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γ-aminobutyric acid (GABA) has various physiological functions and is widely used in medicine, food, and other fields. Glutamate decarboxylase (GAD) is a key enzyme that catalyzes the decarboxylation of L-glutamate to synthesize GABA. However, the industrial application of microorganism-derived GAD is limited by its rapid loss of enzymatic activity with pH approaching neutrality. In this study, a novel glutamate decarboxylase, GAD MSM, from Mycobacterium smegmatis was overexpressed and purified. On the basis of homologous modeling and substrate molecular docking, several GAD MSM mutants were constructed, and their enzymatic properties were analyzed. The results showed that the optimal pH of wild-type GAD MSM is 5.4; at pH 6.2, 22.8% enzymatic activity was retained; the T211 amino acid residue and C-terminal deletion mutant GAD MSMΔC showed relatively high catalytic activity in a pH range of 5.0–7.0. The V max and K m values of GAD MSMΔC were 14.69 and 5.70, respectively, at pH 5.5, and 9.87 and 6.17, respectively, at pH 7.0. Compared with the wild-type GAD, GAD MSMΔC still maintained higher affinity and enzymatic activity of the substrate, maintaining 78.5% of the highest enzymatic activity even at pH 7.0, which is the highest reported activity retention for GAD under neutral pH condition. Therefore, GAD MSMΔC can be used for the transformation of high-yielding strains and industrial production of GABA.

Neutrase-hydrolysates hydrolyzed from mulberry leaf proteins were separated by ion exchange chromatography, gel filtration chromatography, and semipreparative reverse-phase HPLC. Purified fractions were analyzed for their radical scavenging activity, hemolysis inhibition ability, and cellular antioxidant activity (CAA). Three new antioxidant peptides, P1 (SVL, 317 Da), P2 (EAVQ, 445 Da), and P3 (RDY, 452 Da), were obtained from the most active HPLC fraction (R1) and identified using UPLC-QTOF-MS. These three peptides were then synthesized, and their antioxidant activities were analyzed. P1 and P2 had no ability to inhibit hemolysis of erythrocytes but did show antioxidant activity on HepG2 cells. P3 showed the highest hemolysis inhibition ability (92%) and CAA value (2204 μM QE/100 g peptide). The Tyr residues at the C-terminal region play an important role in the antioxidant activity in P3. Thus, the natural peptide R1 and synthesized P3 could be used as antioxidants and might be promising components of functional foods.

Aminolevulinic acid (ALA), a type of natural non-protein amino acid, is a key precursor for the biosynthesis of heme, and it has been broadly applied in medicine, agriculture. Several strategies have been applied to enhance ALA synthesis in bacteria. In the present study, we employed synthetic antisense RNAs (asRNAs) of hemB (encodes ALA dehydratase) to weaken metabolic flux of ALA to porphobilinogen (PBG), and investigated their effect on ALA accumulation. For this purpose, we designed and constructed vectors pET28a-hemA-asRNA and pRSFDuet-hemA-asRNA to simultaneously express 5-ALA synthase (ALAS, encoded by hemA) and PTasRNAs (2 inverted repeat DNA sequences sandwiched with the antisense sequence of hemB), selecting the region ranging from − 57 nt upstream to + 139 nt downstream of the start codon of hemB as a target. The qRT-PCR analysis showed that the mRNA levels of hemB were decreased above 50% of the control levels, suggesting that the anti-hemB asRNA was functioning appropriately. ALA accumulation in the hemB weakened strains were 17.6% higher than that obtained using the control strains while accumulating less PBG. These results indicated that asRNAs can be used as a tool for regulating ALA accumulation in E. coli. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02733-8.

The prevalence of Staphylococcus aureus in 2160 bulk ready-to-eat foods from the Sichuan province of China during 2013&ndash, 2016 was investigated. The antibiotic resistance and the associated genes, as well as biofilm formation capacity of the S. aureus isolates were measured. Furthermore, the relationship between the antibiotic resistance and the resistant genes was discussed. It was found that 54 S. aureus isolates were recovered, and their prevalence in meat products, dairy, fruit and vegetables, and desserts were 31 (2.6%), six (3.0%), nine (2.2%) and eight (2.3%), respectively. Most strains (52/54) were resistant to at least one of the antibiotics, and 21 isolates were identified as multidrug-resistant (MDR) S. aureus. Three isolates were found to be methicillin-resistant S. aureus. Penicillin, erythromycin, clindamycin, tetracycline and inducible clindamycin resistance were determined as the predominant antibiotics, and the isolates with the phenotypic resistance on these five antibiotics were all determined positive for the resistant gene associated. In total, 33 of 54 S. aureus isolates showed biofilm formation capacity, including two strong biofilm producers, one moderate and 30 weak ones. Two S. aureus isolates with strong biofilm formation abilities showed multi-drug resistance, and one moderate biofilm producer was resistant to two categories of antibiotics.

By the standard of today&rsquo, s image-guided surgery (IGS) technology, in order to check and verify the progress of the surgery, the surgeons still require divert their attention from the patients occasionally to check against the display. In this paper, a mixed-reality system for medical use is proposed that combines an Intel RealSense sensor with Microsoft&rsquo, s Hololens head-mounted display system, for superimposing medical data onto the physical surface of a patient, so the surgeons do not need to divert their attention from their patients. The main idea of our proposed system is to display the 3D medical images of the patients on the actual patients themselves by placing the medical images and the patients in the same coordinate space. However, the virtual medical data may contain noises and outliers, so the transformation mapping function must be able to handle these problems. The transform function in our system is performed by the use of our proposed Denoised-Resampled-Weighted-and-Perturbed-Iterative Closest Points (DRWP-ICP) algorithm, which performs denoising and removal of outliers before aligning the pre-operative medical image data points to the patient&rsquo, s physical surface position before displaying the result using the Microsoft HoloLens display system. The experimental results shows that our proposed mixed-reality system using DRWP-ICP is capable of performing accurate and robust mapping despite the presence of noise and outliers.

