The use of bioluminescence stimulant on the immobilized strain, P. putida mt-2 KG1206, with toluene analog inducers and environmental samples

This study was conducted to investigate the applicability of the stimulant conditions for the bioluminescence activity of a recombinant strain of Pseudomonas putida, mt-2 KG1206, when immobilized using alginate polymer. The bioluminescence activity of the immobilized strain was generally approximately three to five times lower than the subcultured strain, and the activity was observed to slowly decrease. These facts may have been caused by several factors, such as the low biomass and the time required for diffusion into the entrapped biomass. Although different inducers produced different degrees of stimulation, immobilized bacteria modified with KNO₃ consistently produced more bioluminescence than those treated with sodium lactate, regardless of the inducer chemical tested. Cells treated with KNO₃ exhibited 2.8 times greater bioluminescence than that of the control activity. This condition also stimulated the bioluminescence activities of the immobilized bacteria exposed to contaminated groundwater samples. Based on these results, the immobilized KG1206 presented in this research can be used as a portable assay for the purpose of preliminary on-site monitoring of specific inducer contaminants, with subsequent off-site instrumental analysis, suggesting the potential of this immobilized cell for preliminary application in a field-ready bioassay.     

An Optimization of a Bioassay for Toluene Analogs using Bioluminescence Reporter Strain KG1206

A genetically engineered strain of P. putida mt-2 KG1206 used in this study contains the intact TOL plasmid and a plasmid with the P _m-lux gene, and bioluminescence was produced by direct (m-toluate and benzoate) and indirect inducers (toluene analogs). Much less bioluminescence was produced by benzoate and o-xylene among the tested inducers. This bioluminescence producing strain was used for the quantification of m-toluate in soil, and a quantification protocol for pollutant was developed for standardization. Values determined by bioluminescence were in the range of 75 (min.) ～158 (max.) % of their true concentration as determined by HPLC analysis. Statistical analysis indicates that this bioluminescence strain is useful for quantifying specific pollutant in environmental system. However, more investigation is required for mixture pollutants in the environment.     

Amplification and cloning of the Chinese hamster glutamine synthetase gene.

A Chinese hamster ovary cell line, KG1MS which is resistant to 5 mM methionine sulphoximine overproduces glutamine synthetase. Overproduction of this 42 000 mol. wt. polypeptide is not seen in either parental KG1 or revertant KG1MSC4-0 cell lines which are resistant to 3 microM and 8 microM methionine sulphoximine, respectively. Restriction endonuclease analysis of DNA from KG1MS cells produces a pattern of amplified DNA fragments not seen in parental KG1 or revertant KG1MSC4-0 digests. Hybridization of cDNA probes complementary to KG1MS poly(A) mRNA against Southern blots of KG1MS restriction digests identifies a specific subset within these amplified sequences which is not detected by cDNA probes made from parental KG1 poly(A) mRNA. One 8.2-kb BglII fragment of KG1MS DNA identified by cDNA hybridization has been cloned to produce recombinant pGS-1. mRNA hybrid selected by pGS-1 translates to a 42 000 mol. wt. polypeptide which co-migrates in polyacrylamide gels with the over-produced protein in KG1MS cells and purified glutamine synthetase. pGS-1 also hybridizes to several mRNA species abundant in KG1MSC4-M, but not KG1, poly(A) mRNA extracts. The high level of resistance to methionine sulphoximine shown by KG1MS cells is due to amplification of a DNA sequence of at least 50 kb which contains the coding region for the enzyme glutamine synthetase.     

