Wuyiencin produced by Streptomyces albulus CK-15 displays biocontrol activities against cucumber powdery mildew.

AIMS: Wuyiencin is a nucleoside antibiotic produced by Streptomyces albulus CK-15. The aim of this study was to determine whether wuyiencin can be used, as a suitable alternative to chemical pesticides, to protect cucumbers (Cucumis sativus L.) from powdery mildew caused by Sphaerotheca fuliginea. Further, the mechanisms underlying the control of cucumber powdery mildew by S. albulus CK-15 were preliminarily elucidated. METHODS AND RESULTS: Wuyiencin solutions of different concentrations were used to treat infected cucumber plants under greenhouse conditions. The results indicated that wuyiencin could significantly reduce powdery mildew disease incidence, with a maximum prevention efficacy of 94·38%. Further, scanning electron micrographs and enzyme assays showed that wuyiencin inhibited S. fuliginea spore growth and elicited the activity of plant systemic resistance-related enzymes. Additionally, real-time quantitative reverse transcription PCR suggested that wuyiencin can activate a salicylic acid-dependent plant defence response. CONCLUSIONS: Wuyiencin produced by S. albulus CK-15 possessed antifungal effects and was able to mitigate cucumber powdery mildew disease via antagonistic action. Wuyiencin also induced defence responses in the plants. SIGNIFICANCE AND IMPACT OF THE STUDY: These results reinforce the biotechnological potential of wuyiencin as both an antagonistic agent and an inducer of plant systemic resistance.

Overexpression of wysR gene enhances wuyiencin production in ΔwysR3 mutant strain of Streptomyces albulus var. wuyiensis strain CK-15.

AIM: The aim of the present work was to investigate the overexpression of the wysR gene in Streptomyces albulus var. wuyiensis strain CK-15 based on the ΔwysR3 mutant strain including the effect on morphological development, wuyiencin production and antibacterial activity. At the same time, we report a new rapid method for producing genetically engineered strains for industrial production of wuyiencin. METHODS AND RESULTS: We developed a method to create a wysR overexpression strain based on the ΔwysR3 mutant strain by direct transformation. In this method, the desired gene fragment to be overexpressed was amplified by polymerase chain reaction (PCR) using Phusion High Fidelity DNA polymerase and fused with the linearized pSETC integrative plasmid by Gibson assembly. The resulting recombinant plasmid was transformed into ΔwysR3 mutant strain by the intergeneric conjugation method. The plasmid was then integrated into the chromosome and the resulting apramycin-resistant overexpression strain was confirmed by PCR using the Apra-F and Apra-R primers. Finally, we successfully screened the genetically engineered strain with overexpression of wysR gene in ΔwysR3 mutant. CONCLUSION: We can conclude that overexpression of wysR gene in ΔwysR3 mutant strain proved to be an effective strategy for significantly increasing wuyiencin production together with faster morphological development. Quantitative real-time RT-PCR analysis showed that wysR regulated wuyiencin biosynthesis by modulating other putative regulatory genes and bld, whi, chp, rdl and ram family genes are crucial for the morphological development. SIGNIFICANCE AND IMPACT OF THE STUDY: Overexpression of wysR gene in the ΔwysR3 mutant strain named OoWysR strain may increase the efficiency in the industrial fermentation processes for wuyiencin production. The mechanism by which wysR overexpression promotes rapid sporulation and a high yield of wuyiencin production is likely related to modulation of other putative regulatory genes.

Cardioprotective effect of Pycnogenol in ischemic-reperfusion injury (IRI) in rats.

Oxidative stress plays a critical task in the biochemical and pathological alteration linked with myocardial ischemic-reperfusion injury (IRI). This warrants identifying agents with a potential for preventing such damage in an effective way. A novel plant based product, Pycnogenol, obtained from the French maritime pine (Pinus pinaster ssp. atlantica) bark extract was known for its tremendous antioxidant potential (both in vivo, in vitro). It was able to attenuate the symptoms of immune dysfunction through restoring a cellular antioxidant status in low micronutrient-induced immune deficient mice. Consequently, the present study was deals with the determination of protective effect of Pycnogenol in ischemic-reperfusion injury (IRI) in rats via Non-recirculating Langendorff's technique. The effect of Pycnogenol  on the level of various key biomarkers in the rat heart homogenate was determined, such as, myocardial thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation), lactic dehydrogenase (LDH) (a marker of tissue injury) and effect on endogenous antioxidants, e.g., superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GPx). The activity of these biomarkers appreciably improved in Pycnogenol-treated group than IRI group (P < 0.05). The effect of Pycnogenol was further confirmed via histopathological examination of cardiac tissues, which suggests that, it considerably improved the injury related to tissue damage through suppression of edema and infiltration of neutrophil compared to IRI group. It also showed modulation of the expression of apoptotic factors, e.g. Bcl-2, bax and caspase-9 as confirmed by western blot analysis.

