J Biol Chem, 2004 Jul 30, 279(31), 32294 - 300 Epub 2004 May 29. Absence of cardiolipin results in temperature sensitivity, respiratory defects, and mitochondrial DNA instability independent of pet56; Zhong Q et al.; Cardiolipin (CL) is a dimeric phospholipid localized primarily in the mitochondrial membrane . Previous studies have shown that yeast cells containing a disruption of CRD1, the structural gene encoding CL synthase, exhibit temperature-sensitive colony formation and multiple mitochondrial defects . A recent report (Zhang, M., Su, X., Mileykovskaya, E., Amoscato, A . A., and Dowhan, W . (2003) J . Biol . Chem . 278, 35204-35210) suggested that defects associated with CL deficiency may result from the reduced expression of PET56 in crd1 Delta mutant backgrounds and should be reevaluated . In the current study, we present evidence that CL deficiency leads to mitochondrial DNA instability, loss of viability, and defects in oxidative phosphorylation at elevated temperatures . The observed mutant phenotypes are characteristic of crd1 Delta mutant cells of both PET56 and pet56 backgrounds and are complemented by an episomal copy of CRD1 but not by expression of the PET56 gene . Phosphatidylglycerol is elevated in crd1 Delta mutant cells when grown in the presence of fermentable and non-fermentable carbon sources, although the extent of the increase is higher in nonfermentable medium . An increase in the ratio of phosphatidylethanolamine to phosphatidylcholine was also apparent in the mutant . These findings demonstrate that CRD1, independent of PET56, is required for optimal mitochondrial function and for an essential cellular function at elevated temperatures. Genetics, 2004 May, 167(1), 523 - 30 Stochastic gene expression in fluctuating environments; Thattai M et al.; Stochastic mechanisms can cause a group of isogenic bacteria, each subject to identical environmental conditions, to nevertheless exhibit diverse patterns of gene expression . The resulting phenotypic subpopulations will typically have distinct growth rates . This behavior has been observed in several contexts, including sugar metabolism and pili phase variation . Under fixed environmental conditions, the net growth rate of the population is maximized when all cells are of the fastest growing phenotype, so it is unclear what fitness advantage is conferred by population heterogeneity . However, unlike ideal laboratory conditions, natural environments tend to fluctuate, either periodically or randomly . Here we use a stochastic population model to show that, during growth in such fluctuating environments, a dynamically heterogenous bacterial population can sometimes achieve a higher net growth rate than a homogenous one . By using stochastic mechanisms to sample several distinct phenotypes, the bacteria are able to anticipate and take advantage of sudden changes in their environment . However, this heterogeneity is beneficial only if the bacterial response rate is sufficiently low . Our results could be useful in the design of artificial evolution experiments and in the optimization of fermentation processes. Yeast, 2004 May, 21(7), 539 - 48 High levels of the mitochondrial large ribosomal subunit protein 40 prevent loss of mitochondrial DNA in null mmf1 Saccharomyces cerevisiae cells; Accardi R et al.; Members of the YERO57c/YJGFc/UK114 protein family have been identified in bacteria and eukaryotes . The budding yeast Saccharomyces cerevisiae contains two different proteins of this family, Hmf1p and Mmf1p . We have previously shown that Mmf1p is a mitochondrial protein functionally related to its human homologue and able to influence the maintenance of mitochondrial DNA . Deletion of Mmf1 results in loss of the mitochondrial genome . Using a multicopy suppression approach, we have identified a protein of the mitochondrial large ribosomal subunit, MRPL40, which stabilizes mtDNA in Deltammf1 cells . Overexpression of MRPL40 did not prevent loss of mtDNA in a mutant strain lacking the mitochondrial protein Abf2p . Thus, MRPL40 does not have a general effect on mtDNA stability, but it may be specific for the mmf1-null strain . We also show that the Deltamrpl40 cells present a similar phenotype to the mmf1-null strain, having reduced mtDNA stability and growth rate . Furthermore, we observed that rho(+)Deltamrpl40 haploid cells can be obtained when tetrads are directly dissected on medium containing a non-fermentable carbon source . Thus, replication and segregation of the mtDNA can occur in the absence of MRPL40 . We also show that another mitochondrial ribosomal protein, MRPL38, is able to overcome the Deltammf1-associated defect . Together, our results suggest a link between Mmf1p and the two mitochondrial ribosomal proteins . Calcif Tissue Int, 2004 Aug, 75(2), 169 - 79 Epub 2004 May 20. Fructooligosaccharides maximize bone-sparing effects of soy isoflavone-enriched diet in the ovariectomized rat; Mathey J et al.; Isoflavones (IF) have been increasingly implicated for use in the prevention of osteoporosis . As their bioavailability could be improved by modulating intestinal microflora, the present study was undertaken to investigate whether IF and fructooligosaccharides (FOS), which are known to modify large-bowel flora and metabolism, may exhibit a cooperative bone-sparing effect . This work was carried out on 3-month-old Wistar rats assigned to 12 groups: 2 SH (sham-operated) and 10 OVX (ovariectomized) . Animals received a diet for 90 days containing total IF (Prevastei HC, Central Soya) at 0 (OVX and SH), 10 (IF10), 20 (IF20), 40 (IF40), or 80 (IF80) microg/g body weight per day . FOS (Actilight, Beghin-Meiji) were orally given to half of the groups, (OVX FOS), (IF10 FOS), (IF20 FOS), (IF40 FOS), (IF80 FOS), and (SH FOS) . Isoflavones exhibited a bone-sparing effect as soon as consumption reached 20 microg/g/day, whereas only the highest dose induced a weak uterotrophic activity . Indeed, total femoral bone mineral density (BMD) was significantly enhanced (compared with that of OVX rats), as was the metaphyseal compartment . Bone strength was improved as well . As far as the FOS diet is concerned, addition of prebiotics significantly raised the efficiency of the IF protective effect on both femoral BMD and mechanical properties . The trend toward higher BMD levels with the lowest IF dose (IF10) even reached a significant level when FOS were added . This effect could be explained by a reduced bone resorption . In conclusion, daily IF consumption prevented castration-induced osteopenia by decreasing bone resorption when given at 20, 40, or 80 microg (total isoflavones)/g/day . Simultaneous FOS consumption improved IF protective effect on the skeleton, with the lowest IF dose becoming efficient . Enhancement of IF bioavailability, following FOS fermentation, is probably involved. Methods Mol Biol, 2004, 276, 77 - 120 Capillary electrophoresis in the analysis and monitoring of biotechnological processes; Klyushnichenko V; Capillary electrophoresis (CE) became a versatile technique for analysis of biological macromolecules . We have applied capillary zone electrophoresis (CZE) and SDS-gel CE for the characterization of recombinant proteins during development of major bioprocessing steps, including fermentation, hybridoma cell cultivation, chromatographic purification, and chemical transformation . Rapid SDS-gel CE was developed for the fast analysis of fermentation broth and hybridoma cell culture . The total analysis time was reduced to 4.5 min . We have developed system for fraction collection, which allows analyzing separated proteins by MALDI-TOF-MS . The main advantages of applied techniques were high resolution and selectivity, fast analysis, and high accuracy. Biotechnol Bioeng, 2004 Jun 30, 86(7), 756 - 64 Rapid chromatography for evaluating adsorption characteristics of cellulase binding domain mimetics; Mosier NS et al.; The cost of cellulolytic enzymes is one barrier to the economic production of fermentable sugars from lignocellulosic biomass for the production of fuels and chemicals . One functional characteristic of cellulolytic enzymes that improves reaction kinetics over mineral acids is a cellulose binding domain that concentrates the catalytic domain to the substrate surface . We have identified maleic acid as an attractive catalytic domain with pK(a) and dicarboxylic acid structure properties that hydrolyze cellulose while producing minimal degradation of the glucose formed . In this study we report results of a rapid chromatographic method to assess the binding characteristics of potential cellulose binding domains for the construction of a synthetic cellulase over a wide range of temperatures (20 degrees to 120 degrees C) . Aromatic, planar chemical structures appear to be key indicators of cellulose adsorption . Indole, the side-chain of the amino acid tryptophan, has been shown to reversibly adsorb to cellulose at temperatures between 30 degrees and 120 degrees C . Trypan blue, a polyaromatic, planar molecule, was shown to be irreversibly adsorbed to cotton cellulose at temperatures of <120 degrees C on the time scale of the experiments . These results confirm the importance of hydrophobic cellulose and the cellulose-binding component of cellulolytic enzymes and cellulolytic enzyme mimetics . J Basic Microbiol, 2004, 44(3), 220 - 31 Production of lignocellulolytic enzymes by Trametes gallica and detection of polysaccharide hydrolase and laccase activities in polyacrylamide gels; Sun X et al.; White rot fungus Trametes gallica was studied for the production of lignocellulolytic enzymes: cellulase, xylanase, laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP) . The results demonstrated that low-nitrogen (2.2 mM N) and surface stationary cultivation favored production of extracellular MnP . MnP activity reached 118.1 UL(-1) while T . gallica was grown in a low-nitrogen culture containing phenylalanine . However, laccase levels observed in high-nitrogen (22 mM N) agitated cultures were much greater than those seen in low-nitrogen . The N source experiments seemed to reveal that NH4+ plays an important role in inducing MnP and laccase of the fungus . Results showed that T . gallica produces a series of the lignocellulolytic enzymes, and needs high N to produce all the enzymes during solid-state fermentation of wheat straw . This paper also presents a modified zymogram procedure to detect xylanase and laccase of T . gallica in polyacrylamide gel . Xylanase in crude enzyme of T . gallica was displayed by contacting protein gel strips with xylan substrate gels and by staining with iodine . By immersing the protein gel strips in o-tolidine solution, the blue-green zones representing laccase activity were visualized against a colorless background . Khirurgiia (Mosk), 2004, (5), 68 - 75 {Combined treatment of acute pancreatitis and its complications}; Blagovestnov DA et al.; Results of combined treatment of 314 patients with acute pancreatitis, including 58 (15.1%) with pancreonecrosis were analyzed . Etiologic factors of acute pancreatitis were alcohol (59% patients), diseases of the bile ducts (31.5%), surgery (2.5%) . Up-to-date diagnostic criteria of severe pancreatitis are presented, character of complications is analyzed . Treatment policy in acute edematous pancreatitis was conservative . In calculous cholecystitis cholecystectomy was performed after regress of acute pancreatitis . Fermentative ascitis-peritonitis was the indication for laparoscopy in aseptic phase of pancreonecrosis . US- and CT-guided puncture and drainage were often used . Surgeries were performed only for complications of pancreonecrosis, more often through mini-approaches . General lethality in acute pancreatitis was 1.9%, in pancreonecrosis - 10.7%, postoperative lethality in pancreonecrosis was 16.6%. Theriogenology, 2004 Jul, 62(1-2), 299 - 310 Effect of undegradable protein supply on testicular size, spermiogram parameters and sexual behavior of mature Assaf rams; Fernandez M et al.; Eighteen mature Assaf rams were used to study the effect of undegradable protein (UDP) supply on testicular size, sperm production and quality, testosterone secretion and reproductive behavior . Animals were allocated to three groups of six animals each and fed during 10 weeks with different diets which were designed to supply approximately 0.5MJ of metabolisable energy (ME)/kg LBW(0.75) and 9g of effective rumen degradable protein (ERDP)/MJ of fermentable ME to all animals and to induce differences in rumen UDP intake (0.97, 1.72 and 3.08g of UDP/kg LBW(0.75) for LP, MP and HP diets, respectively) . Neither plasma testosterone concentration nor reproductive behavior parameters (number of services, number of mounts without ejaculation and reaction time to first mount) were affected (P > 0.05) by protein intake . Nevertheless, there were significant (P < 0.05) differences between diets in both testicular size and sperm production . Scrotal circumference was lower in LP compared to MP and HP groups, no significant differences being observed between these latter two groups at any time . In relation to sperm production, the lowest and the highest values were always observed in LP and HP groups, respectively . MP group showed intermediate values, significantly different from those of LP and HP groups on Week 5 and only from those of LP group on Week 9 . The present results provide a better understanding of the effect of protein nutrition and suggest that UDP should be supplied to Assaf rams during the mating season to improve their reproductive performance. Bioorg Med Chem, 2004 Jun 15, 12(12), 3125 - 33 YM-254890 analogues, novel cyclic depsipeptides with Galpha(q/11) inhibitory activity from Chromobacterium sp . QS3666; Taniguchi M et al.; The structure elucidation and biological activity of novel YM-254890 (1) analogues and semi-synthetic derivatives are described . Three natural analogues, YM-254891 (2), YM-254892 (3), and YM-280193 (4), were isolated from the fermentation broth of Chromobacterium sp . QS3666, and two hydrogenated derivatives, YM-385780 (5) and YM-385781 (6), were synthesized from YM-254890 . Their structures were determined by one- and two-dimensional NMR studies and mass spectrometry . Among these compounds, two natural analogues 2-3 which possessed acyl groups at beta-HyLeu-1 and one derivative 6 whose conformation was similar to that of 1 showed comparable Galpha(q/11) inhibitory activity to that of 1 . This indicates that the acyl beta-HyLeu residue plays an important role in activity and also that the alpha,beta-unsaturated carbonyl group of the N-MeDha residue is not critical to activity . The other hydrogenated derivative 5 had significantly less activity, which could be attributed to conformational differences. Appl Microbiol Biotechnol . 