A synthetic analog of prostaglandin E(1) prevents the production of reactive oxygen species in the intestinal mucosa of methotrexate-treated rats

Administration of methotrexate to rats results in severe enterocolitis and death. Previous our studies showed that a synthetic analog of prostaglandin E(1), OP-1206 [17S, 20-dimethyl-trans-Delta(2)-prostaglandin E(1)] ameliorated the anticancer agent-induced enterocolitis of rats. In the current study, we have focused on the biochemical effect of OP-1206 on the methotrexate-induced intestinal inflammation implicating reactive oxygen species (ROS). Methotrexate (15 mg/kg body weight) was orally administered to rats once daily for 5 days. OP-1206 (0.5 microg/kg body weight) was orally administered to rats twice a day for 5 days. On the 6th day, the chemiluminescence from the jejunum was measured to evaluate the generation of ROS. Spontaneous chemiluminescence from the jejunum of the methotrexate-treated rats increased significantly, compared with the control. Luminol-enhanced chemiluminescence from inflamed mucosal scrapings from the jejunum of the methotrexate-treated rats indicated more remarkable enhancement than the control rats. The treatment of OP-1206 with methotrexate showed significantly lower chemiluminescence of both the jejunum and mucosal scrapings than those of the methotrexate-treated rats. The alkaline phosphatase (ALP) activity, as a marker of small intestinal differentiation, in the intestinal mucosa of the methotrexate-treated rats decreased remarkably, but that of the methotrexate and OP-1206-treated rats was significantly higher than that of the methotrexate-treated rats. Thus, OP-1206 may possibly help the anticancer chemotherapy by protecting the small intestine from the methotrexate-induced damage.     

Effects of orally administered OP-1206 alpha-CD with loxoprofen-Na on walking dysfunction in the rat neuropathic intermittent claudication model

An orally active prostaglandin E1 analogue, OP-1206 alpha-CD improves walking dysfunction in the rat spinal stenosis model. Loxoprofen-Na, a non-steroidal anti-inflammatory drug, is used to relieve chronic pain in patients with lumbar spinal canal stenosis. To determine whether the OP-1206 alpha-CD in combination with loxoprofen-Na could induce a greater therapeutical effect on walking dysfunction and spinal cord blood flow (SCBF) than OP-1206 alpha-CD treatment alone after chronic spinal stenosis in the rat. Spinal stenosis was induced by placing two pieces of silicon rubber strips in the lumbar (L4 and L6) epidural space of rats. After surgery, walking function was measured using a treadmill apparatus and SCBF was measured using a laser-Doppler flow meter. Drugs were administered orally twice a day for 11 days from the day 3 post-surgery. OP-1206 alpha-CD elicited a significant improvement of walking dysfunction on days 7 and 14 post-surgery and significantly increased spinal cord blood flow on day 15, whereas walking dysfunction and SCBF of rats treated with loxoprofen-Na alone remained unchanged. Combined treatment of OP-1206 alpha-CD with loxoprofen-Na did not provide additive therapeutical effect. These results suggest that a significant improvement seen after OP-1206 alpha-CD treatment is primarily mediated by improvement of the local spinal cord blood flow. This effect is not ameliorated or potentiated by a combined treatment with loxoprofen-Na.     

Inhibition of platelet aggregation by the cAMP-phosphodiesterase inhibitor, cilostamide, may not be associated with activation of cAMP-dependent protein kinase.

