Characterisation of Pseudomonas aeruginosa related to bovine mastitis

Pseudomonas aeruginosa is one of the causative pathogens of bovine mastitis. Most P. aeruginosa strains possess the type III secretion system (TTSS), which may increase somatic cell counts (SCCs) in milk from mastitis-affected cows. Moreover, most of P. aeruginosa cells can form biofilms, thereby reducing antibiotic efficacy. In this study, the presence and effect of TTSS-related genotypes on increase of SCCs among 122 P. aeruginosa isolates obtained from raw milk samples from mastitis-affected cows and their antibiotic susceptibility at planktonic and biofilm status were investigated. Based on the presence of TTSS-related genes a total of 82.7% of the isolates were found to harbour exoU and/or exoS genes, including the invasive (exoU-/exoS+, 69.4%), cytotoxic (exoU+/exoS-, 8.3%) and cytotoxic/invasive strains (exoU+/ exoS+, 5.0%). Milk containing exoS-positive isolates had higher SCCs than those containing exoS-negative isolates. The majority of isolates showed gentamicin, amikacin, meropenem and ciprofloxacin susceptibility at planktonic status. However, the susceptibility was decreased at the biofilm status. Based on minimum biofilm eradication concentration (MBEC)/minimum inhibitory concentration (MIC) ratios, the range of change in antibiotic susceptibility varied widely depending on the antibiotics (from ≥ 3.1-fold to ≥ 475.0-fold). In conclusion, most P. aeruginosa isolates studied here had a genotype related to increase in SCCs. The efficiency of antibiotic therapy against P. aeruginosa-related bovine mastitis could be improved by analysing both the MBEC and the MIC of isolates

Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana.

To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed

Saponin Inhibits Hepatitis C Virus Propagation by Up-regulating Suppressor of Cytokine Signaling 2

Saponins are a group of naturally occurring plant glycosides which possess a wide range of pharmacological properties, including anti-tumorigenic and antiviral activities. To investigate whether saponin has anti-hepatitis C virus (HCV) activity, we examined the effect of saponin on HCV replication. HCV replication was efficiently inhibited at a concentration of 10 µg/ml of saponin in cell culture grown HCV (HCVcc)-infected cells. Inhibitory effect of saponin on HCV replication was verified by quantitative real-time PCR, reporter assay, and immunoblot analysis. In addition, saponin potentiated IFN-α-induced anti-HCV activity. Moreover, saponin exerted antiviral activity even in IFN-α resistant mutant HCVcc-infected cells. To investigate how cellular genes were regulated by saponin, we performed microarray analysis using HCVcc-infected cells. We demonstrated that suppressor of cytokine signaling 2 (SOCS2) protein level was distinctively increased by saponin, which in turn resulted in inhibition of HCV replication. We further showed that silencing of SOCS2 resurrected HCV replication and overexpression of SOCS2 suppressed HCV replication. These data imply that saponin inhibits HCV replication via SOCS2 signaling pathway. These findings suggest that saponin may be a potent therapeutic agent for HCV patients

Hypoglycemic effects of vanadium on alloxan monohydrate induced diabetic dogs

The hypoglycemic effects after oral administration of vanadium have been studied previously in many species such as rats, mice and even humans. However, there has been no prior report on the glucose lowering effect of vanadium on diabetic dogs. Therefore, the purpose of this study was to evaluate the hypoglycemic effects of oral vanadium on diabetic dogs. Diabetes mellitus in the dogs studied was induced by alloxan monohydrate intravenous injection. The dogs were divided into two groups, one was the diabetic control (DC) group (n = 4) and the other was the vanadium treated (DV) group (n = 6). Fresh water was supplied to the dogs in the DC group, but sodium metavanadate solution (0.1~0.2 mg/ml) was given to the dogs in DV group from one week after the alloxan injection. The fasting glucose levels, fructosamine and serum chemistry profiles were compared between the two groups weekly for three weeks. The fasting blood glucose levels in DV group were significantly lower than those in the DC group (p < 0.01). Fructosamine levels in the DV group were also lower than those in the DC group (p < 0.05). The serum chemistry profiles were not significantly different in comparisons between the two groups. However, the cholesterol levels were significantly lower in the DV group compared to the DC group (p < 0.05). Our findings showed that oral vanadium administration had a hypoglycemic effect on chemically induced diabetic dogs

