Differential analysis of Bacillus anthracis after pX01 plasmid curing and comprehensive data on Bacillus anthracis infection in macrophages and glial cells

Bacillus anthracis is a gram-positive bacterial organism responsible for anthrax. This organism has two pathogenic plasmids: pX01 and pX02. The genetic function of pX01, which comprises about 198 kb, is not known, except for a region called the pathogenic island, which contains three genes-pag, lef, and cya-that code for three toxic proteins. A 2-D difference gel electrophoresis (2-D DIGE) system was used to verify the existence of proteins controlled by the pX01 plasmid, and protein regulation data were obtained using DeCyder software. A total of 1728 proteins were identified in the wild-type strain of this organism and 1684 in the pX01 plasmid. Twenty-seven of these proteins disappeared and eight appeared when the pX01 plasmid was removed. An additional 52 proteins were downregulated and 15 were upregulated when this plasmid was removed. A total of 102 proteins have been identified using the MALDI-TOF method of analysis, including 49 whose functions are unknown. Among these, 31 participate in metabolic processes, two in cellular processes, 15 in the processing of genetic information, and five in the processing of extracellular information. Another seven proteins participate in bacterial virulence and pathogenesis. We investigated the functions of these proteins in other bacteria, particularly the B. anthracis derivative H9041. Bacterial growth differed between pX01+/pX02+ B. anthracis and its pX01-/pX02+ derivative as did the cytotoxicity of macrophages infected by pX01+/pX02+ B. anthracis and the pX01-pX02+ derivative. We also found that S100B protein levels increased in the host infected with pX01+/pX02+ B. anthracis or its pX01-/pX02+ derivative. These data suggest that the pX01 plasmid plays a key role in the regulation of protein functions in B. anthracis.     

Chemical fingerprinting of Andrographis paniculata using HPLC, HPTLC and densitometry

An attempt has been made to develop a method by which to determine the chemical fingerprint of Andrographis paniculata (Acanthaceae). High-performance thin layer chromatography (HPTLC) was used to analyse hexane, chloroform, methanol and water extracts of leaves of A. paniculata. A computerised densitometer was applied to the two-dimensional spectrographic image analysis of the HPTLC plates. An HPLC equipped with a photodiode array detector was used for the analyses of these different extracts. The analyses showed that andrographolide and neoandrographolide are absent in the hexane extract but are present in greater amounts in the methanol extract as compared with the other extracts. These chromatograms may serve as a chemical fingerprint of the drug A. paniculata for quality control purposes and in the preparation of formulations based on the drug.     

Ligand-dependent structural changes and limited proteolysis of Escherichia coli phosphofructokinase-2

An isoform (rhesus UGT1A01) orthologus to the human UGT1A1 was cloned and sequenced from female rhesus monkey liver cDNA using primers designed from the human nucleotide sequences. Open reading frame analysis of the PCR-generated product encodes a 533-amino acid protein with a proposed 27-residue signal peptide. Nucleotide sequence comparison of rhesus UGT1A01 to other rhesus UGT1A isoforms detected a single-transition mutation at nucleotide 1520 (T-->C), resulting in a neutral F to S substitution at position 507. Rhesus UGT1A01 was greater than 99 and 95% identical to cynomolgus UGT1A01 and human UGT1A1, respectively. The rhesus UGT1A01 was expressed in HK-293 cells for functional analysis. Catalytic activity of UGT1A01 was determined with 7-hydroxy-4-(trifluoromethyl)-coumarin and more specific human UGT1A1 substrates (1-naphthol, beta-estradiol, 17 alpha-ethinylestradiol, and bilirubin). Expression of UGT1A01 protein was also detected by a Western blot utilizing a polyclonal antibody developed against the human UGT1A family.     