Peptide is a promising source of safe hypoglycaemics in nutraceuticals. A biocatalytic approach, isolation and purification procedures, amino acid analysis, and identification of peptides were implemented successively. Results indicated that the hydrolysates with high α-glucosidase inhibitory activity (α-GIA) were obtained from hemp seed protein (HSP) by Alcalase treatment at the degree of hydrolysis (DH) of 27.24 ± 0.88%. Interestingly, the hydrolysates at DH ≤ 9.68 ± 0.45% could be an α-glucosidase activator instead. Two novel α-glucosidase inhibitory peptides with their sequences of Leu-Arg (287.2 Da) and Pro-Leu-Met-Leu-Pro (568.4 Da) were identified. Hydrophobic amino acids in the peptides, particularly Pro and Leu, contribute greatly to the α-GIA, while rich essential amino acids, branched chain amino acids, Pro and Met enlarged markedly the application in functional foods. The present research provided a green route for production of novel α-glucosidase inhibitory peptides as nutrient and functional food ingredients in the management of hyperglycaemia.

An efficient microwave-assisted extraction (MAE) technique was applied to isolate total steroid saponins from Dioscorea zingiberensis C.H. Wright (DZW). The optimal extracting conditions were established as 75% ethanol as solvent, ratio of solid/liquid 1:20 (g/ml), temperature 75 °C, irradiation power 600 W and three extraction cycles of 6 min each. Scanning electron microscopy (SEM) images of DZW processed by four different extractions provided visual evidence of the disruption effect on DZW. Diosgenin was quantified by HPLC and examined further by LC-ESI/MS after acid hydrolysis. Total steroid saponins were calculated using diosgenin from total steroid saponins. The MAE procedure was optimized, validated and compared with other conventional extraction processes. This report provides a convenient technology for the extraction and quantification of total saponins of DZW combining MAE with HPLC and LC-ESI/MS for the first time.

A comprehensive knowledge of the types and ratios of microbes that inhabit the healthy human gut is necessary before any kind of pre-clinical or clinical study can be performed that attempts to alter the microbiome to treat a condition or improve therapy outcome. To address this need we present an innovative scalable comprehensive analysis workflow, a healthy human reference microbiome list and abundance profile (GutFeelingKB), and a novel Fecal Biome Population Report (FecalBiome) with clinical applicability. GutFeelingKB provides a list of 157 organisms (8 phyla, 18 classes, 23 orders, 38 families, 59 genera and 109 species) that forms the baseline biome and therefore can be used as healthy controls for studies related to dysbiosis. This list can be expanded to 863 organisms if closely related proteomes are considered. The incorporation of microbiome science into routine clinical practice necessitates a standard report for comparison of an individual's microbiome to the growing knowledgebase of "normal" microbiome data. The FecalBiome and the underlying technology of GutFeelingKB address this need. The knowledgebase can be useful to regulatory agencies for the assessment of fecal transplant and other microbiome products, as it contains a list of organisms from healthy individuals. In addition to the list of organisms and their abundances, this study also generated a collection of assembled contiguous sequences (contigs) of metagenomics dark matter. In this study, metagenomic dark matter represents sequences that cannot be mapped to any known sequence but can be assembled into contigs of 10,000 nucleotides or higher. These sequences can be used to create primers to study potential novel organisms. All data is freely available from https://hive.biochemistry.gwu.edu/gfkb and NCBI's Short Read Archive.

The metabolism of host cholesterol by Mycobacterium tuberculosis is an important factor for both its virulence and pathogenesis. However, the rationale for this cholesterol metabolism has not been fully understood yet. In the present study, we characterized several previously undescribed acyl-CoA synthetases that are involved in the steroid side-chain degradation in Mycobacterium smegmatis, and an analogue of intermediate from steroid degradation, 5′-O-(lithocholoyl sulfamoyl) adenosine (LCA-AMS), was successfully designed and synthesized to be used as a specific anti-mycobacterial agent. The acyl-CoA synthetases exhibited strong preferences for the length of side chain. FadD19 homologs, including FadD19 (MSMEG_5914), FadD19-2 (MSMEG_2241), and FadD19-4 (MSMEG_3687), are unanimously favorable cholesterol with a C8 alkanoate side chain. FadD17 (MSMEG_5908) and FadD1 (MSMEG_4952) showed high preferences for steroids, containing a C5 alkanoate side chain. FadD8 (MSMEG_1098) exhibited specific activity toward cholestenoate with a C8 alkanoate side chain. An acylsulfamoyl analogue of lithocholate, 5′-O-(lithocholoyl sulfamoyl) adenosine (LCA-AMS), was designed and synthesized. As expected, the intermediate analogue not only specifically inhibited those steroid-activated acyl-CoA synthetases, but also selectively inhibited the growth of mycobacterial species, including M. tuberculosis, M. smegmatis, and Mycobacterium neoaurum. Overall, our research advanced our understanding of mycobacterial steroid degradation and provided new insights to develop novel mechanism-based anti-mycobacterial agents. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-019-1703-y) contains supplementary material, which is available to authorized users.

