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The formation of toxic fermentation inhibitors such as furfural and 5-hydroxy-2-methylfurfural (HMF) during acid (pre-)treatment of lignocellulose, calls for the efficient removal of these compounds. Lignocellulosic hydrolysates can be efficiently detoxified biologically with microorganisms that specifically metabolize the fermentation inhibitors while preserving the sugars for subsequent use by the fermentation host. The bacterium Cupriavidus basilensis HMF14 was isolated from enrichment cultures with HMF as the sole carbon source and was found to metabolize many of the toxic constituents of lignocellulosic hydrolysate including furfural, HMF, acetate, formate and a host of aromatic compounds. Remarkably, this microorganism does not grow on the most abundant sugars in lignocellulosic hydrolysates: glucose, xylose and arabinose. In addition, C. basilensis HMF14 can produce polyhydroxyalkanoates. Cultivation of C. basilensis HMF14 on wheat straw hydrolysate resulted in the complete removal of furfural, HMF, acetate and formate, leaving the sugar fraction intact. This unique substrate profile makes C. basilensis HMF14 extremely well suited for biological removal of inhibitors from lignocellulosic hydrolysates prior to their use as fermentation feedstock. © 2009 The Authors.

A comprehensive taxonomic re-evaluation was performed on the marine, zeaxanthin-producing bacterium formerly classified as [Flavobacterium] sp. strain R-1512 (ATCC 21588). This strain, together with two other previously described marine isolates, [Flavobacterium] strain R-1506 and Paracoccus sp. strain MBIC 3966, were found to comprise a new species of the genus Paracoccus. The name Paracoccus zeaxanthinifaciens sp. nov. is proposed, with ATCC 21588T (=R-1512T =LMG 21293T) designated as the type strain.

Gram-negative sepsis is a major death cause in intensive care units. Accumulating evidence indicates the protective role of plasma lipoproteins such as high-density lipoprotein (HDL) in sepsis. It has recently been shown that septic HDL is almost depleted from apolipoprotein CI (apoCI), suggesting that apoCI may be a protective factor in sepsis. Sequence analysis revealed that apoCI possesses a highly conserved consensus KVKEKLK binding motif for lipopolysaccharide (LPS), an outer-membrane component of gram-negative bacteria. Through avid binding to LPS involving this motif, apoCI improved the presentation of LPS to macrophages in vitro and in mice, thereby stimulating the inflammatory response to LPS. Moreover, apoCI dose-dependently increased the early inflammatory response to Klebsiella pneumoniae-induced pneumonia, reduced the number of circulating bacteria, and protected mice against fatal sepsis. Our data support the hypothesis that apoCI is a physiological protector against infection by enhancing the early inflammatory response to LPS and suggest that timely increase of apoCI levels could be used to efficiently prevent and treat early sepsis. Chemicals / CAS: Apolipoprotein C-I; Apolipoproteins C; Lipopolysaccharides

Inulosucrases catalyze transfer of a fructose moiety from sucrose to a water molecule (hydrolysis) or to an acceptor molecule (transferase), yielding inulin. Bacterial inulin production is rare and a biochemical analysis of inulosucrase enzymes has not been reported. Here we report biochemical characteristics of a purified recombinant inulosucrase enzyme from Lactobacillus reuteri. It displayed Michaelis-Menten type of kinetics with substrate inhibition for the hydrolysis reaction. Kinetics of the transferase reaction is best described by the Hill equation, not reported before for these enzymes. A C-terminal deletion of 100 amino acids did not appear to affect enzyme activity or product formation. This truncated form of the enzyme was used for biochemical characterization. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Molecular Sequence Numbers: GENBANK: AF459437; Chemicals/CAS: Hexosyltransferases, EC 2.4.1.-; inulosucrase, EC 2.4.1.9; Metals; Recombinant Proteins