ε-Caprolactam-degradation by Alcaligenes faecalis for bioremediation of wastewater of a nylon-6 production plant

Alcaligenes faecalis G utilized 95–97% of 5–15 g -caprolactam l^–1 in 24–48 h over a pH range of 6–8.5 and at 23–40 °C, without complex nutrient requirement. In the absence of KH₂PO₄ and K₂HPO₄/MgSO₄ in the medium, only 7.6% and 0.2% of 10 g caprolactam l^–1 was utilized, respectively. The chemical oxygen demand (COD) of the wastewater of nylon-6 plant was mainly due to its caprolactam content. A. faecalis G decreased the caprolactam content and COD of the wastewater by 80–90% of the original in spite of the wastewater having higher caprolactam content (3600 mg l^–1) and COD (7700 mg l^–1) than those of any of the previous reports.     

Free radicals,cytokines and nitric oxide in cardiac failure and myocardial infarction

Myocardial infarction is the most common cause of congestive cardiac failure. Free radicals, cytokines, nitric oxide (NO) and antioxidants play a major role both in atherosclerosis and myocardial damage and preservation. In the early stages of atherosclerosis, neutrophils and monocytes infiltrate the intima and generate free radicals which damage the endothelial cells. As a result, production of NO and prostacyclin by the endothelial cells declines, which have cardioprotective actions. This also has relevance to the beneficial action of aspirin since, it can modulate both prostanoid and l-arginine-NO systems and NF-kB translocation. In both acute myocardial infarction and chronic congestive cardiac failure, the plasma levels of various inflammatory mediators such as interleukins and tumour necrosis factor- (TNF) are elevated. TNF, produced by the inflammatory cells and the myocardium, can suppress myocardial contractility and induce the production of free radicals, which in turn can further damage the myocardium. Transforming growth factor (TGF), polyunsaturated fatty acids and the glucose-insulin-potassium regimen can antagonize the harmful actions of TNF and protect the myocardium. This explains why efforts made to reduce the levels of pro-inflammatory cytokines have beneficial action and preserve the myocardium.     

Removal of CCA from treated wood by oxalic acid extraction, steam explosion, and bacterial fermentation

Most preservative-treated wood produced and consumed in the United States is treated with toxic inorganic compounds containing copper, chromium, and arsenic. Because chromated copper arsenate (CCA) is fixed to the wood, CCA-treated wood has not been considered toxic or hazardous and it is currently disposed of in approved landfills. Growing public concern about environmental contamination from treated wood combined with the removal of greater quantities of CCA-treated wood from service have presented a disposal challenge for this fiber source. In this study, CCA-treated wood was processed by acid extraction, steam explosion, and bacterial fermentation and evaluated for removal of copper, chromium, and arsenic. Copper was the easiest to remove by these treatments and chromium the most resistant to removal. Exposing CCA-treated wood to steady-state bacterial growth by continuous culture with Bacillus licheniformis CC01 did not enhance removal of CCA components compared to standard mixed culture when acid extraction preceded bacterial fermentation. Nor did steam explosion, alone or in conjunction with acid extraction and bacterial fermentation, enhance removal of CCA components; the chromium and arsenic components resisted removal. Grinding CCA-treated wood chips into 20-mesh sawdust provided greater access to and removal of CCA components by all processes. However, grinding the chips was unnecessary if they were treated with acid prior to bacterial fermentation. Extraction with oxalic acid as a precursor to bacterial fermentation with B. licheniformis CC01 removed 90% copper (CuO), 80% chromium (CrO₃), and 100% arsenic (As₂O₅) from treated chips. The combination of acid extraction and bacterial fermentation removed 80–100% of these metals from CCA-treated wood. Received 15 December 1997/ Accepted in revised form 08 March 1998     

Development of a highly sensitive high-performance liquid chromatographic method for measuring an anticancer drug, UCN-01, in human plasma or urine

