The impact of 10-valent pneumococcal vaccine introduction on invasive disease in Fiji.

BACKGROUND: In 2012, Fiji introduced the 10-valent pneumococcal conjugate vaccine (PCV10). We assessed the impact of PCV10 on invasive pneumococcal disease (IPD), probable bacterial or pneumococcal meningitis (PBPM), meningitis and sepsis 3-5 years post-introduction. METHODS: Laboratory-confirmed IPD and PBPM cases were extracted from national laboratory records. ICD-10-AM coded all-cause meningitis and sepsis cases were extracted from national hospitalisation records. Incidence rate ratios were used to compare outcomes pre/post-PCV10, stratified by age groups: 1-23m, 2-4y, 5-9y, 10-19y, 20-54y, ≥55y. To account for different detection and serotyping methods in the pre-and post-PCV10 period, a Bayesian inference model estimated serotype-specific changes in IPD, using pneumococcal carriage and surveillance data. FINDINGS: There were 423 IPD, 1,029 PBPM, 1,391 all-cause meningitis and 7,611 all-cause sepsis cases. Five years post-PCV10 introduction, IPD declined by 60% (95%CI: 37%, 76%) in children 1-23m months old, and in age groups 2-4y, 5-9y, 10-19y although confidence intervals spanned zero. PBPM declined by 36% (95%CI: 21%, 48%) among children 1-23 months old, and in all other age groups, although some confidence intervals spanned zero. Among children <5y of age, PCV10-type IPD declined by 83% (95%CI; 70%, 90%) and with no evidence of change in non-PCV10-type IPD (9%, 95%CI; -69, 43%). There was no change in all-cause meningitis or sepsis. Post-PCV10, the most common serotypes in vaccine age-eligible and non-age eligible people were serotypes 8 and 23B, and 3 and 7F, respectively. INTERPRETATIONS: Our study demonstrates the effectiveness of PCV10 against IPD in a country in the Asia-Pacific of which there is a paucity of data. FUNDING: This study was support by the Department of Foreign Affairs and Trade of the Australian Government and Fiji Health Sector Support Program (FHSSP). FHSSP is implemented by Abt JTA on behalf of the Australian Government.

Clinical and microbiological characteristics of Eggerthella lenta bacteremia.

Eggerthella lenta is an emerging pathogen that has been underrecognized due to historical difficulties with phenotypic identification. Until now, its pathogenicity, antimicrobial susceptibility profile, and optimal treatment have been poorly characterized. In this article, we report the largest cohort of patients with E. lenta bacteremia to date and describe in detail their clinical features, microbiologic characteristics, treatment, and outcomes. We identified 33 patients; the median age was 68 years, and there was no gender predominance. Twenty-seven patients (82%) had serious intra-abdominal pathology, often requiring a medical procedure. Of those who received antibiotics (28/33, 85%), the median duration of treatment was 21.5 days. Mortality from all causes was 6% at 7 days, 12% at 30 days, and 33% at 1 year. Of 26 isolates available for further testing, all were identified as E. lenta by both commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, and none were found to harbor a vanA or vanB gene. Of 23 isolates which underwent susceptibility testing, all were susceptible to amoxicillin-clavulanate, cefoxitin, metronidazole, piperacillin-tazobactam, ertapenem, and meropenem, 91% were susceptible to clindamycin, 74% were susceptible to moxifloxacin, and 39% were susceptible to penicillin.

Improved detection of vanB2-containing enterococcus faecium with vancomycin susceptibility by Etest using oxgall supplementation.

We have isolated a number of vanB-containing Enterococcus faecium isolates on bile esculin screening agar containing 6 mg/liter vancomycin, which on subsequent susceptibility testing using Etest have repeatedly demonstrated vancomycin MICs of

High rates of fecal carriage of nonenterococcal vanB in both children and adults.

We examined the rate of fecal carriage of vanB in the absence of cultivable vancomycin-resistant enterococci in three distinct populations (children, community adults, and hemodialysis patients). Nonenterococcal vanB carriage was similarly high in hemodialysis patients (45%) and community adults (63%; P = 0.066) and significantly more common among community adults than children (27%; P = 0.001).

Regional hemodynamic effects of neutral endopeptidase inhibition and angiotensin (AT(1)) receptor antagonism alone or in combination in conscious spontaneously hypertensive rats.

