Evaluation of a pro-active strategy for managing tuberculosis-HIV co-infection in a UK tertiary care setting.

Management of tuberculosis (TB)-HIV co-infection is complicated by interactions between the diseases and their therapies. We developed and evaluated a strategy to (i) treat co-infected patients in a single co-infection clinic, (ii) maximize use of first-line drugs, (iii) delay antiretroviral therapy (ART) until two months post-TB treatment except in severe immunosuppression, (iv) commence efavirenz at 600 mg daily with therapeutic drug monitoring (TDM) and (v) target treatment completion. We conducted a prospective cohort review over 5.5 years in a UK tertiary referral center where 56 HIV-positive patients treated for TB were followed-up for a median 30 months. Main outcome measures were treatment completion, adverse events, immune reconstitution inflammatory syndrome, immunological and virological parameters, and TDM for efavirenz. Treatment completion rates were 88% (49/56); four patients were lost to local follow-up and three (5.4%) died during treatment; no deaths were TB-related. Adverse events were common (55%), but caused no treatment interruptions. Standard doses (600 mg daily) of efavirenz with rifampicin achieved or exceeded therapeutic levels in 25/28 (89%). This study supports combined management for TB-HIV co-infected patients. Delaying ART to two months post-TB treatment did not seem to result in poor clinical outcomes in this well-resourced context. Although efavirenz 600 mg daily usually achieved satisfactory levels, TDM is recommended.

Characteristic brain hypoperfusion by 99mTc-ECD single photon emission computed tomography (SPECT) in patients with the first-episode schizophrenia.

OBJECTIVE: In this study, we evaluated brain perfusion in patients with first-episode medicated schizophrenia using the new analytical method, statistical parametric mapping (SPM) applied to single photon emission computed tomography (SPECT). METHOD: We performed SPECT with 99-Tc-ethyl cysteinate dimer (99mTc-ECD) of the brain and magnetic resonance imaging (MRI) in patients with schizophrenia (n=30) and control subjects matched for age and gender (n=37). A voxel-by-voxel group analysis was performed using SPM2 (Z>3.0, P<0.001, uncorrected for multiple comparisons). RESULT: In comparison with control subjects, the volumes of the bilateral frontal areas were found to be decreased on MRI. Blood flow was found to be reduced in the bilateral temporal areas in the patients with schizophrenia on SPECT. CONCLUSION: In this study, patients with first-episode schizophrenia appeared to have significant bilateral temporal hypoperfusion, although temporal volumes were not significantly decreased in comparison with control subjects. Abnormality of temporal lobe blood flow in schizophrenia may show that functional changes occur earlier than structural changes, and may assist in the diagnosis of schizophrenia.

Patient perception of cervical screening among women living with human immuno-deficiency virus infection attending an antiretroviral therapy clinic in urban South Africa.

This study aims to ascertain the perception of cervical screening practices among HIV-positive women attending an ART clinic in urban South Africa. It is a prospective cross-sectional study of 100 randomly selected patients using semi-structured interviews. Answers to fixed-response questions were recorded for statistical analysis and themes were identified from responses to open-ended questions. The study found that 59% of women surveyed reported ever having had a Papanicolau (Pap) smear and that 41% of these women had never been notified of the result. Many women surveyed lacked understanding of cervical screening; 78% had never heard of cervical cancer and around 40% had no correct knowledge about Pap smears. The findings suggest that cervical screening practices among HIV-positive women living in urban South Africa do not comply with the recommendations that are based on evidence of increased risk for this population. Systematic cervical screening programmes should be offered to HIV-positive women attending ART clinics in South Africa.

The replication fork trap and termination of chromosome replication.

Bacteria that have a circular chromosome with a bidirectional DNA replication origin are thought to utilize a 'replication fork trap' to control termination of replication. The fork trap is an arrangement of replication pause sites that ensures that the two replication forks fuse within the terminus region of the chromosome, approximately opposite the origin on the circular map. However, the biological significance of the replication fork trap has been mysterious, as its inactivation has no obvious consequence. Here we review the research that led to the replication fork trap theory, and we aim to integrate several recent findings that contribute towards an understanding of the physiological roles of the replication fork trap. Likely roles include the prevention of over-replication, and the optimization of post-replicative mechanisms of chromosome segregation, such as that involving FtsK in Escherichia coli.

Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction.

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.