A novel microbial procedure was proposed for diosgenin production from Dioscorea zingiberensis C.H. Wright (DZW) tubers via employing Aspergillus awamori for the first time. The optimal conditions of fermenter cultivation were established as inoculation dosage of 8%, fermentation temperature of 30 °C, cultivation time of 8 days, initial pH of 7.0 and a stirring rate of 180 rpm when the converted diosgenin content reached a peak value of 74.26 ± 3.23 mg/g substrate. The product was purified by silica gel column and then confirmed as diosgenin (purity: 96.9 ± 2.42%) by nuclear magnetic resonance (NMR). Compared with traditional acid hydrolysis, this new process generated indeed less wastewater with lower chemical oxygen demand (COD) reduced to 500 mg/L from 10,000 mg/L and absence of acid and alkali. This research provided definitely an environmental and high-efficiency microbial technology for diosgenin production.

The presence of redundant genes in a genome might enable bacteria to adapt to varying environmental conditions. The genus Gordonia comprises metabolically diverse bacteria that can degrade a wide range of persistent compounds. This paper reports the multiplicity of genes encoding 3-ketosteroid Δ1-dehydrogenase (KstD) in Gordonia neofelifaecis NRRL B-59395. KstD is one of the key enzymes in microbial steroid catabolism. Five kstD homologues (kstD1–kstD5) distributed over several phylogenetic groups were investigated in G. neofelifaecis NRRL B-59395. Escherichia coli cells expressing kstD homologues were used to transform different steroids, including cholesterol, cholest-4-en-3-one, 4-androstene-3,17-dione (AD), progesterone, and 16α,17α-epoxyprogesterone. All five enzymes displayed KstD activity toward 3-ketosteroids, but none were able to transform 3-hydroxysteroid substrates such as cholesterol. KstD2, KstD3, and KstD5 catalyzed the dehydrogenation reaction of 16α,17α-epoxyprogesterone, while KstD1, KstD3, and KstD4 transformed AD into androsta-1,4-diene-3,17-dione. Transcriptional analyses revealed that the expression of kstD1, kstD3, and kstD4 was upregulated during culture in cholesterol or AD when compared with expression during culture in pyruvic acid. kstD2 and kstD5 were uniquely induced by cholesterol. Thus, the presence of multiple kstD genes in the G. neofelifaecis NRRL B-59395 genome facilitates a dynamic and fine-tuned response to environmental changes.

A novel peptide was extracted from wheat germ globulin and purified using ion-exchange chromatography, gel filtration chromatography, and semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The sequence of the peptide was found to be Glu-Cys-Phe-Ser-Thr-Ala (ECFSTA). Its immunomodulatory effects were evaluated, and the results showed that ECFSTA could enhance phagocytosis of RAW 264.7 cells and significantly increase their secretion of nitric oxide (NO), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and reactive oxygen species (ROS). ECFSTA activated macrophages mainly through the pattern recognition receptors (PRRs) of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4), and the production of ROS simultaneously stimulated macrophages to produce TNF-α. Thus, ECFSTA could be used as an immunomodulator and might be a promising component of functional foods.

3-Ketosteroid-∆1-dehydrogenase (KstD), a key enzyme in microbial steroid catabolism, catalyzes the trans-axial elimination of the C1 and C2 hydrogen atoms of the A-ring from the polycyclic ring structure of 3-ketosteroids, and it was usually used to transform androst-4-ene-3,17-dione (AD) to produce androsta-1,4-diene-3,17-dione. Here, the KstD from Gordonia neofelifaecis was expressed efficiently in Escherichia coli. E. coli cells expressing KstD3gor were subjected to the investigation of dehydrogenation activity for different steroids. The results showed that KstD3gor has a clear preference for steroid substrates with 3-keto-4-ene configuration, and it exhibits higher activity towards steroid substrates carrying a small or no aliphatic side chain than towards substrates having a bulky side chain at the C-17 atom. The recombinant strain could efficiently convert androst-4,9(11)-dien-3,17-dione into androst-1,4,9(11)-trien-3,17-dione (with conversion rate of 96%). 1(2)-Dehydrogenation of androst-4,9(11)-dien-3,17-dione is one of the key steps in glucocorticoid production. To the best of our knowledge, this is the first study reporting on the conversion of androst-4,9(11)-dien-3,17-dione catalyzed by recombinant KstD; the expression system of KstD3gor reported here would have an impact in the industrial production of glucocorticoid in the future.

In our previous study, three novel polysaccharides, named MC-1, MC-2, and MC-3, were separated from the roots of maca (Lepidium meyenii), which is a food source from the Andes region. The structural information and immunomodulatory activity of MC-1 were then investigated. The structure and activity of MC-2 are still unknown. In this study, structural characterization revealed that MC-2 has an average molecular weight of 9.83 kDa and is composed of arabinose (20.9%), mannose (4.5%), glucose (71.9%), and galactose (2.7%). The main linkage types of MC-2 were proven to be (1→5)-α-l-Ara, (1→3)-α-l-Man, (1→)-α-d-Glc, (1→4)-α-d-Glc, (1→6)-α-d-Glc, and (1→6)-β-d-Gal by methylation and NMR analyses. Congo red assay showed that MC-2 possesses a triple-helix conformation. Immunostimulating assays indicated that MC-2 could induce M1 polarization of original macrophages and convert M2 macrophages into M1 phenotype. Although MC-2 could not shift M1 macrophages into M2, it could still inhibit inflammatory reactions induced by lipopolysaccharide. Furthermore, Toll-like receptor 2, tTll-like receptor 4, complement receptor 3, and mannose receptor were confirmed as the membrane receptors for MC-2 on macrophages. These results indicate that MC-2 could potentially be used toward hypoimmunity and tumor therapies.