2004 May 20; {Epub ahead of print} Transcriptional analysis of genes for energy catabolism and hydrolytic enzymes in the filamentous fungus Aspergillus oryzae using cDNA microarrays and expressed sequence tags; Maeda H et al.; Aspergillus oryzae is a fungus used extensively in the fermentation industry . We constructed cDNA microarrays comprising 2,070 highly expressed cDNAs selected from the approximately 6,000 non-redundant expressed sequence tags (ESTs) in the A . oryzae EST database . Using the cDNA microarrays, we analyzed the gene expression profiles of A . oryzae cells grown under the glucose-rich (AC) and glucose-depleted (AN) liquid culture conditions used during the construction of the EST database . The sets of genes identified by the cDNA microarray as highly expressed under each culture condition agreed well with the highly redundant ESTs obtained under the same conditions . In particular, transcription levels of most catabolic genes of the glycolytic pathway (EMP) and tricarboxylic acid (TCA) cycle were higher under AC than AN conditions, suggesting that A . oryzae uses both EMP and TCA for glucose metabolism under AC conditions . We further studied the expression of genes encoding hydrolytic enzymes and enzymes involved in energy catabolism by using three industrial solid-phase biomass media, including wheat-bran . The wheat-bran culture gave the richest gene expression profile of hydrolytic enzymes and the lowest expression levels of catabolic genes (EMP, TCA) among the three media tested . The low expression levels of catabolic genes in the wheat-bran culture may release catabolite repression, consequently leading to the rich expression profiles of the hydrolytic enzymes. Methods Mol Biol, 2004, 268, 481 - 7 Differences between biogenic amine detection by HPLC methods using OPA and dansyl derivates; Alberto MR et al.; Biogenic amines can be formed and degraded as a result of normal metabolic activity in animals, plants, and microorganisms and are usually produced by the decarboxylation of amino acids . Recent trends in food security are promoting an increasing search for trace compounds that can affect human health . Although they are present in fermented foods and beverages in low quantities, they exhibit interactions with normal human metabolism (e.g., having vasoactive or psychoactive properties) that justify the research on their presence in foods and the possible related toxicological effects that they may cause.Estimation of the biogenic amines histamine, tyramine, agmatine, putrescine, and cadaverine is important not only from the point of view of their toxicity, but also because they can be used as indicators of the degree of freshness or spoilage of food . Until recently, because of the difficulty in detecting and quantifying amines reliably we have had insufficient information about their occurrence in different types of foods and beverages . These problems are related to matrix interference (e.g., the presence of free amino acids) and the low levels at which the amines are found.Early techniques for the determination of biogenic amines in foods were based on thin-layer chromatography . More modern analytical techniques have since been developed that allow the acquisition of reliable quantitative data and better separation/resolution of various amines . The quantitative determination of biogenic amines is generally accomplished by overpressure-layer chromatography, high-performance liquid chromatography (HPLC), and gas chromatography.The use of reverse-phase column and precolumn derivatization was more efficient and faster than the conventional ion-exchange techniques . This study was conducted to evaluate two HPLC derivatization methods for quantitative determination of biogenic amines: the method described by Gonzales de Llano et al . for amino acid analysis and the method described by Eerola et al. Antimicrob Agents Chemother, 2004 Jun, 48(6), 2185 - 9 Pyruvate decarboxylase, the target for omeprazole in metronidazole-resistant and iron-restricted Tritrichomonas foetus; Sutak R et al.; The substituted benzimidazole omeprazole, used for the treatment of human peptic ulcer disease, inhibits the growth of the metronidazole-resistant bovine pathogen Tritrichomonas foetus in vitro (MIC at which the growth of parasite cultures is inhibited by 50%, 22 microg/ml {63 microM}) . The antitrichomonad activity appears to be due to the inhibition of pyruvate decarboxylase (PDC), which is the key enzyme responsible for ethanol production and which is strongly upregulated in metronidazole-resistant trichomonads . PDC was purified to homogeneity from the cytosol of metronidazole-resistant strain . The tetrameric enzyme of 60-kDa subunits is inhibited by omeprazole (50% inhibitory concentration, 16 microg/ml) . Metronidazole-susceptible T . foetus, which expresses very little PDC, is only slightly affected . Omeprazole has the same inhibitory effect on T . foetus cells grown under iron-limited conditions . Similarly to metronidazole-resistant cells, T . foetus cells grown under iron-limited conditions have nonfunctional hydrogenosomal metabolism and rely on cytosolic PDC-mediated ethanol fermentation. J Vet Med A Physiol Pathol Clin Med, 2004 Mar, 51(2), 46 - 51 Sodium transport across the isolated epithelium of sheep omasum is influenced by luminal ammonia; Martens H et al.; Ammonia is a physiological fermentation product in the forestomachs and is absorbed from the rumen and omasum . Cellular uptake of ammonia affects the intracellular pH of polar and non-polar cells . The effect of the uptake on the pH of the cytosol depends on the predominant form of ammonia . NH(3) uptake and its intracellular protonation tend to alkalinize the cytoplasm, whereas the uptake of NH(4)(+) acidifies the cytoplasm by reversing this reaction . Consequently, the absorption of ammonia across the omasal epithelium could cause a change of the intracellular pH and pH-dependent transport mechanisms like Na/H exchange . Because no information is available about the form of ammonia absorbed in the omasum and, hence, possible modulation of Na transport by ammonia, the effect of increasing luminal ammonia concentrations (0, 5, 15 and 30 mmol/l) on Na transport were studied . In epithelia of hay-fed animals, ammonia linearly inhibited Na transport in a dose-dependent manner, at a luminal pH of 7.40, but not at a pH of 6.40 . Ammonia did not influence Na transport in epithelia of concentrate-fed animals . Because luminal ammonia did not consistently change the short circuit current or tissue conductance absorption of ammonia as NH(4)(+) appears to be unlikely . The predominant form of ammonia absorbed in the omasum is probably NH(3), which is protonated in the cytosol . The reduced availability of protons may be the cause of inhibition of Na transport via Na/H exchange. Biochem J, 2004 Sep 1, 382(Pt 2), 519 - 26 Isolation and functional expression of human COQ2, a gene encoding a polyprenyl transferase involved in the synthesis of CoQ; Forsgren M et al.; The COQ2 gene in Saccharomyces cerevisiae encodes a Coq2 (p-hydroxybenzoate:polyprenyl transferase), which is required in the biosynthetic pathway of CoQ (ubiquinone) . This enzyme catalyses the prenylation of p-hydroxybenzoate with an all-trans polyprenyl group . We have isolated cDNA which we believe encodes the human homologue of COQ2 from a human muscle and liver cDNA library . The clone contained an open reading frame of length 1263 bp, which encodes a polypeptide that has sequence homology with the Coq2 homologues in yeast, bacteria and mammals . The human COQ2 gene, when expressed in yeast Coq2 null mutant cells, rescued the growth of this yeast strain in the absence of a non-fermentable carbon source and restored CoQ biosynthesis . However, the rate of CoQ biosynthesis in the rescued cells was lower when compared with that in cells rescued with the yeast COQ2 gene . CoQ formed when cells were incubated with labelled decaprenyl pyrophosphate and nonaprenyl pyrophosphate, showing that the human enzyme is active and that it participates in the biosynthesis of CoQ. J Antibiot (Tokyo), 2004 Mar, 57(3), 197 - 204 CE-108, a new macrolide tetraene antibiotic; Perez-Zuniga FJ et al.; In the search for strains producing antifungal compounds, a new tetraene macrolide CE-108 (3) has been isolated from culture broth of Streptomyces diastaticus 108 . In addition, the strain also produces the previously described tetraene rimocidin (1) and also the aromatic polyketide oxytetracycline . Both tetraene compounds, structurally related, are produced in a ratio between 25 to 35% (CE-108 compared to rimocidin), although it can be inverted toward CE-108 production by changing the composition of the fermentation medium . This paper deals with the characterization of the producer strain, fermentation, purification, structure determination and biological properties of the new macrolide tetraene CE-108. J Antibiot (Tokyo), 2004 Mar, 57(3), 180 - 7 Pladienolides, new substances from culture of Streptomyces platensis Mer-11107 . II . Physico-chemical properties and structure elucidation; Sakai T et al.; In the course of our screening using fermented broth from soil microorganisms, novel metabolites (pladienolides), possessing inhibitory activity against vascular endothelial growth factor (VEGF) expression and cancer cell proliferation, were isolated from Streptomyces platensis Mer-11107 . Pladienolides A (1), B (2), C (3), D (4), E (5), F (6), and G (7) were found to be novel 12-membered macrolides by spectroscopic studies including 1H, 13C NMR, HMQC, HMBC, and NOE experiments . Pladienolides are unusual 12-membered macrolides having a long side chain at the carbon that bears a lactone oxygen. J Antibiot (Tokyo), 2004 Mar, 57(3), 173 - 9 Pladienolides, new substances from culture of Streptomyces platensis Mer-11107 . I . Taxonomy, fermentation, isolation and screening; Sakai T et al.; Seven new macrolides having a 12-membered ring, which we termed pladienolides, were isolated from the fermentation broth of Streptomyces platensis Mer-11107 . Six of the seven pladienolides inhibited hypoxia-induced reporter gene expression controlled by human VEGF promoter with IC50 values of 0.0018-2.89 microM . They also demonstrated growth-inhibitory activity against U251 human glioma cells in vitro . Pladienolides are highly potent inhibitors of both hypoxia signals and cancer cell proliferation, and thus may be useful as antitumor agents. Di Yi Jun Yi Da Xue Xue Bao, 2004 May, 24(5), 517 - 20 {Conditions for large-scale production of genetically engineered E.coli bearing humanized anti- HBsAg Fab}; Zheng DY et al.; OBJECTIVE: To define the conditions for large-scale production of genetically engineered E.coli bearing humanized anti-HBsAg Fab . METHOD: Characteristic growth and expression of the engineered E.coli were observed during fermentation in the shaking flask to define the optimal culture conditions to achieve the highest production levels . On the basis of the observation results, the E.coli was cultured in a fermentor using the fed-batch method to determine the optimal production techniques . RESULTS: Observation of the bacterium in the shaking flask showed initiation of the induction procedure in the mid-log growth phase at 25 degrees Celsius; with 0.2% arabinose resulted in the highest production of anti-HBsAg Fab . The D(600) value of the culture reached 55.2, equivalent to 110 g/L wet weight of the bacterium, using the DO-stat fed-batch method . The resultant Fab showed well-preserved biological activity . CONCLUSION: Reliable techniques for rapid and massive production of the Fab have been developed. J Gastroenterol Hepatol, 2004 Jun, 19(6), 632 - 7 Association between diet and esophageal cancer in Taiwan; Hung HC et al.; BACKGROUND: Several studies have reported the importance of dietary factors in the development of esophageal cancer . The purpose of the present study was to evaluate the effects of several common dietary factors on the risk of squamous cell carcinoma of the esophagus in a Taiwanese population . METHODS: The association between diet and esophageal cancer was examined in 284 male patients and 480 male controls, who were recruited during 6 year period . RESULTS: Consumption of preserved and overheated foods was found to be associated with increased risk of esophageal cancer, whereas intake of fresh fruits, vegetables, and tea was inversely associated with this risk . Men who consumed fermented bean products, salted food and preserved/pickled vegetables more than once a week after age 40 years had a 3.4-fold risk (95% confidence interval (CI): 1.9-6.2), 2.3-fold risk (95%CI: 1.2-4.2), and 2.5-fold risk (95%CI: 1.3-4.5), respectively, compared to men eating these items less than once a week . It was further found that these preserved foods were more strongly associated with esophageal cancer among men who consumed fruit less than once per day than those who consumed fruits one or more times per day . CONCLUSIONS: These results suggest that a high intake of preserved foods and overheated drinks might increase the risk of esophageal cancer, and intake of fruit, vegetables, and tea might be negatively associated with risk of esophageal cancer . The results also suggest that diet is an important factor in the development of esophageal cancer in Taiwan. J Antimicrob Chemother, 2004 Jul, 54(1), 56 - 9 Epub 2004 May 18. Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) as a target for anti-amoebic agents; Espinosa A et al.; OBJECTIVES: The current use of metronidazole as an anti-amoebic agent causes significant side-effects . The purpose of this study was to identify alternative compounds with which to treat amoebiasis . METHODS: We tested the effects of cyclopropyl (CPC) and cyclobutyl (CBC) carbinols on the survival of Entamoeba histolytica trophozoites and on the enzymatic activities of E . histolytica alcohol dehydrogenase 2 (EhADH2), a crucial enzyme in the amoebic fermentation pathway . RESULTS: At 72 h, the estimated 50% inhibitory concentrations of CPC and CBC were 38.9 and 11.2 microM, respectively . The EhADH2 alcohol and aldehyde dehydrogenase activities were inhibited by 1.82 microM CPC and 0.89 microM CBC in vitro . CONCLUSIONS: CPC and CBC are expected to be non-toxic to humans at the concentrations required to eliminate E . histolytica trophozoites . Similarities between EhADH2 and the Giardia lamblia AdhE enzyme indicate that CPC and CBC could be effective drugs for treatment of both amoebiasis and giardiasis. Commun Agric Appl Biol Sci, 2003, 68(4 Pt A), 123 - 8 Efficacy of Paecilomyces lilacinus (strain 251) for the control of root-knot nematodes; Kiewnick S et al.; The egg pathogenic fungus Paecilomyces lilacinus (strain 251), is a unique strain with a wide range of activity against the most important plant parasitic nematodes . Due to increased production capacity by solid state fermentation and a new water dispersible granule (WDG) formulation, this biological nematicide may be used in an integrated approach to control plant parasitic nematodes . Dose response experiments were conducted with the root-knot nematode Meloidogyne incognita on tomatoes using the new WDG formulation . The results revealed a clear correlation between rate applied and the degree of control concerning the reduction in damage to the root and multiplication of the nematode . Best control was achieved by applying the biological nematicide at rates of 2 to 4 times 10(9) conidia per plant as a soil treatment one week before planting . Monitoring the P . lilacinus population in the rhizosphere showed a decline after 2 to 3 month which can lead to insufficient control over a full growing season . Repeated application to maintain the antagonist population at a sufficient level could be used to secure long term control of root-knot nematodes. Nahrung, 2004 Apr, 48(2), 91 - 4 Effect of fermentation on the starch digestibility, resistant starch and some physicochemical properties of sorghum flour; Elkhalifa AE et al.; The effect of natural fermentation of Tabat sorghum cultivar (Sorghum bicolor L . Moench) at 37 degrees C for up to 36 h on pH, titratable acidity, starch digestibility, resistant starch and total starch was studied . The pH of the fermenting dough decreased sharply with a concomitant increase in the titratable acidity . In vitro starch digestibility markedly increased as a result of fermentation, while resistant starch and total starch decreased . Results showed that iodine absorption capacity increased during fermentation . Fermented sorghum had more soluble starch and swelling power at 100 degrees C than at 85 degrees C. J Anim Sci, 2004 May, 82(5), 1528 - 35 Daily and alternate-day supplementation of urea or biuret to ruminants consuming low-quality forage: III . Effects on ruminal fermentation characteristics in steers; Currier TA et al.; Five ruminally and duodenally cannulated steers (491 +/- 21 kg BW) were used in an incomplete 5 x 4 Latin square with four 24-d periods to determine the influence of supplemental nonprotein N (NPN) source and supplementation frequency (SF) on the dynamics of ruminal fermentation in steers consuming low-quality grass straw (4% CP) . Treatments (TRT) included an unsupplemented control (CON) and a urea or biuret supplement that were placed directly into the rumen at 0700 daily (D) or every other day (2D) . The NPN treatments were formulated to provide 90% of the estimated degradable intake protein requirement; therefore, the urea and biuret treatments received the same amount of supplemental N over a 2-d period . Daily TRT were supplemented with CP at 0.04% of BW/d, whereas the 2D TRT were supplemented at 0.08% of BW every other day . Forage was provided at 120% of the previous 5-d average intake in two equal portions at 0715 and 1900 . Ruminal fluid was collected 0, 3, 6, 9, 12, and 24 h after supplementation on a day of and a day before supplementation for all TRT . Ruminal NH3-N increased (P < 0.04) with CP supplementation on the day all supplements were provided and on the day on which only daily supplements were provided compared with the CON . However, an NPN source x SF interaction (P = 0.03) on the day all supplements were provided indicated that NH3-N increased at a greater rate for urea as SF decreased compared with biuret . Ruminal NH3-N on the day only daily supplements were provided was greater (P = 0.02) for D compared with 2D . On the day all supplements were provided, D increased (P = 0.05) ruminal indigestible acid detergent fiber passage rate and ruminal fluid volume compared with 2D . These results suggest that urea or biuret can be used effectively as a supplemental N source by steers consuming low-quality forage without adversely affecting ruminal fermentation, even when provided every other day. J Anim Sci, 2004 May, 82(5), 1392 - 400 Effects of Sapindus saponaria fruits on ruminal fermentation and duodenal nitrogen flow of sheep fed a tropical grass diet with and without legume; Abreu A et al.; Six adult African-type hair sheep (BW = 40.3 +/- 6.3 kg) fitted with ruminal and duodenal cannulas were subjected to four treatments . Sheep were offered basal diets at a rate of 80 g of DM/kg of metabolic BW (equivalent to ad libitum access) consisting either of a low-quality grass hay (Brachiaria dictyoneura, 3.7% CP, DM basis) alone or in combination with a forage legume (Cratylia argentea, 18.6% CP, DM basis) in a 3:1 ratio (DM basis) . In addition, 0 or 8 g of DM of Sapindus saponaria fruits (12.0% crude saponins, DM basis) per kilogram of metabolic BW was administered intraruminally . Supplementation of C . argentea increased intakes of OM (+21%; P < 0.01) and CP (+130%; P < 0.001), as well as ruminal fluid ammonia N concentrations (from 2.40 to 8.43 mg/dL; P < 0.001) . Apparent OM and N digestibilities were not affected by legume addition, but ADF digestibility decreased by 10% (P < 0.01) . Total ruminal VFA concentration was unchanged, but acetate:propionate was lower (P < 0.01) and isobutyrate proportion was greater (P < 0.001) with the legume addition . Legume supplementation increased duodenal flows of total N (+56%; P < 0.001), nonammonia N (+52%; P < 0.001), ruminal escape N (+80%; P < 0.001), and microbial N (+28%; P < 0.05) . Microbial efficiency was not affected by legume addition . Supplementation of S . saponaria increased (P < 0.05) dietary OM intake by 14%, but had no effect on CP intake and ruminal fluid ammonia concentration or on OM and N digestion . Digestibility of ADF was decreased (P < 0.01) by 10% with S . saponaria as was acetate:propionate (P < 0.001) and the isobutyrate proportion (P < 0.001) . Ruminal protozoa counts increased (P < 0.01) by 67% with S . saponaria . Duodenal N flows were not significantly affected by S . saponaria supplementation, except for microbial N flow (+34%; P < 0.01) . Microbial efficiency was greater (P < 0.05) by 63% with the addition of S . saponaria . Few interactions between legume and S . saponaria supplementation were observed . The NDF digestibility was decreased with S . saponaria in the grass-alone diet, but not in the legume-supplemented diet (interaction; P < 0.05) . Interactions were absent in ruminal fermentation measures and duodenal N flow, indicating that effects were additive . Results suggest that, even when not decreasing ruminal protozoa count, supplementation of S . saponaria fruits is a beneficial way to improve ruminal VFA profile, microbial efficiency, and duodenal flow of microbial protein in sheep fed tropical grass-alone or grass-legume diets. J Anim Sci, 2004 May, 82(5), 1380 - 91 Prediction of nutritive values in grass silages: II . Degradability of nitrogen and dry matter using digestibility, chemical composition, and fermentation data; Yan T et al.; One hundred thirty-six perennial rye-grass silages with wide variations in quality were evaluated for N and DM degradability in three beef steers offered grass silage of medium quality ad libitum . The silages were incubated in the rumen of each animal in triplicate for 0, 6, 12, 24, 48, and 72 h, respectively . The disappearance rates of N or DM were used to calculate the readily soluble fraction ("a" value), potentially degradable fraction ("b" value), and the fractional degradation rate of"b" ("c" value) . The effective degradability (P) of N or DM was then estimated assuming a ruminal outflow rate of 0.02, 0.05, or 0.08/h (P0.02, P0.05, or P0.08) . The objective was to use these data to develop prediction equations for N and DM degradability in grass silages . There were considerable variations in "a," "b," and "c" values and the P0.02, P0.05, or P0.08 of N and DM (e.g., the P0.02 of N ranged from 75.0 to 93.4% and the P0.02 of DM from 51.5 to 82.5%) . The P0.02, P0.05, or P0.08 of N and DM were negatively related (P < 0.001) to ADF, NDF, and lignin concentrations but positively related (P < 0.001) to protein fractions (CP, soluble CP, and true protein concentrations) and digestibility of DM, OM, GE, CP, and NDF and digestible OM in the total DM (measured with sheep) . The N and DM degradability data were also positively related to silage lactic acid concentration, but the relationships between DM degradability data and pH, ammonia N/total N, and VFA concentration in silages were negative (P < 0.05) . Several sets of prediction equations (linear and multiple) were thus developed for N and DM degradability using CP or NDF concentration, "a" value or digestibility data as primary predictors, together with or without other nutrient concentration and silage fermentation variables . All these relationships were highly significant (P < 0.001), and each predictor had a significant effect on the relationship (P < 0.05) . The R2 values in multiple regression for N and DM degradability were generally over 0.70 and higher than in linear regression equations . Four equations were also developed to convert N and DM degradability at a given ruminal outflow rate, predicted using the above-mentioned equations, to their counterparts at any ruminal outflow rate (0.02 to 0.10/h), respectively. Ukr Biokhim Zh, 2003 Nov-Dec, 75(6), 115 - 22 {Effect of ATP-sensitive potassium channel modulators and intermittent hypoxia on mitochondrial respiration during stress}; Tkachenko HM et al.; The influence of activator of ATP-sensitive potassium channels (KATP) pinacidil and blocker glibenclamide after intermittent hypoxia in rats under stress condition on ADP-stimulated mitochondrial respiration by Chance and lipid peroxidation processes in liver have been investigated . We used next substrates of oxidation--0.35 mM succinate, 1 mM alpha-ketoglutarate, 3 mM glutamate, 3 mM pyruvate, 2.5 mM malate and inhibitor of the mitochondrial fermentative complex I (10 microM rotenone), succinate dehydrogenase inhibitor (2 mM malonate) and inhibitor of transamination (1 mM aminooxiacetate) . We suggest that adaptation by intermittent hypoxia and application of a KATP opener pinacidil possess significant protective effect on mitochondrial energy support under stress condition . Combination of intermittent hypoxia with pinacidil causes more efficient consumption of oxygen and decrease of lipid peroxidation processes comparative to intermittent hypoxia or pinacidil injection used separately . We conclude about the existence of the functional link between nitric oxide which is being increased under intermittent hypoxia and KATP opener . Both intermittent hypoxia and pinacidil effectively decrease the negative results of mitochondrial dysfunction under stress condition. Plant Biol (Stuttg), 2004 May, 6(3), 299 - 306 Interaction of flooding with carbon metabolism of forest trees; Kreuzwieser J et al.; Waterlogging and flooding cause oxygen deprivation in the root system of trees . Since oxygen is essentially for mitochondrial respiration, this process cannot be maintained under anoxic conditions and must be replaced by other pathways . For the roots it is therefore a matter of survival to switch from respiration to alcoholic fermentation . Due to the low efficiency of this process to yield energy equivalents (ATP), energy and carbon metabolism of trees are usually strongly affected by oxygen deprivation, even if a rapid switch from respiration to fermentation is achieved . The roots can compensate for the low energy yield of fermentation either (1) by decreasing the demand for energy by a reduction of energy-dependent processes such as root growth and/or nutrient uptake, or (2) by consuming more carbohydrates per unit time in order to generate sufficient energy equivalents . In the leaves of trees, flooding and waterlogging cause a decline in the rates of photosynthesis and transpiration, as well as in stomatal conductance . It is assumed that, due to reduced phloem transport, soluble sugars and starch accumulate in the leaves of flooded trees, thereby negatively affecting the sugar supply of the roots . Thus, root growth and survival is negatively affected by both changes in root internal carbon metabolism and impaired carbon allocation to the roots by phloem transport . In addition, accumulation of toxic products of fermentation in the roots, such as acetaldehyde, can further impair root metabolism . A main feature of tolerance against flooding and waterlogging of trees seems to be the steady supply of carbohydrates to the roots in order to maintain alcoholic fermentation; in addition, roots of tolerant trees seem to avoid accumulation of fermentation-derived ethanol and acetaldehyde . From studies with flooding tolerant and non-tolerant tree species, it is hypothesized that (1) the transport of ethanol produced in the roots under hypoxic conditions into the leaves via the transpiration stream, (2) its conversion into acetyl-CoA in the leaves, and (3) its use in the plant's general metabolism, are mechanisms of flooding tolerance of trees. Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 739 - 43 Anaerobranca californiensis sp . nov., an anaerobic, alkalithermophilic, fermentative bacterium isolated from a hot spring on Mono Lake; Gorlenko V et al.; A novel, obligately anaerobic, alkalithermophilic, chemo-organotrophic bacterium was isolated from the sediment of an alkaline hot spring located on Paoha Island in Mono Lake, California, USA . This rod-shaped bacterium was motile via peritrichous flagella . Isolated strains grew optimally in 5-25 g NaCl l(-1), at pH 9.0-9.5 and at a temperature of 58 degrees C and were fermentative and mainly proteolytic, utilizing peptone, Casamino acids and yeast extract . Optimal growth was seen in the presence of elemental sulfur, polysulfide or thiosulfate with concomitant reduction to hydrogen sulfide . Sulfite was also formed in an equal ratio to sulfide during reduction of thiosulfate . The novel isolate could also reduce Fe(III) and Se(IV) in the presence of organic matter . On the basis of physiological properties, 16S rRNA gene sequence and DNA-DNA hybridization data, strain PAOHA-1(T) (=DSM 14826(T)=UNIQEM 227(T)) belongs to the genus Anaerobranca and represents a novel species, Anaerobranca californiensis sp . nov. Bioorg Med Chem, 2004 Jun 1, 12(11), 3009 - 17 New dibenzotropolone derivatives characterized from black tea using LC/MS/MS; Sang S et al.; Theaflavins and thearubigins are major pigments in black tea, and it is generally accepted that they are produced by oxidation of flavan-3-ols (catechins) during tea fermentation . In the course of studies on the oxidation mechanism of tea polyphenols, especially the formation of thearubigins, a method combining the enzymatic synthesis and LC/ESI-MS/MS analysis was developed to search for new higher molecular weight polymers from black tea . Three new dibenzotropolones, theadibenzotropolone A, B, and C, together with one new tribenzotropolone, theatribenzotropolone A, were formed by the reaction of theaflavins and tea catechins with horseradish peroxidase in the presence of H(2)O(2) . The structures of these new benzotropolone derivatives were elucidated on the basis of MS and 2D NMR spectroscopic analyses . The existence of these compounds in black tea was characterized by LC/ESI-MS/MS . Theadibenzotropolone A and B were the first benzotropolone-type trimers of catechins found in the black tea extract . The observation that galloyl ester groups of theaflavins can be oxidized to form di- or tri-benzotropolone skeletons strongly implied that this type of oxidation is an important pathway to extend the molecular size of thearubigins. J Pharm Pharmacol, 2004 May, 56(5), 649 - 54 The antioxidant drink effective microorganism-X (EM-X) pre-treatment attenuates the loss of nigrostriatal dopaminergic neurons in 6-hydroxydopamine-lesion rat model of Parkinson's disease; Datla KP et al.; There is continued interest in the assessment and potential use of antioxidants as neuroprotective agents in diseases associated with increased oxidative stress, such as Parkinson's disease . The neuroprotective effect of a natural antioxidant drink, EM-X (a ferment derivative of unpolished rice, papaya and seaweeds with effective microorganisms), was investigated using the 6-hydroxydopamine (6-OHDA)-lesion rat model of Parkinson's disease . The nigrostriatal dopaminergic neurons were unilaterally lesioned with 6-OHDA (8 microg) in rats that were treated with a 10-times diluted EM-X drink (dilEM-X), standard EM-X drink (stdEM-X) or tap water for 4 days . Seven days post lesion, the integrity (no . of tyrosine hydroxylase positive cells (TH+ cells) in the substantia nigra pars compacta (SNpc)) and functionality (dopamine and its metabolites DOPAC and HVA content in the striata) of nigrostriatal dopaminergic neurons were assessed . In the vehicle-treated rats, infusion of 8 microg of 6-OHDA significantly reduced the number of TH+ cells in the SNpc as well as the levels of dopamine, DOPAC and HVA in the striata on the lesion side . The loss of TH+ cells, dopamine and HVA, but not the DOPAC levels, was significantly attenuated by stdEM-X pretreatment, but not by the dilEM-X pretreatment . There were no significant changes in the TH+ cells, or in the monoamine levels with the EM-X pretreatment per se, except for a small but significant fall in the levels of dopamine with the stdEM-X . The evidence presented supports the potential neuroprotective effects of stdEM-X drink, although its effect on dopamine levels needs further investigation. Ital J Biochem, 2003 Dec, 52(4), 145 - 53 Proteomic characterization of a wild-type wine strain of Saccharomyces cerevisiae; Trabalzini L et al.; Saccharomyces cerevisiae is the optimal eukaryotic model system to study mammalian biological responses . At the same time Saccharomyces cerevisiae is also widely utilized as a biotechnological tool in the food industry . Enological Saccharomyces cerevisiae strains have been so far routinely analyzed for their microbiological aspects . Nevertheless, wine yeasts are gaining an increasing interest in the last years since they strongly affect both the vinification process and the organoleptic properties of the final product wine . The protein repertoire is responsible of such features and, consequently, 2D-PAGE can be an useful tool to evaluate and select optimal wine yeast strains . We present here the first proteomic map of a wild-type wine Saccharomyces cerevisiae strain selected for the guided fermentation of very high quality wines. J Appl Microbiol, 2004, 96(6), 1296 - 305 Morphological and rheological properties of the three different species of basidiomycetes Phellinus in submerged cultures; Hwang HJ et al.; AIMS: The objective of this work was to investigate the morphological and rheological properties in submerged culture of the three different basidiomycetes Phellinus (P . baumii, P . gilvus and P . linteus) that produce pharmacologically important exopolysaccharides (EPS) . METHODS AND RESULTS: In flask cultures, pH proved to be a critical factor affecting mycelial growth, morphological change and EPS production . The macroscopic morphologies observed under different pHs in flask cultures were also comparable: i.e . starfish-like pellets with a lesser extent of free mycelium appeared in P . baumii, whereas smooth pellets with higher amounts of free mycelium were observed in P . gilvus and P . linteus . The pelleted fermentations were further characterized in a 5-l stirred-tank fermenter by image analysis with respect to mean diameter, core area and pellet circularity . Phellinus baumii showed the largest pellet size (5.2 mm in diameter), whereas P . linteus had extremely small and spherical pellets . The culture broth of P . baumii and P . gilvus yielded extremely high apparent viscosities, ranging from 5 to 7 Pa s . CONCLUSIONS: Three important species of Phellinus showed significantly different morphological and rheological properties . The morphological variation of the three Phellinus species was closely linked to EPS productivity and the apparent viscosity of the whole broth . SIGNIFICANCE AND IMPACT OF THE STUDY: The morphological change in the three species of Phellinus was a good indicator for identifying cell activity for EPS production . Our finding may be beneficial for further optimization of other fungal fermentation processes for large-scale production of EPS. Zhongguo Zhong Yao Za Zhi, 2003 Jul, 28(7), 650 - 3 {Effect of Cryptoporus volvatus (Peck) Schear on leukotriene production from polymorphonuclear leukocytes in rats}; Jin SH et al.; OBJECTIVE: To study action of Cryptoporus volvatus ferment substance (CVFS) on leukotriene production of polymorphonuclear leukocytes in rats . METHODS: The level of slow reaction substance (SRS) and leukotriene B4 (LTB4) in polymorphonuclear leukocytes (PMNs) in rats in vitro were determined with bioassay and HPLC . RESULTS: CVFS 0.9, 2.7 g.kg-1 by ig significantly inhibited SRS and LTB4 production in PMNs in rats in vivo . CONCLUSION: The inhibition effect of CVFS on SRS and LTB4 release may be related to its mechanism of anti-inflammation and anti-asthma. Int J Mol Med, 2004 Jun, 13(6), 821 - 7 Degradation of wheat allergen in Japanese soy sauce; Kobayashi M et al.; Soy sauce is a traditional fermented seasoning of East Asian countries and is available throughout the world . Wheat and soybeans are the 2 main raw materials of soy sauce, and soy sauce also contains a high concentration of salt . Since wheat allergy is considered a serious problem globally, it is significant to examine the allergenicity of soy sauce . In this study, by immunoblotting, inhibition ELISA and direct ELISA using sera from 5 children with wheat allergy, it was clearly demonstrated that wheat allergens were degraded into amino acids and peptides losing the IgE-binding ability in both salt-soluble and salt-insoluble fractions of soy sauce during fermentation . Furthermore, no wheat allergen was detected in 10 items of commercial soy sauce in Japan, by inhibition ELISA or direct ELISA using the sera of patients . In the brewing process of soy sauce, first salt-insoluble wheat allergen was solubilized to salt water during the koji stage (mold cultivation and enzyme production), and second both the resultant salt-solubilized and initially salt-soluble wheat allergens were completely degraded during the moromi stage (fermentation) by microbial proteolytic enzymes . Therefore, it was concluded that no wheat allergen is contained in soy sauce. Br J Nutr, 2004 May, 91(5), 673 - 81 Relative stability of transgene DNA fragments from GM rapeseed in mixed ruminal cultures; Sharma R et al.; The use of transgenic crops as feeds for ruminant animals has prompted study of the possible uptake of transgene fragments by ruminal micro-organisms and/or intestinal absorption of fragments surviving passage through the rumen . The persistence in buffered ruminal contents of seven different recombinant DNA fragments from GM rapeseed expressing the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) transgene was tracked using PCR . Parental and transgenic (i.e . glyphosphate-tolerant; Roundup Ready, Monsanto Company, St Louis, MO, USA) rapeseed were incubated for 0, 2, 4, 8, 12, 24 and 48 h as whole seeds, cracked seeds, rapeseed meal, and as pelleted, barley-based diets containing 65 g rapeseed meal/kg . The seven transgene fragments ranged from 179 to 527 bp and spanned the entire 1363 bp EPSPS transgene . A 180 bp ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit fragment and a 466 bp 16S rDNA fragment were used as controls for endogenous rapeseed DNA and bacterial DNA respectively . The limit of detection of the PCR assay, established using negative controls spiked with known quantities of DNA, was 12.5 pg . Production of gas and NH3 was monitored throughout the incubation and confirmed active in vitro fermentation . Bacterial DNA was detected in all sample types at all time points . Persistence patterns of endogenous (Rubisco) and recombinant (EPSPS) rapeseed DNA were inversely related to substrate digestibility (amplifiable for 48, 8 and 4 h in whole or cracked seeds, meal and diets respectively), but did not differ between parental and GM rapeseed, nor among fragments . Detection of fragments was representative of persistence of the whole transgene . No EPSPS fragments were amplifiable in microbial DNA, suggesting that transformation had not occurred during the 48 h incubation . Uptake of transgenic DNA fragments by ruminal bacteria is probably precluded or time-limited by rapid degradation of plant DNA upon plant cell lysis. J Agric Food Chem, 2004 May 19, 52(10), 2989 - 96 Extrusion conditions affect chemical composition and in vitro digestion of select food ingredients; Dust JM et al.; An experiment was conducted to determine the effects of extrusion conditions