Role of mitogen-activated protein kinase pathway in reactive oxygen species-mediated endothelin-1-induced β-myosin heavy chain gene expression and cardiomyocyte hypertrophy

Endothelin-1 (ET-1) has been found to increase cardiac -myosin heavy chain (-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced -MyHC gene expression and hypertrophy in cardiomyocytes, however, has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced -MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1, cultured neonatal rat cardiomyocytes were examined for ³H-leucine incorporation and -MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and -MyHC gene expression. ET-1 increased ³H-leucine incorporation and -MyHC promoter activities, which were blocked by the specific ET_A receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced ³H-leucine incorporation, -MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase, p38, and c-Jun NH₂ -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased ³H-leucine incorporation and -MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced -MyHC promoter activities, suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the -MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced -MyHC gene expression. Moreover, ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and -MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways, which culminates in hypertrophic responses and -MyHC expression. Tzu-Hurng Cheng, Neng-Lang Shih: These authors have equally contributed to this work     

Plasmid maintenance and gene expression of a recombinant culture under aerobic and anaerobic conditions

Summary The stability and gene expression of a batch culture ofEscherichia coli, strain C600, carrying the plasmid pKN401 under both aerobic and anaerobic conditions were studied. It was observed that the plasmid was stable under both conditions and during a step change in the environment in which oxygen and nitrogen were supplied alternately. In addition, the -lactamase specific activity was sensitive to the availability of oxygen, with higher activities and a strong dependence on optical density observed under anaerobic conditions.     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.     

Novel effect of aromatic compounds on the iron-dependent expression of theEscherichia coli K12 manganese superoxide dismutase (sodA) gene

Summary InEscherichia coli, the superoxide dismutase genes (sodA andsodB) sense the availability of Fe through the action of thefur locus [E. C. Niederhoffer, C. M. Naranjo, K. L. Bradley, J. A. Fee (1990) Control ofEscherichia coli superoxide dismutases (sodA andsodB) genes by the ferric uptake regulation (fur) locus,J. Bacteriol. 172, 1930–1938]. Previous work from other laboratories has shown that a variety of metal chelators and of redox-active aromatic compounds can dramatically induce expression ofsodA. Here we show that non-redox-active, non-metal-chelating aromatic compounds also enhance expression of a chromosomalsodA gene fusion and that these effects are strongly modulated by the Fur phenotype (Fur^±) and by the availability of iron in the culture medium. The compounds studied were ethidium bromide, hemin, 2,2-bipyridine, 1,10-phenantroline, 4,7-phenantroline, rhodamine B1, rhodamine 6G, and, for comparison to previous studies, Paraquat.Abbreviations DTPA diethylenetriaminepentaacetic acid - Paraquat N,N-dimethyl-1,1-bipyridene - bpy 2,2-bipyridine - phen 1,10-phenanthroline - 4,7-phen 4,7-phenanthroline     

Biodegradation of beta-cyfluthrin by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain S1

-Cyfluthrin [-cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate] pesticide has been in agricultural use in the recent years for controlling Lepidopteran pests affecting solanaceous crops. The extensive use of synthetic pyrethroids like -cyfluthrin has resulted in wide spread environmental contamination. The purpose of this study was to isolate bacteria from soil and to determine their ability to degrade -cyfluthrin and identify the intermediates in culture broth using spectroscopy. An aerobic bacterium capable of degrading -cyfluthrin was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain S1) had 100% identity to the sequence from Pseudomonas stutzeri. Finally products formed during degradation of -cyfluthrin have been identified as -cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (M.W. 341); 4-fluoro-3-phenoxy--cyanobenzyl alcohol (M.W. 243) and 3(2,2-dichlorovinyl)-2,2-dimethyl cyclopropanecarboxylic acid (M.W. 208).     