Background: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents and their fleas. Transmission to humans can cause bubonic, pneumonic or septicemic plague with a high case-fatality rate. Therefore, rapid and reliable diagnostic tools are crucial. The objective of this study was to assess the inter-laboratory reproducibility of in-house developed real-time PCR assays for the identification of Y. pestis. Methods: A total of four samples of quantified Y. pestis DNA and two blank samples were sent blinded to 14 laboratories. To standardize the procedures, oligonucleotides were provided and the same instrument platform and a commercial mastermix were used. The participants were requested to report their results including cycle threshold and melting temperature values. Results: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing Y. pestis DNA correctly. Significant differences between the reference laboratory and participating laboratories were observed in cycle threshold values and melting temperatures. This, however, did not adversely affect the interpretation of results. Conclusions: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification of Y. pestis isolates. Further steps of validation are needed to determine diagnostic accuracy and predictive values with clinical samples. © 2008 by Walter de Gruyter.

It has recently become evident that at least five ceramidase (CDase) isoforms are present in human epidermis, and that specifically acidic CDase (aCDase) and alkaline CDase (alkCDase) activities increase during keratinocyte differentiation, and thus might play a pivotal role(s) in permeability barrier function. Prior to investigating their possible roles in the epidermal barrier function, it is necessary to characterize basic kinetic parameters for these enzymes, as well as to determine the effects of the established CDase inhibitors and their activities. In this study, assays for both aCDase and alkCDase activities in fully differentiated human epidermis were optimized using a radiolabeled substrate. These studies revealed that aCDase activity is substantially higher than alkCDase activity, and that both isoenzymes are inhibited by a CDase inhibitor N-oleylethanolamine. These findings were also confirmed using an in situ enzyme assay. Copyright © 2007 S. Karger AG.

We aimed to identify transcription signal sequences from Streptococcus gordonii strain CH1 by random chromosomal cloning. Five genomic fragments from a Sau3A digest, which constitutively activated transcription of a promoterless spectinomycin resistance gene in this strain, were isolated and characterized. Additionally, one promoter fragment was isolated that was specifically activated under iron-limiting conditions. A sequence motif with similarity to the consensus for Fur-binding regulatory DNA sequences (Fur box) in Escherichia coli was detected within the putative promoter region. The open reading frame downstream of this region possibly encodes a transmembrane protein involved in iron uptake.

To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields. Chemicals/CAS: Ferrous Compounds; Heme, 14875-96-8; lignin peroxidase, EC 1.11.1.-; manganese peroxidase, EC 1.11.1.13; Peroxidases, EC 1.11.1.-; Recombinant Proteins

The question of whether melanins are photoprotecting and/or photosensitizing in human skin cells continues to be debated. To evaluate the role of melanin upon UVA irradiation, DNA single-strand breaks (ssb) were measured in human melanocytes differing only in the amount of pigment produced by culturing at two different concentrations, basic (0.01 mM) or high (0.2 mM), of L-tyrosine, the main precursor of melanin. In parallel, pheo- and total melanin contents of the cells were determined. Identical experiments were performed with two melanocyte cultures derived from a skin type I and a skin type VI individual. For the first time the correlation between UVA-induced genotoxicity and pheo-/total melanin content has been investigated. We observed that cultured in basic medium, the skin type VI melanocytes contained 10 times more total melanin and about seven times more pheomelanin than the skin type I melanocytes. Elevation of tyrosine level in the culture medium resulted in an increase of both pheo- and total melanin levels in both melanocyte cultures; however, the melanin composition of skin type I melanocytes became more pheomelanogenic, whereas that of skin type VI melanocytes remained the same. The skin type VI melanocytes cultured in basic medium demonstrated a very high sensitivity (1.18 ssb per 1010 Da per kJ per m2) toward UVA that is probably related to their high pheo- and total melanin content. Their UVA sensitivity, however, did not change after increasing their melanin content by culturing at high tyrosine concentration. In contrast, the skin type I melanocytes demonstrated a low sensitivity (0.04 ssb per 1010 Da per kJ per m2) toward UVA when cultured in basic medium, but increasing their melanin content resulted in a 3-fold increase in their UVA sensitivity (0.13 ssb per 1010 Da per kJ per m2). These results demonstrate that UVA-irradiated cultured human melanocytes are photosensitized by their own synthesized chromophores, most likely pheomelanin and/or melanin intermediates.