on chemical composition and in vitro hydrolytic and fermentative digestion of barley grits, cornmeal, oat bran, soybean flour, soybean hulls, and wheat bran . Extrusion conditions altered crude protein, fiber, and starch concentrations of ingredients . Organic matter disappearance (OMD) increased for extruded versus unprocessed samples of barley grits, cornmeal, and soybean flour that had been hydrolytically digested . After 8 h of fermentative digestion, OMD decreased as extrusion conditions intensified for barley grits and cornmeal but increased for oat bran, soybean hulls, and wheat bran . Total short-chain fatty acid production decreased as extrusion conditions intensified for barley grits, soybean hulls, and soybean flour . These data suggest that the effects of extrusion conditions on ingredient composition and digestion are influenced by the unique chemical characteristics of individual substrates. J Agric Food Chem, 2004 May 19, 52(10), 2953 - 61 Changes of the metal composition in German white wines through the winemaking process . A study of 63 elements by inductively coupled plasma-mass spectrometry; Castineira Gomez Mdel M et al.; Elemental patterns are often used for the classification or identification of the origin of wines . A prerequisite is that the concentration of the elements is not strongly influenced by the addition of different substances such as yeast and fining products during the winemaking process . Inductively coupled plasma-mass spectrometry (ICP-MS) has been used in this study to determine in total 63 elements (including some nonmetals and the rare earth elements) in five German white wines from five regions of origin . The whole winemaking process was studied, from the must to the ready wine . Microwave acid digestion was used for sample preparation, and indium was added as internal standard for a semiquantitative analysis . Two winemaking processes were compared: with the addition of clarifying agents (bentonites) before and after the fermentation . The concentration of only a few elements such as Li, B, Mg, Ca, Rb, Cs, and Pb seems to be constant throughout the whole winemaking process (changes of <+/-50%) and are independent of the time of addition of the bentonites . When bentonites are added before fermentation, the concentration of other elements, such as V, Co, and Fe, remains constant . If bentonites are added after fermentation, the concentration of some other elements such as Sr, Zn, and Mn is nearly unaffected . These elements are therefore robust elements for origin studies in German white wines. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004 Apr, 39(4), 939 - 49 Enhancing anaerobic fermentation of sewage sludge for increasing biogas generation; Bien JB et al.; The article presents results of biogas generation from sewage sludge after applying two pretreatment methods: sonification and thermal hydrolysis . Original results and literature data of the ultrasonic field influence on biogas generation were compared with literature data concerning effectiveness of the thermal hydrolysis . Sludge pretreatment by the ultrasonic field intensified the biogas production as the amounts of biogas was of ca . 20-24% higher, as compared to the nontreated sludge . The highest generation of biogas was observed after a shorter time . The degree of organic matter reduction was of 45-47% . The content of volatile fatty acids dropped down to 139 mg CH3COOH dm(-3) during 20 days . During the thermal pretreatment of sludge the amounts of generated biogas were ca . 25% higher, as compared to nontreated sludge . The maximum biogas production of 0.92 dm3 was observed in the day 9 of fermentation . To-date results indicate that efficiency of ultrasonic disintegration depends on sonification time, type of heads, as well as power and frequency transmitted to heads. J Environ Sci (China), 2004, 16(2), 194 - 8 Performance evaluation of up-flow anaerobic sludge blanket (UASB) reactor for treatment of paper mill wastewater; Mahadevaswamy M et al.; The present study deals with the performance evaluation of the UASB reactor under varied organic loading rate (OLR) for the treatment of paper mill wastewater . The sludge granulation process started after 120 days from the start-up period . Sludge granules size was found to be 0.8 mm at OLR of 1.72 kgCOD/(m3 x d), which reached maximum size of about 1.0 to 1.2 mm at OLR of 2.1 kgCOD/(m3 x d) . At the end of initial OLR of 1.0 kgCOD/(m3 x d) the VSS concentration was 12.86 gVSS/L, which got increased to 38.05 gVSS/L at the end of an OLR 2.1 kgCOD/( m3 x d) . Most of the times VFA recorded were well within the limit of VFA reported in anaerobic fermentation process . Many times the pH observed was between 6.5 and 7.8, which is more favorable for any anaerobic process . It is also found that pH within the reactor increases along with the height of reactor . The total maximum biogas production was found to be 0.40 L/gCOD removals at OLR of 2.1 kgCOD/(m3 x d) and the maximum BOD removal at this stage was observed to be 90%. Water Sci Technol, 2004, 49(5-6), 223 - 8 Biohydrogen production by mesophilic fermentation of food wastewater; Wu JH et al.; Batch experiments were conducted to convert molasses wastewater (10-160 g COD/L) into hydrogen at 35 degrees C at various pH (4-8) . The maximum hydrogen productivity (HP) and hydrogen production rate (HPR) reached 47.1 mmol-H2/g COD(re) and 97.5 mmol-H2/L-day, respectively, at a substrate concentration of 40 g COD/L and pH 6.0 . The methane-free biogas contained up to 50% (v/v) of hydrogen . Fermentation at wastewater concentrations higher than 60 g COD/L required a long acclimation period (more than 20 h) . Though the fractions were substrate concentration and pH-dependent, acetate and butyrate were the two main liquid fermentation products . A comparison of the HP and HPR data indicates that defining a hydrogen yield indicator to evaluate hydrogen generation efficiency should be taken into consideration in practical fermentation operations. Int J Food Microbiol, 2004 Jun 1, 93(2), 141 - 54 Saccharomyces cerevisiae-Oenococcus oeni interactions in wine: current knowledge and perspectives; Alexandre H et al.; Winemaking can be summarized as the biotransformation of must into wine, which is performed principally by Saccharomyces cerevisiae strains during the primary or alcoholic fermentation . A secondary fermentation, the so-called malolactic fermentation (MLF) is a biodeacidification that is often encouraged, since it improves wine stability and quality . Malolactic fermentation usually occurs either spontaneously or after inoculation with selected bacteria after alcoholic fermentation . The main organism responsible for MLF, the lactic acid bacterium Oenococcus oeni, develops in physicochemically harsh conditions, which may lead to MLF failure . Furthermore, yeast that ferment must before or together with O . oeni can prevent or stimulate the progress of MLF . These phenomena are part of the interactions observed between yeast and bacteria . The mechanisms that govern yeast bacteria interaction are reviewed and the consequences for winemaking are discussed . In the light of recent advances, future prospects are also presented . Regul Toxicol Pharmacol, 2004 Jun, 39(3), 256 - 70 Toxicological studies on Lactose Oxidase from Microdochium nivale expressed in Fusarium venenatum; Ahmad SK et al.; A new carbohydrate oxidase, Lactose Oxidase, with high specificity of oxidizing the disaccharide lactose to lactobionic acid has been found . This enzyme opens up for a variety of applications . A programme of toxicological studies was conducted to establish the safety of Lactose Oxidase to be used as a processing aid in the food industry . The enzyme used in this study was produced by a submerged fermentation of Fusarium venenatum and contained a gene code from Microdochium nivale . Oral administration to rats of up to 10 mL/kg bodyweight (bw)/day (equivalent to a total organic solids dosage of 900 mg/kg bw/day or a Lactose Oxidase dosage of 344 LOXU/kg bw/day) for 13 weeks did not cause any adverse effect . Lactose Oxidase was not found to be mutagenic in the bacterial reverse mutation assay, nor did it cause chromosomal aberrations in cultured human lymphocytes . The maximum recommended dosage of Lactose Oxidase is 50 LOXU/kg liquid whey protein concentrate . The safety margin for exposure is estimated to be at least 6.2 x 10(4) for daily diary product consumption . In conclusion Lactose Oxidase can be considered as safe for use in the food industry. FEBS Lett, 2004 May 7, 565(1-3), 160 - 2 New insights on glycerol transport in Saccharomyces cerevisiae; Neves L et al.; Previous studies evidenced in Saccharomyces cerevisiae the activity of a H(+)/glycerol symport, derepressed by growth on non-fermentable carbon sources, later associated with GUP1 and GUP2 genes . It was also demonstrated that only the combined deletion of GUP1, GUP2 together with GUT1 (glycerol kinase) abolished active transport in ethanol-induced cells . In this work, we show that a glycerol H(+)/symport, with identical characteristics to the previously described, was found in gup1gup2gut1 grown under salt-stress, particularly high in cells collected during diauxic-shift . These results suggest different roles for Gup1/2p than glycerol transport . The gene encoding for glycerol active uptake is thus yet unknown. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Jan, 19(1), 74 - 6 {The preparation of human anti-HBs Fab fragment by bioengineering technique}; An F et al.; AIM: To prepare human anti-HBs Fab by bioengineering technique . METHODS: The specific human anti-HBs Fab gene screened from combinatorial library was cloned into plasmid pBAD/g IIIA, then positive clone was transformed into E.coli Top10 . After the fermentation and expression processes, the soluble Fab fragment in the periplasm were purified by Ni-NTA-agarose affinity chromatography,and the inclusion bodies were in turn denatured, solubilized, purified and renatured . The specificity of Fab protein was confirmed by Western blot, and binding activity to HBsAg was verified by Dot blot . RESULTS: The quantity of soluble Fab protein purified from periplasm with Ni-NTA-Agarose which possessed good specificity as well as excellent binding activity to antigens was up to 80 mg per liter, but the biologically active protein acquired after renaturation of the inclusion bodies was quite small . CONCLUSION: Using pBAD/g IIIA-Top10 expression system, the soluble Fab protein with biological activity could be produced from periplasm of the E.coli Top10, and the strategy is available to prepare human anti-HBsAg Fab fragments in large quantity by gene engineering technique. J Biotechnol, 2004 Mar 4, 108(2), 171 - 8 Rational elimination of Aspergillus terreus sulochrin production; Couch RD et al.; Elimination of undesirable co-metabolites from industrial fermentations is often required due to the toxicities associated with the contaminants and/or due to difficulties in removing the contaminants during downstream processing . Sulochrin is a co-metabolite produced during the Aspergillus terreus lovastatin fermentation . Examination of the sulochrin biosynthetic pathway identifies the emodin anthrone polyketide synthase (PKS) at the origin . Thus, genetically disrupting the emodin anthrone PKS gene was expected to result in the elimination of sulochrin biosynthesis . To perform the disruption by homologous recombination, a fragment of the emodin anthrone PKS gene first needed to be isolated . Analysis of several reported fungal PKS amino acid sequences has identified three subfamilies of related sequences (called the Patulin subfamily, the Pigment subfamily, and the Reduction subfamily) . PCR primers specific for the Pigment subfamily (of which the emodin anthrone PKS is expected to belong) were used to isolate a fragment of a novel PKS gene from A . terreus . Targeted gene disruption identifies the novel gene fragment as that from the emodin anthrone PKS . Consequently, the gene disruption event eliminated the production of metabolites from the sulochrin biosynthetic pathway. J Biotechnol, 2004 Mar 4, 108(2), 115 - 25 Evaluation of the GFP signal and its aptitude for novel on-line monitoring strategies of recombinant fermentation processes; Reischer H et al.; A high number of economically important recombinant proteins are produced in Escherichia coli based host/vector systems . The major obstacle for improving current processes is a lack of appropriate on-line in situ methods for the monitoring of metabolic burden and critical state variables . Here, a pre-evaluation of the reporter green fluorescent protein (GFP) was undertaken to assess its use as a reporter of stress associated promoter regulation . The investigation of GFP and its blue fluorescent variant BFP was done in model fermentations using E . coli HMS 174(DE3)/pET11 aGFPmut3.1 and E . coli HMS174(DE3)/pET1aBFP host/vector systems cultured in fed-batch and chemostat regime . Our results prove the suitability of the fluorescent reporter proteins for the design of new strategies of on-line bioprocess monitoring . GFPmut3.1 variant can be detected after a short lag-phase of only 10 min, it shows a high fluorescence yield in relation to the amount of reporter protein, a good signal to noise ratio and a low detection limit . The fluorescence-signal and the amount of fluorescent protein, determined by ELISA, showed a close correlation in all fermentations performed . A combination of reporter technology with state of the art sensors helps to develop new strategies for efficient on-line