The hydrolysate from duck egg white protein (DEWP) prepared by "SEEP-Alcalase" at degree of hydrolysis (DH) value of 21% (namely HSA21) exhibited high antioxidant capacity in different oxidation systems. A consecutive chromatographic method was then developed for separation and purification of HSA21, including ion-exchange chromatography, macroporous adsorption resin (MAR) and gel filter chromatography. The final peptides "P21-3-75-B" were obtained with significantly enhanced antioxidant activity (p

Ferritin is a hollow sphere protein composed of 24 subunits that can store up to 4500 iron atoms in its inner cavity. It is mainly found in the liver and spleen but also in serum at trace levels. Serum ferritin is considered as the best single indicator in assessing body iron stores except liver or bone marrow biopsy. However, it is confounded by other disease conditions. Ferritin bound iron (FBI) and ferritin saturation have been suggested as more robust biomarkers. The current techniques for FBI determination are limited by low antibody specificity, low instrument sensitivity and possible analyte losses during sample preparation. The need for a highly sensitive and reliable method is widely recognized. Here we describe a novel technique to detect serum FBI using species-specific isotope dilution mass spectrometry (SS-IDMS). [(57)Fe]-ferritin was produced by biosynthesis and in vitro labeling with the (57)Fe spike in the form of [(57)Fe]-citrate after cell lysis and heat treatment. [(57)Fe]-ferritin for sample spiking was further purified by fast liquid protein chromatography. Serum ferritin and added [(57)Fe]-ferritin were separated from other iron species by ultrafiltration followed by isotopic analysis of FBI using negative thermal ionization mass spectrometry. Repeatability of our assay is 8% with an absolute detection limit of 18 ng FBI in the sample. As compared to other speciation techniques, SS-IDMS offers maximum control over sample losses and species conversion during analysis. The described technique may therefore serve as a reference technique for clinical applications of FBI as a new biomarker for assessing body iron status.

Natural and nutrient substances for cardiovascular disease are promising and capture researchers' minds. Two kinds of novel bioactive peptides (high Fischer's ratio oligopeptides and anticoagulant peptides) were obtained from Whitmania pigra protein via enzymatic hydrolysis. An oligopeptide (MW

A number of podophyllotoxin derivatives (3A-3J) had been designed and synthesized, and their biological activities were evaluated in this study. Moreover, the antiproliferation activities of these compounds against four human cancer cell lines (HepG2, HeLa, A549, and MCF-7) were also tested. The results indicated that the most promising compound 3D displayed potent inhibitory activity over the four human cancer cell lines and was further demonstrated to have potent tubulin polymerization inhibitory effects without damaging the non-cancer cells. Additionally, 3D was verified to effectively interfere with tubulin and could prevent the mitosis of cancer cells, leading to cell cycle arrest and eventually inducing apoptosis in a dose- and time-dependent manner. Moreover, the Western blotting and siRNA results showed that Bcl-2 was downregulated in HepG2 cells treated with 3D. Finally, the molecular docking simulation results revealed that 3D could fit well in the colchicine-binding pocket. Taken together, this study has provided certain novel antitubulin agents for possible cancer chemotherapy.

Direct comparisons of the reactivity and mechanistic pathways for anionic systems in the gas phase and in solution are presented. Rate constants and kinetic isotope effects for the reactions of methyl, ethyl, isopropyl, and tert-butyl iodide with cyanide ion in the gas phase, as well as for the reactions of methyl and ethyl iodide with cyanide ion in several solvents, are reported. In addition to measuring the perdeutero kinetic isotope effect (KIE) for each reaction, the secondary alpha- and beta-deuterium KIEs were determined for the ethyl iodide reaction. Comparisons of experimental results with computational transition states, KIEs, and branching fractions are explored to determine how solvent affects these reactions. The KIEs show that the transition state does not change significantly when the solvent is changed from dimethyl sulfoxide/methanol (a protic solvent) to dimethyl sulfoxide (a strongly polar aprotic solvent) to tetrahydrofuran (a slightly polar aprotic solvent) in the ethyl iodide-cyanide ion S(N)2 reaction in solution, as the "Solvation Rule for S(N)2 Reactions" predicts. However, the Solvation Rule fails the ultimate test of predicting gas phase results, where significantly smaller (more inverse) KIEs indicate the existence of a tighter transition state. This result is primarily attributed to the greater electrostatic forces between the partial negative charges on the iodide and cyanide ions and the partial positive charge on the alpha carbon in the gas phase transition state. Nevertheless, in evaluating the competition between S(N)2 and E2 processes, the mechanistic results for the solution and gas phase reactions are strikingly similar. The reaction of cyanide ion with ethyl iodide occurs exclusively by an S(N)2 mechanism in solution and primarily by an S(N)2 mechanism in the gas phase; only approximately 1% of the gas phase reaction is ascribed to an elimination process.