Synthesis of lactococcin 972 is plasmid-encoded. An operon composed of two genes that encode pre-bacteriocin and a putative immunity protein has been identified. The first gene encodes a 91-residue polypeptide that is exported via a sec-dependent system to give the mature 66-aa bacteriocin. The immunity protein is a 563-residue polypeptide with seven potential transmembrane domains. Two transcripts were observed from this region: one comprises the whole operon and is synthesized during the exponential phase of growth while the other, which corresponds just to the bacteriocin structural gene, presents a maximum in exponential cultures but is still present in late-stationary-phase cells.

Microarray technology is being used increasingly to study gene expression of biological systems on a large scale. Both interlaboratory and interplatform differences are known to contribute to variability in microarray data. In this study we have investigated data from different platforms and laboratories on the transcriptomic profile of HepG2 cells exposed to benzo(a)pyrene (BaP). RNA samples generated in two different laboratories were analyzed using both Agilent oligonucleotide microarrays and Cancer Research UK (CR-UK) cDNA microarrays. Comparability of the expression profiles was assessed at various levels including correlation and overlap between the data, clustering of the data and affected biological processes. Overlap and correlation occurred, but it was not possible to deduce whether choice of platform or interlaboratory differences contributed more to the data variation. Principal component analysis (PCA) and hierarchical clustering of the expression profiles indicated that the data were most clearly defined by duration of exposure to BaP, suggesting that laboratory and platform variability does not mask the biological effects. Real-time quantitative PCR was used to validate the two array platforms and indicated that false negatives, rather than false positives, are obtained with both systems. All together these results suggest that data from similar biological experiments analyzed on different microarray platforms can be combined to give a more complete transcriptomic profile. Each platform gives a slight variation in the BaP-gene expression response and, although it cannot be stated which is more correct, combining the two data sets is more informative than considering them individually. © 2009 Mary Ann Liebert, Inc. 2009.

The toxic fermentation inhibitors in lignocellulosic hydrolysates pose significant problems for the production of second-generation biofuels and biochemicals. Among these inhibitors, 5-(hydroxymethyl) furfural (HMF) and furfural are specifically notorious. In this study, we describe the complete molecular identification and characterization of the pathway by which Cupriavidus basilensis HMF14 metabolizes HMF and furfural. The identification of this pathway enabled the construction of an HMF and furfural-metabolizing Pseudomonas putida. The genetic information obtained furthermore enabled us to predict the HMF and furfural degrading capabilities of sequenced bacterial species that had not previously been connected to furanic aldehyde metabolism. These results pave the way for in situ detoxification of lignocellulosic hydrolysates, which is a major step toward improved efficiency of utilization of lignocellulosic feedstock.