Dominant-negative c-Jun inhibits rat cardiac hypertrophy induced by angiotensin II and hypertension.

Cardiac activator protein-1 (AP-1), composed of c-Jun, is significantly activated by hypertension or angiotensin II (AngII). This study was undertaken to elucidate whether c-Jun could be the potential target for treatment of cardiac hypertrophy. We constructed recombinant adenovirus carrying dominant-negative mutant of c-Jun (Ad.DN-c-Jun). Using catheter-based technique of adenoviral gene transfer, we achieved global myocardial transduction of DN-c-Jun in rats, to specifically inhibit cardiac AP-1. (1) AngII (200 ng/kg/min) infusion in rats caused cardiac hypertrophy, increased cardiac p70S6 kinase activity by 1.3-fold (P<0.05) and enhanced the gene expression of cardiac hypertrophic markers. Ad.DN-c-Jun, which was transferred to the heart 2 days before AngII infusion, prevented cardiac hypertrophy (P<0.01), decreased p70S6 kinase phosphorylation (P<0.05), and suppressed cardiac gene expression of brain natriuretic peptide, collagen I, III, and IV, monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) (P<0.01). (2) In genetically hypertensive rats with cardiac hypertrophy, cardiac gene transfer of Ad.DN-c-Jun, without affecting hypertension, regressed cardiac hypertrophy (P<0.05), and suppressed p70S6 kinase phosphorylation by 20% (P<0.05) and suppressed the enhanced expression of collagen I, III, and IV, MCP-1 and PAI-1. These results provided the first evidence that in vivo blockade of cardiac c-Jun inhibits pathologic cardiac hypertrophy.

Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy.

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in 1H-15N correlation spectra of a 15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the 1H-15N correlations was achieved using a suite of triple resonance NMR experiments with 15N,13C,70% 2H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to 1H-15N spectra revealed that the N-termini, alpha3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.

A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis.

Two dimers of the replication terminator protein (RTP) of Bacillus subtilis bind to a chromosomal DNA terminator site to effect polar replication fork arrest. Cooperative binding of the dimers to overlapping half-sites within the terminator is essential for arrest. It was suggested previously that polarity of fork arrest is the result of the RTP dimer at the blocking (proximal) side within the complex binding very tightly and the permissive-side RTP dimer binding relatively weakly. In order to investigate this "differential binding affinity" model, we have constructed a series of mutant terminators that contain half-sites of widely different RTP binding affinities in various combinations. Although there appeared to be a correlation between binding affinity at the proximal half-site and fork arrest efficiency in vivo for some terminators, several deviated significantly from this correlation. Some terminators exhibited greatly reduced binding cooperativity (and therefore have reduced affinity at each half-site) but were highly efficient in fork arrest, whereas one terminator had normal affinity over the proximal half-site, yet had low fork arrest efficiency. The results show clearly that there is no direct correlation between the RTP binding affinity (either within the full complex or at the proximal half-site within the full complex) and the efficiency of replication fork arrest in vivo. Thus, the differential binding affinity over the proximal and distal half-sites cannot be solely responsible for functional polarity of fork arrest. Furthermore, efficient fork arrest relies on features in addition to the tight binding of RTP to terminator DNA.

The midcell replication factory in Bacillus subtilis is highly mobile: implications for coordinating chromosome replication with other cell cycle events.

During vegetative growth, rod-shaped bacterial cells such as Escherichia coli and Bacillus subtilis divide precisely at midcell. It is the Z ring that defines the position of the division site. We previously demonstrated that the early stages of chromosome replication are linked to midcell Z ring assembly in B. subtilis and proposed a direct role for the centrally located replication factory in masking and subsequently unmasking the midcell site for Z ring assembly. We now show that the replication factory is significantly more scattered about the cell centre than the Z ring in both vegetative cells and outgrown spores of B. subtilis. This finding is inconsistent with the midcell replication factory acting as a direct physical block to Z ring assembly. Time-lapse experiments demonstrated that the lower precision of replication factory positioning results from its high mobility around the cell centre. Various aspects of this mobility are presented and the results are discussed in the light of current views on the determinants of positional information required for accurate chromosome segregation and cell division.

The impact of single cysteine residue mutations on the replication terminator protein.

