Acids in coffee: A review of sensory measurements and meta-analysis of chemical composition.

Coffee contains a variety of organic acids (OAs) and chlorogenic acids (CGAs) that contribute to overall sensory properties. Large variations in preparation and measurement methodology across the literature complicate interpretation of general trends. Here, we perform a systematic review and meta-analysis of the published literature to elucidate the concentrations of OAs and CGAs in both Coffea arabica (arabica) and Coffea canephora (robusta), for both green coffee and roasted coffee at multiple roast levels. A total of 129 publications were found to report acid concentration measurements, yielding 8,634 distinct data points. Analysis of the full data set reveals several trends. First, roasted robusta has considerably more acidic compounds than arabica with 2 to 5 times as much total OAs, and much larger amounts of formic and acetic acid. As for CGAs, in both arabica and robusta 5-CQA is the major component, and progressive roasting decreases the concentration of all CGAs. The total amount of CGA present was more dependent on roast level than the type of coffee (arabica vs. robusta). Overall, this meta-analysis suggests that the increases in certain OAs with roast level might play more of a role in the sensory profile of dark roast coffees than previously suspected.

Identification of UAP1L1 as tumor promotor in gastric cancer through regulation of CDK6.

Gastric cancer (GC) is one of the most commonly diagnosed malignancies in digestive tract and its underlying molecular mechanism is still not clear, so we aimed to reveal the relationship between GC and UDP-GlcNAc pyrophosphorylase-1 like 1 (UAP1L1). The detection of UAP1L1 expression in GC tumor and normal tissues was accomplished by immunohistochemistry and demonstrated the upregulation of UAP1L1 in GC, which was statistically associated with tumor grade. GC cell models constructed via transfection of UAP1L1-silencing/overexpressing lentiviruses were employed for evaluating the effects of UAP1L1 knockdown/overexpression on GC in vitro and in vivo. The results indicated that UAP1L1 played important role in development of GC through regulating cell proliferation, colony formation, cell apoptosis and cell migration. Subsequently, CDK6 was identified as a potential target in UAP1L1 induced regulation of GC, downregulation of which exhibited similar inhibition effects on GC with UAP1L1. Moreover, it was demonstrated that the promotion of GC by UAP1L1 overexpression could be significantly attenuated or even reversed by simultaneously silencing CDK6. In conclusion, UAP1L1 was reported to be a tumor promotor in the development and progression of GC which may exert its role through regulating CDK6 and may act as a candidate of therapeutic target in treatment.

Chemoenzymatic Synthesis of C6-Modified Sugar Nucleotides To Probe the GDP-d-Mannose Dehydrogenase from Pseudomonas aeruginosa.

The chemoenzymatic synthesis of a series of C6-modified GDP-d-Man sugar nucleotides is described. This provides the first structure-function tools for the GDP-d-ManA producing GDP-d-mannose dehydrogenase (GMD) from Pseudomonas aeruginosa. Using a common C6 aldehyde functionalization strategy, chemical synthesis introduces deuterium enrichment, alongside one-carbon homologation at C6 for a series of mannose 1-phosphates. These materials are shown to be substrates for the GDP-mannose pyrophosphorylase from Salmonella enterica, delivering the required toolbox of modified GDP-d-Mans. C6-CH3 modified sugar-nucleotides are capable of reversibly preventing GDP-ManA production by GMD. The ketone product from oxidation of a C6-CH3 modified analogue is identified by high-resolution mass spectrometry.

Analysis of the Cellular Roles of MOCS3 Identifies a MOCS3-Independent Localization of NFS1 at the Tips of the Centrosome.

The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the l-cysteine desulfurase NFS1, MOCS3, and MOCS2A. Human MOCS3 is a dual-function protein that was shown to play an important role in Moco biosynthesis and in the mcm5s2U thio modifications of nucleosides in cytosolic tRNAs for Lys, Gln, and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, on the basis of the absence of Moco in these cells. In addition, mcm5s2U thio-modified tRNAs were not detectable. Because the l-cysteine desulfurase NFS1 was shown to act as a sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods, we identified a MOCS3-independent novel localization of NFS1 at the centrosome.

High expression of REV7 is an independent prognostic indicator in patients with diffuse large B-cell lymphoma treated with rituximab.