Expression and genomic organization of a dinoflagellate gene family

The luciferin-binding protein (LBP) of the dinoflagellate Gonyaulax polyedra is encoded by large gene family with at least two different members. To more fully understand the expression and genomic organization of this gene family, 40 full-length LBP cDNAs were isolated and mapped with the restriction enzymes Xho I, Eco RI, Pvu II and Hind III. All cDNAs isolated could be placed into one of two groups called LBP and LBP. Two LBP group cDNAs were completely sequenced and were found to share 99% identity at both the nucleotide and protein levels. One LBP cDNA was sequenced and was found to share only 86% sequence identity with the LBP group at both the nucleotide and protein levels. Both groups of message appear to be expressed at nearly equal levels since (1) two-dimensional gels of purified LBP show two protein isoforms present in roughly equal amounts and (2) northern blots using group-specific probes suggest that cellular levels of LBP and LBP mRNAs are identical. Genomic Southern blots using group-specific probes suggests that the copy number of both gene groups is very similar and that LBP gene loci are organized as tandem repeats of either LBP or LBP sequences.     

Characterization of the nar promoter to use as an inducible promoter

Summary The nar promoter of Escherichia coli was characterized, which is maximally induced under anaerobic conditions in the presence of nitrate. The following results were obtained; Expression of -galactosidase was optimal at 1 % of nitrate and was not affected much by molybdate; the amount of -galactosidase per unit volume was maximal when the nar promoter was induced at OD₆₀₀ = 1.7, and when anaerobic condition was made by supplying nitrogen gas. At the optimal condition, the ratio of -galactosidase between before and after induction was approximately 250 and Miller units were approximately 7,500. The results showed that the nar promoter can be used as an inducible promoter.     

Molecular cloning in Lactobacillus helveticus by plasmid pSA3::pVA797 co-integrate formation and conjugal transfer

Summary A gene encoding -glucanase activity from Bacillus amyloliquefaciens was subcloned in both orientations into plasmid shuttle vector pSA3. In only one orientation could a co-integrate be generated with the conjugative plasmid pVA797. The plasmid co-integrate was conjugated into Lactobacillus helveticus strain CNRZ450, where it was stably maintained without antibiotic selection and exhibited -glucanase activity. This method of introducing cloned DNA into thermophilic lactobacilli will facilitate the study of heterologous gene expression in non-transformable species. Offprint requests to: J. K. Thompson     

Agrobacterium-mediated transformation of white mustard (Sinapis alba L.) and regeneration of transgenic plants

Summary A procedure for the regeneration of fertile transgenic white mustard (Sinapis alba L.) is presented. The protocol is based on infection of stem explants of 7–9 day old plants with an Agrobacterium tumefaciens strain harboring a disarmed binary vector with chimeric genes encoding neomycin phosphotransferase and -glucuronidase. Shoots are regenerated from callus-forming explants within 3–4 weeks. Under selection, 10% of the explants with transgenic embryonic callus develop into fertile transgenic plants. Rooting shoots transferred to soil yield seeds within 14–16 weeks following transformation. Integration and expression of the T-DNA encoded marker genes was confirmed by histochemical glucuronidase assays and Southern-DNA hybridization using primary transformants and S1-progeny. The analysis showed stable integration and Mendelian inheritance of trans-genes in transformed Sinapis lines.Abbreviations BAP 6-benzylaminopurine - CaMV cauliflower mosaic virus - GUS -glucuronidase - IBA indole-3-butyric acid - IM infection medium - NAA 1-naphthalene acetic acid - neo gene encoding NPTII - NPTII neomycin phosphotransferase - RIM root-inducing medium - SEM shoot-elongation medium - SIM shoot-inducing medium - t-nos polyadenylation site of the nopaline synthase gene - uidA gene encoding GUS - WM wash medium - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronide     

Effect of propranolol on cardiac cytokine expression after myocardial infarction in rats