We examined the involvement of cAMP-dependent protein kinase (A kinase)2 in the inhibition by cilostamide, a specific inhibitor of the low Km cAMP-phosphodiesterase (PDE), on 9,11-epithio-11,12-methanothromboxane A2 (STA2)-induced platelet aggregation. For comparative purposes, the PGE1 analogue, 17S-20-dimethyl-trans-delta 2-PGE1 (OP-1206) was used. OP-1206 (IC50 = 18 +/- 0.55 nM) and cilostamide (IC50 = 40 +/- 4.5 nM) were both potent inhibitors of the platelet aggregation induced by STA2 (1 microM). OP-1206 and cilostamide dose-dependently inhibited elevations in intracellular free Ca2+ ([Ca2+]i) caused by STA2. OP-1206 caused an almost complete inhibition of Ca2+ mobilization, but cilostamide did not prevent the STA2-induced elevation in [Ca2+]i to the same extent as OP-1206, even at a high concentration (greater than 200 nM). Cilostamide did not increase the cAMP level at concentrations (5-100 nm) which affected STA2-induced aggregation. OP-1206 significantly increased cAMP contents in platelets, and the degree of aggregation inhibition by OP-1206 appears to be related to the size of increase in cAMP. OP-1206 increased phosphorylation of the 50,000 mol. wt vasodilator-stimulated phosphoprotein, at concentrations of 7.9-79 nM, which inhibited aggregation induced by STA2. Cilostamide treatment resulted in a marginal increase in the 50,000 mol. wt phosphorylation at concentrations (10-100 nM) which completely inhibited the STA2-induced aggregation. (8R*, 9S*, 11S*)-(-)-9-Hydroxy-9-n-hexyloxy-8-methyl-2,3,9,10- tetrahydro-8,11-epoxy-1H, 8H, 11H-2, 7b, 11a-triazadibenzo(a,g)-cycloocta(c,d,e)trinden-1-one (KT-5720), a specific inhibitor of A kinase, not only reversed the inhibition by OP-1206 of STA2-induced platelet aggregation, but also inhibited the OP-1206-induced protein phosphorylation. However, the inhibition by cilostamide of STA2-induced aggregation was not prevented by pretreatment with KT-5720. Inhibition of the STA2-induced aggregation by OP-1206 may be associated with cAMP-dependent protein phosphorylation, while cilostamide may have inhibitory effects on STA2-induced platelet activation through mechanisms other than the activation of A kinase.     

Autocrine GM-CSF transcription in the leukemic progenitor cell line KG1a is mediated by the transcription factor ETS1 and is negatively regulated through SECTM1 mediated ligation of CD7

Background

CD7 expression is found on ~ 30% of acute myeloblastic leukemias (AML). The leukemic progenitor cell line KG1a (CD7 +) constitutively expresses GM-CSF while the parental KG1 (CD7-) cell line does not. This study focuses on the molecular basis of CD7 mediated GM-CSF regulation.

Methods

KG1a cells were treated with recombinant SECTM1-Fc protein, the PI3K kinase inhibitors wortmannin, LY292004, or PI4K activator spermine. Stable KG1-CD7 +, KG1a-shCD7, KG1a-shETS1 as well as KG1a-GFP, KG1a-PKCβII-GFP cell lines were generated and the levels of CD7, GM-CSF and ETS-1 mRNA and protein were compared by real-time-PCR, western blotting, flow cytometry and ELISA.

Results

SECTM1 is expressed in Human Bone Marrow Endothelial Cells (HBMEC) and its expression can be upregulated by both IFN-γ. KG1a cells demonstrated high expression levels of CD7 and ETS-1 allowing a constitutative signaling through the PI3K/Atk pathway to promote GM-CSF expression, while KG1 cells with low expression of CD7 and ETS-1 showed low GM-CSF expression. On KG1a cells GM-CSF expression could be negatively regulated by PI3K inhibitors or by recombinant SECTM1-Fc. Overexpression of CD7 in KG1 cells was insufficient to promote GM-CSF expression, while silencing of CD7 or ETS-1 resulted in reduced GM-CSF expression levels. Differentiation capable KG1a cells overexpressing PKCβII illustrated complete loss of CD7, but maintained normal levels of both ETS-1 and GM-CSF expression.

Conclusion

These findings add an additional layer to the previously described autocrine/paracrine signaling between leukemic progenitor cells and the bone marrow microenvironment and highlight a role for SECTM1 in both normal and malignant hematopoiesis.

General Significance

This work shows that SECTM1 secreted from bone marrow stromal cells may interact with CD7 to influence GM-CSF expression in leukemic cells.     

Construction of a shuttle vector and expression in Streptomyces lividans of the sulfonamide-resistance gene derived from Escherichia coli plasmid pSAS1206

We have constructed a hybrid plasmid using Streptomyces lividans plasmid p1J101 and Escherichia coli plasmid pSAS1206. This plasmid, designated pFSH102, is able to replicate in both hosts and the sulfonamide-resistance gene encoded by pSAS1206 is phenotypically expressed in S. lividans.     

