Distribution of Corticotrophin-Releasing Factor Type 1 Receptor-Like Immunoreactivity in the Rat Pituitary.

Corticotrophin-releasing factor (CRF) regulates the hypothalamic-pituitary-adrenal axis response to stress through its type 1 receptor (CRF1 ) in the corticotrophs of the anterior pituitary. Although CRF1 mRNA expression has been confirmed in the rat pituitary, the distribution pattern of CRF1 protein in the pituitary has not been reported. Therefore, we generated an antiserum against the amino acid fragment corresponding to the 177-188 sequence of the first extracellular loop of the rat CRF1 . Using the antiserum, CRF1 -like immunoreactivity (CRF1 -LI) was detected in the anterior lobe cells of the rat pituitary where some of them expressed intense signals. CRF1 -LI also appeared in the intermediate lobe cells and on the fibre-like elements of the posterior lobe of the pituitary. Dual immunofluorescence labelling showed that corticotrophs exhibited the highest percentage of CRF1 (male: 27.1 ± 3.0%, female: 18.0 ± 3.0%), followed by lactotrophs (male: 6.7 ± 3.0%, female: 12.1 ± 1.3%), gonadotrophs (male: 2.6 ± 1.0%, female: 7.5 ± 0.5%), thyrotrophs (male: 2.9 ± 0.1%, female: 5.3 ± 1.2%) and somatotrophs (male: 1.1 ± 0.3%, female: 1.2 ± 0.5%). The percentage of CRF1 -LI-positive cells that were corticotrophs was significantly higher in male rats than in female rats, whereas CRF1 -LI-positive lactotrophs and gonadotrophs were significantly higher in female rats than in male rats. Almost all of the melanotrophs were positive for CRF1 in the intermediate lobe (98.9 ± 0.2%). CRF1 -LI and the percentage of CRF1 -LI in corticotrophs were decreased in the anterior pituitary, and the distribution patterns were altered from a diffuse to punctate one by adrenalectomy; the changes were restored by treatment with dexamethasone (100 µg/kg bw). These results suggest that CRF1 is involved in the modulation of the functions of the pituitary; moreover, protein expression and the distribution patterns of CRF1 are regulated by glucocorticoids in the rat anterior pituitary.

Molecular characterisation of the quinolone resistance-determining regions (QRDR) including gyrA, gyrB, parC and parE genes in Streptococcus pneumoniae.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia (CAP). Currently, empirical treatment with quinolones is being used due to the emergence of beta-lactam and macrolide resistance in S. pneumonaie. Although the prevalence of quinolone-resistant S. pneumoniae remains low, increasing numbers of resistant isolates are being seen. Genetic mechanisms leading to fluoroquinolone resistance in pneumococci are complex. This study aims to use molecular methods to characterise all isolates through sequence analysis of their QRDR regions. Thirty-two S. pneumoniae isolates were obtained from nasal swabs from adult and paediatric patients attending local general practices in Northern Ireland. Phenotypic minimum inhibitory concentration (MIC) was determined for Clinical and Laboratory Standards Institute (CLSI) broth microdilution against ciprofloxacin, levofloxacin and norfloxacin. Simultaneously, the QRDR regions of gyrA, gyrB, parC and parE were analysed by sequence typing for all pneumococci obtained. Only one isolate (3.1%) showed reduced susceptibility to ciprofloxacin and levofloxacin. Two amino acid positions were discordant in the S. pneumoniae R6 strain and eight (25%) and 23 (71.9%) isolates contained the mutations Ile460Val in gyrA and Lys137Asn in parC (deposited in GenBank, accession numbers GQ999587-GQ999589), respectively. No mutations were found in either the gyrB or parE loci. In conclusion, the study demonstrated increased fluoroquinolone resistance which could not be accounted for simply through QRDR mutations, and, reciprocally, that mutations in the QRDR region do not necessarily result in overt phenotypic resistance.

Molecular analysis and characterization of a urease gene operon from Campylobacter sputorum biovar paraureolyticus.