The pro-inflammatory cytokines interleukin (IL)-1 and IL-6 have been shown to be upregulated in the myocardium after injury and after adrenergic receptor stimulation. Together with other cytokines, such as the transforming growth factor (TGF)-, the pro-inflammatory cytokines have been implicated in the initiation of tissue repair and wound healing after myocardial infarction (MI). In the present study, the effect of -adrenergic receptor blockade with propranolol (2 mg/kg·h s.c. by miniosmotic pumps) on cardiac cytokine expression and on wound healing was analyzed in rats from 6–72 h after MI. IL-1 and IL-6 gene expression strongly increased in the infarcted myocardium 6 h after MI and peaked after 12 h, while TGF-, progressively increased from 12 h onwards. Also, TGF-₂ increased after 12 h, peaked after 24 h and declined thereafter, while TGF-, was only elevated after 72 h. Treatment with propranolol had a negative chronotropic effect throughout the observation period of 72 h. It attenuated the initial elevation in LVEDP and increased cardiac output ultimately. Furthermore, propranolol attenuated IL-1 mRNA expression, but had not effect on the other cytokines. Moreover, MMP-9 gelatinolytic activity was markedly attenuated by propranolol indicating a delayed resorption of the necrotic tissue and, possibly, collagen turnover. Replacement by scar tissue, however, was not affected as indicated by normal collagen expression.     

The Role of Antioxidant Systems in the Response of Escherichia colito Heat Shock

Shifting the temperature from 30 to 45°C in an aerobic Escherichia coliculture inhibited the expression of the antioxidant genes katG, katE, sodA, and gor.The expression was evaluated by measuring -galactosidase activity in E. colistrains that contained fusions of the antioxidant gene promoters with the lacZoperon. Heat shock inhibited catalase and glutathione reductase, lowered the intracellular level of glutathione, and increased its extracellular level. It also suppressed the growth of mutants deficient in the katG-encoded catalase HPI, whereas the sensitivity of the wild-type andsodA sodBmutant cells to heat shock was almost the same. In the E. coliculture adapted to growth at 42°C, the content of both intracellular and extracellular glutathione was two times higher than in the culture grown at 30°C. The temperature-adapted cells grown aerobically at 42°C showed an increased ability to express the fused katG–lacZgenes.     

Inhibin/activin subunits alpha, beta-A and beta-B are differentially expressed in normal human endometrium throughout the menstrual cycle

Inhibins are dimeric glycoproteins composed of an alpha () subunit and one of two possible beta (-) subunits (A or B). The aims of this study were to assess the frequency and tissue distribution patterns of the inhibin subunits in normal human endometrium. Samples from human endometrium from proliferative phase (PP; n=32), early secretory phase (ES; n=10) and late secretory phase (LS; n=12) were obtained. Immunohistochemistry, immunofluorescence and a statistical analysis were performed. All three inhibin subunits were expressed by normal endometrium by immunohistochemistry and immunofluorescence. Inhibin- was primarily detected in glandular epithelial cells, while inhibin- subunits were additionally localised in stromal tissue. Inhibin- staining reaction increased significantly between PP and ES (P<0.05), PP and LS (P<0.01), and ES and LS (P<0.02). Inhibin-A and -B were significant higher in LS than PP (P<0.05) and LS than ES (P<0.05). All three inhibin subunits were expressed by human endometrium varying across the menstrual cycle. This suggests substantial functions in human implantation of inhibin- subunit, while stromal expression of the subunits could be important in the paracrine signalling for adequate endometrial maturation. The distinct expression in human endometrial tissue suggests a synthesis of inhibins into the lumen and a predominant secretion of activins into the stroma.I. Mylonas and U. Jeschke contributed equally to this work     

Effects of glycine supplement on protein production and release in recombinant Escherichia coli

Summary The effect of glycine supplement to growth media on protein expression and release in a recombinant strain RR1 of E. coli was investigated. Addition of glycine to the growth media in moderate amount (up to 1%) was observed to enhance significantly the release of periplasmic proteins from the cell to the broth. The extracellular activities of the model enzymes -amylase and -lactamase were increased by a factor of 16.3 and 3.8 respectively in the presence of glycine. These activities corresponded to about 50% of the total production for each protein. Furthermore, with glycine supplement the total enzyme activity of both -amylase, -lactamase as well as -galactosidase were increased by a factor of about 2.5. Cell growth characteristics and low extracellular activity of the cytoplasmic protein -galactosidase are indicative that glycine does not cause significant cell-lysis for a concentration below 0.7%.     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Isolation of a rat immune response gene identical to an alleged mouse A class II β-chain pseudogene

A human HLA-DQ -chain cDNA was used as a probe to identify and isolate a rat major histocompatibility antigen -chain gene from a genomic library constructed in the vector Charon 28 using Wistar rat DNA (RT1 ^u). The isolated exon of the rat gene (RT1.B ₂) encoding a -chain second domain was found to share 93% nucleotide homology with a mouse A ₂ exon. Although the genomic organization of this gene is consistent with the hypothesis that it represents a pseudogene, the remarkable preservation of a specific sequence favors the view that this class II antigen -chain gene has retained its coding function.     