Performance of genotype imputations using data from the 1000 Genomes Project

Genotype imputations based on 1000 Genomes (1KG) Project data have the advantage of imputing many more SNPs than imputations based on HapMap data. It also provides an opportunity to discover associations with relatively rare variants. Recent investigations are increasingly using 1KG data for genotype imputations, but only limited evaluations of the performance of this approach are available. In this paper, we empirically evaluated imputation performance using 1KG data by comparing imputation results to those using the HapMap Phase II data that have been widely used. We used three reference panels: the CEU panel consisting of 120 haplotypes from HapMap II and 1KG data (June 2010 release) and the EUR panel consisting of 566 haplotypes also from 1KG data (August 2010 release). We used Illumina 324,607 autosomal SNPs genotyped in 501 individuals of European ancestry. Our most important finding was that both 1KG reference panels provided much higher imputation yield than the HapMap II panel. There were more than twice as many successfully imputed SNPs as there were using the HapMap II panel (6.7 million vs. 2.5 million). Our second most important finding was that accuracy using both 1KG panels was high and almost identical to accuracy using the HapMap II panel. Furthermore, after removing SNPs with MACH Rsq <0.3, accuracy for both rare and low frequency SNPs was very high and almost identical to accuracy for common SNPs. We found that imputation using the 1KG-EUR panel had advantages in successfully imputing rare, low frequency and common variants. Our findings suggest that 1KG-based imputation can increase the opportunity to discover significant associations for SNPs across the allele frequency spectrum. Because the 1KG Project is still underway, we expect that later versions will provide even better imputation performance.     

A comparative study of the formation of 2-keto-D-gluconic acid by free and immobilized cells of Pseudomonas putida

The effect of glucose, oxygen and 2-keto-D-gluconic acid (2KG) concentrations on the 2KG production by free and immobilized cells of Pseudomonas putida was studied. The effect of these factors was found to be similar in case of both free and immobilized cells, but the rate of the 2KG production by the free cells was a little higher as compared to the immobilized cells.     

Cell-surface properties of Lactococcus garvieae strains and their immunogenicity in the yellowtail Seriola quinqueradiata

The cell-surface properties of strains of Lactococcus garvieae were examined. Two capsular types were found, one with a highly developed capsule (KG9408) and one with a micro-capsule (MS93003) carrying fimbriae-like components projecting from the cell surface. One strain (NSS9310) had neither cell capsular nor fimbriae-like structures on its cell surface. The strains with the highly developed capsule were more virulent to fish than either the micro-capsular or non-capsular strains. The KG9408, MS93003 and NSS9310 strains could be clearly differentiated by their susceptibility to bacteriophages. Protection against L. garvieae infection was induced in the yellowtail Seriola quinqueradiata by immunization with formalin-killed L. garvieae KG9408 and MS93003 cells. Although protection was also induced by immunization with NSS9310, the level of protection was significantly lower than that with KG9408 and MS93003 vaccines. Passive immunization with yellowtail immune sera raised against KG9408 and MS93003 conferred strong protection on yellowtail with rapid bacterial clearance after challenge with L. garvieae. Immunoblotting analysis of protein antigens extracted from L. garvieae strains using rabbit anti-KG9408 and anti-MS93003 sera and yellowtail anti-KG9408 and anti-MS93003 sera indicated that some bands in KG9408 and MS93003 strains were not detectable in NSS9310.     