Monoclonal antibodies (mAbs) have the potential to be a very powerful tool in proteomics research to determine protein expression, quantification, localization and modification, as well as protein-protein interactions, especially when combined with microarray technology. Thus, a large amount of well-characterized and highly qualified antibodies are needed in proteomics. Purified antigen, which is not always available, has proven to be one of the rate-limiting steps in mAb large-scale generation. Here we describe our strategies to establish a murine hybridoma cell bank for human liver mitochondria using unknown native proteins as the immunogens. The antibody-recognized mitochondrial proteins were identified by MS following immunoprecipitation (IP), and by screening of human liver cDNA expression library. We found that the established antibodies reacted specifically with a number of important enzymes in mitochondria. The subcellular localization of these antigens in mitochondria was further confirmed by immunohistocytochemistry. A panel of antibodies was also tested for their ability to capture and deplete the targeting proteins and complexes from the total mitochondrial proteins. We believe these well-characterized antibodies would be useful in various applications for Human Liver Proteome Project (HLPP) when the scale of this hybridoma cell bank is enlarged significantly in the near future.

Apoptosis induced by high concentrations of nicotinamide in tobacco suspension cells was observed. When cells were treated with 250 mM nicotinamide for 24 h, the hallmarks of apoptosis were detected, including DNA fragments increasing in size by multiples of 180–200 bp, condensation and peripheral distribution of nuclei chromatin and positive reaction to the TUNEL assay. In addition, the degradation of poly (ADP-ribose) polymerase (PARP) was also detected. This indicates that caspase-3-like activity is involved in apoptosis in cultured tobacco cells induced by high-concentration nicotinamide. However, as an inhibitor of PARP, nicotinamide has a contrary effect on apoptosis at low concentrations, which suggests that nicotinamide plays a dual role depending on to its concentration in cells.

The Sterile Alpha Motif Domain-containing 9 (SAMD9) gene has been recently emphasized during the discovery that it is expressed at a lower level in aggressive fibromatosis and some cases of breast and colon cancer, however, the underlying mechanisms are poorly understood. Here, we found that SAMD9 is down-regulated in human non-small cell lung cancer (NSCLC). Furthermore, knockdown of SAMD9 expression is increased the invasion, migration and proliferation in H1299 cells in vitro and overexpression of SAMD9 suppressed proliferation and invasion in A549 cells. Finally, depletion of SAMD9 increases tumor formation in vivo. Our results may provide a strategy for blocking NSCLC tumorigenesis and progression.

Gordonia neofelifaecis NRRL B-59395 was initially isolated from the fresh feces of a clouded leopard based on its ability to degrade cholesterol. The transcriptome profiles of G. neofelifaecis NRRL B-59395 grown with cholesterol, androstenedione (AD), and pyruvic acid were compared by RNA-Seq. The sterol catabolic genes are highly conserved in G. neofelifaecis, Rhodococcus jostii RHA1, and Mycobacterium tuberculosis. The RNA-Seq results indicated that the genes involved in the sterol side chain cleavage were exclusively induced by cholesterol, while the genes involved in the degradation of rings A/B and C/D were up-regulated by both cholesterol and AD. It appears that the induction mechanisms for the genes responsible for side chain cleavage and those for degradation of rings are different. There are approximately 21 genes encoding transporter proteins that are differentially expressed in cholesterol or AD compared with pyruvic acid. The genes camABCD and camM encode two systems that take up cholate, and they have been shown to be cholesterol- and AD-inducible. The potential biological functions of other differentially expressed genes are also discussed. These results will promote the functional characterization of the sterol catabolic genes and also provide important clues in understanding the mechanisms of their gene expression, and they may help us understand the mechanism underlying microbial cholesterol catabolism.

An enzymatic hydrolysis approach was proposed for the preparation of bioactive hydrolysate of scorpion Buthus martensii Karsch (BmK) protein. Results showed that the anticoagulant activity of the hydrolysates of BmK protein was closely related to both the enzyme type and the degree of hydrolysis. Alcalase AF showed to be the best enzymes for the hydrolysis. And the hydrolysis degree (DH) was closely related with the anticoagulant activity of the hydrolyzate. At a DH value of 18% with Alcalase AF, the hydrolyzate exhibited the highest activity. The hydrolysate was then separated and purified by consecutive chromatographic procedures, giving a novel anticoagulant peptide consisting of ten amino acids (MW: 1119.8Da) with its sequence of Val-Glu-Pro-Val-Thr-Val-Asn-Pro- His-Glu identified by MALDI-TOF/TOF MS. The negatively charged amino acids and hydrophobic amino acids may contribute to the anticoagulant activity of the prepared peptides. And the Val residue in N-terminal was also critical for the anticoagulant activity of the BmK peptide. Furthermore, the anticoagulant activity kept stable after in vitro digestive simulation. This research provided a promising bioprocessing route for production of anticoagulant peptides using BmK protein as a potentially valuable bioresource.

In tumor cells telomerase activity is associated with resistance to apoptosis and the introduction of the human telomerase reverse transcriptase (hTERT) subunit into normal human cells is associated with life span extension of the cells. To determine the role of telomerase in regulating apoptosis, telomerase negative human embryo lung fibroblasts were transfected with the hTERT gene. Unlike the control fibroblasts, the telomerase-expressing cells had elongated telomeres and were resistant to apoptosis induced by hydroxyl radicals. The results indicate that expression of telomerase and, thus, the maintenance of telomere length in normal human somatic cells caused resistance to not only cellular senescence but also apoptosis. Moreover, we found that hydroxyl radical-induced apoptosis in telomerase-expressing and control fibroblasts was caspase-3 independent. These findings have revealed a new type of interrelation between telomerase and caspase-3, which may indicate that in this case the expressed telomerase may inhibit apoptosis at a site not related to the caspase-3 cascade.