The border fresidues of the dihydrofolate reductase domain inEscherichia coli β-galactosidase correspond to the positions of introns 1 and 5 of dihydrofolate reductase of chicken

Summary In-galactosidase ofEscherichia coli residues 820–934 are similar to residues in dihydrofolate reductase ofE. coli. Dihydrofolate reductase ofE. coli and chicken are also similar and have identical tertiary structures. I used the similarity of the three-dimensional structure of prokaryotic and eukaryotic dihydrofolage reductases to align the chicken dihydrofolate reductase and the similar residues of-galactosidase. The positions of introns 1 and 5 of the chicken dihydrofolate reductase gene correspond exactly to the start and the end of the dihydrofolate reductase-like domain in the-galactosidase polypeptide chain. This equivalence of intron positions in a eukaryotic gene and domain structure in a prokaryotic protein was interpreted as evidence for a common origin of both genes.     

Synthesis of methyl α- and β-N-dansyl-d-galactosaminides,probes for the combining sites ofN-acetyl-d-galactosamine-specific lectins

The synthesis of the methyl - and -N-dansyl-d-galactosaminides is described using methyl ,-2-azido-2-deoxy-d-galactopyranoside as starting material. This was reduced to the corresponding methyl ,-2-amino-2-deoxy-d-galactopyranoside and then treated with dansyl chloride to yield a mixture of methyl ,-N-dansyl-d-galactosaminides which was separated into individual anomeric forms by flash chromatography on silica gel. Methyl -N-dansyl-d-galactosaminide was used as a fluorescent indicator ligand in continuous substitution titrations to determine the association constants of nonchromophoric carbohydrates with theN-acetyl-d-galactosamine specific lectin fromErythrina corallodendron.Abbreviations ECorL Erythrina corallodendron lectin - MeGalNDns methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside - MeGalNDns methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside Dedicated to Hilde De Boeck (1958–1991).     

A pH-regulated promoter for the expression of recombinant proteins inEscherichia coli

Summary A series of plasmid vectors have been developed which allows control of recombinant gene expression by altering the pH of the growth medium. Expression is controlled by the regulatory region of thecadA gene ofE. coli. Experiments using -galactosidase as the expressed gene have resulted in an induced expression of up to 60-fold when the pH of the growth medium is lowered from pH 7 to 5.5. Expression can also be induced by switching from aerobic to anaerobic growth environment. The pH and anaerobic effects are additive boosting the expression level of -galactosidase to a dramatic value of 200 fold. Finally, the pH-induction effect is fully reversible, a unique property which allows continuous control of gene expression using exiting pH monitoring and control equipment.     

Histochemistry of non-specific phosphatases: the use of p-nitrophenyl phosphate and -glycerophosphate as substrates

Synopsis It has been found that the acid and alkaline phosphatases in homogenates of respectively intestine and hypodermis ofAscaris suum hydrolyse sodiump-nitrophenyl phosphate at about twice the rate of sodium -glycerophosphate. This difference was also observed histochemically. Thus, when sections of intestine were incubated for acid phosphatase withp-nitrophenyl phosphate as substrate, the intensity of staining was about twice as great as that obtained after incubation in -glycerophosphate. Further, alkaline phosphatase was evident in sections of hypodermis after only 2 hr incubation inp-nitrophenyl phosphate but was not apparent after 10 hr incubation with -glycerophosphate. Hence biochemical assays and histochemical studies both indicate thatp-nitrophenyl phosphate is a superior substrate to -glycerophosphate for the visualization of acid and alkaline phosphatases in tissues.This paper was presented in part at the 1969 Aberdeen meeting of the British Society for Parasitology.