REV7 is a multifunctional protein involved in DNA damage tolerance, cell-cycle regulation, gene expression, and carcinogenesis. Although its expression is reportedly associated with poor prognosis in human solid tissue cancers, the significance of REV7 expression in hematopoietic malignancies is unclear. This study evaluated the prognostic significance of REV7 expression in patients with diffuse large B-cell lymphoma (DLBCL) treated with rituximab-combined chemotherapy. Using immunohistochemistry, we analyzed 83 specimens of de novo DLBCL [38 germinal center B-cell-like (GCB) and 45 non-GCB DLBCLs] treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone for REV7 expression. Aberrant REV7 expression was detected in DLBCL cell nuclei. High REV7 expression was associated with significantly shorter overall survival (OS) and progression-free survival (PFS) using Kaplan-Meier analysis and log-rank tests (P < 0.01 and P < 0.01, respectively). Multivariate analysis revealed that REV7 expression is an independent prognostic factor for both OS and PFS. Additionally, when patients were divided into four groups using a combination of REV7 expression and international prognostic index (IPI) or Bcl-2 expression, REV7(High)/IPI(Poor) and REV7(High)/Bcl-2(High) patients showed the poorest outcome. These results indicate that REV7 may be a useful biomarker to predict the prognosis of patients with DLBCL treated with rituximab.

Authentication of official Da-huang by sequencing and multiplex allele-specific PCR of a short maturase K gene.

Rhubarb (official Da-huang) is an important medicinal herb in Asia. Many adulterants of official Da-huang have been discovered in Chinese markets in recent years, which has resulted in adverse effects in medicinal treatment. Here, novel molecular markers based on a short maturase K (matK) gene were developed for authenticating official Da-huang. This study showed that all the species from official Da-huang were clustered together in one clade in the polygenetic trees based on short matK. Two highly conserved single nucleotide polymorphisms of short matK were mined in the species from official Da-huang. Based on these polymophisms, four improved specific primers of official Da-huang were successfully developed that generated reproducible specific bands. These results suggest that the short matK sequence can be considered as a favorable candidate for distinguishing official Da-huang from its adulterants. The established multiplex allele-specific PCR was determined to be simple and accurate and may serve as a preferable tool for authentication of official Da-huang. In addition, we suggest that short-sized specific bands be developed to authenticate materials used in traditional Chinese medicine.

Development of a colorimetric assay and kinetic analysis for Mycobacterium tuberculosis D-glucose-1-phosphate thymidylyltransferase.

dTDP-L-rhamnose as a sugar donor provides L-rhamnosyl residue in the synthesis of disaccharide linker (D-N-acetylglucosamine-L-rhamnose), the key structure of the Mycobacterium tuberculosis cell wall. Four enzymes are involved in the formation of dTDP-L-rhamnose and D-glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the first step of D-glucose-1-phosphate and dTTP to dTDP-D-glucose and PPi. The previous studies on RmlA essentiality proved RmlA as a potential target for antituberculosis drugs. However, there has not been a suitable assay for RmlA to screen inhibitors currently. In this study, the authors reported a microtiter plate-based colorimetric assay for RmlA enzyme activity. Using this assay, the kinetic properties of M. tuberculosis RmlA including initial velocity, optimal temperature, optimal pH, the effect of Mg(2+), and kinetic parameters were determined. The establishment of the accurate and rapid colorimetric assay and kinetic analysis of M. tuberculosis RmlA will facilitate high-throughput screening of RmlA inhibitors.

Plant mitochondria use two pathways for the biogenesis of tRNAHis.

All tRNA(His) possess an essential extra G(-1) guanosine residue at their 5' end. In eukaryotes after standard processing by RNase P, G(-1) is added by a tRNA(His) guanylyl transferase. In prokaryotes, G(-1) is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G(-1) we find here that both maturation pathways can be used. Indeed, tRNA(His) with or without a G(-1) are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNA(His) precursors at both positions G(+1) and G(-1). The G(-1) is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNA(His) without G(-1) has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNA(His) with a G(-1) are recovered. This shows that a previously unreported tRNA(His) guanylyltransferase activity is present in plant mitochondria.

Mitochondrial localization of human FAD synthetase isoform 1.

