0 . 5) were 0.2-0.4 mbar for ethanol, acetate, and succinate, and 1 mbar for formate . The pO0.5 for the expression of the adhE gene encoding alcohol dehydrogenase was approximately 0.8 mbar . Thus, the pO2 for the onset of fermentation was distinctly lower than that for anaerobic respiration (pO0.5 </= 5 mbar), which was determined earlier . An essential role for quinol oxidase bd in microaerobic growth was demonstrated . A mutant deficient for quinol oxidase bd produced lactate as a fermentation product during growth at microoxic conditions (approximately 10 mbar O2), in contrast to the wild-type or a quinol-oxidase-bo-deficient strain . In the presence of nitrate, the amount of lactate was largely decreased . Therefore, under microoxic conditions, the pO2 appears to be too high for (mixed acid) fermentation to function and too low for aerobic respiration by quinol oxidase bo. Appl Environ Microbiol, 1997 Sep, 63(9), 3399 - 404 Metabolic responses of pyruvate decarboxylase-negative Saccharomyces cerevisiae to glucose excess; Flikweert MT et al.; In Saccharomyces cerevisiae, oxidation of pyruvate to acetyl coenzyme A can occur via two routes . In pyruvate decarboxylase-negative (Pdc-) mutants, the pyruvate dehydrogenase complex is the sole functional link between glycolysis and the tricarboxylic acid (TCA) cycle . Such mutants therefore provide a useful experimental system with which to study regulation of the pyruvate dehydrogenase complex . In this study, a possible in vivo inactivation of the pyruvate dehydrogenase complex was investigated . When respiring, carbon-limited chemostat cultures of wild-type S . cerevisiae were pulsed with excess glucose, an immediate onset of respiro-fermentative metabolism occurred, accompanied by a strong increase of the glycolytic flux . When the same experiment was performed with an isogenic Pdc- mutant, only a small increase of the glycolytic flux was observed and pyruvate was the only major metabolite excreted . This finding supports the hypothesis that reoxidation of cytosolic NADH via pyruvate decarboxylase and alcohol dehydrogenase is a prerequisite for high glycolytic fluxes in S . cerevisiae . In Pdc- cultures, the specific rate of oxygen consumption increased by ca . 40% after a glucose pulse . Calculations showed that pyruvate excretion by the mutant was not due to a decrease of the pyruvate flux into the TCA cycle . We therefore conclude that rapid inactivation of the pyruvate dehydrogenase complex (e.g., by phosphorylation of its E1 alpha subunit, a mechanism demonstrated in many higher organisms) is not a relevant mechanism in the response of respiring S . cerevisiae cells to excess glucose . Consistently, pyruvate dehydrogenase activities in cell extracts did not exhibit a strong decrease after a glucose pulse. J Endocrinol, 1997 Aug, 154(2), 275 - 83 Effects of season, protein and nutritional state on glucose tolerance during an annual cycle of growth in young red deer stags; McMahon CD et al.; Two hypotheses were tested in gonad-intact, young (aged 6-18 months), growing red deer stags during an annual growth cycle . First, that glucose clearance rate is faster during summer than during winter . Secondly, that increased dietary protein availability will enhance winter growth . Stags were randomly assigned into one of two groups: group 1 (n = 5) had 16% while group 2 (n = 6) had 48% of dietary protein naturally protected against fermentative degradation in the rumen . Total crude protein and energy remained similar for each diet (12 and 14% respectively for protein and 11 MJ metabolisable energy/kg dry matter) . Stags were kept indoors in individual pens for 12 months and given monthly intravenous glucose tolerance tests (IVGTT), at a dose of 200 mg/kg, in the fed and fasted (48 h) states to determine both growth and steady-state tissue requirements . Protein level had no effect on food intake, weight gain, insulin kinetics, or glucose clearance rate . In the fed state, insulin peak (highest level' after IVGTT) increased (P < 0.01) from October (139 pmol/l) to December (247 pmol/l) (S.E.D . = 42) and remained elevated during the summer, before declining (P < 0.01) from February (223 pmol/l) to April (130 pmol/l) (S.E.D . = 25) . Glucose clearance rate was faster (P < 0.05) in December (1.69 litres/min) than June (0.61 litres/min) in the fed state (S.E.D . = 0.30), and decreased (P < 0.05) from February (1.75 litres/min) to April (0.92 litres/min) (S.E.D . = 0.39) . During fasting, the pattern of glucose clearance was similar to that observed in the fed state, but the amplitude was lower, while the pattern for insulin peak was similar to that of the fed state . We concluded first, that additional protected protein does not benefit growth during winter . Secondly, we concluded from the fasted, steady-state data that stags are insulin resistant during summer . Thirdly, despite insulin resistance, data on the fed state demonstrated that stags have higher tissue energy requirements during summer growth. Cell Tissue Res, 1997 Oct, 290(1), 61 - 9 Distribution of enteroglucagon- and peptide YY-immunoreactive cells in the intestinal mucosa of germ-free and conventional mice; Arantes RM et al.; There are evidences that microflora modulates endocrine cells in the gastrointestinal tract . In the present study we investigated the distribution of EG- and PYY-immunoreactive cells throughout the intestine of adult male NMRI conventional and germ-free mice . EG-immunoreactive cells were significantly more frequent in the proximal and middle colon than in the remainder of the intestine in both groups . In germ-free animals, these cells were more frequent in the cecum and less frequent in the distal ileum compared to conventional mice . PYY-immunoreactive cells were more frequent in the distal colon than in the remainder of the intestine in both groups, but they were significantly more frequent in the middle and distal colon of germ-free animals than in that of conventional counterparts . The number of EG-immunoreactive cells was 4.5-fold higher than the number of PYY-immunoreactive cells in the cecum of germ-free mice . The present results indicate the existence of an inverse gradient of EG- and PYY-immunoreactive cells along the colon, which is not significantly changed in the absence of a microflora . PYY production seems to be more significant in the distal colon . The cecum and the proximal portion of the colon are probably the regions of greatest functional importance for EG production, which is related to the microflora and probably to fermentation products, whether or not the effect of this peptide is trophic or antitrophic. Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9608 - 13 Pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon, Pyrococcus furiosus, functions as a CoA-dependent pyruvate decarboxylase; Ma K et al.; Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from the hyperthermophilic archaeon, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C by fermenting carbohydrates and peptides . The enzyme contains thiamine pyrophosphate and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 and reduces P . furiosus ferredoxin . Here we show that this enzyme also catalyzes the formation of acetaldehyde from pyruvate in a CoA-dependent reaction . Desulfocoenzyme A substituted for CoA showing that the cofactor plays a structural rather than a catalytic role . Ferredoxin was not necessary for the pyruvate decarboxylase activity of POR, nor did it inhibit acetaldehyde production . The apparent Km values for CoA and pyruvate were 0.11 mM and 1.1 mM, respectively, and the optimal temperature for acetaldehyde formation was above 90 degrees C . These data are comparable to those previously determined for the pyruvate oxidation reaction of POR . At 80 degrees C (pH 8.0), the apparent Vm value for pyruvate decarboxylation was about 40% of the apparent Vm value for pyruvate oxidation rate (using P . furiosus ferredoxin as the electron acceptor) . Tentative catalytic mechanisms for these two reactions are presented . In addition to POR, three other 2-keto acid ferredoxin oxidoreductases are involved in peptide fermentation by hyperthermophilic archaea . It is proposed that the various aldehydes produced by these oxidoreductases in vivo are used by two aldehyde-utilizing enzymes, alcohol dehydrogenase and aldehyde ferredoxin oxidoreductase, the physiological roles of which were previously unknown. Cancer Res, 1997 Sep 1, 57(17), 3697 - 707 Induction of caspase-3 protease activity and apoptosis by butyrate and trichostatin A (inhibitors of histone deacetylase): dependence on protein synthesis and synergy with a mitochondrial/cytochrome c-dependent pathway; Medina V et al.; The induction of apoptosis of tumor cells by the colonic fermentation product butyrate is thought to be an important mechanism in protection against colorectal cancer . Because a major action of butyrate is to inhibit histone deacetylase (leading to chromatin relaxation and altered gene expression), butyrate may induce apoptosis by derepression of specific cell death genes . Here we show that butyrate and trichostatin A (a more selective inhibitor of histone deacetylase) induce the same program of apoptosis in Jurkat lymphoid and LIM 1215 colorectal cancer cell lines that is strictly dependent on new protein synthesis (within 10 h) and that leads to the conversion of the proenzyme form of caspase-3 to the catalytically active effector protease (within 16 h) and apoptotic death (within 24 h) . Cells primed with a low concentration of butyrate that itself did not induce activation of caspase-3 or apoptosis were, nevertheless, rendered highly susceptible to induction of apoptosis by staurosporine (an agent that has recently been shown to act by causing mitochondrial release of cytochrome c) . Synergy between butyrate and staurosporine was due to the presence of a factor in the cytosol of butyrate-primed cells which enhanced over 7-fold the activation of caspase-3 induced by the addition of cytochrome c and dATP to isolated cytosol . We propose that changes at the level of chromatin structure, induced by a physiological substance butyrate, lead to the expression of a protein that facilitates the pathway by which mitochondria activate caspase-3 and trigger apoptotic death of lymphoid and colorectal cancer cells. Infect Immun, 1997 Sep, 65(9), 3799 - 805 Shiga toxin-producing Escherichia coli isolates from cases of human disease show enhanced adherence to intestinal epithelial (Henle 407) cells; Paton AW et al.; Shiga toxin-producing Escherichia coli (STEC) strains are a diverse group of organisms which are known to cause diarrhea and hemorrhagic colitis in humans . We have recently described a large food-borne outbreak of STEC disease caused by contaminated semidry fermented sausage (A . W . Paton, R . Ratcliff, R . M . Doyle, J . Seymour-Murray, D . Davos, J . A . Lanser, and J . C . Paton, J . Clin . Microbiol . 34:1622-1627, 1996) . STEC strains belonging to several O serotypes were isolated from the contaminated food source, but of these, only a subset were isolated from patients with diarrhea or hemolytic-uremic syndrome (HUS) . In the present study, we characterized these STEC isolates with respect to the presence of putative virulence-associated genes and the capacity to adhere to a human intestinal epithelial cell line (Henle 407) . The O111:H- STEC strain 95NR1 (isolated from one of the outbreak HUS patients) was shown to adhere to Henle 407 cells in a dose-dependent, mannose-resistant fashion . Microscopic examination revealed a diffuse pattern of adherence for this as well as several other STEC strains . Interestingly, the adherence of STEC strains from HUS cases (both outbreak related and sporadic) was significantly greater than that of STEC strains found in the contaminated food source but not found in any patients . These studies support the hypothesis that an enhanced capacity to adhere to intestinal cells is one of the factors which distinguishes human-virulent STEC strains from those of lesser clinical significance. Poult Sci, 1997 Sep, 76(9), 1220 - 6 Preservation of hatchery waste by lactic acid fermentation . 