Purification, identification, and characterization of two GTP-binding proteins with molecular weights of 25,000 and 21,000 in human platelet cytosol. One is the rap1/smg21/Krev-1 protein and the other is a novel GTP-binding protein

We have purified, characterized, and identified two GTP-binding proteins with Mr of 25,000 (c25KG) and 21,000 (c21KG) from the cytosol fraction of human platelets. These two proteins were not copurified with the beta gamma subunits of heterotrimeric GTP-binding proteins. Amino acid sequences of tryptic fragments of c21KG completely matched with those of rap1 protein (Pizon, V., Chardin, P., Lerosey, I., Olofsson, B., and Tavitian, A. (1988) Oncogene 3, 201-204), smg p21 (Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y., and Takai, Y. (1988) J. Biol. Chem. 263, 18965-18971), and Krev-1 protein (Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) Cell 56, 77-84). The partial amino acid sequence analysis of c25KG revealed that this protein was different from any low Mr GTP-binding proteins already reported. c25KG bound about 1 mol of [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein, with a Kd value of about 45 nM. [35S]GTP gamma S-binding to c25KG was specifically inhibited by guanine nucleotides, GTP and GDP, but not by adenine nucleotides such as ATP and adenyl-5'-yl beta, gamma-imidodiphosphate. The binding activity was not inhibited by pretreatment with N-ethylmaleimide. c25KG hydrolyzed GTP to librate Pi with the specific activity of 1.8 mmol of Pi/mol of protein/min, which are different from the activities of the already purified low Mr GTP-binding proteins. We conclude that c25KG is a novel GTP-binding protein and c21KG is a rap1/smg p21/Krev-1 product.     

A dysfunctional Mo1 glycoprotein is present on a subline of the KG1 acute myelogenous leukemia cell line

Mo1 is a glycoprotein heterodimer found on the surface of phagocytic cells. By use of monoclonal antibodies directed against various epitopes of the 155 kd alpha-chain of Mo1, two distinct functions of this glycoprotein have been identified. Mo1 serves as the receptor (CR3) for iC3b, one of the breakdown products of the third component of complement, and in addition appears to be involved in promoting cell-cell adhesion of granulocytes. In studies performed with a subline of the acute myelogenous leukemia tumor cell line KG1, we found cells from this subline (KG1m) incapable of iC3b-mediated binding despite these cells having surface Mo1. Five distinct epitopes on Mo1 identifiable on normal cells by a panel of anti-Mo1 alpha-chain monoclonal antibodies (including OKM10, thought to be identical to or close to the iC3b binding site) were equally present on KG1m by indirect immunofluorescence. The electrophoretic mobilities of both the alpha- and beta-chains of Mo1 derived from KG1m were identical to those isolated from normal granulocytes. To determine whether altered receptor mobility or decreased surface density of Mo1 was responsible for the lack of Mo1-related functions, binding to EiC3b of isolated Mo1 derived from KG1m lysate was measured. KG1m-derived Mo1 did not bind to EiC3b when compared with normal granulocyte-derived Mo1, suggesting that the lack of iC3b binding is not secondary to decreased surface receptor number or mobility. This subline of KG1 provides an excellent model for the study of the relationship between surface receptor structure and function.     

Correlation between methotrexate-induced intestinal damage and decrease in polyamine content

A synthetic analog of prostaglandin E(1), OP-1206 [17S, 20-dimethyl-trans-Delta(2)-prostaglandin E(1)] protects the small intestine from the methotrexate (MTX)-induced damage. The purpose of this study is to evaluate the protective effect of OP-1206 on the methotrexate-induced small intestinal damage in rats from the biochemical point of view. MTX (15 mg/kg body weight) was orally administered to rats once daily for 5 days. OP-1206 (0.5 microg/kg body weight) was orally administered to rats twice a day for 5 days, and on the 6th day biochemical components in the jejunal mucosa of the treated rats were determined. The contents of DNA, RNA, proteins and polyamines (spermine and spermidine) in the jejunal mucosa of rats were markedly decreased by the MTX treatment. The coadministration of OP-1206 with MTX prevented such decreases caused by the MTX treatment. The MTX treatment decreased the incorporation of 3H-thymidine into DNA in the jejunal mucosa, while the coadministration of OP-1206 with MTX prevented it. These results indicated that OP-1206 could protect the intestinal mucosa against the biochemical effects of MTX through a trophic action on intestinal villi. Further, it should be noted that polyamines may possibly play an important role of modulation action on intestinal mucosa.     