Classification of rice (oryza sativa l. japonica nipponbare) immunophilins (fkbps, cyps) and expression patterns under water stress

<p>Abstract</p> <p>Background</p> <p>FK506 binding proteins (FKBPs) and cyclophilins (CYPs) are abundant and ubiquitous proteins belonging to the peptidyl-prolyl <it>cis/trans </it>isomerase (PPIase) superfamily, which regulate much of metabolism through a chaperone or an isomerization of proline residues during protein folding. They are collectively referred to as immunophilin (IMM), being present in almost all cellular organs. In particular, a number of IMMs relate to environmental stresses.</p> <p>Results</p> <p>FKBP and CYP proteins in rice (<it>Oryza sativa </it>cv. Japonica) were identified and classified, and given the appropriate name for each IMM, considering the ortholog-relation with <it>Arabidopsis </it>and <it>Chlamydomonas </it>or molecular weight of the proteins. 29 FKBP and 27 CYP genes can putatively be identified in rice; among them, a number of genes can be putatively classified as orthologs of <it>Arabidopsis </it>IMMs. However, some genes were novel, did not match with those of <it>Arabidopsis </it>and <it>Chlamydomonas</it>, and several genes were paralogs by genetic duplication. Among 56 IMMs in rice, a significant number are regulated by salt and/or desiccation stress. In addition, their expression levels responding to the water-stress have been analyzed in different tissues, and some subcellular IMMs located by means of tagging with GFP protein.</p> <p>Conclusion</p> <p>Like other green photosynthetic organisms such as <it>Arabidopsis </it>(23 FKBPs and 29 CYPs) and <it>Chlamydomonas </it>(23 FKBs and 26 CYNs), rice has the highest number of IMM genes among organisms reported so far, suggesting that the numbers relate closely to photosynthesis. Classification of the putative FKBPs and CYPs in rice provides the information about their evolutional/functional significance when comparisons are drawn with the relatively well studied genera, <it>Arabidopsis </it>and <it>Chlamydomonas</it>. In addition, many of the genes upregulated by water stress offer the possibility of manipulating the stress responses in rice.</p

Gene Expression Profiling of Breast Cancers with Emphasis of β-Catenin Regulation

To gain molecular understanding of carcinogenesis of breast cancer, gene expression profiles were analyzed using cDNA microarray representing 4,600 cDNAs in 10 breast cancer samples and the adjacent noncancerous breast tissues from the same patients. The alterations in gene expression levels were confirmed by reverse-transcription PCR in four randomly selected genes. Genes that were differently expressed in cancer and noncancerous tissues were identified. 106 (of which 55 were known) and 49 (of which 28 were known) genes were up- or down-regulated, respectively, in greater than 60% of the breast cancer samples. In cancer tissues, genes related to cell cycle, transcription, metabolism, cell structure/motility and signal transduction were mostly up-regulated. Furthermore, three cancer tissues showing immunohistochemically aberrant accumulation of β-catenin in the nucleus and/or cytoplasm revealed down-regulation of Siah and Axin genes and up-regulation of Wnt and c-myc genes. These findings were highly consistent with Wnt signaling pathway associated with β-catenin regulation previously suggested by others. Our studies, therefore, provide not only a molecular basis to understand biological processes of breast cancer but also useful resources to define the mechanism of β-catenin expression in tumorigenesis of breast cancer

Therapeutic genome editing for Charcot-marie-tooth disease type 1a

Charcot-Marie-Tooth 1A (CMT1A) is the most common inherited neuropathy without a known therapy, which is caused by a 1.4 Mb duplication on human chromosome 17, which includes the gene encoding the peripheral myelin protein of 22 kDa (PMP22). Overexpressed PMP22 protein from its gene duplication is thought to cause demyelination and subsequently axonal degeneration in the peripheral nervous system (PNS). Here, we targeted regulatory region of human PMP22 to normalize overexpressed PMP22 level in C22 mice, a mouse model of CMT1A harboring multi copies of human PMP22. Direct local intraneural delivery of CRISPR/Cas9 designed to target TATA-box of PMP22 before the onset of disease, downregulates gene expression of PMP22 and preserves both myelin and axons. Notably, the same approach was effective in partial rescue of demyelination even after the onset of disease. Collectively, our data present a potential therapeutic efficacy of CRISPR/Cas9-mediated targeting of regulatory region of PMP22 to treat CMT1A. Please click Additional Files below to see the full abstract