FAD synthetase or ATP:FMN adenylyl transferase (FADS or FMNAT, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. We face here the still controversial sub-cellular localization of FADS in eukaryotes. First, by western blotting experiments, we confirm the existence in rat liver of different FADS isoforms which are distinct for molecular mass and sub-cellular localization. A cross-reactive band with an apparent molecular mass of 60 kDa on SDS-PAGE is localized in the internal compartments of freshly isolated purified rat liver mitochondria. Recently we have identified two isoforms of FADS in humans, that differ for an extra-sequence of 97 amino acids at the N-terminus, present only in isoform 1 (hFADS1). The first 17 residues of hFADS1 represent a cleavable mitochondrial targeting sequence (by Target-P prediction). The recombinant hFADS1 produced in Escherichia coli showed apparent K(m) and V(max) values for FMN equal to 1.3+/-0.7 microM and 4.4+/-1.3 nmol x min(-1) x mg protein(-1), respectively, and was inhibited by FMN at concentration higher than 1.5 microM. The in vitro synthesized hFADS1, but not hFADS2, is imported into rat liver mitochondria and processed into a lower molecular mass protein product. Immunofluorescence confocal microscopy performed on BHK-21 and Caco-2 cell lines transiently expressing the two human isoforms, definitively confirmed that hFADS1, but not hFADS2, localizes in mitochondria.

Dual SP/ALDH functionalities refine the human hematopoietic Lin-CD34+CD38- stem/progenitor cell compartment.

Identification of prevalent specific markers is crucial to stem/progenitor cell purification. Determinants such as the surface antigens CD34 and CD38 are traditionally used to analyze and purify hematopoietic stem/progenitor cells (HSCs/HPCs). However, the variable expression of these membrane antigens poses some limitations to their use in HSC/HPC purification. Techniques based on drug/stain efflux through the ATP-binding cassette (ABC)G2 pump (side population [SP] phenotype) or on detection of aldehyde dehydrogenase (ALDH) activity have been independently developed and distinguish the SP and ALDH(Bright) (ALDH(Br)) cell subsets for their phenotype and proliferative capability. In this study, we developed a multiparametric flow cytometric method associating both SP and ALDH activities on human lineage negative (Lin(-)) bone marrow cells and sorted different cell fractions according to their SP/ALDH activity level. We find that Lin(-)CD34(+)CD38(Low/-) cells are found throughout the spectrum of ALDH expression and are enriched especially in ALDH(Br) cells when associated with SP functionality (SP/ALDH(Br) fraction). Furthermore, the SP marker identified G(0) cells in all ALDH fractions, allowing us to sort quiescent cells regardless of ALDH activity. Moreover, we show that, within the Lin(-)CD34(+)CD38(-)ALDH(Br) population, the SP marker identifies cells with higher primitive characteristics, in terms of stemness-related gene expression and in vitro and in vivo proliferative potential, than the Lin(-)CD34(+) CD38(-)ALDH(Br) main population cells. In conclusion, our study shows that the coexpression of SP and ALDH markers refines the Lin(-)CD34(+)CD38(-) hematopoietic compartment and identifies an SP/ALDH(Br) cell subset enriched in quiescent primitive HSCs/HPCs.

Studies of enzymes that cause resistance to aminoglycosides antibiotics.

Aminoglycoside antibiotics are highly potent, wide-spectrum bactericidals (1, 2). Bacterial resistance to aminoglycosides, however, is a major problem in the clinical use of aminoglycosides. Enzymatic modification of aminoglycosides is the most frequent resistance mode among several resistance mechanisms employed by resistant pathogens (1,3). Three families of aminoglycoside modifying enzymes, O-phosphotransferases, N-acetyltransferases, and N-nucleotidyltransferases, are known to have more than 50 enzymes (1,3,4). In this chapter, determination of enzymatic activity of a single enzyme from each family in the presence and absence of an inhibitor is described.

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase.

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.

In vitro binding of purified NahR regulatory protein with promoter Psal.

The NahR regulatory protein activates the naphthalene catabolic operon through binding to the Psal promoter in the presence of salicylate. Here, we investigated in vitro binding interaction between NahR and Psal using purified functional recombinant NahR. The T7-tagged NahR was shown to exist as a monomer in solution. Electrophoretic mobility shift assay (EMSA) showed that purified NahR bound to Psal in 3 different forms, whereas surface plasmon resonance (SPR) showed on an SPR chip at ratios ranging from 1:1 (at 0.42 microM NahR) to 8:1 (at 6.8 microM NahR). The binding was slightly inhibited by salicylate, suggesting that salicylate may not be involved in the binding of NahR to the promoter, but rather may be important in the activation of prebound NahR. An examination of the binding kinetics by SPR for the interaction between NahR and Psal revealed that the equilibrium dissociation constant was approximately 2.44 x 10(-6) M and the association and dissociation rates were 7.82 x 10(4) M(-1) s(-1) and 0.191 s(-1), respectively. These results demonstrate for the first time that purified NahR binds as a monomer to Psal and undergoes multimerization. In addition, we present novel data on the kinetics of NahR binding.

A colorimetric assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase activity.