We tested the hypothesis that angiotensin (AT(1)) receptor antagonism (with losartan) would enhance the cardiovascular actions of neutral endopeptidase (NEP) inhibition [with candoxatrilat or (2S)-2-{[1-({[(1S)-1-carboxy-2-(5-phenyl-1,3-oxazol-2-yl)ethyl]amino}carbonyl)cyclopentyl]methyl}-4-methoxybutanoic acid (UK-489,329)] in conscious spontaneously hypertensive rats (SHR). Four-day continuous intravenous infusion of candoxatrilat (1.9 microg kg(-1) min(-1)) or UK-489,329 (0.15 microg kg(-1) min(-1)) had no significant cardiovascular effects, whereas candoxatrilat (6.4 microg kg(-1) min(-1)) had a modest antihypertensive effect (-10.9 mm Hg on day 4) but no significant sustained effects on regional hemodynamics. Losartan caused a fall in blood pressure (maximum -29.2 mm Hg on day 4) that was associated with renal, mesenteric, and, to a lesser extent, hindquarters vasodilatation. The combination of losartan with either dose of candoxatrilat had no greater antihypertensive or vasodilator effects than losartan alone, with the exception of the increase in renal vascular conductance, which was greater with the combination of the drugs than with either drug alone (significant only in the lower dose study). Losartan combined with UK-489,329 showed a greater antihypertensive effect than losartan alone (-14.6 mm Hg greater on day 4), although the effects of the combination were not significantly greater than the sum of the effects of both agents administered separately. However, losartan combined with UK-489,329 caused increases in renal and hindquarters vascular conductance that were significantly greater with the combination than with either agent given alone. Thus, in conscious SHR, the renin-angiotensin system may act to oppose a vasodilator action of NEP inhibition, particularly in the renal vascular bed.

Molecular characterization of vanB elements in naturally occurring gut anaerobes.

Previously, we reported the isolation of 10 vancomycin-resistant gram-positive anaerobic bacilli carrying the vanB ligase gene from nine hemodialysis patients (S. A. Ballard et al., Antimicrob. Agents Chemother. 49:77-81, 2005; T. P. Stinear et al., Lancet 357:855-856, 2001). In the present study, the molecular and evolutionary relationship of the vanB resistance element within these 10 anaerobes and two vancomycin-resistant Enterococcus faecium strains were examined. PCR analysis and nucleotide sequencing demonstrated that all 12 isolates carried the vanB operon associated with an element identical to Tn1549 and Tn5382 of Enterococcus. Restriction fragment length polymorphism analysis of the vanB operon in these isolates revealed two distinct patterns, and sequencing showed that minor base differences existed. PCR amplification of the joint region of a circular intermediate was demonstrated in nine of these organisms, a finding indicative of an ability to excise and circularize, an intermediate step in transposition and conjugative transfer. Southern hybridization with a vanB-vanX(B) probe suggests that there is one insert of the transposon in all isolates. Sequence analysis of the integration site revealed distinct sequences: the Tn1549/5382 element within E. faecium was inserted within the host chromosome, whereas nucleotide sequences surrounding the Tn1549/5382 element in the 10 anaerobes showed no significant homology to sequences in the GenBank database. We demonstrate considerable similarity between the Tn1549/5382 element identified in 10 anaerobe isolates with that found in enterococci. The homology and potential to transpose suggest a recent horizontal transfer event may have occurred. However, the original direction of transposition and the mechanism involved remains unknown.

Comparison of three PCR primer sets for identification of vanB gene carriage in feces and correlation with carriage of vancomycin-resistant enterococci: interference by vanB-containing anaerobic bacilli.