Development of a Fluorescence Multiplex Amplification System with 24 Y-STR Loci.

OBJECTIVES: To establish a novel multiplex amplification system which comprises 24 Y-STR loci. METHODS: otal 24 Y-STR gene loci, concluding DYS531, DYS630, DYS622, DYS552, DYS510, DYS449, DYS459a/b, DYS446, DYS443, DYS635, DYS587, DYS527a/b, DYS460, Y-GATA-A10, DYS520, DYS557, DYS522, DYS481, DYS570, DYS385a/b, DYS444, were chosen for establishing the fluorescence multiplex amplification system. The specificity, identity, sensitivity, balance of the amplification, anti-interference and accuracy of the system were detected and the gene diversity was investigated in the population of Guangdong. RESULTS: No band was found in nonhuman and female samples that were tested by the established multiplex amplification system. The same genotyping results were obtained from different tissues of the same person. Complete profiles could be obtained from more than 0.1 ng of the standard sample 9948. The loss of alleles was found when the common inhibitors such as hemoglobin and calcium ion were added 120-200 µmol/L and 1.5-2.0 mmol/L respectively to the system which with a strong anti-interference to the indigo, humic acid and EDTA. The typing of 24 Y-STR system could give the reliable results when 146 unrelated male individuals were detected and compared with the Yfiler system parallelly. The haplotype diversity （HD） of the population in Guangdong reached 0.999 72 that was better than the result retained from Yfiler system, which the HD was 0.998 58. CONCLUSIONS: The fluorescence amplification system with 24 Y-STR loci established in present study has a wildly application prospect and can be used for cases inspection, paternity tests and Y-STR database construction.

Biological control of Botrytis cinerea on tomato plants using Streptomyces ahygroscopicus strain CK-15.

UNLABELLED: We developed a real-time PCR assay to specifically detect and quantify the efficacy of a biological fungicide from Streptomyces ahygroscopicus var. wuyiensis on tomato leaves. This fungicide, the natural secondary metabolite wuyiencin, is an antifungal agent against Botrytis cinerea. Specific primers were designed based on the ß-actin gene sequences, which were used to detect a 303 bp fragment from B. cinerea isolates. Our assay is highly sensitive and can be used to reliably detect and quantify as little as 1·75 pg of B. cinerea DNA. We used this detection method to monitor the progression of B. cinerea infection in inoculated plant material under preventive (wuyiencin) and nonpreventive treatment. After 5 days, plants under preventive treatment exhibited a sharp decrease in fungal biomass and no symptoms, whereas plants under nonpreventive treatment displayed severe disease symptoms. The results demonstrate that wuyiencin has significant effects on B. cinerea in tomato plants and that real-time PCR is a reliable method for evaluating the effects of Streptomyces wuyiensis CK-15 on B. cinerea. SIGNIFICANCE AND IMPACT OF THE STUDY: Botrytis cinerea commonly produces latent or nonsymptomatic infection on and within plant tissues, which can develop into symptomatic infection when triggered by changes in environmental conditions or host plant physiology. In this study, we develop a specific, sensitive real-time PCR assay for detecting and quantifying B. cinerea on tomato leaves to determine the control efficacy of Streptomyces ahygroscopicus var. wuyiensis as a biological fungicide. Our findings demonstrate that wuyiencin has significant effects on B. cinerea in tomato plants and that real-time PCR is a reliable method for evaluating the effects of biological fungicides on plant pathogens.

Robustness of Salmonella loop-mediated isothermal amplification assays for food applications.