We have established a highly sensitive high-performance liquid chromatographic method for the determination of an anticancer drug, UCN-01, in human plasma or urine. Using a fluorescence detector set at an excitation wavelength of 310 nm and emission monitored at 410 nm, there was a good linearity for UCN-01 in human plasma (r=0.999) or urine (r=0.999) at concentrations ranging from 0.2 to 100 ng/ml or 1 to 400 ng/ml, respectively. For intra-day assay, in plasma samples, the precision and accuracy were 1.8% to 5.6% and −10.0% to 5.2%, respectively. For inter-day assay, the precision and accuracy were 2.0% to 18.2% and 2.4% to 10.0%, respectively. In urine samples, the intra- and inter-day precision and accuracy were within 3.9% and ±2.7%, respectively. The lower limit of quantification (LLOQ) was set at 0.2 ng/ml in plasma and 1 ng/ml in urine. UCN-01 in plasma samples was stable up to two weeks at −80°C and also up to four weeks in urine samples. This method could be very useful for studying the human pharmacokinetics of UCN-01.     

CTRP3/cartducin is induced by transforming growth factor‐β1 and promotes vascular smooth muscle cell proliferation

CTRP3 (C1q and tumour necrosis factor‐related protein 3)/cartducin, a novel serum protein, is a member of the CTRP superfamily. Although the CTRP3/cartducin gene is markedly up‐regulated in rat carotid arteries after balloon injury, little is known about its biological roles in arterial remodelling and neointima formation in injured blood vessels. We have investigated the mechanisms underlying CTRP3/cartducin up‐regulation and the in vitro effects of CTRP3/cartducin on vascular smooth muscle cells. CTRP3/cartducin expression in cultured p53LMAC01 vascular smooth muscle cells was induced by TGF‐β1 (transforming growth factor‐β1), but not by bFGF (basic fibroblast growth factor) or PDGF‐BB (platelet‐derived growth factor‐BB). Exogenous CTRP3/cartducin promoted the proliferation of p53LMAC01 cells in a dose‐dependent manner via ERK1/2 (extracellular signal‐regulated kinase 1/2)‐ and MAPK (p38 mitogen‐activated protein kinase)‐signalling pathways. In contrast, CTRP3/cartducin exhibited no effect on the migration of p53LMAC01 cells. Taken together, the results of the present study demonstrate a novel biological role of CTRP3/cartducin in promoting vascular smooth muscle cell proliferation in blood vessel walls after injury.     

Biosynthesis of eicosanoids and transcellular metabolism of leukotrienes in murine bone marrow cells

Leukotriene B(4) (LTB(4)) biosynthesis by polymorphonuclear leukocytes (PMNs) is an important factor of inflammatory responses. PMNs also release LTA(4), an unstable intermediate that can be taken up by neighboring cells and metabolized into LTC(4). Most studies of LT synthesis have been carried out using human PMNs, but very little information is available about mouse PMNs. Mouse bone marrow PMNs were found to synthesize eicosanoids upon stimulation with A23187, fMLP, or zymosan. The major eicosanoids produced are LTB(4) and 5-hydroxyeicosatetraenoic acid, with some nonenzymatic products of LTA(4) hydrolysis. No cysteinyl leukotrienes were produced, in contrast to what was observed with human blood neutrophil preparations. Human megakaryoblast-like MEG-01 cells synthesized thromboxane B(2) and prostaglandin E(2) in response to A23187 but produced no 5-lipoxygenase (5-LO)-derived eicosanoids. When mouse bone marrow cells (mBMCs) and MEG-01 cells were stimulated during coincubation, LTC(4) and LTD(4) were produced. Mouse peritoneal macrophages from 5-LO-deficient mice were able to synthesize LTC(4) when incubated with mBMCs from wild-type mice, demonstrating transcellular exchange of LTA(4) from mBMCs into murine peritoneal macrophages. These data demonstrate that murine bone marrow PMNs are a valid model for the study of LT biosynthesis, which now offers the possibility to investigate specific biochemical pathways through the use of transgenic mice.