We assessed the sensitivities and specificities of three previously described PCR primers on enrichment broth cultures of feces for the accurate detection of fecal carriage of vancomycin-resistant enterococci (VRE). In addition, we investigated specimens that were vanB PCR positive but VRE culture negative for the presence of other vanB-containing pathogens. Feces from 59 patients (12 patients carrying vanB Enterococcus faecium strains and 47 patients negative for VRE carriage) were cultured for 36 h in aerobic brain heart infusion (BHI) broth, anaerobic BHI (AnO(2)BHI) broth, or aerobic Enterococcosel (EC) broth. DNA was extracted from the cultures and tested for the presence of vanB by using the PCR primers of Dutka-Malen et al. (S. Dutka-Malen, S. Evers, and P. Courvalin, J. Clin. Microbiol. 33:24-27, 1995), Bell et al. (J. M. Bell, J. C. Paton, and J. Turnidge, J. Clin. Microbiol. 36:2187-2190, 1998), and Stinear et al. (T. P. Stinear, D. C. Olden, P. D. R. Johnson, J. K. Davies, and M. L. Grayson, Lancet 357:855-856, 2001). The sensitivity (specificity) of PCR compared with the results of culture on BHI, AnO(2)BHI, and EC broths were 67% (96%), 50% (94%), and 17% (100%), respectively, with the primers of Dutka-Malen et al.; 92% (60%), 92% (45%), and 92% (83%), respectively, with the primers of Bell et al.; and 92% (49%), 92% (43%), and 100% (51%) respectively, with the primers of Stinear et al. The primers of both Bell et al. and Stinear et al. were significantly more sensitive than those of Dutka-Malen et al. in EC broth (P = 0.001 and P < 0.001, respectively). The poor specificities for all primer pairs were due in part to the isolation and identification of six anaerobic gram-positive bacilli, Clostridium hathewayi (n = 3), a Clostridium innocuum-like organism (n = 1), Clostridium bolteae (n = 1), and Ruminococcus lactaris-like (n = 1), from five fecal specimens that were vanB positive but VRE culture negative. All six organisms were demonstrated to contain a vanB gene identical to that of VRE. VanB-containing bowel anaerobes may result in false-positive interpretation of PCR-positive fecal enrichment cultures as VRE, regardless of the primers and protocols used.

Hemodynamic effects of phosphodiesterase 5 and angiotensin-converting enzyme inhibition alone or in combination in conscious SHR.

The regional hemodynamic responses to continuous 4-day infusion of UK-357,903 [1-ethyl-4-{3-[3-ethyl-6,7-dihydro-7-oxo-2-(2-pyridylmethyl)-2H-pyrazolo[4,3-d]pyrimidin-5-yl]-2-(2-methoxyethoxy)-5-pyridylsulphonyl}piperazine] (266 microg kg(-1) h(-1)) alone and in combination with a low dose of enalapril (10 microg kg(-1) h(-1)) were measured in conscious spontaneously hypertensive rats to test the hypothesis that the renin-angiotensin system may influence the cardiovascular consequences of inhibition of phosphodiesterase 5 (PDE5) by UK-357,903 or vice versa. UK-357,903 alone caused a fall in mean blood pressure (-12.1 mm Hg) associated with vasodilatation in the mesenteric and hindquarters vascular beds. The only way in which the effects of enalapril given alone differed significantly from those of the vehicle was in causing mesenteric vasodilatation, which developed over the 4 days of infusion. UK-357,903 given in combination with enalapril caused hypotension (-17.8 mm Hg) and vasodilatation in the renal, mesenteric, and hindquarter vascular beds. There was evidence of a significant interaction between the effects of the two compounds on renal Doppler shift and vascular conductance with the combined action of the two compounds being greater than the sum of their individual effects. However, although there was a trend for the combination to have greater effects than either of the individual agents on blood pressure and mesenteric vascular conductance, there was no statistical evidence of an interaction. The results indicate that inhibition of the renin-angiotensin system uncovers additional renal vasodilator effects of UK-357,903, and/or inhibition of PDE5 enhances the renal vasodilator effects of angiotensin-converting enzyme inhibition.

Inhibition of cyclic GMP-binding cyclic GMP-specific phosphodiesterase (Type 5) by sildenafil and related compounds.

The cGMP-binding cGMP-specific phosphodiesterase (PDE5) degrades cGMP and regulates the intracellular level of cGMP in many tissues, including the smooth muscle of the corpus cavernosum of the penis. Sildenafil (Viagra), a specific PDE5 inhibitor, promotes penile erection by blocking the activity of PDE5, which causes cGMP to accumulate in the corpus cavernosum. In the present study, sildenafil, like other PDE5 inhibitors, stimulates cGMP binding to the allosteric sites of PDE5 by interacting at the catalytic site of this enzyme, but the drug does not compete with cGMP for binding at the allosteric sites. Both sildenafil and zaprinast are competitive inhibitors of PDE5, and double-inhibition analysis shows that these two inhibitors added together interact with the catalytic site of PDE5 in a mutually exclusive manner. After site-directed mutagenesis of each of 23 conserved amino acid residues in the catalytic domain of PDE5, the pattern of changes in the IC50 values for sildenafil or UK-122764 is similar to that found for zaprinast. However, among the three inhibitors, sildenafil exhibits the most similar pattern of changes in the IC50 to that found for the affinity of cGMP, implying similar interactions with the catalytic domain. This may explain in part the stronger inhibitory potency of sildenafil for wild-type PDE5 compared with the other inhibitors [sildenafil (Ki = 1 nM) > UK-122764 (Ki = 5 nM) > zaprinast (Ki = 130 nM)]. The affinity of each of these inhibitors for PDE5 is much higher than that of cGMP itself (Km = 2000 nM). It is concluded that residues such as Tyr602, His607, His643, and Asp754 may form important interactions for sildenafil in PDE5, but because these amino acids are conserved in all mammalian PDEs, the selectivity and potency of sildenafil is likely to be provided by a nonconserved residue or residues in the PDE5 catalytic domain.