When recombinant plasmid DNA from a genomic DNA library and inverse PCR products of Campylobacter sputorum biovar paraureolyticus LMG17591 strain were analyzed, an approximate 6.5-kb pair region, encoding a urease gene operon, was identified. Within the operon, seven closely spaced and putative open reading frames for ureG, ureH(D), ureA, ureB, ureC, ureE, and ureF were detected in order. A possible overlap was detected between ureG and ureH(D), ureH(D) and ureA, and ureE and ureF. In addition, two putative promoter structures, probable ribosome-binding sites and a putative ρ-independent transcriptional terminator structure were identified. The urease gene operon transcription in the cells was confirmed by the reverse transcription-PCR analysis. A neighbor-joining tree constructed based on the nucleotide sequence information of urease genes showed that C. sputorum biovar paraureolyticus formed a cluster with Arcobacter butzleri, urease-positive thermophilic Campylobacter and some Helicobacter spp., separating those from the other urease-producing bacteria, suggesting a commonly shared ancestry among these organisms.

Occurrence and characterisation of intervening sequences (IVSs) within 16S rRNA genes from two atypical campylobacter species, c. sputorum and c. curvus.

A polymerase chain reaction (PCR) method was carried out on 21 isolates of atypical Campylobacter sputorum (n=14) and C. curvus (n=7) using a primer pair to amplify the helix 11 region within 16S ribosomal RNA (rRNA) gene sequences. Following sequencing and alignment analysis, 14 C. sputorum (100%) and six C. curvus (86%) isolates were shown to carry intervening sequences (IVSs) in this region. Interestingly, the nucleotide sequences of all the IVSs were identical among the 14 C. sputorum isolates (n=5 C. sputorum biovar [bv] paraureolyticus; n=5 by fecalis; n=4 by sputorum). In addition, two different nucleotide lengths and sequences of IVSs were identified among the six C. curvus isolates. On the first prediction of the secondary structure model of the IVSs in 16S rRNA genes, stem and loop structures were identified. In the purified RNA fractions from the 20 Campylobacter isolates carrying IVSs, no 16S rRNA was evident. Instead, other smaller RNA fragments were identified. Thus, the primary 16S rRNA transcripts may have been fragmented in the 20 isolates. This is the first demonstration of atypical C. sputorum and C. curvus isolates carrying IVSs in the helix 11 region in 16S rRNA genes.

Molecular characterisation of urease genes from urease-positive thermophilic campylobacters (UPTC).

This study aims to clarify the molecular characteristics of the urease gene operon from urease-positive thermophilic campylobacters (UPTC) obtained from different sources and in various countries. Sequence heterogeneity was observed for the promoter structures at the -35-like region among the 12 isolates examined. The most probable TTG start codon was suggested for the ureB and ureH genes, and for the ureA, E, F and G genes, ATG was suggested among all the isolates examined. Overlap was detected between ureA and ureB and between ureB and ureE among all the isolates examined. UPTC is the first example of an overlap between the two structural genes ureA and ureB. When the completely sequenced open reading frames (ORFs) for ureE, ureF, ureG and ureH were identified, non-coding regions between ureE and ureF, ureF and ureG, and ureG and ureH were also demonstrated. All six start codons of the six urease genes were demonstrated to be preceded by Shine-Dalgarno sequences among all the isolates examined. The Cys-His sequence corresponding to urease active sites were aligned perfectly and fully conserved among the three UPTC isolates examined. A putative and intrinsic p-independent transcriptional terminator was identified to be identical among all the isolates examined. A partial and putative ORF of about 200 bp in length showing high sequence similarity to GTP cyclohydrolase I was observed downstream of ureH.

Cloning, sequencing and characterization of a urease gene operon from urease-positive thermophilic Campylobacter (UPTC).