2 . Large-scale fermentation and feeding trial to evaluate feeding value; Deshmukh AC et al.; Two waste streams from a Leghorn hatchery were preserved and recycled by fermentation with a by-product carbohydrate and extrusion processing into new feed ingredients that were evaluated with broiler chickens . Cockerel chicks (CC) and a 60:40 ratio of CC:shell waste (CC:SW) were fermented in 189-L barrels for 21 d following grinding, then mixing with a liquid culture (0.2%) and carbohydrate source at 15 and 16.66%, respectively . At 2 wk, pH was 4.44 and 5.09 for the CC and CC:SW products compared with higher values of 6.54 and 6.98 for the raw ingredients at the onset . Negligible hydrogen sulfide and no ammonia gas were recorded during the fermentation period . At 21 d, the fermented CC and CC:SW were extruded, dried, and ground to meals containing CP and TMEn levels of 47.4%, 3,187 kcal/kg, and 33.1%, 2,696 kcal/kg, respectively . Broiler chickens were fed a control diet and the CC (5 and 10%) and CC:SW (2.5 and 5%) ingredient diets with corn and soybean meal for 6 wk to evaluate feeding value and carcass yield . Body weight, gain and feed conversion at 42 d for birds fed diets supplemented with CC or CC:SW at all levels were comparable to those of the control . Diets supplemented with hatchery by-product had no negative effect on carcass measurements except ready to cook carcass and wing yield, which were significantly greater for the 10% CC:SW birds than for the control . These data indicate that nutrient dense hatchery by-products can be preserved with fermentation up to 21 d and support broiler live performance and carcass yield as dietary ingredients equal to or better than a corn-soybean meal control. Mol Biochem Parasitol, 1997 Sep, 88(1-2), 163 - 74 Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae; Dowd AJ et al.; Cathepsin L2 is a major cysteine proteinase secreted by adult Fasciola hepatica . The enzyme differs from other reported cathepsin Ls in that it can cleave peptide substrates that contain proline in the P2 position . A cDNA was isolated from an expression library by immunoscreening with antiserum prepared against purified native cathepsin L2 . This cDNA was sequenced and shown to encode a complete preprocathepsin L proteinase . Functionally active recombinant cathepsin L proteinase was expressed and secreted by Saccharomyces cerevisiae transformed with the cDNA . The recombinant enzyme was purified from large-scale fermentation broths using ultrafiltration and gel filtration chromatography on Sephacryl S200 HR columns . NH2-terminal amino acid sequencing showed that the cleavage point for activation of the recombinant pro-enzyme is identical to that of the F . hepatica-produced cathepsin L2 . The mature active recombinant proteinase behaved similarly to the native enzyme when analysed by SDS-PAGE, immunoblotting and zymography and also cleaved peptides containing proline in the P2 position . Finally, the recombinant cathepsin L2 cleaved fibrinogen to form a fibrin clot, a property we described for F . hepatica cathepsin L2. J Med Chem, 1997 Aug 15, 40(17), 2755 - 61 6A-O-{(4-biphenylyl)acetyl}-alpha-, -beta-, and -gamma-cyclodextrins and 6A-deoxy-6A-{{(4-biphenylyl)acetyl}amino}-alpha-, -beta-, and -gamma-cyclodextrins: potential prodrugs for colon-specific delivery; Uekama K et al.; Cyclodextrins (CyDs) are known to be fermented to small saccharides by colonic microflora, whereas they are only slightly hydrolyzable and thus are not easily absorbed in the stomach and small intestine . This property of CyDs is particularly useful for colon-specific delivery of drugs . In this study, an antiinflammatory 4-biphenylylacetic acid (BPAA) was selectively conjugated onto one of the primary hydroxyl groups of alpha-, beta-, and gamma-CyDs through an ester or amide linkage, 6A-O-{(4-biphenylyl)acetyl{-alpha-, -beta-, and -gamma-CyDs (1-3) and 6A-deoxy-6A-{{(4-biphenylyl)acetyl}amino}-alpha-, -beta-, and -gamma-CyDs (4-6) . In rat cecal and colonic contents (10%, w/v), 1 and 3 released more than 95% of BPAA within 1-2 h, and 2 released about 50% of the drug within 12 h . The amide prodrugs, 4-6, did not release BPAA in the cecal contents, but gave BPAA/maltose or BPAA/triose conjugates linked through an amide bond . On the other hand, these prodrugs were found to be stable in the contents of rat stomachs and small intestines, in intestinal or liver homogenates, and in rat blood . The serum levels of BPAA increased about 3 h after oral administration of 1 and 3 to rats, accompanying a marked increase in the serum levels, whereas 2 and 4-6 resulted in little increase of the serum levels . These facts suggest that BPAA is released after the ring opening of CyDs followed by the ester hydrolysis, and the BPAA activation takes place site-specifically in the cecum and colon . Therefore, the present CyD prodrug approach provides a versatile means of constructing a novel colon-specific drug delivery system. Wien Klin Wochenschr, 1997 Aug 8, 109(14-15), 578 - 83 Genital mycoplasma infections; Taylor-Robinson D et al.; Since 1937, 13 Mycoplasma species, two Acholeplasma species, and one Ureaplasma species have been isolated from humans . Six of these have the urogenital tract as the primary site of colonisation but others, which have the oropharynx and respiratory tract as the primary site, are found occasionally in the urogenital tract because of orogenital contact . Mycoplasma hominis was the first to be isolated and is most strongly associated with bacterial vaginosis (BV), together with a variety of other bacteria . Its involvement in pelvic inflammatory disease (PID) and other conditions may be as part of BV, although when isolated in pure culture from the blood of women who have postpartum or postabortal fever there is no reason to suspect its aetiological role . There is evidence for an aetiological role for Ureaplasma urealyticum organisms (ureaplasmas) in acute non-gonococcal urethritis (NGU) and particularly chronic NGU in men, but they rank third to Chlamydia trachomatis and M . genitalium . Whether the association of ureaplasmas with miscarriage and preterm labour is in the context of BV is not clear . Of no doubt, however, is the ability of ureaplasmas to cause septic arthritis in hypogammaglobulinaemic patients and there is evidence that they may cause some cases of sexually acquired reactive arthritis . The advent of polymerase chain reaction technology has seen an advance in the understanding of the role of M . genitalium; there is strong evidence that it is one of the causes of both acute and chronic NGU independent of C . trachomatis . There is some support for the role of M . genitalium in PID, but this needs to be substantiated . Other mycoplasmas, for example M . fermentans, M . pivum, M . primatum, M . penetrans, M . spermatophilum and even M . pneumoniae have the capacity to cause urogenital tract disease but there is no evidence to indicate that they do so. JAMA, 1997 Aug 6, 278(5), 412 - 7 Biological warfare . A historical perspective; Christopher GW et al.; The deliberate use of microorganisms and toxins as weapons has been attempted throughout history . Biological warfare has evolved from the crude use of cadavers to contaminate water supplies to the development of specialized munitions for battlefield and covert use . The modern development of biological agents as weapons has paralleled advances in basic and applied microbiology . These include the identification of virulent pathogens suitable for aerosol delivery and industrial-scale fermentation processes to produce large quantities of pathogens and toxins . The history of biological warfare is difficult to assess because of a number of confounding factors . These include difficulties in verification of alleged or attempted biological attacks, the use of allegations of biological attacks for propaganda purposes, the paucity of pertinent microbiological or epidemiologic data, and the incidence of naturally occurring endemic or epidemic diseases during hostilities . Biological warfare has been renounced by 140 nations, primarily for strategic and other pragmatic reasons . International diplomatic efforts, including the 1972 Biological Weapons Convention, have not been entirely effective in preventing the enhancement and proliferation of offensive biological warfare programs . The threats posed by biological weapons are likely to continue into the future. Dig Dis Sci, 1997 Aug, 42(8), 1571 - 9 Colonic sulfide in pathogenesis and treatment of ulcerative colitis; Roediger WE et al.; A role for colonic sulfide in the pathogenesis and treatment of ulcerative colitis (UC) has emerged based on biochemical, microbiological, nutritional, toxicological, epidemiological, and therapeutic evidence . Metabolism of isolated colonic epithelial cells has indicated that the bacterial short-chain fatty acid n-butyrate maintains the epithelial barrier and that sulfides can inhibit oxidation of n-butyrate analogous to that observed in active UC . Sulfur for fermentation in the colon is essential for n-butyrate formation and sulfidogenesis aids disposal of colonic hydrogen produced by bacteria . The numbers of sulfate-reducing bacteria and sulfidogenesis is greater in UC than control cases . Sulfide is mainly detoxified by methylation in colonic epithelial cells and circulating red blood cells . The enzyme activity of sulfide methylation is higher in red blood cells of UC patients than control cases . Patients with UC ingest more protein and thereby sulfur amino acids than control subjects . Removing foods rich in sulfur amino acids (milk, eggs, cheese) has proven therapeutic benefits in UC . 5-Amino salicylic acid reduces fermentative production of hydrogen sulfide by colonic bacteria, and aminoglycosides, which inhibit sulfate-reducing bacteria, are of therapeutic benefit in active UC . Methyl-donating agents are a category of drugs of potential therapeutic use in UC . A correlation between sulfide production and mucosal immune responses in UC needs to be undertaken . Control of sulfidogenesis and sulfide detoxification may be important in the disease process of UC, although whether their roles is in an initiating or promoting capacity has yet to be determined. Int J Biol Macromol, 1997 Aug, 21(1-2), 115 - 21 Structural studies of CV-70 polysaccharide; Scamparini A et al.; The goal of this paper is the characterization of the chemical structure of the water-soluble polysaccharide, CV-70, produced by bacteria Beijerinckia sp . Beijerinckia sp . is a genus of gram-negative, aerobic bacteria, usually found in sugar cane root . The CV-70 polysaccharide was produced in a fermentation medium containing 5% sucrose as the carbon source, tryptose and salts, at 25 degrees C {1} . The polysaccharide was hydrolyzed with 2 N trifluoroacetic acid at 100 degrees C for 16 h, purified, and analyzed by HPLC . Index of refraction was used for the detection of sugars . For GC-MS analysis, the CV-70 polysaccharide was derivatized through methylation and acetylation . Together with the GC-MS data, periodate oxidation studies were used to determine the possible glucosidic linkages . Carbon-13 NMR studies were carried out with hydrolyzed and silylated samples . Glucose, galactose and fucose were identified as the components in the CV-70 polysaccharide, in a 3:1:3 ratio. J Dairy Sci, 1997 Aug, 80(8), 1666 - 73 Nutrient fluxes in splanchnic tissue of dairy cows: influence of grass quality; De Visser H et al.; A crossover design was used to investigate the effects of high (450 kg of N/ha) or low (150 kg of N/ha) N fertilization of ryegrass on fermentation and nutrient fluxes in splanchnic tissue of dairy cows fed those grasses . Grass that was fertilized with the high amount of N contained more N and less sugar than did grass that was fertilized with less N . In rumen fluid, the concentration of NH3 N was lower for ryegrass that was fertilized with the low amount of N . The NH3 release by portal-drained viscera and urea synthesis in the liver were higher for cows fed ryegrass that was fertilized with the high amount of N . The concentration of NH3 N in rumen fluid, NH3 N release in portal-drained viscera, urea synthesis in the liver, urea release from the liver, and urea concentrations in milk were highly correlated . The release of acetate and propionate in portal-drained viscera was similar for both grasses and was well correlated with the proportion of volatile fatty acids in rumen fluid . The proportion of butyrate in rumen fluid was closely correlated with the release of butyrate and beta-hydroxybutyrate in portal-drained viscera . Glucose synthesis in the liver indicated gluconeogenesis from amino acids, which corresponded well with urea synthesis in the liver . For the grass fertilized with more N, availability of energy sources for rumen microbes was low, and, therefore, cows did not use the N in that grass efficiently. J Dairy Res, 1997 Aug, 64(3), 453 - 7 Effect of yogurt and bifidus yogurt fortified with skim milk powder, condensed whey and lactose-hydrolysed condensed whey on serum cholesterol and triacylglycerol levels in rats; Beena A et al.; The possible hypocholesterolaemic properties of milk and fermented milk products have been investigated in groups of albino rats given a basal diet, basal diet plus cholesterol, and basal diet plus cholesterol together with whole milk or standard or bifidus yogurt . The yogurts were fortified with skim milk powder, condensed whey or lactose-hydrolysed condensed whey . After 30 d, triacylglycerols, total cholesterol, HDL-cholesterol and LDL-cholesterol were measured in serum . Whole milk and ordinary yogurt had no hypocholesterolaemic effect, but standard yogurt containing lactose-hydrolysed condensed whey and all bifidus yogurts lowered serum cholesterol . In general, yogurts