Molecular analysis of the dnaK locus of Leptospira interrogans serovar Copenhageni.

Analysis of the dnaK locus of Leptospira interrogans serovar Copenhageni identified four genes in the order hrcA, grpE, dnaK and dnaJ. This is the first time a homologue of hrcA has been identified in a spirochete. The hrcA gene and a regulatory sequence, designated CIRCE, play a significant role in the regulation of the dnaK locus of several Gram+ organisms. Their presence upstream of dnaK in Leptospira suggested a similiar regulatory mechanism. Transcriptional analysis using reverse transcriptase-PCR demonstrated transcription of all four genes and indicated that hrcA and grpE were co-transcribed, as were grpE and dnaK. Whilst hrcA, grpE and dnaK were closely linked on the chromosome, transcription terminators between dnaK and dnaJ and downstream of dnaJ suggested that this latter gene exists in its own operon. Primer extension analysis located functional promoters upstream of hrcA and grpE; however, no evidence of a functional promoter could be found for dnaJ. Moreover, transcripts encompassing the first three genes or the entire locus could not be demonstrated, suggesting that the four genes are regulated independently at the transcriptional level. These results indicate that the regulation of the dnaK locus of Leptospira differs somewhat from that observed in other organisms.

Effect of the selective phosphodiesterase type 5 inhibitor sildenafil on erectile dysfunction in the anesthetized dog.

PURPOSE: The effects of sildenafil, a highly selective inhibitor of cyclic guanosine monophosphate-specific phosphodiesterase type 5, on erectile function in the anesthetized dog were evaluated. MATERIALS AND METHODS: In pentobarbital-anesthetized dogs, increases in intracavernosal pressure in the corpus cavernosum and penile blood flow were induced by pelvic nerve stimulation over a frequency range of 1 to 16 hertz. The effects of increasing doses of sildenafil on electrically stimulated intracavernosal pressure, penile blood flow, blood pressure, and heart-rate were evaluated. In parallel experiments, the effects of the nitric oxide synthase inhibitor N omega-Nitro-L-Arginine (L-NOArg) on these same parameters also were assessed. RESULTS: The effects of nerve stimulation on intracavernosal pressure and blood flow to the penis were blocked by L-NOArg, 0.1-3 mg./kg., in a dose-related manner, confirming the important role of nitric oxide in producing erections. Sildenafil, 1-100 microg./kg administered intravenously, had no direct effect on intracavernosal pressure but potentiated the increase in intracavernosal pressure induced by nerve stimulation. This potentiation occurred at sildenafil plasma concentrations consistent with its relaxation effect on isolated human cavernosal tissue and its inhibition of phosphodiesterase type 5 in vitro. Sildenafil had no significant effect on blood pressure or heart rate. CONCLUSIONS: By inhibiting cyclic guanosine monophosphate-specific phosphodiesterase type 5, sildenafil augments the neuronal mechanism responsible for penile erection. This mechanism explains the significant improvements reported in the rigidity and duration of erections seen in patients with erectile dysfunction who have been treated with oral sildenafil.

Effects of sildenafil on the relaxation of human corpus cavernosum tissue in vitro and on the activities of cyclic nucleotide phosphodiesterase isozymes.