monitoring needed for industrial process optimisation . The development of efficient monitoring will contribute to advanced control of recombinant protein production and accelerate the development of optimised production processes. Biotechnol Bioeng, 2004 Jun 5, 86(5), 520 - 30 Physiological significance of the cytometric distribution of fluorescent yeasts after viability staining; Bouchez JC et al.; This article describes a new method for the early detection of alcoholic fermentation arrest . This methodology is based on the flow cytometric assessment of Saccharomyces cerevisiae yeasts stained with a carboxyfluorescein diacetate fluorescent viability probe . Multicomponent analysis of viable cell distribution constitutes a promising new tool to describe physiological and dynamic changes to heterogeneous viable populations during alcoholic fermentation, through its ability to discriminate between successful processes and those ending prematurely . This framework, which is based on the comparison of cytometric histogram descriptors' combinations that can be related to simple physiological significance comparison, quickly and simply, allows testing yeasts for their fermentation ability and can be used to detect any kind of viability loss so that fermentation arrest can be avoided . Appl Environ Microbiol, 2004 May, 70(5), 3005 - 12 Studies of single-chain antibody expression in quiescent Escherichia coli; Mukherjee KJ et al.; Quiescent Escherichia coli cells are generated by overexpressing the Rcd transcript in an hns-205 mutant host . The resulting nongrowing, metabolically active cells were used here to express a single-chain antibody fragment (scFv) in shake flask and fermentor cultures . The expression system is based on two plasmids; one carries the product gene expressed from lambdaP(L) under the control of the cI857 temperature-sensitive repressor, while the second expresses Rcd from lambdaP(R) . Shifting the culture from 30 to 42 degrees C induces Rcd expression and product expression simultaneously . Our scFv carried a PelB leader, and 90% of the protein was secreted into the culture supernatant . In a batch culture, the supernatant concentration of scFv in the quiescent-cell culture (optical density at 600 nm {OD(600)} of 3.5) was 37 mg x liter(-1), compared to a maximum of 13 mg x liter(-1) in the control culture (final OD(600) of 20) . In a fed-batch fermentor culture, quiescent cells were held at an OD(600) of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg x liter(-1) . A control culture with a similar feed reached an OD(600) of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg x liter(-1) . Quiescent cells at an OD(600) of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg x liter(-1) was achieved in 8 h . The rate of extracellular accumulation was 10-fold greater in the quiescent culture than in the control culture . This study demonstrates that it is possible to establish high-density quiescent E . coli cultures that are capable of efficient synthesis, folding, and export of proteins. Appl Environ Microbiol, 2004 May, 70(5), 2898 - 905 Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: possible consequence of energy-dependent lactate export; van Maris AJ et al.; Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing . The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents . Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27) . Like all pyruvate decarboxylase-negative S . cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A . Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product . Ethanol formation was absent . However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen . In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type . Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast . This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export. Appl Environ Microbiol, 2004 May, 70(5), 2892 - 7 Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae; Sonderegger M et al.; Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae . A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH . Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway . By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c . Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed . The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation . Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate . By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered. Appl Environ Microbiol, 2004 May, 70(5), 2771 - 8 Two distinct pathways for trehalose assimilation in the yeast Saccharomyces cerevisiae; Jules M et al.; The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth . However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear . By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose . We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route . First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains . Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease . Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0 . The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast. Appl Environ Microbiol, 2004 May, 70(5), 2748 - 55 Effect of adaptation to ethanol on cytoplasmic and membrane protein profiles of Oenococcus oeni; Silveira MG et al.; The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into mechanisms of ethanol toxicity and of acquired ethanol tolerance in this organism . Therefore, the site-specific location of proteins involved in ethanol adaptation, including cytoplasmic, membrane-associated, and integral membrane proteins, was investigated . Ethanol triggers alterations in protein patterns of O . oeni cells stressed with 12% ethanol for 1 h and those of cells grown in the presence of 8% ethanol . Levels of inosine-5'-monophosphate dehydrogenase and phosphogluconate dehydrogenase, which generate reduced nicotinamide nucleotides, were decreased during growth in the presence of ethanol, while glutathione reductase, which consumes NADPH, was induced, suggesting that maintenance of the redox balance plays an important role in ethanol adaptation . Phosphoenolpyruvate:mannose phosphotransferase system (PTS) components of mannose PTS, including the phosphocarrier protein HPr and EII(Man), were lacking in ethanol-adapted cells, providing strong evidence that mannose PTS is absent in ethanol-adapted cells, and this represents a metabolic advantage to O . oeni cells during malolactic fermentation . In cells grown in the presence of ethanol, a large increase in the number of membrane-associated proteins was observed . Interestingly, two of these proteins, dTDT-glucose-4,6-dehydratase and D-alanine:D-alanine ligase, are known to be involved in cell wall biosynthesis . Using a proteomic approach, we provide evidence for an active ethanol adaptation response of O . oeni at the cytoplasmic and membrane protein levels. Extremophiles, 2004 Aug, 8(4), 309 - 16 Epub 2004 May 01. Anaerobic, alkaliphilic, saccharolytic bacterium Alkalibacter saccharofermentans gen . nov., sp . nov . from a soda lake in the Transbaikal region of Russia; Garnova ES et al.; Three strains of new obligately anaerobic alkaliphilic bacteria have been isolated as a saccharolytic component from the cellulolytic community of alkaline Lake Nizhnee Beloe (Transbaikal region, Russia), a lake with low salt concentration . DNA analysis of these strains showed an interspecies level of DNA similarity of 96-100% . Strain Z-79820 was selected for further investigations . Cells were Gram-positive, asporogenous, nonmotile short rods with pointed ends . The strain was a true alkaliphile: growth occurred from pH 7.2 to 10.2 with the optimum at pH 9.0 . Strain Z-79820 was halotolerant and could grow in medium with up to 10% (w/v) NaCl, with the optimum between 0 and 4% NaCl . The new isolate obligately depended on Na+ ions in the form of carbonates or chlorides . Total Na+ content needed for optimal growth was 0.46 M Na+, with a wide range from 0.023-0.9 M Na+ at which growth also occurred . The isolate was a mesophile and grew at temperatures from 6 to 50 degrees C (slow growth at 6 and 15 degrees C) with an optimum at 35 degrees C . The organotrophic organism fermented ribose, xylose, glucose, mannose, fructose, sucrose, mannitol, and peptone . The products of glucose fermentation were acetate, ethanol, formate, H2, and CO2 . Yeast extract was required for some anabolic needs . The DNA G+C content of the type strain Z-79820 was 42.1 mol% . The new bacterium fell into the 16S rRNA gene cluster XV of the Gram-positive bacteria with low G+C content, where it formed an individual branch . Based on its growth characteristics and genotype traits, we propose the new genus and species named Alkalibacter saccharofermentans with the type strain Z-79820 (=DSM14828), Uniqem-218 (Institute Microbiology, RAS; http://inmi.da.ru). Prikl Biokhim Mikrobiol, 2004 Mar-Apr, 40(2), 186 - 93 {Immobilization of champagne yeasts by inclusion into cryogels of polyvinyl alcohol for preventing cell escape from carrier matrix}; Martynenko NN et al.; Wine champagnizing, a process involving the use of champagne yeasts immobilized by inclusion into cryogels of polyvinyl alcohol, has been studied . Treatment of yeast cells with the autoregulatory factor d1 was proposed as a means of preventing the cell escape from the carrier matrix . Such a treatment inhibited growth and proliferation processes in yeasts cells, without affecting the activity of fermentation; the resulting champagne had the same organoleptic and chemical characteristics as its counterparts obtained using traditional techniques. Arch Latinoam Nutr, 2003 Dec, 53(4), 348 - 54 {Germinated or fermented legumes: food or ingredients of functional food}; Davila MA et al.; Epidemiological research has shown a positive association between certain diseases and dietary intake of food components found in fruits, grains, legumes, fish oil among others . Food that may provide a health benefit beyond the traditional nutrients that it contains, are named functional food . In addition to the varied nutrients, legumes contain compounds such as polyphenols, soluble fiber, alpha-galactosides and isoflavones which confer propierties of functional foods . Do to the cuse of flatus production in some people, long cooking periods, or anti-nutritional factors, legume consumption levels are limited . In this review, germination and fermentation processes will be presented as alternatives that are able to reduce or inactivate anti-nutritional factors, preserve and even improve the content of the isoflavones, or better the potencial of the legumes as functional food or as ingredients for the formulation of functional foods. J Biol Chem, 2004 Jul 16, 279(29), 30449 - 58 Epub 2004 Apr 30. Sue1p is required for degradation of labile forms of altered cytochromes C in yeast mitochondria; Wei J et al.; Previous studies on certain altered holo-isocytochromes c revealed a rho(-)-dependent degradation (RDD) phenotype, in which certain altered holo-iso-1-cytochromes c are at normal or nearly normal levels in rho+ strains, but are at low levels or absent in rho- strains, although wild-type holo-iso-1-cytochrome c is present at normal levels in both rho+ and related rho- strains . The diminished levels of altered holo-iso-1-cytochrome c are due to the rapid degradation that is carried out by a novel proteolytic pathway in the IMS of mitochondria . SUE1, a nuclear gene that encodes a mitochondrial protein, was identified with a genetic screen for mutants that diminish RDD . The levels of RDD and certain other types of altered holo-iso-1-cytochrome c were elevated in rho- sue1 strains . Also, rho+ sue1 strains containing certain altered holo-iso-1-cytochromes c grew better on non-fermentable carbon sources than the corresponding rho+ SUE1 strains . These results indicate that Sue1p may play an important role in the degradation of abnormal holo-iso-1-cytochrome c in the mitochondria. J Ethnopharmacol, 2004 Apr, 91(2-3), 231 - 5 Cholesterol lowering effect of SG-GN3, the extract of salted and fermented small shrimps, Acetes japonicus, in Triton WR-1339 or high cholesterol-diet induced hypercholesterolemic rats; Seok SH et al.; The cholesterol lowering effect of SG-GN3, the extract of salted and fermented small shrimps, Acetes japonicus, was investigated in hypercholesterolemic animal models . Hypercholesterolemia was induced with Triton WR-1339 (nonionic detergent) or high cholesterol (HC)-diet . SG-GN3 significantly decreased total cholesterol (TC) in Triton WR-1339 model at 30 post-treatment hour (549.80 +/- 152.46 mg/dl) compared to the control which induced by only Triton WR-1339 (798.84 +/- 94.98 mg/dl), whereas high-density lipoprotein (HDL) content did not decrease (P < 0.05) . In HC-diet model, TC content significantly decreased by SG-GN3 treatment at 3 post-treatment day (P < 0.05) . These results suggest that SG-GN3 effectively decreased serum TC level in hypercholesterolemic animal models. Physiol Res, 2004, 53 Suppl 1, S99 - 102 Down-regulation of model yeast proteins by ubiquitin-dependent proteolysis; Horak J; Ubiquitination is a versatile tool used by all eukaryotic organisms for controlling the stability, function, and intracellular localization of a multitude of proteins . I will attempt to bring together our recent data on the down-regulation of two yeast model proteins, the galactose transporter Gal2 and fructose-1,6-bisphosphatase, by ubiquitin-dependent proteolysis triggered by the addition of easily fermentable carbon sources. Curr Issues Mol Biol, 2004 Jul, 6(2), 159 - 88 Molecular biology of microbial hydrogenases; Vignais PM et