OBJECTIVES. The purpose of this work was to assess the costs of 4 neonatal screening strategies for cystic fibrosis in relation to health effects. In each strategy, the first test was the measurement of serum concentration of immunoreactive trypsin. The second step consisted of either a second immunoreactive trypsin test (strategy 1) or a multiple mutation analysis (strategy 2). In strategies 3 and 4, a third step was added to strategy 2: a second immunoreactive trypsin test (strategy 3) or an extended mutation analysis of the cystic fibrosis gene, that is, a denaturing gradient gel electrophoresis analysis (strategy 4). METHODS. We conducted an economic-modeling exercise in the Netherlands based on published data and expert opinions. Subjects were a hypothetical cohort of 200 000 neonates, the approximate number of children born annually in the Netherlands, and we assessed the costs and number of life-years gained as a result of neonatal screening for cystic fibrosis. The costs and effects of changes in reproductive decisions because of neonatal screening were also assessed. RESULTS. Immunoreactive trypsin + immunoreactive trypsin had the most favorable cost-effectiveness ratio of €24 800 per life-year gained. Immunoreactive trypsin + DNA + denaturing gradient gel electrophoresis achieved more health effects than immunoreactive trypsin + DNA + immunoreactive trypsin at lower cost. The incremental costs per life-year gained of the immunoreactive trypsin + DNA + denaturing gradient gel electrophoresis strategy compared with the immunoreactive trypsin + immunoreactive trypsin strategy were €130 700, whereas the incremental costs of the immunoreactive trypsin + DNA strategy compared with the immunoreactive trypsin + DNA + denaturing gradient gel electrophoresis strategy were €2 154 300. When changes in reproductive decisions as a result of neonatal screening are also taken into account, neonatal screening for cystic fibrosis may lead to financial savings of approximately €1.8 million annually, depending on the screening strategy used. CONCLUSIONS. Cystic fibrosis screening for neonates is a good economic option, and positive health effects can also be expected. Immunoreactive trypsin + immunoreactive trypsin and immunoreactive trypsin + DNA + denaturing gradient gel electrophoresis are the most cost-effective strategies. Copyright © 2006 by the American Academy of Pediatrics. Chemicals / CAS: DNA, 9007-49-2; trypsin, 9002-07-7; Trypsin, EC 3.4.21.4

Associations between lipoxygenases (Lox) and 14-3-3 proteins were demonstrated by two different methods. First, immunoprecipitation experiments, using isoenzyme-specific monoclonal Lox antibodies, showed that 14-3-3 proteins co-precipitate with 13-Lox, but not with the 9-Lox from barley. Second, interactions between 13-Lox and 14-3-3 were established by surface plasmon resonance studies, showing that 13-Lox binds with 14-3-3 proteins in a concentration-dependent manner. The interactions between 14-3-3 proteins and 13-Lox may reveal their role during plant development. Copyright (C) 2000 Federation of European Biochemical Societies. Molecular Sequence Numbers: GENBANK: L35931, L37358, X62388, X93170, Y14200; Chemicals/CAS: 13-lipoxygenase, EC 1.13.11.-; 14-3-3 Proteins; Antibodies, Monoclonal; Isoenzymes; Lipoxygenase, EC 1.13.11.12; Proteins; Recombinant Proteins; Tyrosine 3-Monooxygenase, EC 1.14.16.2

Background: A vaginal microbiota dominated by lactobacilli (particularly Lactobacillus crispatus) is associated with vaginal health, whereas a vaginal microbiota not dominated by lactobacilli is considered dysbiotic. Here we investigated whether L. crispatus strains isolated from the vaginal tract of women with Lactobacillus-dominated vaginal microbiota (LVM) are pheno- or genotypically distinct from L. crispatus strains isolated from vaginal samples with dysbiotic vaginal microbiota (DVM). Results: We studied 33 L. crispatus strains (n = 16 from LVM; n = 17 from DVM). Comparison of these two groups of strains showed that, although strain differences existed, both groups degraded various carbohydrates, produced similar amounts of organic acids, inhibited Neisseria gonorrhoeae growth, and did not produce biofilms. Comparative genomics analyses of 28 strains (n = 12 LVM; n = 16 DVM) revealed a novel, 3-fragmented glycosyltransferase gene that was more prevalent among strains isolated from DVM. Most L. crispatus strains showed growth on glycogen-supplemented growth media. Strains that showed less-efficient (n = 6) or no (n = 1) growth on glycogen all carried N-terminal deletions (respectively, 29 and 37 amino acid deletions) in a putative pullulanase type I protein. Discussion: L. crispatus strains isolated from LVM were not phenotypically distinct from L. crispatus strains isolated from DVM; however, the finding that the latter were more likely to carry a 3-fragmented glycosyltransferase gene may indicate a role for cell surface glycoconjugates, which may shape vaginal microbiota-host interactions. Furthermore, the observation that variation in the pullulanase type I gene is associated with growth on glycogen discourages previous claims that L. crispatus cannot directly utilize glycogen. © 2019 The Author(s). CAS galamustine, 105618-02-8, 107811-63-2; glycogen, 9005-79-2; glycosyltransferase, 9033-07-2