AIMS: To clone, sequence and characterize the genetic organization of urease genes within urease-positive thermophilic Campylobacter (UPTC). METHODS AND RESULTS: An approx. 5.1-kbp region encoding a urease gene operon was identified, when recombinant plasmid DNAs from a genomic DNA library of a Japanese isolate (CF89-12) of UPTC were analysed. CONCLUSIONS: Six closely spaced and putative open reading frames (ORFs) for ureA, ureB, ureE, ureF, ureG and ureH were detected. ATG codons initiated each ORF of the UPTC urease operon except for ureB and ureH, which commenced with the most probable TTG codon. Overlaps were detected between ureA and ureB and also between ureB and ureE. Probable ribosome-binding sites and a putative rho-independent transcriptional termination region were identified. Two putative promoter structures, consisting of consensus sequences at the -35 like and -10 regions were also identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Construction of a neighbour-joining tree based on the nucleotide sequence data of urease genes indicated that UPTC formed a cluster with some Helicobacter organisms separate from the other urease-producing bacteria, suggesting a commonly shared ancestry between UPTC and Helicobacter urease genes.

Endothelin-1 stimulates cardiomyocyte injury during mitochondrial dysfunction in culture.

To understand the pathophysiological role of endothelin-1 in the failing heart, we constructed a cellular mitochondrial impairment model and demonstrated the effect of endothelin-1. Primary cultured cardiomyocytes from neonatal rats were pretreated with rotenone, a mitochondrial complex I inhibitor, and the cytotoxic effect of endothelin-1 on the cardiomyocytes was demonstrated. Rotenone gradually decreased the pH of the culture medium with incubation time and caused slight cell injury. Endothelin-1 markedly enhanced the effect of rotenone that decreased the pH of the medium and enhanced cellular injury. The enhancement of the decrease in pH and cell injury induced by endothelin-1 was counteracted by the endothelin ET(A) receptor antagonist BQ123 or by maintaining the pH of the medium by the addition of 50 mM HEPES. Endothelin-1 markedly increased the uptake of 2-deoxyglucose and lactic acid production when the cardiomyocytes were pretreated with rotenone. These findings suggest that the stimulation of glucose uptake and anaerobic glycolysis followed by the increase in lactic acid accumulation in cardiomyocytes under the condition of mitochondrial impairment may be involved, at least in part, in the cellular injury by endothelin-1. Moreover, these findings suggest the possibility that the effect of endothelin-1 on myocardium is reversed by the condition of the mitochondria, and endogenous endothelin-1 may deteriorate cardiac failure with mitochondrial dysfunction. This may contribute to clarify the beneficial effect of endothelin receptor blockade in improving heart failures.

Endothelin-1 production is enhanced by rotenone, a mitochondrial complex I inhibitor, in cultured rat cardiomyocytes.

In chronic heart failure and acute myocardial infarction, the tissue level of endothelin (ET)-1 in the heart, as well as its plasma level, has been reported to increase markedly. There is, however, little information about what in these pathologic conditions leads to increased production of ET-1, and which type of cell in the heart produces ET-1. We examined the mRNA and peptide expression of ET-1 using cultured rat neonatal cardiomyocytes, in which mitochondrial dysfunction was induced by rotenone, a mitochondrial respiratory chain complex I inhibitor, because one of the common features in failing or ischemic hearts is an alteration in energy metabolism due to mitochondrial dysfunction. Rotenone increased glucose use by the culture cells within 12 h of addition without affecting cell viability, and depressed the mitochondrial membrane potential after 72 h, indicating the induction of mitochondrial dysfunction in cardiomyocytes. Rotenone induced significant increase in the expression level of mRNA for ET-1 within 1 h of addition. In accordance with this finding, immunoreactive ET-1 in culture medium increased 3 times after 24 h of incubation, suggesting active secretion of ET-1 from cultured cells treated with rotenone. Immunocytochemical analysis verified significant increase of ET-1 peptide in cardiomyocytes, confirming the production of ET-1 by cardiomyocytes. These results suggest that derangement of mitochondrial function in cardiomyocytes itself could lead to the increased production of ET-1 in cardiomyocytes, and that this mechanism may contribute to the increased production of ET-1 in failing and ischemic hearts.

ATP-dependent affinity change of Na+-binding sites of V-ATPase.