Influence of pentoxifylline on gene expression of PAG1/ miR-1206/ SNHG14 in ischemic heart disease

The regulation by immune checkpoint is able to prevent excessive tissue damage caused by ischemia reperfusion (I/R); therefore, the study aims to investigate the behavior of phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) mRNA, miR-1206 and small nucleolar RNA host gene 14 (SNHG14) during I/R and intake of pentoxifylline (PTX) as a protective drug. The relative expression level of PAG1/miR-1206/SNHG14 was determined by qRT-PCR. Cardiac tissue levels of cytotoxic T-lymphocyte associated antigen 4 (CTLA4) and PAG1 protein expression were determined by ELISA technique. The regulatory T cells achieved by the flow cytometry. The results found that the relative expression of SNHG14 was significantly upregulated in I/R, but suppressed in PTX treated groups with enhancement of the relative expression level of miR-1206. The gene and protein expression of PAG1 were downregulated with effective doses of PTX. The results showed that (30 and 40 mg/kg bwt) PTX dose suppressed the CTLA4 development significantly. The mean of the regulatory T cell in PTX protective groups is significantly reduced at (p < 0.001) in a comparison with I/R group. Spearman's correlation analysis revealed a significant negative correlation between SNHG14 and miR-1206, but a significant positive correlation between SNHG14 and PAG1 in I/R heart tissue. The results indicated that miR-1206 and SNHG14 can be used as biomarkers with perfect sensitivity and specificity. Using PTX reduced cardiac tissue damage. SNHG14 and miR-1206 can be used as a diagnostic tool in I/R.     

Identification of a proliferation-related transcript with an elevated expression in the mid-gestation mouse embryo

Polyadenylated RNA enriched in transformation specific sequences from hamster embryo fibroblast cells transformed by HSV-2 was used to construct a cDNA library. A cDNA clone (pKG4) contained a sequence which was upregulated in HSV-2 transformed cells and also in fibroblastic cell lines transformed by SV40 and 3-methylcholanthrene. The expression of the KG4 sequences in HSV-2-transformed cells was found to be modulated by the growth state of the cells. In confluent cells its level was reduced 5-times compared to the homologous RNAs from exponentially growing cells. Expression of the KG4 sequence was also examined in mouse embryos from day 8 onwards and in adult tissues. During development, KG4 is expressed at all times examined. However, there is a dramatic increase in expression on day 11. In adult tissues, a low and variable level of expression was observed. These findings suggest that the KG4 sequence is related to cellular proliferation.     

Novel Toxoplasma gondii inhibitor chemotypes

We profiled three novel T. gondii inhibitors identified from an antimalarial phenotypic high throughput screen (HTS) campaign: styryl 4-oxo-1,3-benzoxazin-4-one KG3, tetrahydrobenzo[b]pyran KG7, and benzoquinone hydrazone KG8. These compounds inhibit T. gondii in vitro with IC₅₀ values ranging from 0.3 to 2 μM, comparable to that of 0.25 to 1.5 μM for the control drug pyrimethamine. KG3 had no measurable cytotoxicity against five mammalian cell lines, whereas KG7 and KG8 inhibited the growth of 2 of 5 cell lines with KG8 being the least selective for T. gondii. None of the compounds were mutagenic in an Ames assay. Experimental gLogD_7.4 and calculated PSA values for the three compounds were well within the ranges predicted to be favorable for good ADME, even though each compound had relatively low aqueous solubility. All three compounds were metabolically unstable, especially KG3 and KG7. Multiple IP doses of 5 mg/kg KG7 and KG8 increased survival in a T. gondii mouse model. Despite their liabilities, we suggest that these compounds are useful starting points for chemical prospecting, scaffold-hopping, and optimization.