The primary hydrogen−deuterium incoming nucleophile KIEs for the SN2 reactions between para-substituted benzyl chlorides and borohydride ion in DMSO at 30.000 ± 0.002 °C are small (≤1.14) and insensitive to a change in substituent at the α-carbon. The small Hammett ρ (0.51) and ρr (−0.52) values found when the para substituent on the benzene ring of the substrate is altered indicate there is very little charge on the α-carbon in the transition state. The large, constant secondary α-deuterium KIEs of 1.089 ± 0.002 and the large chlorine leaving group KIEs of 1.0076, 1.0074, and 1.0078 found for the p-methyl-, the p-hydrogen-, and the p-chlorobenzyl chloride reactions suggest that the transition states for these reactions are unsymmetric with short H−Cα and long B−Η and Cα−Cl bonds. The decrease in the chlorine leaving group KIE from 1.0076 ± 0.0003 for the p-methylbenzyl chloride reaction to 1.0036 ± 0.0003 for the p-nitrobenzyl chloride reaction indicates the Cα−Cl bond shortens markedly when a strongly e...

In recent years, apoptosis has been reported to exist in plants during normal development and in response to stress. However, little is known about the relation of hormones to this form of programmed cell death. Here, we report examination of characteristics of apoptosis in carrot protoplasts induced by ethylene evolved from ethrel (2-chloroethylphosphonic acid). Nucleus condensation and DNA ladders were observed, and neutral comet assay, which detects DNA cleavage, also provided evidence that ethrel treatment resulted in nuclear DNA fragmentation. Strikingly, a close correlation between the incidence of DNA comets and the percentage of apoptotic protoplasts was shown in ethrel-treated carrot protoplasts. In conclusion, this study demonstrated that ethylene is an active inducer of apoptosis in carrot protoplasts, and that ethylene-induced plant cell death showed characteristics similar to those of apoptosis in animals.

The secondary α deuterium and heavy atom kinetic isotope effects found for two different SN2 reactions suggest that the magnitude of secondary α deuterium kinetic isotope effects can be determined by the length of only the shorter (stronger) reacting bond in an unsymmetrical SN2 transition state rather than by the usual nucleophile−leaving group distance. Although this means the interpretation of these isotope effects is more complex than has been recognized, the results suggest that they can be used to determine whether an SN2 transition state is symmetrical or unsymmetrical.

A large incoming group 11C/14C kinetic isotope effect of 1.0104 ± 0.0023 has been measured for the SN2 reaction between benzyl chloride and labeled cyanide ion in 20% aqueous DMSO at 30.00 °C. These large incoming group kinetic isotope effects have also been measured for the SN2 reactions of other para-substituted benzyl chlorides with cyanide ion. A combination of the incoming group 11C/14C and chlorine (Hill J. W.; Fry, A., J. Am. Chem. Soc. 1962, 84, 2763) leaving group kinetic isotope effects have been used to model the SN2 transition states for these reactions. Adding a more electron-withdrawing para substituent to the substrate does not affect the stronger N⋮C- - -Cα reacting bond significantly but shortens the weaker Cα- - -Cl reacting bond markedly.

The catecholamines (50 μM dopamine, 50 μM norepinephrine and 100 μM epinephrine) and phenylethylamine (200 μM) were found to stimulate ethylene production in potato suspension cultures. When 100 μM amino-oxyacetic acid was added together with epinephrine, ethylene release returned to control levels. The endogenous 1-aminocyclopropane-1-carboxylic acid levels were increased in parallel with the release of ethylene, suggesting that the observed effect probably occurs via regulation of aCC synthase. Our results suggest that there is a link between these naturally occurring monoamines and ethylene in plants.

In this work, an ordered mesoporous titania film was introduced to coat a capillary by means of the sol-gel technique. Its electroosmotic flow (EOF) property was investigated in a variety of nonaqueous media (methanol, formamide and N,N'-dimethylformamide and mixtures of methanol and acetonitrile). The titania-coated capillary exhibited a distinctive EOF behavior, the direction and magnitude of which were strongly dependent on various parameters such as the solvent composition, apparent pH (pH*) and the electrolytes. The nonaqueous capillary electrophoresis separation of several alkaloids was investigated in the positively charged titania-coated capillary. Comparison of separation between coated and uncoated capillaries under optimal nonaqueous conditions was also carried out.

Reactive oxygen species (ROS) have been found to trigger apoptosis in tumor cells. At the same time, telomerase is found to be associated with malignancy and reduced apoptosis. However little is known about the linkage between ROS such as *OH and telomerase/telomere. To address the interrelations between *OH and telomerase/telomere in tumor cell killing, HeLa, 293 and MW451 cells were induced to undergo apoptosis with *OH radicals generated via Fe(2+)-mediated Fenton reactions (0.1 mM FeSO(4) plus 0.3-0.9 mM H2O2) and telomerase activity, telomere length were measured during apoptosis. We found that during *OH-induced apoptosis, telomere shortening took place while no changes in telomerase activity were observed. Our results suggest that *OH-induced telomere shortening is not through telomerase inhibition but possibly a direct effect of *OH on telomeres themselves indicating that telomere shortening but not telomerase inhibition is the primary event during *OH-induced apoptosis. Strikingly, we also found that *OH-induced apoptosis in HeLa cells is caspase-3-independent but is associated with reduction of mitochondrial transmembrane potential. Our results indicate that *OH triggers apoptotic tumor cell death through a telomere-related, caspase-independent pathway.