PURPOSE: Sildenafil, an inhibitor of cGMP-specific phosphodiesterase 5 (PDE5), is currently undergoing evaluation as an oral therapy for penile erectile dysfunction. The aims of this study were to investigate the mechanism of action of sildenafil on the neurogenic relaxation of human corpus cavernosum (HCC) in vitro and to determine the activity of sildenafil against a full range of PDE isozymes. MATERIALS AND METHODS: Strips of HCC tissue were precontracted with phenylephrine. Relaxation responses resulting from electrical field stimulation (EFS) were then determined in the presence and absence of sildenafil. The effects of sildenafil on PDE1 to 5 prepared from human tissues and PDE6 from bovine retina were determined by measuring the conversion of [3H]-cGMP or [3H]-cAMP to their respective [3H]-5'-mononucleotides. RESULTS: Sildenafil (0.001 to 1 microM) enhanced the EFS-induced, nitric oxide (NO) dependent, relaxation of HCC in a concentration-dependent manner to a maximum of 3 times the pretreatment level at 1 microM sildenafil. Compared with zaprinast, an early PDE5 inhibitor, sildenafil was approximately 240-fold more potent, inhibiting PDE5 from HCC with a geometric mean IC50 of 3.5 nM. For sildenafil, IC50 values for inhibition of PDE1 to 4 were 80 to more than 8500 times greater than that for PDE5 and the IC50 for PDE6 (33 nM) was approximately 9-fold greater. CONCLUSIONS: The data support the proposal that enhancement of penile erection by sildenafil in patients with erectile dysfunction involves potentiation of the NO-stimulated cGMP signal mediating relaxation of cavernosal smooth muscle during sexual stimulation. Sildenafil is a potent inhibitor of PDE5 from HCC, with high selectivity for PDE5 relative to other PDE isozymes.

Effects of sildenafil, a type-5 cGMP phosphodiesterase inhibitor, and papaverine on cyclic GMP and cyclic AMP levels in the rabbit corpus cavernosum in vitro.

OBJECTIVE: To investigate further the mechanisms of action of sildenafil, a highly selective and potent inhibitor of type 5 cGMP phosphodiesterase (PDE5) that has proved effective in the treatment of erectile dysfunction, by assessing its effect on the in vitro formation of cGMP and cAMP in the corpus cavernosum of the rabbit. MATERIALS AND METHODS: Male New Zealand White rabbits (2.5 kg) were killed and their penises rapidly excised, cut into segments and pooled. Penile segments were then incubated with various concentrations of sildenafil or papaverine. The formation of cGMP was stimulated with increasing concentrations of sodium nitroprusside (SNP) and the cGMP and cAMP concentrations measured by radioimmunoassay. Responses were compared to those obtained with papaverine, which is used therapeutically as an erectogen. RESULTS: In the presence or absence of SNP, sildenafil increased cGMP concentrations in rabbit penile tissue with increasing dose; the increase was greatest (about 28-fold) when cGMP was stimulated with SNP (up to 10 mumol/L). At all stimulatory concentrations of SNP, the effective concentrations for 50% stimulation (EC50) of sildenafil were 430-520 nmol/L. Concentrations of cAMP were unaltered by sildenafil. Papaverine enhanced cGMP formation in response to SNP, but at much higher concentrations than did sildenafil (> or = 10 mumol/L). CONCLUSIONS: Sildenafil specifically increases cGMP levels in rabbit corpora cavernosa; the increase is greater in the presence of SNP indicating that, in vivo, sildenafil may enhance erection by the augmentation of nitric oxide-mediated relaxation pathways. The erectogenic effect of sildenafil is mediated by a specific enhancement of cGMP accumulation in the corpus cavernosum, consistent with the known activity of sildenafil as a potent and highly selective inhibitor of cGMP-specific PDE.

Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction.

Sildenafil (Viagra, UK-92,480) is a novel oral agent under development for the treatment of penile erectile dysfunction. Erection is dependent on nitric oxide and its second messenger, cyclic guanosine monophosphate (cGMP). However, the relative importance of phosphodiesterase (PDE) isozymes is not clear. We have identified both cGMP- and cyclic adenosine monophosphate-specific phosphodiesterases (PDEs) in human corpora cavernosa in vitro. The main PDE activity in this tissue was due to PDE5, with PDE2 and 3 also identified. Sildenafil is a selective inhibitor of PDE5 with a mean IC50 of 0.0039 microM. In human volunteers, we have shown sildenafil to have suitable pharmacokinetic and pharmacodynamic properties (rapid absorption, relatively short half-life, no significant effect on heart rate and blood pressure) for an oral agent to be taken, as required, prior to sexual activity. Moreover, in a clinical study of 12 patients with erectile dysfunction without an established organic cause, we have shown sildenafil to enhance the erectile response (duration and rigidity of erection) to visual sexual stimulation, thus highlighting the important role of PDE5 in human penile erection. Sildenafil holds promise as a new effective oral treatment for penile erectile dysfunction.