V-type Na(+)-ATPase of Enterococcus hirae binds about six (6 +/- 1) Na(+) ions/enzyme molecule with a high affinity (Murata, T., Igarashi, K., Kakinuma, Y., and Yamato, I. (2000) J. Biol. Chem. 275, 13415-13419). After the addition of 5 mm ATP, the binding capacity dropped to about 2 (1.8 +/- 0.3) Na(+) ions/enzyme molecule, returning to the initial value concomitant with the decrease of ATP hydrolysis rate. These findings suggest that the affinity of four of six Na(+)-binding sites of the enzyme changes (lowers) in enzyme reaction. The ATP analogs (adenosine 5'-O-(3-thiotriphosphate) or 5'-adenylylimido-diphosphate), ADP, or aluminum fluoride that is postulated to trap ATPases at their transition state did not inhibit the Na(+) binding capacity significantly. Therefore, the affinity decrease of Na(+)-binding sites was unlikely to be due to ATP binding alone or at the transition state of ATP hydrolysis. In the presence of 5 mm ATP, the ATPase showed strong negative cooperativity (n(H) = 0.16 +/- 0.03) for Na(+) stimulation of ATPase activity. The Hill coefficient (n(H)) increased to 1 in parallel to the decrease of ATP concentration in the reaction mixture. Thus, the ATP-dependent affinity change cooperatively occurs in continuous enzyme reaction.

[Endothelin receptor antagonist, phosphodiesterase inhibitor, thromboxane inhibitor].

According to the advanced comprehension of pathophysiology of primary pulmonary hypertension (PPH), a therapeutical approach to PPH has changed recently. One of the breakthrough to the treatment of PPH is application of prostacyclin. It has been revealed that intravenous administration of prostacyclin has improved the prognosis and patient's quality of life. Another development of endothelin receptor antagonists and phosphodiesterase inhibitors have provided a novel pulmonary-specific effect. An endothelin receptor antagonist has a great inhibitory effect against pulmonary vasculature remodeling. In this regard, this regard, this receptor antagonist has superior effect to other medicines. Furthermore, a phosphodiesterase inhibitor shows a great decreasing effect on pulmonary hypertension with less effect on systemic blood pressure. These drugs will provide a great potential to the treatment of pulmonary hypertension.

Novel molecular mechanism of increased myocardial endothelin-1 expression in the failing heart involving the transcriptional factor hypoxia-inducible factor-1alpha induced for impaired myocardial energy metabolism.

BACKGROUND: Hypoxia-inducible factor (HIF)-1alpha is an important transcriptional factor that activates the gene expression of glycolytic enzymes, which are activated as compensation for impaired beta-oxidation of fatty acid in the failing heart. We reported that cardiac endothelin (ET)-1 expression is markedly increased in heart failure. The mechanism, however, is unknown. Because we found an HIF-1alpha binding site in the 5'-promoter region of the ET-1 gene, we hypothesized that HIF-1alpha is involved in this mechanism. METHODS AND RESULTS: In rat cardiomyocytes, luciferase assay and electrophoretic mobility shift assay showed that HIF-1alpha transcriptionally activates ET-1 gene expression by direct interaction with the predicted DNA binding site in the 5'-promoter region. HIF-1alpha mRNA and ET-1 mRNA in the failing heart increased during the aggravation of heart failure in vivo in animal models, ie, rats with myocardial infarction and hamsters with cardiomyopathy. In cultured cardiomyocytes treated with a mitochondrial inhibitor, HIF-1alpha mRNA and ET-1 mRNA were markedly increased with activated glycolysis, and antisense oligonucleotide for HIF-1alpha largely inhibited the increased gene expression of ET-1. CONCLUSIONS: The present study revealed a novel molecular mechanism of upregulation of myocardial ET-1 in heart failure, indicating that induction of HIF-1alpha to stimulate glycolysis as an adaptation in heart failure against impaired energy metabolism alternatively causes an elevation of cardiac ET-1 gene expression as a maladaptation.

Potassium uptake with low affinity and high rate in Enterococcus hirae at alkaline pH.