Apoptosis in heat shock-treated tobacco protoplasts was evidenced by DNA fragmentation, flow cytometric analysis and activation of caspase 3-like protease. Furthermore, an in vitro apoptosis system was established which reproduced the apoptotic events. Western blotting analysis using an antibody against lamin A and C showed that in both in vivo and in vitro systems lamin-like proteins were cleaved into a 35-kDa fragment, and that lamin-like protein degradation precedes DNA fragmentation. Moreover, we found a 22.8-fold increase in caspase 6-like activity in cytosol of heat-treated protoplasts as compared with the control.

The acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex catalyzes the reversible cleavage and synthesis of acetyl-CoA in methanogens. This report of the enzyme complex in Archaeoglobus fulgidus demonstrates the existence of a functional ACDS complex in an organism that is not a methanogen. The A. fulgidus enzyme complex contained five subunits of 89, 72, 50, 49.5, and 18.5 kDa, and it catalyzed the overall synthesis of acetyl-CoA according to the following reaction: CO2 + 2 Fdred(Fe2+) + 2 H+ + CH3 - H4SPt + CoA acetyl-CoA + H4SPt + 2 Fdox(Fe3+) + H2O where Fd is ferredoxin, and CH3-H4SPt and H4SPt denote N5-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively.

Addition of catecholamines at micromolar concentrations caused a dramatic stimulation of growth of tobacco (Nicotiana tabacum) thin cell layers (TCLs) and Acmella oppositifolia "hairy" root cultures. A threefold increase in the rate of ethylene evolution was observed in the catecholamine-treated explants. Aminooxyacetic acid and silver thiosulfate, inhibitors of ethylene biosynthesis and action, respectively, reduced the growth-promoting effect of dopamine. However, these compounds alone could also inhibit the growth of the TCL explants. When ethylene in the culture vessel was depleted by trapping with mercuric perchlorate, dopamine-stimulated growth was still obtained, suggesting that ethylene does not mediate the dopamine effect. Dopamine potentiated the growth of TCLs grown in Murashige and Skoog medium supplemented with indoleacetic acid (IAA) and kinetin. When IAA was replaced by 2,4-dichlorophenoxyacetic acid, dopamine addition showed no growth-promoting effect. Instead, 2,4-dichlorophenoxyacetic acid stimulated the growth of TCL explants to the same extent as that obtained with IAA plus dopamine. Because synthetic auxins do not appear to be substrates for IAA oxidizing enzymes, we hypothesized that catecholamines exert their effect by preventing IAA oxidation. Consistent with this explanation, dopamine (25 micromolar) inhibited IAA oxidase activity by 60 to 100% in crude enzyme extracts from tobacco roots and etiolated corn coleoptiles, but had no effect on peroxidase activity in the same extracts. Furthermore, addition of dopamine to TCL cultures resulted in a fourfold reduction in the oxidative degradation of [1-(14)C]IAA fed to the explants. Because the growth enhancement by catecholamines is observed in both IAA-requiring and IAA-independent cultures, we suggest that these aromatic amines may have a role in the regulation of IAA levels in vivo.

Vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligands, vascular endothelial growth factor-C (VEGF-C) and D (VEGF-D), are the major molecules involved in the development of the embryonic vascular system and pathological lymphangiogenesis. Throughout embryogenesis, VEGFR-3 is expressed in most endothelial cells, whilst being restricted to lymphatic vessels later in development. This receptor plays a significant role in the normal development of blood and lymphatic vessels. In the present studies, we generated a novel panel of 17 monoclonal antibodies (mAbs) against the human VEGFR-3 and characterized their ability to inhibit the proliferation of human erythroleukemia (HEL) cells and angiogenesis of chick embryo chorioallantoic membrane (CAM). Among these mAbs, BDD073 was demonstrated to inhibit the interaction of soluble VEGFR-3 with VEGF-D and the proliferation of HEL cells. In CAM angiogenesis experiments, the angiogenesis induced by recombinant GST-VEGF-D was decreased in the presence of antibody BDD073. These data indicate that this novel neutralizing antibody against human VEGFR-3 not only might be a potential compounds in blocking the VEGF-D-induced angiogenesis and lymphangiogenesis, but also be a tool for the investigations of biology of VEGFR-3 and analysis of lymphatic vessels in malignant tumors and their metastases.

Polyamine concentration in cotyledons of Pharbitis nil Choise, strain Violet during a single inductive or a parallel non-inductive photoperiod was determined. A general decline in putrescine titer was found in induced cotyledons as compared with the non-induced ones. The most noticeable drop in putrescine titer occurred at the 12th hour of the inductive dark period. In contrast, spermidine and spermine content in the cotyledons of short-day treated plants fluctuated dramatically at crucial time points of the dark period (i.e. the 8th, 12th and 16th h), whereas the titers of these two polyamines in the cotyledons of the non-induced plants showed much less fluctuation. In addition, the concentration of spermidine and spermine in induced cotyledons peaked alternately with a priority for spermidine in the time sequence. These data suggest an involvement of polyamines in floral induction.