A new method for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase, the enzyme catalyzing the third reaction of the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesis of isoprenoids, is described. This is an end-point assay based on the transformation of inorganic pyrophosphate, one of the products of the reaction, to phosphate by using inorganic pyrophosphatase as auxiliary enzyme. The phosphate formed is reacted then with the dye malachite green to yield a colored product which can be determined spectrophotometrically. The method is easy to perform, sensitive, and robust and can be used in automated high-throughput screening analyses for the search of inhibitors of the enzyme.

REV1 accumulates in DNA damage-induced nuclear foci in human cells and is implicated in mutagenesis by benzo[a]pyrenediolepoxide.

The REV1 gene encodes a Y-family DNA polymerase that has been postulated to have both catalytic and structural functions in translesion replication past UV photoproducts in mammalian cells. To examine if REV1 is implicated in DNA damage tolerance mechanisms after exposure of human cells to a chemical carcinogen, we generated a plasmid expressing REV1 protein fused at its C-terminus with green fluorescent protein (GFP). In transient transfection experiments, virtually all of the transfected cells had a diffuse nuclear pattern in the absence of carcinogen exposure. In contrast, in cells exposed to benzo[a]pyrenediolepoxide, the fusion protein accumulated in a focal pattern in the nucleus in 25% of the cells, and co-localized with PCNA. These data support the idea that REV1 is present at stalled replication forks. We also examined the mutagenic response at the HPRT locus of human cells that had greatly reduced levels of REV1 mRNA due to the stable expression of gene-specific ribozymes, and compared them to wild-type cells. The mutant frequency was greatly reduced in the ribozyme-expressing cells. These data indicate that REV1 is implicated in the mutagenic DNA damage tolerance response to BPDE and support the development of strategies to target this protein to prevent such mutations.

General assay for sugar nucleotidyltransferases using electrospray ionization mass spectrometry.

An electrospray ionization mass spectrometry-based assay has been developed to study the class of enzymes called sugar nucleotidyltransferases that couple sugar-1-phosphates and nucleotide triphosphates to form Leloir pathway glycosyl donors. The recombinant Escherichia coli and the commercially available yeast uridine-diphosphoglucose pyrophosphorylases were used as model systems. This technique allows the simultaneous and direct detection of the substrates and products without separation and, as described, is as sensitive as traditional coupled techniques. More importantly, the assay is capable of easily measuring kinetic values and inhibition constants for a range of natural and nonnatural substrates. This new assay was used to show for the first time that the reaction of the commercially available yeast uridine-diphosphoglucose pyrophosphorylase preparation is competitively inhibited by adenosine 5'-triphosphate (ATP), an observation that indicates a single active site that accepts both uridine 5'-triphosphate and ATP substrates.

An assay for adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase.

Adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5'-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S(0.5) determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.

Isolation and characterization of bluensomycin biosynthetic genes from Streptomyces bluensis.

The biosynthetic gene cluster for bluensomycin, a member of the aminoglycoside family of antibiotics, was isolated and characterized from the bluensomycin producing strain, Streptomyces bluensis ATCC27420. PCR primers were designed specifically to amplify a segment of the dTDP-glucose synthase gene based on its conserved sequences among several actinomycete strains. By screening a cosmid library using amplified PCR fragments, a 30-kb DNA fragment was isolated. Sequence analysis identified 15 open reading frames (ORFs), eight of which had previously been identified by Piepersberg et al. But seven are novel to this study. We demonstrated that one of these ORFs, blmA, confers resistance against the antibiotic dihydrostreptomycin, and another, blmD, encodes a dTDP-glucose synthase. These findings suggest that the isolated gene cluster is very likely to be responsible for the biosynthesis of bluensomycin.

Role of glycosylation in hyperphosphorylation of tau in Alzheimer's disease.

In Alzheimer's disease (AD) brain, microtubule-associated protein tau is abnormally modified by hyperphosphorylation and glycosylation, and is aggregated as neurofibrillary tangles of paired helical filaments. To investigate the role of tau glycosylation in neurofibrillary pathology, we isolated various pools of tau protein from AD brain which represent different stages of tau pathology. We found that the non-hyperphosphorylated tau from AD brain but not normal brain tau was glycosylated. Monosaccharide composition analyses and specific lectin blots suggested that the tau in AD brain was glycosylated mainly through N-linkage. In vitro phosphorylation indicated that the glycosylated tau was a better substrate for cAMP-dependent protein kinase than the deglycosylated tau. These results suggest that the glycosylation of tau is an early abnormality that can facilitate the subsequent abnormal hyperphosphorylation of tau in AD brain.