Dietary quercetin intake is suggested to be health promoting, but this assumption is mainly based on mechanistic studies performed in vitro. Previously, we identified rat lung as a quercetin target tissue. To assess relevant in vivo health effects of quercetin, we analyzed mechanisms of effect in rat lungs of a chronic (41 weeks) 1% quercetin diet using whole genome microarrays. We show here that fatty acid catabolism pathways, like beta-oxidation and ketogenesis, are up-regulated by the long-term quercetin intervention. Up-regulation of genes (Hmgcs2, Ech1, Acox1, Pcca, Lpl and Acaa2) was verified and confirmed by quantitative real time PCR. In addition, free fatty acid levels were decreased in rats fed the quercetin diet, confirming that quercetin affects fatty acid catabolism. This in vivo study demonstrates for the first time that fatty acid catabolism is a relevant process that is affected in rats by chronic dietary quercetin. © Birkhäuser Verlag, 2006. Molecular Sequence Numbers: GENBANK: AF217591, AI014074, AI236795, AW530584, AW917667, AY234182, AY331040, BC061782, BC092586, BF281337, BF284618, BF406174, CA510796, CB546044, M84148, NM_012588, NM_012598, NM_013168, NM_013224, NM_017321, NM_017340, NM_019179, NM_022594, NM_057102, NM_130433, NM_138896, NM_173094, S81289, X55180, XM_213425, XM_232194, XM_234686, XM_234745, XM_234749, XM_234878, XM_341195, XM_341383, XM_342500, XM_342742, XM_345742, XM_345750, XM_345754, XM_345756, XM_575533, XM_575534, XM_578308, XM_578339, Z75902, Z93359, Z93363; Chemicals / CAS: quercetin, 117-39-5; Fatty Acids; Quercetin, 117-39-5

Limited information is available about homopolysaccharide synthesis in the genus Lactobacillus. Using efficient screening techniques, extracellular glucosyltransferase (GTF) enzyme activity, resulting in α-glucan synthesis from sucrose, was detected in various lactobacilli. PCR with degenerate primers based on homologous boxes of known glucosyltransferase (gtf) genes of lactic acid bacteria strains allowed cloning of fragments of 10 putative gtf genes from eight different glucan producing Lactobacillus strains (five Lactobacillus reuteri strains, one Lactobacillus fermentum strain, one Lactobacillus sake strain and one Lactobacillus parabuchneri strain). Sequence analysis revealed that these lactobacilli possess a large variation of (putative) gtf genes, similar to what has been observed for Leuconostoc and Streptococcus strains. Homologs of GTFA of Lb. reuteri 121 (synthesizing reuteran, a unique glucan with α(1 → 4) and α-(1 → 6) glycosidic bonds) (Kralj et al., 2002) were found in three of the four other Lb. reuteri strains tested. The other Lactobacillus GTF fragments showed the highest similarity with GTF enzymes of Leuconostoc spp.

The rapid identification of existing and emerging respiratory viruses is crucial in combating outbreaks and epidemics. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid and reliable identification method in bacterial diagnostics, but has not been used in virological diagnostics. Mass spectrometry systems have been investigated for the identification of respiratory viruses. However, sample preparation methods were laborious and time-consuming. In this study, a reliable and rapid sample preparation method was developed allowing identification of cultured respiratory viruses. Tenfold serial dilutions of ten cultures influenza A strains, mixed samples of influenza A virus with human metapneumovirus or respiratory syncytial virus, and reconstituted clinical samples were treated with the developed sample preparation method. Subsequently, peptides were subjected to MALDI-TOF MS and liquid chromatography tandem mass spectrometry (LC-MS/MS). The influenza A strains were identified to the subtype level within 3. h with MALDI-TOF MS and 6. h with LC-MS/MS, excluding the culturing time. The sensitivity of LC-MS/MS was higher compared to MALDI-TOF MS. In addition, LC-MS/MS was able to discriminate between two viruses in mixed samples and was able to identify virus from reconstituted clinical samples. The development of an improved and rapid sample preparation method allowed generic and rapid identification of cultured respiratory viruses by mass spectrometry.