changed HDL-cholesterol little, but tended to raise triacylglycerols . There was marked lowering of LDL-cholesterol in rats given either type of yogurt fortified with whey proteins . This study has demonstrated in a rat model that bifidus yogurts and yogurts fortified with whey proteins can reduce total and LDL-cholesterol, and suggests that if they have the same effect in human subjects they have potential value in cholesterol-lowering diets. Microbiology, 1997 Aug, 143 ( Pt 8), 2627 - 37 The Sch9 protein kinase in the yeast Saccharomyces cerevisiae controls cAPK activity and is required for nitrogen activation of the fermentable-growth-medium-induced (FGM) pathway; Crauwels M et al.; In cells of the yeast Saccharomyces cerevisiae, trehalase activation, repression of CTT1 (catalase), SSA3 (Hsp70) and other STRE-controlled genes, feedback inhibition of cAMP synthesis and to some extent induction of ribosomal protein genes is controlled by the Ras-adenylate cyclase pathway and by the fermentable-growth-medium-induced pathway (FGM pathway) . When derepressed cells are shifted from a non-fermentable carbon source to glucose, the Ras-adenylate cyclase pathway is transiently activated while the FGM pathway triggers a more lasting activation of the same targets when the cells become glucose-repressed . Activation of the FGM pathway is not mediated by cAMP but requires catalytic activity of cAMP-dependent protein kinase (cAPK; Tpk1, 2 or 3) . This study shows that elimination of Sch9, a protein kinase with homology to the catalytic subunits of cAPK, affects all target systems in derepressed cells in a way consistent with higher activity of cAPK in vivo . In vitro measurements with trehalase and kemptide as substrates confirmed that elimination of sch9 enhances cAPK activity about two- to threefold, in both the absence and presence of cAMP . In vivo it similarly affected the basal and final level but not the extent of the glucose-induced responses in derepressed cells . The reduction in growth rate caused by deletion of SCH9 is unlikely to be responsible for the increase in cAPK activity since reduction of growth rate generally leads to lower cAPK activity in yeast . On the other hand, deletion of SCH9 abolished the responses of the protein kinase A targets in glucose-repressed cells . Re-addition of nitrogen to cells starved for nitrogen in the presence of glucose failed to trigger activation of trehalase, caused strongly reduced and aberrant repression of CTT1 and SSA3, and failed to induce the upshift in RPL25 expression . From these results three conclusions can be drawn: (1) Sch9 either directly or indirectly reduces the activity of protein kinase A; (2) Sch9 is not required for glucose-induced activation of the Ras-adenylate cyclase pathway; and (3) Sch9 is required for nitrogen-induced activation of the FGM pathway . The latter indicates that Sch9 might be the target of the FGM pathway rather than cAPK itself. Yeast, 1997 Aug, 13(10), 931 - 43 Surface properties of top- and bottom-fermenting yeast; Dengis PB et al.; The surface physico-chemical properties (hydrophobicity, electrophoretic mobility, chemical composition) of a large set of top- and bottom-fermenting brewing yeasts, harvested in the exponential and stationary growth phases, have been investigated . Bottom- and top-fermenting strains showed different surface properties . Top strains were generally more hydrophobic than bottom strains, due to higher surface protein concentrations . Bottom strains possessed higher surface phosphate concentrations . The different profiles of electrophoretic mobility versus pH for top and bottom strains could be explained by modelling the surface charge according to the surface chemical composition as given by X-ray photoelectron spectroscopy . For bottom strains, the electrical properties were mainly controlled by phosphate, resulting in a low isoelectric point (pH 2 or below) and an electrophoretic mobility that did not become much more negative above pH 4 . For the top strains, they were mainly determined by the balance of protonated amino- and carboxylate groups in proteins, which gave a high isoelectric point (pH 4) and an electrophoretic mobility changing greatly with pH in the range of 2 to 7 . No difference in surface properties was found between flocculating and non-flocculating strains, or between cells from the exponential and stationary growth phases, even for strains where flocculation occurred during the transition from one growth phase to the other. Yeast, 1997 Aug, 13(10), 903 - 15 Stationary-phase gene expression in Saccharomyces cerevisiae during wine fermentation; Riou C et al.; Genetic engineering of wine yeast strains requires the identification of gene promoters specifically activated under wine processing conditions . In this study, transcriptional activation of specific genes was followed during the time course of wine fermentation by quantifying mRNA levels in a haploid wine strain of Saccharomyces cerevisiae grown on synthetic or natural winery musts . Northern analyses were performed using radioactive probes from 19 genes previously described as being expressed under laboratory growth conditions or on molasses in S . cerevisiae during the stationary phase and/or under nitrogen starvation . Nine genes, including members of the HSP family, showed a transition-phase induction profile . For three of them, mRNA transcripts could be detected until the end of the fermentation . Expression of one of these genes, HSP30, was further studied using a HSP30::lacZ fusion on both multicopy and monocopy expression vectors . The production of beta-galactosidase by recombinant cells was measured during cell growth and fermentation on synthetic and natural winery musts . We showed that the HSP30 promoter can induce high gene expression during late stationary phase and remains active until the end of the wine fermentation process . Similar expression profiles were obtained on five natural winery musts. Trends Biotechnol, 1997 Aug, 15(8), 315 - 20 Heterologous biopharmaceutical protein expression in Streptomyces; Binnie C et al.; The commercial production of human proteins in recombinant microorganisms for therapeutic use is well established . Systems have been developed to exploit the natural ability of certain bacteria to secrete properly folded, bioactive proteins into the extracellular medium . The streptomycetes are a relatively well-characterized group of nonpathogenic filamentous bacteria that have the capacity to secrete large amounts of protein . In particular, Streptomyces lividans has the ability to secrete human proteins at a commercially viable level, thanks to relatively well-established plasmid-based expression system, a high-biomass fermentation process and a low level of endogenous protease activity. J Anim Sci, 1997 Aug, 75(8), 2277 - 83 Ruminal fermentation and nutrient digestion in sheep fed hydroxyethylsoyamide; Jenkins TC; Hydroxyethylsoyamide (HESA) was reported previously to protect soybean oil from ruminal biohydrogenation and increase plasma unsaturated fatty acids in sheep . Two digestibility trials with sheep and a rumen in vitro trials were conducted in this study to determine the effects of HESA on ruminal VFA and nutrient digestibility . Trial 1 was a 4 x 4 Latin square with 17-d periods in which four wethers were fed either a control diet (CON) with no added fat, 2.5% soybean oil (SBO), 5% butylsoyamide (BuSA), or 5% HESA . The HESA diet was ground with a mortar and pestle before feeding to disperse fat lumps that formed during diet mixing . Compared with the CON diet, the HESA diet reduced DMI, acetate/ propionate (A/P), and total tract fiber digestibility, but these were not affected by SBO or BuSA . Trial 2 was a 24-h rumen in vitro study showing that total VFA concentration and A/P in cultures were reduced by 10% linoleic acid but not by 10% ethanolamine or 10% HESA . In Trial 3, four wethers were fed the CON and HESA diets in a replicated 2 x 2 Latin square to determine digestibility responses to HESA when grinding was avoided . Fiber digestibilities and A/P were not affected by HESA in Trial 3 . The HESA in this study had variable effects on fiber digestibility that may have been related to physical attributes of the diet, including particle size . Substitution of ethanolamine for butylamine during synthesis of the amide increased fatty acid digestibility but reduced dry matter intake. J Anim Sci, 1997 Aug, 75(8), 2248 - 55 Considerations for gastrointestinal cannulations in ruminants; Harmon DL et al.; The complexity of ruminant digestion necessitates a greater variety and complexity of experimental methods than with any other species . The fact that dietary ingredients are first subjected to microbial fermentation requires elaborate measures to ascertain nutrients presented for absorption . Numerous approaches have been attempted to obtain representative samples of digesta at sites throughout the gastrointestinal tract . The choices of a researcher before an experiment include animal(s), site(s) for cannula placement, style of cannula, cannula material, and numerous other more subtle factors that may contribute to the success of an experiment . This review compares the advantages and disadvantages of various approaches, cannula types, and cannula materials that should be considered before experiments are conducted. J Anim Sci, 1997 Aug, 75(8), 2161 - 4 Technical note: pig model for studying nutrient assimilation by the intestine and colon; Kien CL et al.; We have developed a system for chronically catheterizing 10- to 25-d-old pigs that permits stable isotope tracer studies of intestinal or colonic assimilation of nutrients . This model also can be used to ensure constant enteral feeding or to assess the rate of entry into the terminal ileum of carbohydrates, fats, and amino acids . A plastic cannula with a luminal flange can be surgically placed in the stomach for tracer studies of sugar digestion or for controlled infusion of any formula diet . A similar cannula can be placed in the cecum for infusion of tracer and(or) substrates for studies of fermentation . The cannula has been machined so that a washer and nut can be threaded onto it, allowing the entire apparatus to be fixed to the abdominal wall . The distal end protruding above the skin was tapered to fit standard i.v . extension tubing . A carotid arterial catheter was used to sample substrates for isotopic enrichment measurements. J Gastroenterol, 1997 Aug, 32(4), 453 - 6 Small bowel transit time and colonic fermentation in young and elderly women; Kagaya M et al.; Small bowel transit time (SBTT) in 15 young and 13 elderly women was assessed by measuring breath hydrogen concentrations after they had consumed a solid test meal . The meal consisted of 200 g cooked rice, 50 ml miso (made from fermented soy bean curd) soup, a boiled egg, and 95.5 g of cooked soy beans with mixed vegetables . This meal provided 17 g protein, 14.1 g fat, 92.9 g carbohydrate, 7 g dietary fiber, and 565 kcal total energy . The SBTT, calculated by a mean 3 ppm increase in breath hydrogen, was 191 +/- 14.9 (mean +/- SE) min in the young and 188.1 +/- 16.8 min in the elderly group; the difference was not significant . Breath hydrogen levels, however, were higher in the young than in the elderly group (39.1 +/- 6.3 ppm, vs 22.2 +/- 4.3 ppm, P < 0.05) . There was an initial peak of hydrogen concentration, reached almost immediately after the ingestion of the meal, and then a decline to baseline within 60 min . This initial peak was not as pronounced in the elderly subjects . A second peak, indicating the entry of the test meal into the cecum, was more pronounced in the young than in the elderly group . SBTT did not differ significantly between the two groups, but colonic fermentation was more pronounced in the young, both in the fasting and the postprandial state. J Biotechnol, 1997 Jul 23, 56(1), 57 - 61 Biotechnological potential of P450 monooxygenases high-level production of bovine cytochrome P450c17 monooxygenase during medium cell density culture of a recombinant yeast, Saccharomyces cerevisiae GRF 18 (YEp-Toku1); Nishihara H et al.; Bovine cytochrome P450c17 monooxygenase was produced in a 30-L fermenter by Saccharomyces cerevisiae GRF18 (YEp-Toku1), harboring the GAL10 promoter, and using conditions of medium cell density culture . Upon addition of D-galactose as an inducer and FeCl3 as a cofactor, cells began to produce the P450 hemoprotein . The yield of this enzyme reached a maximum after 31 h but its formation continued for more than 60 h after induction . The amount of P450c17 produced was 4.7-fold as compared to shake flask experiments. FEBS Lett, 1997 Jul 7, 411(1), 97 - 101 A novel fluorescent marker for assembled mitochondria ATP synthase of yeast . OSCP subunit fused to green fluorescent protein is assembled into the complex in vivo; Prescott M et al.; We have shown that OSCP, a subunit of yeast mitochondrial ATP synthase, can be incorporated into the