5 alpha-reductase expression by prostate cancer cell lines and benign prostatic hyperplasia in vitro.

5 alpha-reductase (5 alpha R) activity in two human prostate cancer cell lines was compared to that in benign prostatic hyperplasia (BPH) tissue and COS cells transfected with and expressing the human genes for 5 alpha-reductase type 1 (5 alpha R1) and type 2 (5 alpha R2). Comparisons were based on pH profiles and sensitivities to selective inhibitors of 5 alpha-reductase In the cancer lines, activity was greatest over the pH range 7-8, compared to a sharp peak of activity between pH 5-5.5 in BPH tissue and COS cells expressing 5 alpha R2. Finasteride and SKF105,657 were potent inhibitors of 5 alpha-reductase activity in BPH tissue and COS cells expressing 5 alpha R2, but weak inhibitors in the cancer lines and in COS cells expressing 5 alpha R1. In contrast, UK117,026 was a more potent inhibitor of 5 alpha-reductase activity in the prostate cancer cell lines and in COS cells expressing 5 alpha R1. These data indicate that human prostate cancer cell lines express 5 alpha-reductase activity similar to that in COS cells transfected with 5 alpha R1, but different from that in BPH tissue. This may be a consequence of in vitro culture. Alternatively, it may reflect a change occurring as a result of neoplastic transformation in which case it will be important to select appropriate inhibitors in the clinic.

Comparison of testosterone metabolism in benign prostatic hyperplasia and human prostate cancer cell lines in vitro.

Pathways of testosterone metabolism in tissue slices and cell suspensions of human benign hyperplastic prostate (BPH) tissue and human prostate cancer cell lines (DU145, HPC-36M, PC-3/MA2 and LNCaP) were investigated. Thin layer chromatography analysis was used to identify the following tritiated metabolites: testosterone, 5 alpha-dihydrostestosterone (DHT), 5 alpha-androstane-3 alpha/3 beta-17 beta-diol (androstanediols), 4-androstene-3,17-dione (androstenedione) and 5 alpha-androstanedione. The predominant pathway for testosterone metabolism in BPH was via 5 alpha-reductase producing 5 alpha-dihydrotestosterone (71% and 75% total metabolites in slices and suspensions incubated for 24 h, respectively). The cancer cell lines DU145 and HPC-36M resembled BPH by metabolizing testosterone predominantly to DHT (68% and 82% total metabolites, respectively), although the rate of metabolism was much lower in the cell lines (0.099 and 0.05 pmol testosterone/mg protein/h in DU145 and HPC-36M) compared to the BPH cell suspensions (6.4 pmol testosterone/mg protein/h). In contrast, PC-3/MA2 contained high 17 beta-HSD activity forming large amounts of 4-androstene-3,17-dione (84% total metabolites), converting testosterone at a rate faster (12.8 pmol testosterone/mg protein/h) than the BPH cell suspensions. LNCaP rapidly converted testosterone exclusively to a glucuronide conjugate (7.4 pmol testosterone/mg protein/h), although after incubation with [3H]-4-androstene-3,17-dione, 5 alpha-reductase activity was demonstrated. LNCaP was the only cell line whose growth and colony-forming ability was stimulated by testosterone and DHT. BPH and all the cell lines tested had 5 alpha-reductase activity, but only the prostate tissue and the cell lines DU145 and HPC-36M converted testosterone predominantly to DHT.

Molecular analysis of the hsp (groE) operon of Leptospira interrogans serovar copenhageni.

A chromosomal gene library of Leptospira interrogans serovar copenhageni strain Wijnberg was constructed in phage lambda gt11. Plaque immunoassay with R alpha P64 antiserum identified one clone expressing a putative groEL homologue. DNA sequence analysis of the 2.4 kb EcoRI-Bam HI cloned fragment from strain Wijnberg revealed two open reading frames encoding polypeptides of 10.5 kDa (Hsp10) and 58 kDa (Hsp58). Sequence comparison of the deduced amino acid sequences of these ORFs confirmed the operon as the groE equivalent of Leptospira. Transcriptional analysis suggested that this operon is primarily under the control of an E sigma 70 promoter element. However, both Hsp10 and Hsp58 were overexpressed under heat-shock conditions as determined by [35S]-methionine pulse labelling experiments. As no functional heat-shock promoter could be identified, a 9bp inverted repeat, located between the transcription and translation start sites, may play a role in the upregulation of this operon under heat-shock conditions, similar to mechanisms described for several Gram-positive organisms.