AIMS: Loop-mediated isothermal amplification (LAMP) assays have been developed recently for Salmonella detection. This study aimed at evaluating the robustness of two Salmonella LAMP assays in comparison with PCR and real-time quantitative PCR for food applications. METHODS AND RESULTS: Performance of the assays was examined under abusive preparation conditions, running temperatures and pH, and with the addition of various inhibitors and food rinses. LAMP achieved robust detection under abusive assay preparation conditions (holding at 22 and 37°C for up to 30 min) and running temperatures (57-68°C). With a hot-start DNA polymerase, PCR obtained comparable results under these temperature ranges. However, PCR performed markedly poorer under abusive pH. LAMP also showed greater tolerance to potential inhibitors than PCR. When food rinses including meat juice, chicken rinse, egg homogenate and produce homogenate were added at 20% of the reaction mix, PCR amplifications were completely inhibited, but LAMP reactions were not. CONCLUSIONS: Our results demonstrated that LAMP is a robust alternative to PCR in Salmonella detection for food applications. SIGNIFICANCE AND IMPACT OF THE STUDY: This study filled important knowledge gaps regarding the robustness of Salmonella LAMP assays. The findings will help bring Salmonella LAMP assays closer to wider applications in food testing.

Evaluation of diffusion and dilution methods to determine the antimicrobial activity of water-soluble chitosan derivatives.

AIMS: Chitosan has gained wide applications in the food industry and biomedical field owing to its biodegradability, biocompatibility, nontoxicity and its antimicrobial activity against a wide spectrum of micro-organisms. However, the methods used to investigate antimicrobial effects of chitosan vary considerably among studies, making comparisons difficult. METHODS AND RESULTS: One diffusion (disc diffusion) and two dilution (agar dilution and broth microdilution) methods commonly used in clinical laboratories to assess microbial susceptibility/resistance to antimicrobial agents were comparatively used to determine the antimicrobial activity of two water-soluble chitosan derivatives (molecular weights of 43 and 67 kDa) against 31 representative foodborne pathogens. When tested at 1.6% for the 43-kDa chitosan and 3.2% for the 67-kDa chitosan, by disc diffusion, approximately 10- to 11-mm-diameter inhibition zones were observed for all of the bacterial groups, except for Salmonella tested for the 67-kDa chitosan where no inhibition zone was observed. By agar dilution and broth microdilution, the minimal inhibitory concentration (MIC) values varied largely dependent upon the molecular weight of chitosan, bacterial genus/species and the testing method. The agreement between MIC values obtained by the two methods was poor, with broth microdilution generally having lower MIC values than agar dilution. Regardless of the testing method, Salmonella strains were the least susceptible among Gram-negative strains for both chitosans, followed by Escherichia coli and Vibrio. CONCLUSIONS: Besides chitosan's molecular weight and bacterial genus/species, the antimicrobial activity of chitosan was also influenced largely by the susceptibility testing method used. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that comparatively evaluated these diffusion and dilution methods, particularly two quantitative methods (agar dilution and broth microdilution), to assess the antimicrobial activity of two water-soluble chitosans against a large number of foodborne pathogens. The study highlights the need for standardized methods to be used in evaluating chitosan's antimicrobial properties in future studies.

Radiation hybrid map of the mouse genome.

Radiation hybrid (RH) maps are a useful tool for genome analysis, providing a direct method for localizing genes and anchoring physical maps and genomic sequence along chromosomes. The construction of a comprehensive RH map for the human genome has resulted in gene maps reflecting the location of more than 30,000 human genes. Here we report the first comprehensive RH map of the mouse genome. The map contains 2,486 loci screened against an RH panel of 93 cell lines. Most loci (93%) are simple sequence length polymorphisms (SSLPs) taken from the mouse genetic map, thereby providing direct integration between these two key maps. We performed RH mapping by a new and efficient approach in which we replaced traditional gel- or hybridization-based assays by a homogeneous 5'-nuclease assays involving a single common probe for all genetic markers. The map provides essentially complete connectivity and coverage across the genome, and good resolution for ordering loci, with 1 centiRay (cR) corresponding to an average of approximately 100 kb. The RH map, together with an accompanying World-Wide Web server, makes it possible for any investigator to rapidly localize sequences in the mouse genome. Together with the previously constructed genetic map and a YAC-based physical map reported in a companion paper, the fundamental maps required for mouse genomics are now available.