intact enzyme as a fusion protein representing OSCP fused at its C-terminus to the green fluorescent protein (GFP) of Aequorea victoria . The relevant fusion OSCP-GFP-h6 additionally contains a hexahistidine tag at the C-terminus . Expression of OSCP-GFP-h6 in yeast cells lacking endogenous OSCP led to the efficient restoration of growth of cells on the non-fermentable substrate, ethanol . Confocal laser scanning microscopy revealed fluorescence due to GFP in mitochondria of cells expressing OSCP-GFP-h6 . Use of immobilised metal ion affinity chromatography enabled the recovery of assembled ATP synthase complexes which contained OSCP-GFP-h6 identified by its mobility on SDS-PAGE and immunoreactivity to anti-OSCP and anti-GFP antibodies . The successful isolation of the assembled multisubunit ATP synthase containing GFP fused to one of the essential subunits of the complex widely expands the potential applications of GFP . In principle, these include the spatial and temporal monitoring of ATP synthase complexes in vivo, and the exploration of interactions involving ATP synthase subunits by fluorescence resonance energy transfer (FRET). J Ind Microbiol Biotechnol, 1997 Jul, 19(1), 12 - 7 Ethanol production from spent cherry brine; Park H et al.; Spent cherry brine is an acidic byproduct of maraschino cherry processing and typically consists of variable amounts of glucose and fructose of up to 11% fermentable solids, 0.5-1.5% CaCl2, up to 0.4% sulfur dioxide, sorbitol, and lesser amounts of other cherry constituents . Disposal of brine represents a significant cost to processors because of its high biological oxygen demand . As an alternative, brine was tested as a substrate for ethanol production . Initially, the toxic level of sulfur dioxide was reduced by raising brine pH to 8.0 to precipitate calcium sulfite . Because alkalinization was subsequently found to result in a 10-fold reduction in phosphorous, brines were titrated with phosphoric acid to pH 6.0 prior to inoculation with Saccharomyces cerevisiae . All strains of Saccharomyces cerevisiae tested were able to ferment all lots of Ca(OH)2-treated and phosphorous-enriched brines efficiently . One lot of brine containing 10% (w/v) fermentable sugar yielded 4.7% (w/v) ethanol in 4 days. Appl Microbiol Biotechnol, 1997 Jul, 48(1), 23 - 6 Effect of soybean oil and glucose on sophorose lipid fermentation by Torulopsis bombicola in continuous culture; Kim SY et al.; The effect of soybean oil and glucose on the growth of Torulopsis bombicola and sophorose lipid production in continuous culture was investigated . As the dilution rate in 100 g/l glucose and 100 g/l soybean oil medium was increased, the dry cell weight and sophorose lipid concentration decreased . Sophorose lipid productivity, however, was maximum at a dilution rate of 0.03 h-1 . The cell yield from glucose and the sophorose lipid production from soybean oil were approximately constant regardless of the dilution rate . The specific consumption rate of soybean oil was closely related to the specific production rate of sophorose lipid . These results suggest that soybean oil was used only for sophorose lipid production whereas glucose was used only for cell mass and maintenance . When the soybean oil concentration was varied at fixed dilution rate in 100 g/l glucose medium, a high concentration of soybean oil was found to inhibit sophorose lipid production. Biotechnol Prog, 1997 Jul-Aug, 13(4), 503 - 5 Reverse micellar extraction of antibiotics from aqueous solutions; Fadnavis NW et al.; Several antibiotics such as erythromycin, oxytetracyclin, benzylpenicillin, and actidione were extracted from aqueous buffers into reverse micellar solution of bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT) in isooctane and recovered with high efficiency under mild conditions . Preliminary experiments with oxytetracycline dissolved in a fermentation broth indicate that the antibiotic can be selectively extracted from the broth and recovered efficiently without serious loss of potency. Biotechnol Prog, 1997 Jul-Aug, 13(4), 374 - 9 Ubiquitin fusion technology: bioprocessing of peptides; Pilon A et al.; Ubiquitin fusion technology represents an emerging method for economically producing peptides and small proteins in the bacterium Escherichia coli . Our focus is on peptide production where the need for cost-effective, scaleable processes has recently been highlighted by Kelley (1996) . There are two principal features: (1) the expression system consists of a suitable E . coli host strain paired with a plasmid that encodes the ubiquitin fusion and (2) an ubiquitin-specific protease, UCH-L3, which cleaves only C-terminal extensions from ubiquitin . In this work, multigram yields were obtained of four ubiquitin fusions derived from cell paste generated in single 10-L fermentations . All were expressed intracellularly and remained soluble at extremely high levels of expression . Bacterial freeze--thaw lysates contained over 95% pure ubiquitin fusion protein . All four fusions were efficiently cleaved to ubiquitin and the peptide products . In one case, the final yield of peptide was 1.08 g from 3 L of low cell density bacterial culture . The combination of exceptional overexpression of the ubiquitin--peptide fusion proteins and a robust and specific protease are unique advantages contributing to a cost-effective, scaleable, and generic bioprocess for peptide production. J Pediatr, 1997 Jul, 131(1 Pt 2), S5 - 7 Pioneering recombinant growth hormone manufacturing: pounds produced per mile of height; Cronin MJ; The first efforts to produce recombinant human growth hormone (GH) for clinical use were begun by scientists at Genentech, Inc., almost a generation ago, late in 1979 . The very small market for GH that was predicted at the time led to this manufacturing effort being done as a demonstration project . Among the early issues was whether the Escherichia coli host cell could be routinely produced in a stable manner and be inactivated after the GH production run (as required by Federal guidelines) without the GH being permanently denatured . A 10 L E . coli process was developed, and phase I testing began in early 1981 . The approval of this recombinant GH product by the FDA in 1985 paved the way for many improvements and a sustained production effort in the next decade . The more than 1990 fermentation runs have produced tons of E . coli and more than 130 pounds of GH for both clinical research and the treatment of severely short children. Dig Dis Sci, 1997 Jul, 42(7), 1354 - 61 Gastroesophageal reflux in achalasia . When is reflux really reflux? Crookes PF, Corkill S, DeMeester TR. An abnormal score during 24-hr esophageal pH monitoring in achalasia may be associated either with a slow steady drift to below pH 4, or else multiple sharp dips characteristic of typical gastroesophageal reflux . To test the hypothesis that the former pattern was due to food fermentation and not reflux, samples of chewed bland food (N = 22) were incubated with saliva at 37 degrees C for 24 hr and the pH monitored (in vitro study) . Further, the pH tracings of 20 patients with achalasia before operation and 12 patients after operation were studied (in vivo study) . The pH of chewed food fell to a median of pH 4.0 during incubation and in seven of 22 samples fell to below pH 4 . Preoperatively, four of the five patients with an abnormal pH score showed a slow steady drift, and all of these had evidence of retained food at endoscopy . Postoperatively, three of the six patients with an abnormal pH score had a slow steady drift to below pH 4 . Use of pH 3 as a threshold clearly distinguished true reflux from food fermentation, since the patients with reflux all had an abnormal percentage of time below pH 3. Biochem J, 1997 Jul 1, 325 ( Pt 1), 101 - 9 Three spinach leaf nitrate reductase-3-hydroxy-3-methylglutaryl-CoA reductase kinases that are required by reversible phosphorylation and/or Ca2+ ions; Douglas P et al.; In spinach (Spinacea oleracea L.) leaf extracts, three protein kinases (PKI, PKII and PKIII) were identified each of which phosphorylated spinach nitrate reductase on serine-543, and inactivated the enzyme in the presence of nitrate reductase inhibitor, 14-3-3 . PKIII was also very active in phosphorylating and inactivating Arabidopsis (Landsberg erecta) 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR1) . PKI and PKII phosphorylated HMGR1 more slowly than PKIII, compared with their relative rates of phosphorylation of nitrate reductase . HMGR1 identical with those that are seen after phosphorylation of serine-577 by the sucrose non-fermenting (SNF1)-like PK, 3-hydroxy-3-methylglutaryl-Co A reductase kinase A (HRK-A), from cauliflower {Dale, Arro, Becerra, Morrice, Boronat, Hardie and Ferrer (1995) Eur . J . Biochem . 233, 506-513} . PKI was Ca2+-dependent when prepared in the absence of protein phosphatase (PP) inhibitors, and largely Ca2+-dependent when prepared in the presence of PP inhibitors (NaF and EGTA) . The Ca2+-independent portion of PKI was inactivated by either PP2A or PP2C, while the Ca2+-dependent portion of PKI became increasingly activated during storage, which we presume was mimicking the effect of an unidentified PP . These findings indicate that PK1 is regulated by two functionally distinct phosphorylations . PKI had a molecular mass of 45 kDa on gel filtration and was active towards substrate peptides that terminated at the +2 residue from the phosphorylation site, whereas PKIII was inactive towards these peptides . PKII was Ca2+-stimulated under all conditions tested . PKIII was Ca2+-indepdented, inactivated by PP2A or PP2C, had a requirement for a hydrophobic residue in the +4 position of peptide substrates, had a molecular mass by gel filtration of approximately 140 kDa, and an antibody against the rye SNF1-related PK (RKIN1) recognized a 58 kDa subunit in fractions containing PKIII . These properties of PKIII are identical with those reported previously for the SNF1-like enzyme, HRK-A . Our results indicate a considerable complexity of kinase cascades mediating the regulation of assimilatory and biosynthetic pathways in response to environmental stimuli in plants. J Dairy Sci, 1997 Jul, 80(7), 1463 - 81 Creating a system for meeting the fiber requirements of dairy cows; Mertens DR; Current NRC recommendations for dairy cattle provide limited guidance to nutritionists for meeting the fiber and carbohydrate needs of lactating cows . The NRC provide only minimum recommendations for fiber and no accommodation for factors such as physical effectiveness of fiber, interactions with nonfibrous carbohydrates, or animal attributes, which can affect the optimality of dairy rations . To be an improvement, any new system for meeting the fiber requirements of dairy cows must be based on 1) feed characteristics that can be defined and preferably be determined quantitatively using routine laboratory methods and 2) animal requirements that correspond to critical feed characteristics and vary with feeding situation, ration composition, and attributes of the animal . Published data were used to develop coefficients for defining the physical effectiveness or roughage value of feeds and the fiber requirements of dairy cows . Information in this paper is intended to provide practical guidelines for improving current fiber recommendations and to serve as an idealized framework for future research on meeting the fiber requirements of dairy cows . The system is based on NDF as the measure of total chemical fiber in feeds . Adjustments for the effectiveness of NDF in maintaining milk fat production and optimizing ruminal fermentation are based on the particle size and inherent characteristics of NDF that affect chewing activity, ruminal pH, and milk fat production. J Dairy Sci, 1997 Jul, 80(7), 1447 - 62 Relationship between fermentation acid production in the rumen and the requirement for physically effective fiber; Allen MS; The content of ruminally fermented OM in the diet affects the fiber requirement of dairy cattle . Physically effective fiber is the fraction of feed that stimulates chewing activity . Chewing, in turn, stimulates saliva secretion . Bicarbonate and phosphate buffers in saliva neutralize acids produced by fermentation of OM in the rumen . The balance between the production of fermentation acid and buffer secretion is a major determinant of ruminal pH . Low ruminal pH may decrease DMI, fiber digestibility, and microbial yield and thus decrease milk production and increase feed costs . Diets should be formulated to maintain adequate mean ruminal pH, and variation in ruminal pH should be minimized by feeding management . The fraction of OM that is fermented in the rumen varies greatly among diets . This variation affects the amount of fermentation acids produced and directly affects the amount of physically effective fiber that is required to maintain adequate ruminal pH . Acid production in the rumen is due primarily to fermentation of