When gibberellic acid (GA 3 ; 5-35 micrograms per milliliter) is sprayed on 9-day-old light-grown dwarf Progress pea ( Pisum sativum ) seedlings, it causes a marked increase in the activity of arginine decarboxylase (ADC; EC 4.1.1.9) in the fourth internodes. The titer of putrescine and spermidine, polyamines produced indirectly as a result of ADC action, also rises markedly, paralleling the effect of GA 3 on internode growth. Ammonium (5-hydroxycarvacryl) trimethyl chloride piperidine carboxylate (AMO-1618; 100-200 micrograms per milliliter) causes changes in the reverse direction for enzyme activity, polyamine content, and growth. GA 3 also reverses the red-light-induced inhibition of ADC activity in etiolated Alaska pea epicotyls; this is additional evidence for gibberellin-light interaction in the control of polyamine biosynthesis. The enzyme ornithine decarboxylase (ODC; EC 4.1.1.17), an alternate source of putrescine arising from arginine, is not increased by GA 3 or by AMO-1618. The results support the hypothesis that ADC and polyamine content are important regulators of plant growth.

The specific activity of arginine decarboxylase (ADC; l-arginine carboxylase; EC 4.1.1.19) rises steadily over an 8 hour experimental period in the growing buds and subapical epicotyl internodes of 6-day-old totally etiolated pea seedlings. Treatment with red light (R) completely annuls this rise in epicotyls but increases it in buds, thus paralleling the opposite effects of R on the growth of these two organs. Far red light (FR) reverses both effects of R on ADC and is, in turn, reversed by R, indicating phytochrome control. Effects in both organs are clearly seen within 2 hours. By 6 hours after R, the post-irradiation rise in ADC specific activity in buds is 3 times greater than that of the dark controls. Over the same period, ADC specific activity in epicotyls is inhibited by 56% relative to dark controls, reflecting zero net change after R and a continued rise in the dark. Cycloheximide inhibits the rise in ADC activity in both rapidly growing organs (epicotyls in dark and buds after R) but is without effect in both slower growing organs. Actinomycin D inhibits only in dark grown epicotyls, whereas chloramphenicol produces no inhibition in any system tested. ADC is the first enzyme to show a two-way, organ-specific response to phytochrome conversion from Pr to Pfr. This finding is discussed in relation to the growing evidence that polyamines formed from arginine may be important growth regulators in plants, as well as in microbial and animal cells.

The cleavage of poly(ADP-ribose) polymerase (PARP) by caspase (casp)-3 is an essential link in the apoptotic pathway in animal cells. In plant cells, however, there is no authentic evidence for the similar role that PARP may play during apoptosis. Using a heat shock (HS)-induced apoptosis system of tobacco cells, we found that immediately after a 4 h heat treatment, PARP was cleaved to form an 89 kDa signature fragment, while DNA laddering appeared only after a 20 h recovery following the HS. An activation of casp-3-like protease was also observed. The results suggest that apoptosis in plants and animals may share common mechanisms. On the other hand, when cells were preincubated with 4 mM 3-aminobenzamide or 2–8 mM nicotinamide, the specific inhibitors of PARP, before HS treatment, apoptotic cell death was reduced significantly. Our results thus imply that PARP may also be involved in apoptosis in a different way from the casp-related events.

When gibberellic acid (GA3) is sprayed on 9-day-old light-brown dwarf Progress pea (Pisum sativum) seedlings, arginine decarboxylase (ADC; EC 4.1.1.9) activity increases within 3 h and peaks at about 9 h after GA3 application. This is followed by a second lower peak at about 30 h; both peaks were higher than the corresponding peaks in the controls. In contrast, no appreciable effect of GA3 on internode length was observed until about 12 h, after which time a dramatic increase in growth rate occurred and persisted for about 12 h. Specific (DL-alpha-difluoromethylarginine) and non-specific (D-arginine and L-canavanine) inhibitors of ADC strongly inhibited ADC activity and to a lesser extent internode growth. The inhibition was reversed only slightly by the addition of polyamines. Actinomycin D and cycloheximide inhibited the rise in ADC activity induced by GA3. The half-life of the enzyme was increased by GA3 treatment. The results suggest that part of the GA3-induced increase in internode growth may result from enhanced polyamine biosynthesis through the ADC pathway. Furthermore, the GA3 induced increase in ADC activity probably requires de novo synthesis of both RNA and protein.

Background The use of biocatalysts has become an increasingly attractive alternative to traditional chemical methods, due to the high selectivity, mild reaction conditions and environmentally-friendly processes in nonaqueous catalysis of nucleosids. However, the extensive use of organic solvents may generally suffer from sever drawbacks such as volatileness and toxicity to the environment and lower activity of the biocatalyst. Recently, ionic liquids are considered promising solvents for nonaqueous biocatalysis of polyhydroxyl compounds as ILs are environmental-friendly. Results In this research, we developed new IL-containing reaction systems for synthesis of long chain nucleoside ester catalyzed by Pseudomonas fluorescens whole-cells. Various ILs exerted significant but different effects on the bio-reaction. And their effects were closely related with both the anions and cations of the ILs. Use of 10% [BMI][PF6]/THF gave high reaction efficiency of arabinocytosine laurate synthesis, in which the initial rate, product yield and 5′-regioselectivity reached 2.34 mmol/L·h, 81.1% and >99%, respectively. Furthermore, SEM analysis revealed that ILs can alter the cell surface morphology, improve the permeability of cell envelopes and thus facilitate the mass transfer of substrates to the active sites of cell-bound enzymes. Conclusion Our research demonstrated the potential of ILs as promising reaction medium for achieving highly efficient and regioselective whole-cell catalysis.