ARSACS, a spastic ataxia common in northeastern Québec, is caused by mutations in a new gene encoding an 11.5-kb ORF.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS or SACS) is an early onset neurodegenerative disease with high prevalence (carrier frequency 1/22) in the Charlevoix-Saguenay-Lac-Saint-Jean (CSLSJ) region of Quebec. We previously mapped the gene responsible for ARSACS to chromosome 13q11 and identified two ancestral haplotypes. Here we report the cloning of this gene, SACS, which encodes the protein sacsin. The ORF of SACS is 11,487 bp and is encoded by a single gigantic exon spanning 12,794 bp. This exon is the largest to be identified in any vertebrate organism. The ORF is conserved in human and mouse. The putative protein contains three large segments with sequence similarity to each other and to the predicted protein of an Arabidopsis thaliana ORF. The presence of heat-shock domains suggests a function for sacsin in chaperone-mediated protein folding. SACS is expressed in a variety of tissues, including the central nervous system. We identified two SACSmutations in ARSACS families that lead to protein truncation, consistent with haplotype analysis.

First Report of Tomato mosaic virus Infecting Pepino in China.

Pepino (Solanum muricatum L.) is a vegetatively propagated plant that is native to South America and is commercially grown in many countries (4) including China for its juicy and fragrant fruits. An unusual disease of pepino characterized by a yield reduction of fruits and mosaic, puckering, and distortion of leaves was observed in surveys conducted during 2010 to 2011 in pepino growing regions of Gansu, Jilin, and Yunnan provinces. These symptoms were similar to a disease caused by the Tomato mosaic virus (ToMV) first reported in Spain in 1998 (3). The sap from infected pepino was mechanically inoculated to 12 plants each of Chenopodium quinoa, Lycopersicon esculentum, and Gomphrena globosa, and the resulting symptoms were similar to those incited by ToMV (1). Symptoms included yellowish local lesions on inoculated leaves of C. quinoa and systemic mosaic in L. esculentum. Necrotic or semi-necrotic local lesions developed on inoculated leaves of G. globosa followed by systemic mosaic 7 dpi. Symptomatic pepino and indicator plants were tested for the presence of ToMV using commercial double-antibody sandwich-ELISA diagnostic kits (Agdia, Elkhart, IN). The virus incidence ranged from 33.3 to 75% in pepino and the other three indicator plants. To further confirm the presence of ToMV, ELISA-positive samples were subjected to total RNA extraction using Trizol (Invitrogen) followed by reverse transcription (RT)-PCR with ToMV-specific forward (5'-ATGTCTTACTCAATCACTTC-3') and reverse primers (5'-TTAA(G)GAT(C) GCA(T)GGTGCAG(C)AGG-3'), designed to amplify a 480-bp fragment of the coat protein. Amplicons of the expected size were obtained from all ELISA-positive samples, but not from healthy pepino plants. The amplicons were cloned into the pMD18-T (TaKaRa, Da Lian, China) vector and transformed into E. coli DH-5α competent cells. The sequences obtained (GenBank Accession Nos. JX025562, JX025563, JX025565, and JX025566) shared 99.58 to 99.79% similarity with the ToMV reference sequence (Accession No. NC002692). To our knowledge, this is the first report of ToMV infecting pepino in China. ToMV is an important pathogen that is mechanically transmitted with high efficiency as a result of agricultural practices. The disease is difficult to control once infection occurs in the field. This disease has caused serious economic loss in Spain (2), thus it is important to survey and monitor the incidence and distribution of ToMV in China. References: (1) I. Kamenova et al. Acta Hort. 722:277, 2006. (2) L. Pérez-Benlloch et al. Euphytica. 120:247, 2001. (3) J. Prohens et al. Plant Dis. 82:1281, 1998. (4) J. Prohens et al. Acta Hort. 523:53, 2000.

Population genetic structure and historical demography of Oratosquilla oratoria revealed by mitochondrial DNA sequences.

Genetic diversity, population genetic structure and molecular phylogeographic pattern of mantis shrimp Oratosquilla oratoria in Bohai Sea and South China Sea were analyzed by mitochondrial DNA sequences. Nucleotide and haplotype diversities were 0.00409-0.00669 and 0.894-0.953 respectively. Neighbor-Joining phylogenetic tree clustered two distinct lineages. Both phylogenetic tree and median-joining network showed the consistent genetic structure corresponding to geographical distribution. Mismatch distributions, negative neutral test and "star-like" network supported a sudden population expansion event. And the time was estimated about 44000 and 50000 years ago.

The Association of Age at Diagnosis of Hypertension With Brain Structure and Incident Dementia in the UK Biobank.

[Figure: see text].

Effects of Folic Acid and Vitamin B12 Supplementation on Cognitive Impairment and Inflammation in Patients with Alzheimer's Disease: A Randomized, Single-Blinded, Placebo-Controlled Trial.