carbohydrates, which represent over 65% of the DM in diets of dairy cows and have the most variable ruminal degradation across diets . The non-fiber carbohydrate content of the diet is often used as a proxy for ruminal fermentability, but this measure is inadequate . Ruminal fermentation of both nonfiber carbohydrate and fiber is extremely variable, and this variability is not related to the nonfiber carbohydrate content of the diet . The interaction of ruminally fermented carbohydrate and physically effective fiber must be considered when diets for dairy cattle are evaluated and formulated. J Dairy Sci, 1997 Jul, 80(7), 1366 - 73 Influence of time of feeding a protein meal on ruminal fermentation and forestomach digestion in dairy cows; Robinson PH et al.; Four ruminally and duodenally cannulated dairy cows in midlactation were fed twice daily a mixed diet of alfalfa silage and whole-crop oat silage and a concentrate consisting of primarily barley grain . A high protein supplement was fed at approximately 15% of the estimated dry matter intake of the mixed diet once daily at 0830 h, 0.5 h after the morning meal (day), or at 0030 h, 7.5 h after the evening meal (night) . Cows fed the protein supplement during the night had higher apparent forestomach digestion of organic matter and crude protein . Ruminal concentrations of all volatile fatty acids, except isobutyrate, were higher for cows fed the protein supplement during the night . Although ruminal pH and concentrations of ammonia N did not differ between treatments, time by treatment interactions indicated that the feeding times of the protein supplement influenced diurnal patterns of ruminal fermentation . The flow of nonbacterial nonammonia N at the duodenum, as a proportion of N intake, was lower for cows fed the protein supplement during the night, but production of milk fat was higher . Results were consistent with a mechanism whereby protein fed during the night stimulated ruminal fermentation, particularly during the night, resulting in greater forestomach digestion of organic matter and less escape of dietary protein from the forestomach . Clearly, the different feeding times of this protein supplement changed the nutritional value of the overall diet. J Dairy Sci, 1997 Jul, 80(7), 1296 - 314 Comparison of mechanistic rumen models on mathematical formulation of extramicrobial and microbial processes; Bannink A et al.; This study investigated the consequences of differences in applied concepts and individual mathematical formulations on steady-state behavior of three important mechanistic rumen models . In the models of Baldwin et al . (2) and Danfaer (6), the formulation of passage rate, nondietary inputs, defined rumen substrate pools, absorption rates, degradation rates, molecular weights, parameterization of VFA production, and physical compartmentalization were sequentially exchanged for the formulation of the model of Dijkstra et al . (9) . Most of these adaptations had a considerable influence on model behavior, indicating large qualitative differences in formulation and sensitivity to concept choice . Because microbial substrate environments were similar after all adaptations, the microbial mechanisms could be compared objectively without being concealed by differences in extramicrobial formulation . None of the microbial functions were altered except for substrate degradation, which gave rise to a similar rate of substrate entrance to soluble rumen pools that are available for microbial utilization . Large differences remained in microbial functions of substrate fermentation, substrate incorporation, and microbial synthesis . Differences in extramicrobial rumen functions and microbial mechanisms had important consequences for simulated nutrient outputs from the rumen, illustrating the necessity for further validation of individual formulations. Yeast, 1997 Jul, 13(9), 783 - 93 Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cerevisiae strains overexpressed or disrupted for GPD1 encoding glycerol 3-phosphate dehydrogenase; Michnick S et al.; The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated . Variations in the glycerol 3-phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol-3-phosphatase were found when low and high glycerol-forming wine yeast strains were compared . GPDH is thus a limiting enzyme for glycerol production . Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose-rich medium . Engineered strains fermented glucose with a strongly modified {glycerol} : {ethanol} ratio . gpd1delta mutants exhibited a 50% decrease in glycerol production and increased ethanol yield . Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production ( x 4) at the expense of ethanol . Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH . Accumulation of by-products such as pyruvate, acetate, acetoin, 2,3 butane-diol and succinate was observed, with a marked increase in acetoin production. Eur J Clin Nutr, 1997 Jul, 51(7), 417 - 23 A new look at dietary carbohydrate: chemistry, physiology and health . Paris Carbohydrate Group; Cummings JH et al.; The current view of dietary carbohydrates as simply providing us with energy is outdated . Because of their varied chemistry and physical form the rate and extent to which the different types are digested in and absorbed from the small intestine varies . This in turn leads to affects on satiety, blood glucose and insulin, protein glycosylation, lipids and bile acids . Some carbohydrates reach the colon where they are fermented and affect many aspects of large bowel function, colonocyte and hepatic metabolism . A new framework for classifying and measuring food carbohydrates is needed to allow a greater understanding of the role of individual species in health and to inform the public of their importance . A classification based primarily on molecular size (degree of polymerisation) into sugars, oligosaccharides and polysaccharides, is suggested, with sub-groups identified by the nature of the monosaccharides . Greater knowledge of the chemical and physical properties of carbohydrates allow a more precise relation with physiology and health to be drawn . The Carbohydrate Group met in Paris in December 1995 at the invitation of Gerard Pascal, Director of CNERNA . Financial support for the meeting was provided by CNERNA. Lab Anim, 1997 Jul, 31(3), 254 - 63 The morphological changes of intestinal mucosa in growing rabbits; Yu B et al.; The study aimed to increase understanding of digestive function from the development of the digestive tract from suckling to maturity in rabbits . The relative weights of the digestive tract (in relation to body weight) in different segments increase linearly during the rapid growth period between 2 and 8 weeks of age, thereafter intestinal weight gain is slower . An underdeveloped mucosal histology was observed in the hindgut of suckling rabbits at 2 weeks compared with 4 weeks of age . From SEM micrographs, the small intestinal mucosal villi look more slender and finger-like in the suckling period, thereafter becoming broader or tongue-like or plate-shaped in mature rabbits . The micrographs showed a compact arrangement in the underdeveloped hindgut mucosa at 2 weeks, but after weaning as hindgut fermentation becomes significant the mucosa increased in surface area. FEMS Microbiol Lett, 1997 Jul 1, 152(1), 183 - 8 Cloning and sequencing of the cDNA encoding lipase I from Trichosporon fermentans WU-C12; Arai T et al.; A cDNA clone encoding extracellular lipase I (TFL I) from Trichosporon fermentans WU-C12 was isolated and characterized . The TFL I cDNA was isolated from a lambda gt10-based cDNA library using as a probe a 0.8 kb fragment, amplified by PCR with synthetic oligonucleotide corresponding to the partial amino acid sequences of TFL I . The cDNA encodes a protein consisting of 563 amino acids containing a putative signal peptide of 19 amino acids . The deduced amino acid sequence shares 99.5% overall identity with that of lipase II (GCL II) from Geotrichum candidum ATCC 34614, whereas TFL I is a trimer enzyme and GCL II monomer . Southern hybridization with the TFL I cDNA as a probe revealed that WU-C12 contained two different lipase genes. Int J Syst Bacteriol, 1997 Jul, 47(3), 742 - 6 Mycoplasma crocodyli sp . nov., a new species from crocodiles; Kirchhoff H et al.; Organisms with the typical characteristics of mycoplasmas were isolated from joints and lungs of crocodiles . The results of growth inhibition tests and immunobinding assays showed that the 24 mycoplasma strains isolated were identical and distinct from previously described Mycoplasma, Entomoplasma, Mesoplasma, and Acholeplasma species . These organisms represent a new species, for which the name Mycoplasma crocodyli is proposed . M . crocodyli ferments glucose and maltose, does not produce films and spots, does not hydrolyze arginine, esculin, and urea, reduces tetrazolium chloride, and possesses phosphatase activity . It lyses and adsorbs bovine, ovine, and rabbit erythrocytes . Cholesterol or serum is required for growth . The optimum growth temperature is 37 degrees C . The G + C content of the DNA is 27.6 mol% . This organism causes exudative polyarthritis in crocodiles . The type strain of M . crocodyli is strain MP145 (= ATCC 51981). Protein Expr Purif, 1997 Jul, 10(2), 214 - 25 Stable expression and purification of a secreted human recombinant prethrombin-2 and its activation to thrombin; Russo G et al.; A human prothrombin cDNA has been engineered to obtain a cDNA coding for a secreted form of human prethrombin-2 . The secreted prethrombin-2 has been produced in a mammalian expression system using DXB11 cells, a mutant strain of CHO cells in which the dihydrofolate reductase gene has been deleted, and an expression vector carrying the dihydrofolate reductase cDNA . Methotrexate-induced gene amplification favored an efficient production of the recombinant protein which accumulated in the culture medium of the DXB11 cells . Growth in suspension of the stable transformants in an airlift fermenter resulted in the production of 25 mg/L recombinant prethrombin-2 . The recombinant protein was purified using single-step affinity chromatography on a recombinant-hirudin column and activated by agarose gel-immobilized ecarin . All purified recombinant prethrombin-2 was activated and the generated recombinant thrombin showed catalytic properties identical to those of plasma-derived alpha-thrombin . This expression system can be used to prepare mutants of prethrombin-2 for structure-function studies investigating thrombin interactions with substrate proteins, inhibitors, and cell membranes. Metabolism, 1997 Jul, 46(7), 805 - 11 Time of day and glucose tolerance status affect serum short-chain fatty acid concentrations in humans; Wolever TM et al.; Short-chain fatty acids (SCFA) are derived from endogenous (metabolism of fat, carbohydrate, and amino acids) and exogenous (colonic fermentation) sources . To see how time of day and glucose tolerance status influenced serum SCFA concentrations, we determined serum SCFA throughout the day in 22 subjects with impaired glucose tolerance (IGT) and 10 young and eight middle-aged normal controls . On 1 day, insulin sensitivity was assessed as the steady-state plasma glucose (SSPG) level achieved during intravenous infusion of glucose insulin, and somatostatin . On another day, plasma glucose and insulin and serum SCFA levels were measured 12 times over 12 hours with subjects eating a standard diet . SSPG in young controls (5.5 +/- 1.1 mmol/L) was less than in middle-aged controls (9.3 +/- 1.6 mmol/L), which in turn was less than in IGT subjects (13.7 +/- 0.6 mmol/L; P < .01) . Mean plasma glucose in IGT subjects was greater than in normal controls, and mean plasma insulin in IGT subjects was higher than in young controls but similar to the levels in middle-aged controls . Mean 12-hour serum acetate in young controls (143 +/- 13 mumol/L) was greater than in middle-aged controls (104 +/- 11 mumol/L) and IGT subjects (113 +/- 5 mumol/L; P < .05) . Mean 12-hour serum propionate in young controls (3.8 +/- 0.5 mumol/L) was less than in IGT subjects (5.4 +/- 0.3 mumol/L; P < .01), with middle-aged controls being intermediate (4.6 +/- 0.3 mumol/L) . Both young (1.6 +/- 0.3 mumol/L) and middle-aged (1.0 +/- 0.2) controls had lower mean butyrate than IGT subjects (3.1 +/- 0.4 mumol/L; P < .05) . Levels of all three SCFA varied significantly during the day, tending to decrease after breakfast and increase transiently after lunch and dinner . It is concluded that both time of day and glucose tolerance status affect serum SCFA levels in nondiabetic humans . The results suggest that serum acetate is derived primarily from colonic fermentation, serum butyrate primarily from endogenous fatty acid metabolism, and serum propionate from both exogenous and endogenous sources. J Anim Sci, 1997 Jul, 75(7), 1704 - 7 Comparative ruminal and total tract digestion of a finishing diet containing fresh vs air-dry steam-flaked corn; Zinn RA et al.; Ten Holstein steers (465 +/- 6 kg) with cannulas in the rumen and proximal duodenum were used in a crossover design experiment to evaluate the influence of air-dry vs fresh steam-flaked corn on characteristics of ruminal and total tract digestion . The basal diet contained 77% steam-flaked corn (DM basis) . Air-dry steam-flaked corn (SFC-AD) was obtained from a single batch that had been allowed to air-dry for 5 d before beginning the trial . Fresh steam-flaked corn (SFC-F) was produced daily Monday through Friday . Following production, the SFC-F was placed in air-tight polybags and stored at 4 degrees C until the time of feeding . There was little difference (P > .20) between SFC-AD and SFC-F with respect to site and extent of digestion of OM, starch, and fiber . Moreover, the two treatments did not differ (P > .20) in ruminal degradability of feed N . Apparent total tract N digestion was slightly greater (2.4%, P < .05) for SFC-F than for SFC-AD . Treatments did not affect ruminal pH (P > .20); however, VFA concentration of ruminal fluid tended to be greater (8.3%, P < .10) for SFC-F than for SFC-AD, indicating that the initial rate of fermentation may have been greater with SFC-F . Ruminal molar proportions of acetate were not affected by treatments (P > .20), but ruminal molar proportions of propionate tended to be greater (9.7%, P < .10) and molar proportions of butyrate tended to be less (10.0%, P < .10) for SFC-F than for SFC-AD . We conclude that the characteristics of digestion and the feeding value of steam-flaked corn is not altered by air drying before feeding. Genetics, 1997 Jul, 146(3), 1131 - 41 Cloning of the Arabidopsis and rice formaldehyde dehydrogenase genes: implications for the origin of plant ADH enzymes; Dolferus R et al.; This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH) . Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P) . These data provide further evidence that plant class P genes have evolved from class III genes by gene duplication and acquisition of new substrate specificities . The position of introns and similarities in the nucleic acid and amino acid sequences of the different classes of ADH enzymes in plants and humans suggest that plant and animal class III enzymes diverged before they duplicated to give rise to plant and animal ethanol-active ADH enzymes . Plant class P ADH enzymes have gained substrate specificities and evolved promoters with different expression properties, in keeping with their metabolic function as part of the alcohol fermentation pathway. Arthritis Rheum, 1997 Jul, 40(7), 1219 - 28 Mycoplasma infection and rheumatoid arthritis: analysis of their relationship using immunoblotting and an ultrasensitive polymerase chain reaction detection method; Hoffman RW et al.; OBJECTIVE: To examine the relationship between infection with Mycoplasma and the development of rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA) . METHODS: Immunoblotting of patient synovial fluid and sera on detergent-phase membrane protein extracts of various Mycoplasma species was carried out to learn whether patients exhibited serologic evidence of previous exposure to mycoplasmas . Moreover, an ultrasensitive polymerase chain reaction (PCR) method was developed for assessing whether Mycoplasma DNA could be detected in synovial fluid from patients and controls . RESULTS: Immunoblotting provided serologic evidence of previous Mycoplasma exposure in patients and controls . The genus-specific PCR detected known human Mycoplasma species and could reliably detect <5 copies of Mycoplasma hominis, Mycoplasma fermentans, or a molecular mimic control in synovial fluid . Repeat testing revealed no evidence of Mycoplasma DNA in patient synovial samples . CONCLUSION: This study provided serologic evidence suggesting that, while previous exposure to Mycoplasma was common, there was no detectable persistence of Mycoplasma DNA in the synovial fluid or tissue of patients with RA or JRA. Appl Environ Microbiol, 1997 Jul, 63(7), 2844 - 9 A spontaneous change in the intracellular cyclic AMP level in Aspergillus niger is influenced by the sucrose concentration in the medium and by light; Gradisnik-Grapulin M et al.; A spontaneous rise in intracellular cyclic AMP (cAMP) levels was observed in the early stages of Aspergillus niger growth under conditions yielding large amounts of citric acid . The amount of cAMP formed was found to depend on the initial concentration of sucrose in the medium . Under higher-sucrose conditions, the cAMP peak appeared earlier and was higher, while in lower-sucrose media a flattened peak was observed later in fermentation . Since in media with higher concentrations of sucrose intracellular citric acid starts to accumulate earlier and more rapidly, cAMP synthesis may be triggered by intracellular acidification, which is caused by the dissociation of citric acid . No spontaneous increase in cAMP concentrations could be detected when the cells were grown in continuously illuminated cultures, suggesting that A . niger phosphodiesterase (PDE) is photoregulated . More evidence for the activation of PDE by light was obtained from morphological studies under light and dark conditions in the presence of cAMP or N6,O2'-dibutyryl cAMP, and this idea was additionally supported by experiments in which PDE inhibitors were tested. Appl Environ Microbiol, 1997 Jul, 63(7), 2821 - 5 Nitrogen availability of grape juice limits killer yeast growth and fermentation activity during mixed-culture fermentation with sensitive commercial yeast strains; Medina K et al.; The competition between selected or commercial killer strains of type K2 and sensitive commercial strains of Saccharomyces cerevisiae was studied under various conditions in sterile grape juice fermentations . The focus of this study was the effect of yeast inoculation levels and the role of assimilable nitrogen nutrition on killer activity . A study of the consumption of free amino nitrogen (FAN) by pure and mixed cultures of killer and sensitive cells showed no differences between the profiles of nitrogen assimilation in all cases, and FAN was practically depleted in the first 2 days of fermentation . The effect of the addition of assimilable nitrogen and the size of inoculum was examined in mixed killer and sensitive strain competitions . Stuck and sluggish wine fermentations were observed to depend on nitrogen availability when the ratio of killer to sensitive cells was low (1:10 to 1:100) . A relationship between the initial assimilable nitrogen content of must and the proportion of killer cells during fermentation was shown . An indirect relationship was found between inoculum size and the percentage of killer cells: a smaller inoculum resulted in a higher proportion of killer cells in grape juice fermentations . In all cases, wines obtained with pure-culture fermentations were preferred to mixed-culture fermentations by sensory analysis . The reasons why killer cells do not finish fermentation under competitive conditions with sensitive cells are discussed. Appl Environ Microbiol, 1997 Jul, 63(7), 2779 - 84 Glucose metabolism in the yeast Schwanniomyces castellii: role of phosphorylation site I and an alternative respiratory pathway; Zimmer E et al.; Glucose metabolism in a Crabtree-negative yeast, Schwanniomyces castellii, and a cytochrome b-deficient mutant of this strain was investigated in chemostat culture . The wild-type and mutant strains exhibited the same behavior . Oxidative metabolism was observed when the substrate uptake rate (qS) was low . Fermentative metabolites were excreted when the qS value was higher than 0.40 g.g-1.h-1, indicating the occurrence of a respirofermentative metabolism; however, the respiratory quotient (RQ) remained near 1 . When fermentation occurred, the cytochrome pathway was repressed but not the salicylhydroxamic acid (SHAM)-sensitive pathway . The presence of an alternative SHAM-sensitive respiratory pathway and the presence of phosphorylation site I in all metabolic conditions explained the RQ value of 1 and accounted for high biomass yields in oxidative metabolism conditions (0.62 g.g-1 for the wild-type strain and 0.31 g.g-1 for the cytochrome b-deficient mutant strain). Appl Environ Microbiol, 1997 Jul, 63(7), 2695 - 701 Production of succinic acid through overexpression of NAD(+)-dependent malic enzyme in an Escherichia coli mutant; Stols L et al.; NAD(+)-dependent malic enzyme was cloned from the Escherichia coli genome by PCR based on the published partial sequence of the gene . The enzyme was overexpressed and purified to near homogeneity in two chromatographic steps and was analyzed kinetically in the forward and reverse directions . The Km values determined in the presence of saturating cofactor and manganese ion were 0.26 mM for malate (physiological direction) and 16 mM for pyruvate (reverse direction) . When malic enzyme was induced under appropriate culture conditions in a strain of E . coli that was unable to ferment glucose and accumulated pyruvate, fermentative metabolism of glucose was restored . Succinic acid was the major fermentation product formed . When this fermentation was performed in the presence of hydrogen, the yield of succinic acid increased . The constructed pathway represents an alternative metabolic route for the fermentative production of dicarboxylic acids from renewable feedstocks. Appl Environ Microbiol, 1997 Jul, 63(7), 2637 - 46 Identification of a laccase gene family in the new lignin-degrading basidiomycete CECT 20197; Mansur M et al.; A new lignin-degrading basidiomycete, strain I-62 (CECT 20197), isolated from decayed wood exhibited both a high dephenolization activity and decolorization capacity when tested on effluents from the sugar cane by-product fermentation industry . It has been classified as a member of the Polyporaceae family . The major ligninolytic activity detected in culture supernatants of basidiomycete I-62 was a phenoloxidase (laccase), in conjunction with small amounts of manganese peroxidase . No lignin peroxidase was detected . Laccase activity was produced in either defined or complete media . Addition of veratryl alcohol as the inducer, in defined medium, enhanced laccase production 10-fold . The use of fructose instead of glucose as a carbon source resulted in a 100-fold increase in laccase specific activity . Native isoelectrofocusing gels stained with guaiacol revealed the presence of at least seven laccase isozymes, with the most intense band being detected at pI 3 . Southern hybridization analysis indicated the presence of a laccase gene family in strain I-62 . Three different genes coding for phenoloxidases, lcc1, lcc2, and lcc3, were cloned and characterized . The high degree of homology between laccases from strain I-62 and laccases from Trametes species suggests a phylogenetic proximity between this new isolated fungus and the genus Trametes. Am J Clin Nutr, 1997 Jul, 66(1), 62 - 6 Maldigestion and colonic fermentation of wheat bread in humans and the influence of dietary fat; Olesen M et al.; A fraction of wheat bread is malabsorbed in healthy humans . The malabsorbed fraction is bigger than what can be accounted for by in vitro measurements of dietary fibers and resistant starch . To determine whether it is a specific fraction defined by the structure of the starch molecule or a variable amount--which depends on the individual, the amount ingested, and other components of the meal--we performed a dose-response study on wheat bread in healthy human volunteers . Malabsorption was evaluated by using the breath-hydrogen test . Test meals were as follows: 20 g wheat bran mixed in 100 mL water; bread made from 25, 75, 100, 150, and 200 g white wheat flour (WWF); bread made from 0 g WWF and 20 g wheat bran; and bread made from 100 g WWF served with 11 or 26 g butter, corresponding to 20% or 35% of energy from fat in the meals . Three of seven volunteers malabsorbed a fraction of the bread made from 25 g WWF and five of seven a fraction of the bread made from 75 g WWF . All volunteers malabsorbed a fraction of the 100-g WWF bread, Bread made from 180 g WWF and 20 g wheat bran resulted in a breath-hydrogen response of the same magnitude as that from bread made from 200 g WWF alone . The 100-g WWF bread + 11 g butter resulted in a significantly higher breath-hydrogen response than did the bread alone, whereas the 100-g WWF bread + 26 g butter resulted in an average response of the same magnitude as that from bread alone . We conclude that the malabsorbed fraction of wheat bread was dependent on the amount ingested, the composition of the meal, and individual gastrointestinal handling . Fermentation of wheat bran resulted in a very low breath-hydrogen response compared with lactulose or wheat bread . Addition of 11 g butter to the bread seemed to increase the malabsorbed fraction of the starch, an effect that was abolished when the amount of butter was increased to 26 g. J Nutr, 1997 Jul, 127(7), 1349 - 56 Soluble