Two high-affinity K+ uptake systems, KtrI and KtrII, have been reported in Enterococcus hirae. A mutant, JEMK1, defective in these two systems did not grow at pH 10 in low-K+ medium (less than 1 mM K+), but grew well when supplemented with 10 mM KCl. In this mutant, we found an energy-dependent K+ uptake at pH 10 with a low affinity for K+ (Km of approximately 20 mM) and an extremely high rate [Vmax of 1.6 micromol x min(-1) (mg protein)(-1)]. Rb+ uptake [Km of approximately 40 mM and Vmax of 0.5 micromol x min(-1) (mg protein)(-1)], which was inhibited competitively by K+ and less prominently by Cs+, was also observed. The specificity of this transport is likely to be K+>Rb+>Cs+. This peculiar K+ transport plays a role as a salvage mechanism against defects in high-affinity systems in the K+ homeostasis of this bacterium.

Catalytic properties of Na(+)-translocating V-ATPase in Enterococcus hirae.

V-ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. We found a variant of this family, a Na(+)-translocating ATPase, in a Gram-positive bacterium, Enterococcus hirae. The Na(+)-ATPase was encoded by nine ntp genes from F to D in an ntp operon (ntpFIKECGABDHJ): the ntpJ gene encoded a K(+) transporter independent of the Na(+)-ATPase. Expression of this operon, encoding two transport systems for Na(+) and K(+) ions, was regulated at the transcriptional level by intracellular Na(+) as the signal. Structural aspects and catalytic properties of purified Na(+)-ATPase closely resembled those of other V-type H(+)-ATPases. Interestingly, the E. hirae enzyme showed a very high affinity for Na(+) at catalytic reaction. This property enabled the measurement of ion binding to this ATPase for the first time in the study of V- and F-ATPases. Properties of Na(+) binding to V-ATPase were consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. We propose here a structure model of Na(+) binding sites of the enzyme.

Multiple polyamine transport systems on the vacuolar membrane in yeast.

We recently identified a gene (TPO1, YLL028w) that encodes a polyamine transport protein on the vacuolar membrane in yeast [Tomitori, Kashiwagi, Sakata, Kakinuma and Igarashi (1999) J. Biol. Chem. 274, 3265-3267]. Because the existence of one or more other genes for a polyamine transport protein on the vacuolar membrane was expected, we searched sequence databases for homologues of the protein encoded by TPO1. Membrane proteins encoded by the open reading frames YGR138c (TPO2), YPR156c (TPO3) and YOR273c (TPO4) were postulated to be polyamine transporters and, indeed, were subsequently shown to be polyamine transport proteins on the vacuolar membrane. Cells overexpressing these genes were resistant to polyamine toxicity and showed an increase in polyamine uptake activity and polyamine content in vacuoles. Furthermore, cells in which these genes were disrupted showed an increased sensitivity to polyamine toxicity and a decrease in polyamine uptake activity and polyamine content in vacuoles. Resistance to polyamine toxicity in cells overexpressing the genes was overcome by bafilomycin A(1), an inhibitor of the vacuolar H(+)-ATPase. Among the four polyamine transporters, those encoded by TPO2 and TPO3 were specific for spermine, whereas those encoded by TPO1 and TPO4 recognized spermidine and spermine. These results suggest that polyamine content in the cytoplasm of yeast is elaborately regulated by several polyamine transport systems in vacuoles. Furthermore, it was shown that Glu-207, Glu-324 (or Glu-323) and Glu-574 of TPO1 protein were important for the transport activity.

Impairment of cardiac energy metabolism in vivo causes hemodynamic abnormality and increases cardiac expression of preproendothelin-1 mRNA.