OBJECTIVES: To evaluate the combined action of folic acid and vitamin B12 supplementation on cognitive performance and inflammation in patients with Alzheimer's disease (AD). DESIGN: This was a randomized, single-blind, placebo-controlled trial. PARTICIPANTS: Patients (n=120) diagnosed clinically as probable AD and in stable condition from Tianjin Key Laboratory of Cerebrovascular and Neurodegenerative Diseases. MEASUREMENTS: Individuals were randomly divided into the intervention group (n=60, folic acid 1.2 mg/d + vitamin B12 50 µg/d) and the placebo group (n=60). Cognitive performance, blood folate, vitamin B12, one carbon cycle metabolite, and inflammatory cytokine levels were measured at baseline and after 6 months. The data were analyzed using linear mixed models for repeated measures. RESULTS: A total of 101 participants (51 in the intervention group and 50 in the placebo group) completed the trial. Folic acid plus vitamin B12 supplementation had a beneficial effect on the MoCA total scores (P=0.029), naming scores (P=0.013), orientation scores (P=0.004), and ADAS-Cog domain score of attention (P=0.008), as compared to those of the control subjects. Moreover, supplementation significantly increased plasma SAM (P<0.001) and SAM/SAH (P<0.001), and significantly decreased the levels of serum Hcy (P<0.001), plasma SAH (P<0.001), and serum TNFα (P<0.001) compared to in the control subjects. CONCLUSIONS: Folic acid and vitamin B12 supplementation showed a positive therapeutic effect in AD patients who were not on a folic acid-fortified diet. The findings of this study help to delineate nutrient intervention as far as public health management for the prevention of dementia is concerned.

Multiplex PCR assays for simultaneous detection and characterization of Vibrio vulnificus strains.

AIMS: Vibrio vulnificus is a major cause of seafood-related deaths in the United States. Several biomarkers, e.g. the virulence-correlated gene (vcg), 16S rRNA, and the capsular polysaccharide operon (CPS) have been used to differentiate virulent- from nonvirulent-type V. vulnificus strains. In this study, we combined the use of these biomarkers with a species-specific V. vulnificus cytolysin/haemolysin gene (vvhA) to develop two pairs of multiplex PCR assays that simultaneously detect and characterize V. vulnificus strains. METHODS AND RESULTS: The first multiplex PCR pair amplified four genes (vvhA, vcg, 16S rRNA, and CPS), with one for virulent-type and the other one for nonvirulent-type V. vulnificus strains, while the second pair targeted three of those genes excluding CPS. Primer concentration and annealing temperature were optimized for the four multiplex PCR assays. When testing ten V. vulnificus reference strains and 80 field oyster isolates, results from each multiplex PCR matched 100% with known strain characteristics for these target genes. CONCLUSIONS: The optimized multiplex PCR assays were capable of simultaneously detecting and characterizing V. vulnificus with high specificity and speed. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiplex PCR assays designed in this study are valuable tools for microbial ecology and epidemiology studies. They may facilitate better control of V. vulnificus risks in oysters, thereby reducing the number of illnesses and deaths because of V. vulnificus in the long run.

MicroRNA-150-5p inhibits proliferation and invasion of osteosarcoma cells by down-regulating VEGFA.

OBJECTIVE: The aim of this study was to investigate whether microRNA-150-5p was involved in osteosarcoma cell proliferation and invasiveness via modulating vascular endothelial growth factor A (VEGFA) expression. PATIENTS AND METHODS: 10 pairs of osteosarcoma tissues and para-cancerous tissues were collected from patients with osteosarcoma in our center from February 2012 to July 2018. Relative expression levels of microRNA-150-5p and VEGFA in tissues and cell lines were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Luciferase reporter gene assay was conducted to illustrate the binding interplay between microRNA-150-5p and VEGFA. Furthermore, proliferative and invasive potentials in HOS and MG-63 cells regulated by both microRNA-150-5p and VEGFA were determined using Cell Counting Kit-8 (CCK-8), colony formation assay, and transwell assay, respectively. RESULTS: MicroRNA-150-5p was remarkably downregulated, while VEGFA was upregulated in osteosarcoma tissues compared with para-cancerous tissues (p<0.05). Similar results were observed in osteosarcoma cells and normal osteoblasts. Overexpression of microRNA-150-5p significantly inhibited the proliferation and invasion of osteosarcoma cells (p<0.05). Luciferase reporter gene assay demonstrated that microRNA-150-5p could target to VEGFA to negatively modulate its expression. In addition, the knockdown of VEGFA remarkably weakened osteosarcoma cell proliferative and invasive capacities (p<0.05). CONCLUSIONS: MicroRNA-150-5p weakens proliferative and invasive potentials in osteosarcoma cells by downregulating VEGFA level. All our findings suggest that microRNA-150-5p/VEGFA axis is a promising target for osteosarcoma treatment.