amylose cornstarch is more digestible than soluble amylopectin potato starch in rats; Zhou X et al.; In liquid enteral formulations, high molecular weight soluble starches may be able to replace glucose and low molecular weight glucose polymers that have high glycemic indices . Male rats were fed either commercial cornstarch, dextrose, modified soluble potato (70-75% amylopectin) starch, or modified soluble amylomaize-7 (70% amylose) starch for 4 wk . Body weights did not differ among the groups . Food consumption was significantly higher in the two modified starch-fed groups than in the two control groups . Commercial cornstarch, dextrose, modified potato starch and modified amylomaize-7 starch were 100 +/- 0, 100 +/- 0, 69.0 +/- 1.0 and 91.5 +/- 0.8% digestible, respectively (n = 9, mean +/- SEM) . The modified potato starch-fed group deposited the least fat, protein and energy . In both modified starch-fed groups, liver weights were significantly greater than in the two control groups . In food-deprived rats, serum free fatty acid concentrations in the modified potato starch-fed group were significantly higher than in the two control groups, and serum glucose concentrations were significantly higher in the two modified starch-fed groups than in the controls . The insulin to glucagon ratios were significantly lower in the modified potato starch-fed and amylomaize-7 starch-fed groups than in the dextrose-fed control group . Serum protein concentrations, measured after food deprivation, were significantly lower in the modified potato starch-fed group than in the other three groups . Gluconeogenesis from fermentation products might account for the high serum glucose concentrations in the two experimental groups . These data indicate that only the modified amylomaize-7 starch may be useful in the development of food products for liquid nutritional supplements because of the high digestibility and the low resultant insulin levels. FEBS Lett, 1997 Jun 30, 410(2-3), 145 - 9 HIV-1 protein Vpr causes gross mitochondrial dysfunction in the yeast Saccharomyces cerevisiae; Macreadie IG et al.; The biological effects of the HIV-1 accessory protein, Vpr, have been studied in yeast expression systems . In our previous study {1}, employing the pCUP1-vpr copper-inducible expression cassette, Vpr was shown to cause growth arrest and structural defects . In this study yeast constitutively expressing vpr, through elevated copy number and/or elevated transcription levels, displayed no growth arrest in fermentative growth conditions while Vpr was produced at much lower levels than in the inducible expression system . However, such cells were respiratory deficient and unable to utilise ethanol or glycerol as the sole carbon source . They exhibited gross mitochondrial dysfunction displayed in the loss of respiratory chain complex I, II, III, IV and citrate synthase activities . The effects on mitochondria required a C-terminal domain of Vpr that contains a conserved amino acid sequence motif HFRIGCRHSRIG . These results suggest that the widely observed phenomenon of 'Vpr-induced growth arrest' in human cells could be due to mitochondrial dysfunction. Int J Food Microbiol, 1997 Jun 17, 37(1), 21 - 5 The effects of bakery processing on natural deoxynivalenol contamination; Neira MS et al.; The aim of this study was the evaluation of the influence of the breadmaking process on initial deoxynivalenol (DON) contamination . Samples (92) were taken from four batches of eight different types of products in a low-technology bakery . The final products, as well as the corresponding flours, doughs and fermented doughs were analyzed . Extracts were obtained with acetonitrile:water (84:16), the clean up was made with a multifunctional column and DON was quantified by thin layer chromatography by visual comparison with standards . Confirmation was made by electron capture gas chromatography . The contamination levels in flour samples ranged from 500 micrograms/kg to 2000 micrograms/kg on dry weight basis . The results showed a positive correlation between the initial contamination level and the reduction of DON after fermentation . A significant reduction was observed as a consequence of the breadmaking process. J Biotechnol, 1997 Jun 13, 55(2), 113 - 24 Biosorption of lead and zinc from solutions using Streptoverticillium cinnamoneum waste biomass; Puranik PR et al.; Mycelial wastes of microbial origin from fermentation industries have been recognized as potential biosorbents for decontamination of waste waters containing heavy metals . Dried, nonliving, granulated biomass of Streptoverticillium cinnamoneum was used for the recovery of lead and zinc from solutions . It was found that pretreatment of the biomass with boiling water for 15 min increased the biosorption of lead and zinc by 52 and 41%, respectively . The optimum pH range for lead uptake was 3.5-4.5 while for zinc it was 5.0-6.0 . The lead and zinc adsorption data when applied to Freundlich and Langmuir isotherm equations showed good correlation (r2 = 0.97) and hence equal conformity to both models . The Scatchard plots indicated clearly that more than one type of binding sites were involved in the adsorption of lead and zinc by the biomass . The maximum loading capacity of S . cinnamoneum biomass was found to be 57.7 mg/g for lead and 21.3 mg/g for zinc with boiling water pretreatment . The loaded metals could be desorbed effectively with dilute hydrochloric acid, nitric acid and 0.1 M EDTA . Treatment with 0.1 M sodium carbonate permitted reuse of the desorbed biomass although the metal loading capacity in the subsequent cycles decreased by 14-37% . The metal biosorbent granules prepared are a value-added product that has the potential for removal/recovery of lead and zinc from dilute solutions on a commercial scale. J Exp Med, 1997 Jun 2, 185(11), 1951 - 8 Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration; Muhlradt PF et al.; Macrophages are typically stimulated by components of microbial cell walls . Surprisingly, cell wall-less mycoplasmas can also very efficiently stimulate macrophages . We showed recently that mycoplasma-derived lipopeptides constitute the active principle . We have now isolated a clone of Mycoplasma fermentans expressing mainly one macrophage-stimulating lipopeptide . This lipopeptide was detergent-extracted and isolated by reversed-phase high-performance liquid chromotography, using nitric oxide release from C3H/HeJ mouse macrophages as bioassay for detection . In contrast to "conventional" bacterial lipoproteins, this lipopeptide had a free NH2 terminus . Amino acid composition, sequence, and the molecular weight of 2,163 . 3 are consistent with the following structure: S-(2, 3-bisacyloxypropyl)cysteine-GNNDESNISFKEK with one mole C16:0, and a further mole of a mixture of C18:0 and C18:1 fatty acid per lipopeptide molecule . The sequence could not be found in either the protein identification resource nor the Swiss Prot data bank . We named this 2-kD lipopeptide, macrophage-activating lipopeptide-2 (MALP-2) . Synthetic dipalmitoyl MALP-2 and mycoplasma-derived MALP-2 were compared with the bioassay . Both lipopeptides showed an identical dose dependency with a half-maximal response at 10(-11) M concentration . MALP-2 may be one of the most potent natural macrophage stimulators besides endotoxin. Comp Immunol Microbiol Infect Dis, 1997 Jun, 20(3), 271 - 9 Isolation of verotoxigenic Escherichia coli from the Tasmanian environment; Manandhar R et al.; Growing concerns on the emergence of verotoxin producing Escherichia coli (VTEC) in Australia have focused our attention on the possible sources of VTEC within the island state of Tasmania . An analysis of 156 food samples and 194 water samples obtained from various areas revealed evidence of eight possible sources . Six strains, with serotypes Ont:Hnt, O86:H-, O88:H-, O126:H21 and O134:H-, were isolated from water samples . Two VTEC of serotypes Ont:H8, 081:H- were isolated from raw meat samples . The waterborne isolates produced verocytotoxin . VT1, while both foodborne isolates were strong producers of VT2 . Three VTEC isolates produced haemolysins, only one produced enterohaemolysin (EntHly) and the remaining were reported with alpha-haemolysin (alpha-Hly) activity . An important feature in the majority of isolates from water was their lack of ability to ferment lactose these isolates are routinely overlooked in public health laboratories. Appl Biochem Biotechnol, 1997 Jun, 66(3), 249 - 62 Analysis of biomass cellulose in simultaneous saccharification and fermentation processes; Chung YC et al.; A direct method for determining the cellulose content of biomass residues resulting from simultaneous saccharifiaction and fermentation (SSF) experiment has been developed and evaluated . The method improves on classical cellulose assays by incorporating the enzymatic removal of yeast glucans from the biomass residue prior to acid hydrolysis and subsequent quantification of cellulose-derived glucose . An appropriate cellulase-free, commercially available, yeast-lysing enzyme preparation from Cytophaga was identified . A freeze-drying step was identified as necessary to render the SSF yeast cells susceptible to enzymatic lysis . The method was applied to the analysis of cellulose and yeast-associated glucans in SSF residues from three pretreated feedstocks; hybrid poplar, switchgrass, and cornstover . Cellulose assays employing the lysing-enzyme preparation demonstrated relative errors up to 7.2% when yeast-associated glucans were not removed prior to analysis of SSF residues . Enzymatic lysis of SSF yeast cells may be viewed as a general preparatory procedure to be used prior to subsequent chemical and physical analysis of SSF residues. Vet Med (Praha), 1997 Jun, 42(6), 165 - 9 Survival of model helminth eggs and larvae (Ascaris suum, Oesophagostomum sp.) in the ensilaging process; Juris P et al.; Ascaris suum nonembryonated eggs remained viable for the most part even after 42 days of ensilaging . At the end of the anaerobic fermentation, mean of damaged eggs was 15.2 +/- 4.02 (min . 11, max . 21), 32.9% . Conversely, the viability of Oesophagostomum sp . nonembryonated eggs and infective L3 larvae was reduced-eggs: mean number 23.6 +/- 3.64 (min . 20 . max . 28) specimens (93.3%), L3 larvae: mean number 24.2 +/- 4.38 (min . 19, max . 28) specimens (96.7%), during the period of study (42 days) . Control group of the same helminth propagative stages, was kept under optimum aerobic conditions . After 42 days of exposition, 9.0 +/- 3.46 (min . 5, max . 11) nonembryonated Ascaris suum eggs (12.9%), 17.33 +/- 2.51 (min . 15, max . 20) Oesophagostomum sp . eggs (36.4%) and 3.66 +/- 1.15 (min . 3, max . 5) Oesophagostomum sp . larvae L3 (6.3%) were damaged on average . Helminth eggs, thick-walled and more resistant to the environment in particular, are able to survive the anaerobic process of ensilaging . To protect animals against parasitic diseases, it is necessary to consider the epidemiological hazard of silages and silage juices, which are potentially contaminated by helminth propagative stages . Silages and silage juices under certain conditions may become harmful to polygastric animals. J Antibiot (Tokyo), 1997 Jun, 50(6), 490 - 5 UCE6, a new antitumor antibiotic with topoisomerase I-mediated DNA cleavage activity produced by actinomycetes: producing organism, fermentation, isolation and biological activity; Fujii N et al.; A novel antitumor antibiotic, UCE6 (1,3,8,10,11-pentahydroxy-2-methyl-10-(2-oxo-4-hydroxypentyl)na phthacene-5, 12-dione) with topoisomerase I-mediated DNA cleavage activity, was isolated from the culture broth of actinomycetes strain UOE6 . Addition of silicone oil antifoam agent, KS69 (2%), to the fermentation enhanced the production of UCE6 by approximately 3 fold . A total of 1.15 g of UCE6 was recovered as reddish orange crystals from a 100 liter fermentation supplemented with 2% KS69 . UCE6 exhibited growth inhibitory activity against HeLa S3, HCT116 and Lu-65 cells comparable to that of camptothecin. J Antibiot (Tokyo), 1997 Jun, 50(6), 474 - 8 MR566A and MR566B, new melanin synthesis inhibitors produced by Trichoderma harzianum . II . Physico-chemical properties and structural elucidation; Lee CH et al.; New melanin synthesis inhibitors (MR566A and B) and six related known isocyanocyclopentenes were isolated from the fermentation broth of Trichoderma harzianum, and their structures were elucidated by spectroscopic methods . The structures of novel isocyanides, MR566A (1) and B (2), were elucidated as 1-(3-chloro-1,2-dihydroxy-4-isocyano-4-cyclopenten-1-yl)etha nol, 1-(1,2,3-trihydroxy-3-isocyano-4-cyclopenten-1-yl)ethanol, respectively . The structure of novel oxazole, MR93B (9), was elucidated as 4-{(1Z)-3-hydroxy-2-hydroxymethyl-1-propen-1-yl}oxazole. J Antibiot (Tokyo), 1997 Jun, 50(6), 469 - 73 MR566A and MR566B, new melanin synthesis inhibitors produced by Trichoderma harzianum . I . Taxonomy, fermentation, isolation