We investigated whether impairment of myocardial energy metabolism attenuates cardiac function and increases cardiac endothelin-1 (ET-1) gene expression in rats. Three weeks after commencing administration of cobalt chloride (CoCl2), an inhibitor of mitochondrial function, the peak positive first derivative of left ventricular (LV) pressure, an indicator of myocardial contractility, was significantly decreased in the CoCl2-treated rats. LV end-diastolic pressure and right ventricular systolic pressure were increased in the CoCl2-treated rats. Echocardiography showed that fractional shortening was significantly decreased in the CoCl2-treated rats. Myocardial expressions of acyl-CoA synthase mRNA, an enzyme involved in fatty acid utilization, was markedly decreased in the CoCl2-treated rats. Under such conditions, myocardial expression of preproendothelin-1 mRNA and atrial natriuretic peptide (ANP) mRNA, molecular markers of heart failure, was markedly increased in the CoCl2 rats. In conclusion, the data suggest that impairment of myocardial energy metabolism causes hemodynamic abnormality and increases molecular markers of heart failure (ET-1, ANP mRNA). These data suggest that myocardial energy metabolism is one of the factors involved in the upregulation of ET-1 gene expression in the failing heart.

Mitochondrial dysfunction of cardiomyocytes causing impairment of cellular energy metabolism induces apoptosis, and concomitant increase in cardiac endothelin-1 expression.

It has been reported that at the end stage, apoptosis is involved in the progression of heart failure. It is suggested that cardiac energy metabolism is impaired during the progression of heart failure. Although the mechanism of induction of apoptosis in the failing heart varies according to the model of heart failure, it is not known whether an impairment of energy metabolism in cardiomyocytes is a primary cause of apoptosis. In this study, we applied mitochondrial inhibitors, such as rotenone, cobalt chloride and antimycin A, which inhibit mitochondrial function at different sites of the mitochondrial respiratory chain, to cardiomyocytes. All these reagents markedly decreased 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay (MTT) reduction activity, an indicator of mitochondrial function, of cardiomyocytes and greatly increased glucose consumption, suggesting that cardiac energy metabolism is switched from beta-oxidation of fatty acid to glycolysis. It was shown that after 48-72 h of treatment with each reagent, apoptosis was shown to occur by DNA laddering and increase in caspase activity. Interestingly, each reagent with a different action site greatly activated caspase-3, but not caspase-8 activity, suggesting that mitochondria are involved in induction of apoptosis. On the other hand, within 24 h of the treatment, when apoptosis of cardiomyocytes was not observed, the treated cardiomyocytes showed a marked increase in preproendothelin-1 and atrial natriuretic peptide (ANP) gene expressions. In conclusion, the present study suggests that mitochondrial dysfunction with impaired energy metabolism elevates gene expression of cardiac ET-1, an aggravating factor in heart failure, and then finally induces apoptosis in cardiomyocytes. The finding of marked increases in expression of molecular markers (ET-1 mRNA and ANP mRNA) in the failing heart, followed by apoptosis in the treated cardiomyocytes suggests that the inhibition of mitochondrial function of cultured cardiomyocytes provides a possible new in vitro model of heart failure.

Mitochondrial dysfunction increases expression of endothelin-1 and induces apoptosis through caspase-3 activation in rat cardiomyocytes in vitro.

We have reported that the expression of endothelin-1 (ET-1) increases in the failing heart. With the progress of heart failure, it has been reported that energy metabolism switches from mitochondrial b-oxidation to glycolysis. Furthermore, it has been reported that apoptosis is induced in the failing heart. However, it is not known how the gene expression of preproendothelin-1 and cellular apoptosis are affected by the mitochondrial dysfunction. Therefore, in order to elucidate this problem, we developed an in vitro model of mitochondrial dysfunction using rotenone, a mitochondrial respiratory chain complex I inhibitor, and studied preproendothelin-1 gene expression and apoptosis. Rotenone greatly increased the gene expression of pre-proendothelin-1 in cardiomyocytes. This result suggests that the gene expression of preproendothelin-1 is induced by the mitochondrial dysfunction. Furthermore, treatment of cardiomyocytes with rotenone induced an elevation of caspase-3 activity, and caused a marked increase in DNA laddering, an indication of apoptosis. In conclusion, it is suggested that mitochondrial impairment in primary cultured cardiomyocytes induced by rotenone in vitro, mimics some of the pathophysiological features of heart failure in vivo, and that ET-1 may have a role in myocardial dysfunction with impairment of mitochondria in the failing heart.