al.; Hydrogenases (H2ases) are metalloproteins . The great majority of them contain iron-sulfur clusters and two metal atoms at their active center, either a Ni and an Fe atom, the {NiFe}-H2ases, or two Fe atoms, the {FeFe}-H2ases . Enzymes of these two classes catalyze the reversible oxidation of hydrogen gas (H2 <--> 2 H+ + 2 e-) and play a central role in microbial energy metabolism; in addition to their role in fermentation and H2 respiration, H2ases may interact with membrane-bound electron transport systems in order to maintain redox poise, particularly in some photosynthetic microorganisms such as cyanobacteria . Recent work has revealed that some H2ases, by acting as H2-sensors, participate in the regulation of gene expression and that H2-evolving H2ases, thought to be involved in purely fermentative processes, play a role in membrane-linked energy conservation through the generation of a protonmotive force . The Hmd hydrogenases of some methanogenic archaea constitute a third class of H2ases, characterized by the absence of Fe-S cluster and the presence of an iron-containing cofactor with catalytic properties different from those of {NiFe}- and {FeFe}-H2ases . In this review, we emphasise recent advances that have greatly increased our knowledge of microbial H2ases, their diversity, the structure of their active site, how the metallocenters are synthesized and assembled, how they function, how the synthesis of these enzymes is controlled by external signals, and their potential use in biological H2 production. Biosci Biotechnol Biochem, 2004 Apr, 68(4), 978 - 80 Unusual transcription regulation of the niaD gene under anaerobic conditions supporting fungal ammonia fermentation; Takasaki K et al.; The niaD gene of the fungus Aspergillus nidulans encodes an assimilatory nitrate reductase and exogenous ammonium represses its expression . Under anoxic conditions, however, A . nidulans expressed niaD even in the presence of ammonium and used the gene product for dissimilatory nitrate reduction (ammonia fermentation) . This transcription regulation mechanism under anaerobiosis is critical for the fungus to ferment ammonium. Biosci Biotechnol Biochem, 2004 Apr, 68(4), 968 - 72 Effect of ethanol on cell growth of budding yeast: genes that are important for cell growth in the presence of ethanol; Kubota S et al.; The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds of alcoholic beverages . But the effect of ethanol on the cell growth of this yeast is poorly understood . This study shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion of F-actin cytoskeleton, resulting in an increase in cell size . We found that the tyrosine kinase Swe1, the negative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol . Indeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and the amount of Swe1 protein increased transiently in the presence of ethanol . These results indicated that Swe1 is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes a transient up-regulation of Swe1 . Further we investigated comprehensively the ethanol-sensitive strains in the complete set of 4847 non-essential gene deletions and identified at least 256 genes that are important for cell growth in the presence of ethanol. Biosci Biotechnol Biochem, 2004 Apr, 68(4), 820 - 6 New brasiliamide congeners, brasiliamides C, D and E, from Penicillium brasilianum Batista JV-379; Fujita T et al.; Three new brasiliamide congeners, brasiliamides C, D and E, were isolated from okara fermented with Penicillium brasilianum Batista JV-379 . Their structures were elucidated on the basis of spectral data and chemical evidence . NMR spectra of these brasiliamides exhibited a mixture of four or two conformers due to the restricted rotation of an amide bond in a solution . The (1)H- and (13)C-NMR spectral data were analyzed for a major rotamer at an appropriate temperature, since the signals were broadened at room temperature . Both brasiliamides C and D showed convulsive activity against silkworms with an ED(50) value of 400 microg/g of diet, whereas brasiliamide E showed less activity than the others. J Biol Chem, 2004 Jul 2, 279(27), 28174 - 81 Epub 2004 Apr 26. Asr1p, a novel yeast ring/PHD finger protein, signals alcohol stress to the nucleus; Betz C et al.; During fermentation, yeast cells are exposed to increasing amounts of alcohol, which is stressful and affects both growth and viability . On the molecular level, numerous aspects of alcohol stress signaling remain unresolved . We have identified a novel yeast Ring/PHD finger protein that constitutively shuttles between nucleus and cytoplasm but accumulates in the nucleus upon exposure to ethanol, 2-propanol, or 1-butanol . Subcellular localization of this protein is not altered by osmotic, oxidative, or heat stress or during nitrogen or glucose starvation . Because of its exclusive sensitivity to environmental alcohol, the protein was called Asr1p for Alcohol Sensitive Ring/PHD finger 1 protein . Nuclear accumulation of Asr1p is rapid, reversible, and requires a functional Ran/Gsp1p gradient . Asr1p contains two N terminally located leucine-rich nuclear export sequences (NES) required for nuclear export . Consistently, it accumulates in the nucleus of xpo1-1 cells at restrictive temperature and forms a trimeric complex with the exportin Xpo1p and Ran-GTP . Deletion of ASR1 leads to sensitivity in growth on medium containing alcohol or detergent, consistent with a function of Asr1p in alcohol-related signaling . Asr1p is the first reported protein that changes its subcellular localization specifically upon exposure to alcohol and therefore represents a key element in the analysis of alcohol-responsive signaling. J Biol Chem, 2004 Jul 23, 279(30), 31429 - 39 Epub 2004 Apr 26. Identification and activity of a series of azole-based compounds with lactate dehydrogenase-directed anti-malarial activity; Cameron A et al.; Plasmodium falciparum, the causative agent of malaria, relies extensively on glycolysis coupled with homolactic fermentation during its blood-borne stages for energy production . Selective inhibitors of the parasite lactate dehydrogenase (LDH), central to NAD(+) regeneration, therefore potentially provide a route to new antimalarial drugs directed against a novel molecular target . A series of heterocyclic, azole-based compounds are described that preferentially inhibit P . falciparum LDH at sub-micromolar concentrations, typically at concentrations about 100-fold lower than required for human lactate dehydrogenase inhibition . Crystal structures show these competitive inhibitors form a network of interactions with amino acids within the active site of the enzyme, stacking alongside the nicotinamide ring of the NAD(+) cofactor . These compounds display modest activity against parasitized erythrocytes, including parasite strains with known resistance to existing anti-malarials and against Plasmodium berghei in BALB/c mice . Initial toxicity data suggest the azole derivatives have generally low cytotoxicity, and preliminary pharmoco-kinetic data show favorable bioavailability and circulation times . These encouraging results suggest that further enhancement of these structures may yield candidates suitable for consideration as new therapeutics for the treatment of malaria . In combination these studies also provide strong support for the validity of targeting the Plasmodium glycolytic pathway and, in particular, LDH in the search for novel anti-malarials. J Med Food, 2004 Spring, 7(1), 13 - 8 Differentiation-promoting activity of pomegranate (Punica granatum) fruit extracts in HL-60 human promyelocytic leukemia cells; Kawaii S et al.; Differentiation refers to the ability of cancer cells to revert to their normal counterparts, and its induction represents an important noncytotoxic therapy for leukemia, and also breast, prostate, and other solid malignancies . Flavonoids are a group of differentiation-inducing chemicals with a potentially lower toxicology profile than retinoids . Flavonoid-rich polyphenol fractions from the pomegranate (Punica granatum) fruit exert anti-proliferative, anti-invasive, anti-eicosanoid, and pro-apoptotic actions in breast and prostate cancer cells and anti-angiogenic activities in vitro and in vivo . Here we tested flavonoid-rich fractions from fresh (J) and fermented (W) pomegranate juice and from an aqueous extraction of pomegranate pericarps (P) as potential differentiation-promoting agents of human HL-60 promyelocytic leukemia cells . Four assays were used to assess differentiation: nitro blue tetrazolium reducing activity, nonspecific esterase activity, specific esterase activity, and phagocytic activity . In addition, the effect of these extracts on HL-60 proliferation was evaluated . Extracts W and P were strong promoters of differentiation in all settings, with extract J showing only a relatively mild differentiation-promoting effect . The extracts had proportional inhibitory effects on HL-60 cell proliferation . The results highlight an important, previously unknown, mechanism of the cancer preventive and suppressive potential of pomegranate fermented juice and pericarp extracts. Crit Rev Food Sci Nutr, 2004, 44(2), 113 - 37 The relative effect of milk base, starter, and process on yogurt texture: a review; Sodini I et al.; Yogurt is a milk curd produced all over the world, obtained by a lactic fermentation of a milk base enriched with milk proteins, and sometimes sugars and thickeners . One of the most important sensorial attributes for yogurt is texture, which could be assessed by sensory or instrumental analysis . A lot of work has been published in studying the contribution of milk base, starter, and process on yogurt texture in order to develop new textures, or simply to reduce fat content, or the level of addition of protein and thickener in milk . However, these studies are limited to only a few factors . The topic of this review was to synthesize the data of literature, with the aim of extracting and classifying factors on the basis of their influence on yogurt texture . Three factors, milk base heating, starter, and yogurt shearing after fermentation, respectively, play a key role in the elaboration of texture . The control of these three parameters allows the improvement of the textural attributes of yogurts by 2 to 15 times. Br Poult Sci, 2004 Feb, 45(1), 76 - 84 Influence of phytase and xylanase, individually or in combination, on performance, apparent metabolisable energy, digestive tract measurements and gut morphology in broilers fed wheat-based diets containing adequate level of phosphorus; Wu YB et al.; 1 . The aim of the present study was to examine the influence of microbial phytase and xylanase, individually or in combination, on performance, apparent metabolisable energy, digesta viscosity, digestive tract measurements and gut morphology in broilers fed on wheat-soy diets containing adequate phosphorus (P) . The wheat-soy basal diet was formulated to contain 4.5 g/kg non-phytate P and the experimental diets were formulated by supplementing the basal diet with xylanase (1000 xylanase units/kg diet), phytase (500 phytase units/kg diet) or a combination of phytase and xylanase . 2 . Supplemental phytase improved the weight gains and feed efficiency by 17.5 and 2.9%, respectively . Corresponding improvements due to the addition of xylanase were 16.5 and 4.9%, respectively . The combination of phytase and xylanase caused no further improvements in broiler performance . 3 . Individual additions of xylanase or phytase resulted in numerical improvements in apparent metabolisable energy (AME), but the differences were not significant . The combination of the two enzymes significantly increased AME . Addition of xylanase and the combination of the two enzymes reduced the viscosity of digesta in all sections of the intestine . Phytase supplementation reduced digesta viscosity in the duodenum and ileum, but not in the jejunum . 4 . Enzyme supplementation lowered the relative weight and length of the small intestine . Additions of xylanase and phytase reduced the relative weight of the small intestine by 15.5 and 11.4%, respectively, while the corresponding reductions in the relative length of the small intestine were 16.5 and 14.1%, respectively . The combination of phytase and xylanase had no further effects on the relative weight and length of the small intestine compared with the xylanase group . 5 . The addition of phytase increased villus height in the duodenum and decreased the number of goblet cells in the jejunum compared with those on the unsupplemented basal diet . Xylanase supplementation tended to increase goblet cell numbers in the duodenum and decreased crypt depth in thejejunum . The combination of phytase and xylanase increased villus height in the ileum and crypt depth in thejejunum and ileum . 