and biological activities; Lee CH et al.; New melanin synthesis inhibitors (MR566A and B) and six related known isocyanocyclopentenes were isolated from the fermentation broth of Trichoderma harzianum . The IC50 values of MR566A and B against mushroom tyrosinase were 1.72 and 47 microM, respectively . They inhibited melanin biosynthesis in B16 melanoma cells with MIC values of 0.1 and 2.2 microM, respectively . Also isolated from the same culture extract of T . harzianum was a new oxazole (MR93B), which showed no inhibitory activity against mushroom tyrosinase at a concentration of 1,000 microg/ml. J Antibiot (Tokyo), 1997 Jun, 50(6), 457 - 68 N-type calcium channel blockers from a marine bacterium, Cytophaga sp . SANK 71996; Morishita T et al.; N-(3-Acyloxyacyl)glycines were isolated as N-type calcium channel blockers from a marine bacterium Cytophaga sp . SANK 71996 . The identification and fermentation of the producing strain and structure characterization of N-(3-acyloxyacyl)glycines by spectral analyses and chemical syntheses are described together with their antagonistic activities. Appl Microbiol Biotechnol, 1997 Jun, 47(6), 625 - 9 Development of a low-cost fermentation medium for ethanol production from biomass; Kadam KL et al.; Nutrient cost is an important aspect in the fermentation of biomass to ethanol . With a goal of developing a cost-effective fermentation medium, several industrially available nutrient sources were evaluated for their effectiveness in the simultaneous saccharification and fermentation of pretreated poplar with Saccharomyces cerevisiae D5A . These studies showed that a low-cost medium containing 0.3% corn steep liquor and 2.5 mM MgSO4 7H2O was similar in performance to a nutrient-rich medium . Besides its low cost, this alternative medium consists of components that are available on a commercial scale, thereby making it industrially relevant. Nahrung, 1997 Jun, 41(3), 150 - 4 Influence of white light, near-UV irradiation and other environmental conditions on production of aflatoxin B1 by Aspergillus flavus and ochratoxin A by Aspergillus ochraceus; Aziz NH et al.; The effects of illumination, near-ultraviolet, incubation temperature pH and some minor elements on the growth rate and production of aflatoxin B1 by A . flavus and ochratoxin A by A . ochraceus were investigated . Aflatoxin B1 and ochratoxin A production was considerably higher in the light than in the dark . The greatest aflatoxin B1 and ochratoxin A production was occurred after 11 days of fermentation with light- and dark-grown cultures at 25 degrees C . The mycelial dry weight was also greater in the light than in the dark for both A . flavus and A . ochraceus . Exposure of conidia to near-UV irradiation increased mycelial dry weight and mycotoxins by both fungi more than white light . The greatest aflatoxin B1 and ochratoxin A was at 25 degrees C with UV-grown culture (24 h exposure) producing a mean of 400 and 260 micrograms/50 ml of medium, respectively . The maximum aflatoxin B1 and ochratoxin A yield was obtained at pH 5.5 and with increasing the initial pH to near neutrality, both mycotoxins yield decreased . Iron, copper and zinc were observed to stimulate aflatoxin B1 and ochratoxin A production and enhanced the growth rate of both A . flavus and A . ochraceus. Mol Microbiol, 1997 Jun, 24(5), 1049 - 60 'Locked-on' and 'locked-off' signal transduction mutations in the periplasmic domain of the Escherichia coli NarQ and NarX sensors affect nitrate- and nitrite-dependent regulation by NarL and NarP; Chiang RC et al.; The Escherichia coli NarX, NarQ, NarL and NarP proteins comprise a two-component regulatory system that controls the expression of many anaerobic electron-transport and fermentation-related genes in response to nitrate and nitrite . Either of the two sensor-transmitter proteins, NarX and NarQ, can activate the response-regulator proteins, NarL and NarP, which in turn are able to bind at their respective DNA regulatory sites to modulate gene expression . NarX contains a conserved 17 amino acid sequence, designated the 'P-box' element, that is essential for nitrate sensing . In this study we characterize narQ mutants that also confer altered nitrate control of NarL-dependent nitrate reductase (narGHJI) and fumarate reductase (frdABCD) gene expression . While some narQ mutations cause the constitutive activation or repression of reporter-gene expression even when the cells are grown in the absence of the nitrate signal (i.e . a 'locked-on' phenotype), other mutations abolish nitrate-dependent control (i.e . a 'locked-off' phenotype) . Interestingly the narQ (A42-->T) and narQ (R50-->Q) mutations along with the analogous narX18 (A46-->T) and narX902 (R54-->E) mutations also confer a 'locked-on' or a 'locked-off' phenotype in response to nitrite, the second environmental signal detected by NarQ and NarX . Furthermore, these narQ and narX mutations also affect NarP-dependent gene regulation of nitrite reductase (nrfABCDEFG) and aeg-46.5 gene expression in response to nitrite . We therefore propose that the NarQ sensor-transmitter protein also detects nitrate and nitrite in the periplasmic space via its periplasmic domain . A signal transduction model, which we previously proposed for NarX, is now extended to NarQ, in which a nitrate- or nitrite-detection event in the periplasmic region of the cell is followed by a signal transduction event through the inner membrane to the cytoplasmic domain of NarQ and NarX proteins to modulate their protein kinase/phosphatase activities. Can J Cardiol, 1997 Jun, 13(6), 577 - 82 Muscle characteristics in effort angina before and after CABG; Karlsson J et al.; Seven males with effort angina undertook graded ergometer tests and had muscle biopsies taken from their vastus lateralis muscle before, and three and six months after coronary bypass surgery . Muscle fibre composition (percentage of slow twitch fibres), ubiquinone (vitamin Q), and oxidative and fermentative enzyme activities were determined . After six months, muscle ubiquinone and oxidative enzymes were still depressed, indicating sustained muscle trauma . The only peripheral changes were that muscle lactate dehydrogenase and its skeletal muscle-specific subunits and isozymes were increased 35% to 40% (P < 0.001) three to six months postsurgery . Onset of blood lactate accumulation (2.0 mmol/L), symptom-limited ('maximal') exercise and peak blood lactate increased linearly over time (r = 0.52, P < 0.05; r = 0.63, P < 0.01; and r = 0.76, P < 0.001, respectively) . It is suggested that the initial physical performance increase was due to improved circulatory capacity, oxygen delivery and lactate efflux, whereas the increased fermentative capacity ('anaerobic power') first contributed after a lag of three or more months . Whether the muscle histochemical changes reflected a healing process (recovery) is speculative. J Dairy Sci, 1997 Jun, 80(6), 1179 - 84 Influence of tallow and Aspergillus oryzae fermentation extract in dairy cattle rations; Bertrand JA et al.; Objectives were to determine the effects of adding 3 g/d of Aspergillus oryzae fermentation extract to diets with or without 5.6% added tallow . Twenty-eight Holstein cows (mean = 98 d of lactation) were assigned to a randomized block experiment in a 2 x 2 factorial arrangement of treatments . Treatments were the basal diet 1) without tallow or extract, 2) with extract but no tallow, 3) with tallow but no extract, and 4) with tallow and extract . Milk production, dry matter intake, 3.5% fat corrected milk, digestibility of neutral detergent fiber in the total tract were depressed for cows fed tallow . Addition of fermentation extract did not stimulate fiber digestion or milk production of cows fed diets with or without fat . Addition of extract did not overcome depression of fiber digestibility by cows fed tallow. J Dairy Sci, 1997 Jun, 80(6), 1150 - 9 Effects of amount and ruminal degradability of protein on nutrient digestibility and production by cows fed tallow; Weigel DJ et al.; Five cows with ruminal cannulas were used in a 5 x 5 Latin square design to determine the effects of fat and amount and ruminal degradability of dietary crude protein (CP) on nutrient digestibility and production of milk and milk components . Treatments were 1) control; 2) 15% CP, soybean meal; 3) 15% CP, by-product proteins; 4) 18% CP, soybean meal; and 5) 18% CP, soybean meal and by-product proteins . Diets 2 through 5 contained 3.5% tallow . Diets consisted of 28% alfalfa haylage, 22% corn silage, and 50% concentrate on a dry matter (DM) basis . Fat did not affect dry matter intake or percentages and yields of fat and CP in milk but increased milk yield 2.5 kg/d . Fat did not affect N fractions in milk but decreased the percentages of short- and medium-chain fatty acids (C6:0 to C16:0) and increased the percentages of long-chain fatty acids (C18:0 and C18:1) in milk fat . Fat did not affect ruminal fermentation characteristics or the percentages of dietary DM, organic matter, CP, acid detergent fiber, neutral detergent fiber, starch, ether extract, and energy that were digested . An increase in dietary CP from 15 to 18% increased dry matter intake 1.7 kg/d; increased intake of gross energy 8 Mcal/d; increased the percentages and quantities of DM, organic matter, CP, and energy digested; increased the quantities of acid detergent fiber and neutral detergent fiber digested; decreased ruminal pH; increased concentrations of total volatile fatty acids; and increased NH3 N in ruminal fluid . However, the difference in dietary CP did not affect milk yield or composition . Replacement of soybean meal in the diet with a mixture of by-product proteins decreased NH3 N in ruminal fluid, tended to decrease concentrations of total volatile fatty acids and increase pH of ruminal fluid, but did not affect milk yield or percentages and yields of milk components. J Dairy Sci, 1997 Jun, 80(6), 1126 - 35 Effects of cellobiose and monensin on in vitro fermentation of organic acids by mixed ruminal bacteria; Callaway TR et al.; The objective of this study was to determine the effects of cellobiose and monensin on the in vitro fermentation of organic acids (L-aspartate, fumarate, and DL-malate) by mixed ruminal bacteria . Ruminal fluid was collected from a steer fed 36.7 kg of forage and 4.5 kg of concentrate supplement once per day . Ruminal fluid was centrifuged to sediment feed particles and protozoa, and the resulting supernatant, which contained bacteria, was added (33%, vol/vol) to anaerobic media (500 ml) . Incubations (n = 2) were performed in batch culture at 39 degrees C and sampled at 0, 2, 4, 6, 8, 12, and 24 h . Organic acids were added to achieve a final concentration of 7.5 mM . Cellobiose was added to obtain a final concentration of 5 mM, and monensin dissolved in ethanol was included at concentrations of 0 or 5 ppm . Addition of cellobiose to organic acid fermentations increased the rate of organic acid utilization by the mixed bacterial population . Total concentrations of volatile fatty acids were increased by the addition of cellobiose to all fermentations . A lag period (< or = 8 h) occurred in fermentations that were treated with monensin before organic acids were utilized . Total concentrations of volatile fatty acids were increased, and the acetate to propionate ratio was decreased, by monensin treatment . When cellobiose and monensin were added together, propionate production and organic acid utilization were increased . Both cellobiose and monensin affected the in vitro fermentation of organic acids by mixed ruminal bacteria by providing a carbon and energy source and by influencing electron disposal. Biotechnol Appl Biochem, 1997 Jun, 25 ( Pt 3), 235 - 42 Sources and nature of heterogeneity in recombinant phenol hydroxylase derived from the basidiomycetous soil yeast Trichosporon cutaneum; Waters S et al.; Preparations of the dimeric flavoenzyme phenol hydroxylase derived from Trichosporon cutaneum were found to contain an active tetrameric form when the enzyme was produced in Escherichia coli . The relative content of the tetramer was estimated from scans of silver-stained native PAGE gels and/or size-exclusion chromatography (SEC) . Proportions of up to 22% of the enzyme protein, depending on the growth temperature and the level of added inducer, were observed in independent cultures as well as in purified preparations . No tetramer was ever seen in cell extracts or purified preparations from T . cutaneum . Traces of higher multimers and of possibly deamidated species were also detected in preparations of the recombinant enzyme . The rate of enzyme production seems to be the major factor in promoting formation of the tetramer, whereas the specific growth rate of the fermentor culture appears to be of minor importance . The dimeric and the tetrameric forms were purified using either SEC or ion-exchange chromatography as a final step . The two purified species did not interchange under a variety of