Background: Understanding biochemical and structural changes of the extracellular matrix in Achilles tendinosis might be important for developing mechanism-based therapies. Hypothesis: In Achilles tendinosis, changes occur in biochemical composition and collagen turnover rate. Study Design: Descriptive laboratory study. Methods: From 10 patients undergoing surgery for Achilles tendinopathy, 1 tendinosis biopsy specimen and 1 biopsy specimen of macroscopically healthy tendon tissue adjacent to the lesion were collected. Furthermore, biopsy samples were collected from 3 donors with asymptomatic Achilles tendons. Water content, collagen content, percentage of denatured collagen, amount of lysine hydroxylation, number of enzymatic and nonenzymatic crosslinks, matrix metalloproteinase activity, and matrix metalloproteinase and collagen gene-expression levels were analyzed. Results: In tendinotic lesions, the water content was highest, and collagen content was subnormal with higher amounts of denatured/damaged collagen. Low pentosidine levels in tendinotic tissue indicated the presence of relatively young collagenous matrix. More hydroxylated lysine residues were present in tendinotic samples, but enzymatic crosslinks revealed no differences between tendinotic, adjacent, and healthy samples. In tendinotic specimens, matrix metalloproteinase activity was higher, matrix metalloproteinase gene-expression profile was altered, and collagen type I and III gene expression were upregulated. Conclusion: In Achilles tendinosis, the collagen turnover rate is increased, and the natural biochemical composition of the collagenous matrix is compromised. Clinical Relevance: Although tendon tissue directly adjacent to an Achilles tendinosis lesion looks macroscopically healthy, histological and biochemical degenerative changes in adjacent tissue are evident, which may have implications for surgical interventions. © 2007 American Orthopaedic Society for Sports Medicine.

Polycyclic aromatic hydrocarbons (PAHs) cover a wide range of structurally related compounds which differ greatly in their carcinogenic potency. PAH exposure usually occurs through mixtures rather than individual compounds. Therefore, we assessed whether the effects of binary PAH mixtures on gene expression, DNA adduct formation, apoptosis and cell cycle are additive compared with the effects of the individual compounds in human hepatoma cells (HepG2). Equimolar and equitoxic mixtures of benzo[a]pyrene (B[α]P) with either dibenzo[a,l]pyrene (DB[a,l]P), dibenzo[a,h]anthracene (DB[a,h]A), benzo[b]fluoranthene (B[b]F), fluoranthene (FA) or 1-methylphenanthrene (1-MPA) were studied. DB[a,l]P, B[α]P, DB[a,h]A and B[b]F dose-dependently increased apoptosis and blocked cells cycle in S-phase. PAH mixtures showed an additive effect on apoptosis and on cell cycle blockage. DNA adduct formation in mixtures was higher than expected based on the individual compounds, indicating a synergistic effect of PAH mixtures. Equimolar mixtures of B[α]P and DB[a,l]P (0.1, 0.3 and 1.0 μM) were assessed for their effects on gene expression. Only at 1.0 μM, the mixture showed antagonism. All five compounds were also tested as a binary mixture with B[α]P in equitoxic concentrations. The combinations of B[α]P with B[b]F, DB[a,h]A or FA showed additivity, whereas B[α]P with DB[a,l]P or 1-MPA showed antagonism. Many individual genes showed additivity in mixtures, but some genes showed mostly antagonism or synergism. Our results show that the effects of binary mixtures of PAHs on gene expression are generally additive or slightly antagonistic, suggesting no effect or decreased carcinogenic potency, whereas the effects on DNA adduct formation show synergism, which rather indicates increased carcinogenic potency. © The Author 2007. Published by Oxford University Press. All rights reserved.