6 . In summary, the present results showed that the addition of a microbial phytase, produced by solid state fermentation and containing significant activities of beta-glucanase and xylanase, was as effective as xylanase in improving the performance of broiler chickens fed on wheat-based diets containing adequate levels of P . Improved performance with enzyme supplementation was generally associated with reduced digesta viscosity, increased AME, and reduced relative weight and length of small intestine. J Nutr, 2004 May, 134(5), 1193 - 6 A combined 13CO2/H2 breath test can be used to assess starch digestion and fermentation in humans; Symonds EL et al.; Ingestion of starch from corn (naturally enriched with (13)C) should produce (13)CO(2) after small intestinal digestion and (13)CO(2) and H(2) from colonic fermentation . This study used a combined (13)CO(2)/H(2) breath test to assess the digestion and fermentation of resistant starch and to show that the test could detect changes in digestibility due to cooking . Volunteers consumed 40 g digestible cornstarch with water (n = 8), or 40 g resistant cornstarch in liquid (n = 12) or cooked into a pancake (n = 4) . Interval breath sampling was performed and analyzed for (13)CO(2) and H(2) . Ingestion of resistant starch produced a double-peaked (13)CO(2) excretion curve . The first increase in (13)CO(2) occurred at the same time as excretion from digestible starch (55 +/- 9 and 68 +/- 9 min, respectively), which was due to small intestinal digestion . The second increase in (13)CO(2) was accompanied by an increase in H(2) excretion (432 +/- 15 and 428 +/- 48 min, respectively), which was indicative of colonic bacterial fermentation . Cooking resistant starch increased its degree of digestion from 36 to 72% . The (13)CO(2)/H(2) breath test can be used to estimate digestion and fermentation of starches in different physiologic and pathologic conditions. Metab Eng, 2004 Apr, 6(2), 155 - 63 Engineering of Saccharomyces cerevisiae for the production of L-glycerol 3-phosphate; Nguyen HT et al.; L-glycerol 3-phosphate (L-G3P) was accumulated in Saccharomyces cerevisiae by pathway engineering . Intracellular concentration of this metabolic intermediate could be increased more than 20 times compared to the wild type by overexpressing GPD1 encoding the glycerol 3-phosphate dehydrogenase in a gpp1 Delta gpp2 Delta mutant which lacks both isoenzymes of glycerol 3-phosphatase . Investigation of cellular pattern of triacylglycerols and glycerophospholipids did not reveal considerable changes due to accumulation of their precursor L-G3P . Hyperosmotic stress did not affect the L-G3P pool in the gpp1 Delta gpp2 Delta mutant overexpressing GPD1 despite an about 4-fold increase of specific GPD activity . In contrast, oxygen limitation improved intracellular L-G3P concentration by enhancing the availability of cytosolic NADH . The reduction of pyruvate decarboxylase activity by deleting PDC2 led to an additional increase . In fact, the triple mutant gpp1 Delta gpp2 Delta pdc2 Delta overexpressing GPD1 accumulated 17 mg L-G3P/g dry weight during glucose batch fermentation under oxygen limitation . This value corresponds to an about 100-fold increase compared to that found in the wild type. J Agric Food Chem, 2004 May 5, 52(9), 2652 - 8 Identification and occurrence of tryptamine- and tryptophan-derived tetrahydro-beta-carbolines in commercial sausages; Herraiz T et al.; The identification and occurrence of tetrahydro-beta-carbolines were studied in different kinds of commercial sausages including cooked, fresh, dry-fermented, and ripened sausages, such as salamis and Spanish chorizo, salchichon, fuet, and morcilla, both smoked and unsmoked . Four compounds were identified in several sausages by high-performance liquid chromatography-mass spectrometry (HPLC-MS): 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (1), 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid diastereoisomers (2a,b), 1,2,3,4-tetrahydro-beta-carboline (3), and 1-methyl-1,2,3,4-tetrahydro-beta-carboline (4) . The latter two (3 and 4) are now reported for the first time in meat products . The presence and occurrence of tetrahydro-beta-carbolines were highly variable depending on each particular sample of sausage, and it did not follow a single specific pattern . The concentration range taken as a sum of the four carbolines varied from undetectable levels to 33 microg/g, with the highest content found in ripened, dry-fermented, and smoked sausages (salami, chorizo, and morcilla) and the lowest in cooked sausages (Frankfurt) . Formation of tetrahydro-beta-carbolines might occur during elaboration and the ripening process from a chemical condensation between tryptophan or tryptamine and aldehydes (formaldehyde and acetaldehyde) . Smoked samples had higher concentrations of formaldehyde-derived 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (1) and 1,2,3,4-tetrahydro-beta-carboline (tryptoline) (3) than those unsmoked . Also, 1 and 3 were more concentrated in the outer part of the sausage, likely to be in contact with smoke . It is concluded that some dry-fermented and/or smoked sausages may be significant dietary sources of tetrahydro-beta-carbolines. J Antibiot (Tokyo), 2004 Feb, 57(2), 136 - 42 Exploitation of heparanase inhibitors from microbial metabolites using an efficient visual screening system; Ishida K et al.; In this paper we describe the establishment of an efficient visual method for screening heparanase inhibitors, and we present the results of screening 10,000 microbial culture broths . Heparanase-overexpressing stable clones of the human hepatocellular carcinoma HepG2 cells were established and used as an enzyme source . Digestion of heparan sulfate (HS) was detected using novel HS-containing tablets or SDS-polyacrylamide gel electrophoresis . This method was able to find suramin, a known heparanase inhibitor, from a library of typical enzyme inhibitors . By screening 10,000 culture broths of microorganisms (actinomycetes, fungi, and bacteria) an actinomycete strain, RK99-A234, was found to have heparanase inhibitory activity . RK-682 was identified in the fermentation broth as a heparanase inhibitor, IC50 = 17 microM. J Antibiot (Tokyo), 2004 Feb, 57(2), 97 - 103 ICM0301s, new angiogenesis inhibitors from Aspergillus sp . F-1491 . I . Taxonomy, fermentation, isolation and biological activities; Kumagai H et al.; In the course of screening program for inhibitors of angiogenesis, novel substances designated as ICM0301A approximately H (1 approximately 8) were isolated from the culture broth of Aspergillus sp . F-1491 . ICM0301s inhibited the growth of human umbilical vein endothelial cells (HUVECs) induced by basic fibroblast growth factor (bFGF) with IC50 values of 2.2 approximately 9.3 microg/ml . ICM0301A (1) showed significant anti-angiogenic activity at lower than 10 microg/ml in the angiogenesis model using rat aorta cultured in fibrin gel . ICM0301s showed very low cytotoxicity against various tumor cells . Furthermore, 1CM0301A did not show any toxic symptom in mice by intraperitoneal injection at 100 mg/kg. J Antibiot (Tokyo), 2004 Feb, 57(2), 89 - 96 SF2809 compounds, novel chymase inhibitors from Dactylosporangium sp . 2 . Structural elucidation; Tani M et al.; Novel chymase inhibitors, SF2809-I, II, III, IV, V and VI, were isolated from the fermentation broth of Dactylosporangium sp . SF2809, and their structures were determined by spectroscopic analyses . SF2809 compounds commonly contain a substituted indole moiety and a quinolinone moiety . The two moieties are connected to a methylene carbon in SF2809-I and III . The other compounds, SF2809-II, IV, V and VI, have an additional moiety, a p-hydroxyphenyl group or a phenyl group . In these compounds, all of three moieties are connected to a methine carbon . Furthermore, studies concerning the stereochemistry of SF2809-V revealed that the isolated compound was racemic, and the isomer possessing (R)-configuration was about thirty times more potent than another isomer. J Antibiot (Tokyo), 2004 Feb, 57(2), 83 - 8 SF2809 compounds, novel chymase inhibitors from Dactylosporangium sp . 1 . Taxonomy, fermentation, isolation and biological properties; Tani M et al.; Six novel chymase inhibitors, SF2809-I, SF2809-II, SF2809-III, SF2809-IV, SF2809-V and SF2809-VI, were isolated from the fermentation broth of an actinomycete strain SF2809 . The strain was identified as Dactylosporangium sp . by morphological, chemotaxonomical and phylogenetic studies . These six novel compounds inhibited recombinant human chymase in the range between IC50 of 0.014 and 7.3 microM . However, they showed little or no inhibitory activity against chymotrypsin or cathepsin G, even though these two and chymase belong to the chymotryptic serine protease family . This result indicates that these compounds work as specific chymase inhibitors. Biotechnol Bioeng, 2004 May 20, 86(4), 414 - 24 Morphologically structured model for antitumoral retamycin production during batch and fed-batch cultivations of Streptomyces olindensis; Giudici R et al.; A morphologically structured model is proposed to describe trends in biomass growth, substrate consumption, and antitumoral retamycin production during batch and fed-batch cultivations of Streptomyces olindensis . Filamentous biomass is structured into three morphological compartments (apical, subapical, and hyphal), and the production of retamycin, a secondary metabolite, is assumed to take place in the subapical cell compartment . Model accounts for the effect of glucose as well as complex nitrogen source on both the biomass growth and retamycin production . Laboratory data from bench-scale batch and fed-batch fermentations were used to estimate some model parameters by nonlinear regression . The predictive capability of the model was then tested for additional fed-batch and continuous experiments not used in the previous fitting procedure . The model predictions show fair agreement to the experimental data . The proposed model can be useful for further studies on process optimization and control . Biotechnol Bioeng, 2004 May 20, 86(4), 405 - 13 Combined discrete particle and continuum model predicting solid-state fermentation in a drum fermentor; Schutyser MA et al.; The development of mathematical models facilitates industrial (large-scale) application of solid-state fermentation (SSF) . In this study, a two-phase model of a drum fermentor is developed that consists of a discrete particle model (solid phase) and a continuum model (gas phase) . The continuum model describes the distribution of air in the bed injected via an aeration pipe . The discrete particle model describes the solid phase . In previous work, mixing during SSF was predicted with the discrete particle model, although mixing simulations were not carried out in the current work . Heat and mass transfer between the two phases and biomass growth were implemented in the two-phase model . Validation experiments were conducted in a 28-dm3 drum fermentor . In this fermentor, sufficient aeration was provided to control the temperatures near the optimum value for growth during the first 45-50 hours . Several simulations were also conducted for different fermentor scales . Forced aeration via a single pipe in the drum fermentors did not provide homogeneous cooling in the substrate bed . Due to large temperature gradients, biomass yield decreased severely with increasing size of the fermentor . Improvement of air distribution would be required to avoid the need for frequent mixing events, during which growth is hampered . From these results, it was concluded that the two-phase model developed is a powerful tool to investigate design and scale-up of aerated (mixed) SSF fermentors . J Ind Microbiol Biotechnol, 2004 Mar, 31(3), 99 - 108 Epub 2004 Apr 27. Strain improvement for fermentation and biocatalysis processes by genetic engineering technology; Chiang SJ; Twenty years ago, the first complete gene cluster encoding the actinorhodin biosynthetic pathway was cloned and characterized . Subsequently, the gene clusters encoding the biosynthetic pathways for many antibiotics were isolated . In the past decade, breakthroughs in technology brought that generation of rationally designed or new hybrid metabolites to fruition . Now, the development of high-throughput DNA sequencing and DNA microarray techniques enables researchers to identify the regulatory mechanisms for the overproduction of secondary metabolites and to monitor gene expression during the fermentation cycle, accelerating the rational application of metabolic pathway engineering . How are the new tools of biotechnology currently being applied to improve the production of secondary metabolites? Where will this progress lead us tomorrow? The use of whole cells or partially purified enzymes as catalysts has been increased significantly for chemical synthesis in pharmaceutical and fine-chemical industries . The development of PCR technologies for protein engineering and DNA shuffling is leading to the generation of new enzymes with increased stability to a wide range of pHs, temperatures and solvents and with increased substrate specificity, reaction rate and enantioselectivity . Where will this emerging technology lead us in the twenty-first century? FEBS Lett, 2004 Apr 30, 564(3), 289 - 93 Automated large-scale purification of a G protein-coupled receptor for neurotensin; White JF et al.; Structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis . Here, we describe a protocol for the purification of a G protein-coupled neurotensin receptor fusion protein at the 3-mg or 10-mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode . Fermentation at a 200-l scale of Escherichia coli expressing functional receptors provides the material needed to feed into the purification routine . Constructs with tobacco etch virus protease recognition sites at either end of the receptor allow the isolation of neurotensin receptor devoid of its fusion partners . The presented expression and purification procedures are simple and robust, and provide the basis for crystallization experiments of receptors on a routine basis. Food Chem Toxicol, 2004 Jun, 42(6), 935 - 44 Safety evaluation of phosphodiesterase derived from Leptographium procerum; Steensma A et al.; 5'-Phosphodiesterase is produced by fermentation of the fungus Leptographium proce