High-glucose promotes proliferation of human bladder cancer T24 cells by activating Wnt/ß-catenin signaling pathway.

OBJECTIVE: Bladder cancer is the most prevalent genitourinary malignant disorder worldwide. We aimed to observe effects of high-glucose on bladder cancer proliferation and explore the associated mechanisms. MATERIALS AND METHODS: Human bladder cancer cell line, T24, was divided into Blank, Control (Ctrl), 10 mmol/l, 20 mmol/l and 30 mmol/l group. T24 cell proliferation was evaluated by using multiple table tournament (MTT) assay and colony formation analysis, respectively. Quantitative Real-time PCR (qRT-PCR) assay was employed to examine mRNA expression of Wnt-5a and ß-catenin. Meanwhile, Western blot assay was used to evaluate expression of Wnt-5a and ß-catenin protein. The linear regression analysis was utilized to analyze correlation between Wnt-5a/ß-catenin expression and T24 cell proliferation. RESULTS: High-glucose significantly enhanced proliferation of T24 cells compared to that of Blank and Ctrl group (p < 0.05). High-glucose significantly promoted colony formation of T24 cells compared to that of Blank and Ctrl group (p < 0.05). High-glucose significantly up-regulated Wnt-5a mRNA and protein expression compared to that of Blank and Ctrl group (p < 0.01). High-glucose significantly increased ß-catenin mRNA and protein expression compared to that of Blank and Ctrl group (p < 0.01). Effects of high-glucose on T24 cell proliferation were increased following with the enhanced glucose concentration. Wnt/ß-catenin signaling pathway molecules were correlated with colony formation of T24 cells (p < 0.05). CONCLUSIONS: High-glucose promoted the proliferation of T24 cells by activating the Wnt/ß-catenin signaling pathway. This study would provide the novel targets for bladder cancer therapy.

BCL3 is induced by IL-6 via Stat3 binding to intronic enhancer HS4 and represses its own transcription.

BCL3 is a proto-oncogene affected by chromosomal translocations in some patients with chronic lymphocytic leukemia. It is an IkappaB family protein that is involved in transcriptional regulation of a number of NF-kappaB target genes. In this study, interleukin (IL)-6-induced BCL3 expression and its effect on survival of multiple myeloma (MM) cells were examined. We demonstrate the upregulation of BCL3 by IL-6 in INA-6 and other MM cell lines. Sequence analysis of the BCL3 gene locus revealed four potential signal transducer and activator of transcription (Stat) binding sites within two conserved intronic enhancers regions: one located within enhancer HS3 and three within HS4. Chromatin immunoprecipitation experiments showed increased Stat3 binding to both enhancers upon IL-6 stimulation. Silencing Stat3 expression by small interfering RNA (siRNA) abrogated BCL3 expression by IL-6. Using reporter gene assays, we demonstrate that BCL3 transcription depends on HS4. Mutation of the Stat motifs within HS4 abolished IL-6-dependent BCL3 induction. Furthermore, BCL3 transcription was inhibited by its own gene product. This repressive feedback is mediated by NF-kappaB sites within the promoter and HS3. Finally, we show that overexpression of BCL3 increases apoptosis, whereas BCL3-specific siRNA does not affect the viability of INA-6 cells suggesting that BCL3 is not essential for the survival of these cells.

Identification of amygdalin and its major metabolites in rat urine by LC-MS/MS.

Amygdalin and its metabolites in rat urine were identified using liquid chromatography-electrospray ionization (ESI) tandem ion-trap mass spectrometry. The purified rat urine sample was separated using a reversed-phase C18 column with 10 mM sodium phosphate buffer (pH 3.1) containing 30% methanol as the mobile phase, amygdalin and its metabolites were detected by on-line mass detector in selected ion monitoring (SIM) mode. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. At least seven metabolites and the parent drug were found in rat urine after i.v. injection of 100 mg/kg doses of amygdalin. Among them, six metabolites were reported for the first time.