We report the structural and biophysical consequences of cysteine substitutions in the DNA-binding replication terminator protein (RTP) of Bacillus subtilis, that resulted in an optimised RTP mutant suitable for structural studies. The cysteine residue 110 was replaced with alanine, valine or serine. Protein secondary structure and stability (using circular dichroism spectropolarimetry), self-association (using analytical ultracentrifugation), and DNA-binding measurements revealed RTP.C110S to be the most similar mutant to wild-type RTP. The C110A and C110V.RTP mutants were less soluble, less stable and showed lower DNA-binding affinity. The structure of RTP.C110S, solved to 2.5A resolution using crystallographic methods, showed no major structural perturbation due to the mutation. Heteronuclear NMR spectroscopic studies revealed subtle differences in the electronic environment about the site of mutation. The study demonstrates the suitability of serine as a substitute for cysteine in RTP and the high sensitivity of protein behaviour to single amino acid substitutions.

The Min system is not required for precise placement of the midcell Z ring in Bacillus subtilis.

In bacteria, the Min system plays a role in positioning the midcell division site by inhibiting the formation of the earliest precursor of cell division, the Z ring, at the cell poles. However, whether the Min system also contributes to establishing the precise placement of the midcell Z ring is unresolved. We show that the Z ring is positioned at midcell with a high degree of precision in Bacillus subtilis, and this is completely maintained in the absence of the Min system. Min is therefore not required for correct midcell Z ring placement in B. subtilis. Our results strongly support the idea that the primary role of the Min system is to block Z ring formation at the cell poles and that a separate mechanism must exist to ensure cell division occurs precisely at midcell.

Could tourist boots act as vectors for disease transmission in Antarctica?

BACKGROUND: Over the last decade there has been a rapid increase in the number of visitors landing at wildlife sites on the Antarctic continent, and concern has been raised that tourists may transmit important pathogens to or between wildlife colonies. The aim of this study was to determine if tourist activities pose a potential threat to Antarctic wildlife, or possibly to human populations through carriage of pathogens on boots. METHODS: In two trips conducted to Antarctica in the summer season of 2000/2001, swabs were collected from tourist boots: prior to landing, to determine baseline level of bacterial flora on the boots (A isolates); immediately on return to the ship, to quantify the level of contamination (B isolates); and after the boots were washed in seawater to determine the recovery of the organisms after cleaning (C isolates). Swabs were cultured for coliforms, and isolates identified using the API system. RESULTS: Twenty organisms resembling coliforms were isolated from 15 of 72 pairs of boots. Two isolates were recovered from group A, 4 from group B, and 14 from group C. Of these 20 isolates, 11 could be identified using the API identification method. The remaining 9 isolates all produced an unknown but identical profile number. CONCLUSION: These results indicate that current practices for cleaning the boots of tourists visiting Antarctic wildlife colonies may not be sufficient to prevent the transmission of pathogens, and indicate that further studies are needed to define the best method of disinfection.

Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest.

The coordinated termination of DNA replication is an important step in the life cycle of bacteria with circular chromosomes, but has only been defined at a molecular level in two systems to date. Here we report the structure of an engineered replication terminator protein (RTP) of Bacillus subtilis in complex with a 21 base pair DNA by X-ray crystallography at 2.5 A resolution. We also use NMR spectroscopic titration techniques. This work reveals a novel DNA interaction involving a dimeric 'winged helix' domain protein that differs from predictions. While the two recognition helices of RTP are in close contact with the B-form DNA major grooves, the 'wings' and N-termini of RTP do not form intimate contacts with the DNA. This structure provides insight into the molecular basis of polar replication fork arrest based on a model of cooperative binding and differential binding affinities of RTP to the two adjacent binding sites in the complete terminator.

Mid-cell Z ring assembly in the absence of entry into the elongation phase of the round of replication in bacteria: co-ordinating chromosome replication with cell division.

We have shown previously that, when spores of a thymine-requiring strain of Bacillus subtilis were grown out in the absence of thymine, mid-cell Z rings formed over the nucleoid and much earlier than might be expected with respect to progression into the round of replication. It is now shown that such conditions allow no replication of oriC. Rather than replication, partial degradation of the oriC region occurs, suggesting that the status of this region is connected with the 'premature' mid-cell Z ring assembly. A correlation was observed between entry into the replication elongation phase and a block to mid-cell Z rings. The conformation of the nucleoid under various conditions of DNA replication inhibition or limitation suggests that relief of nucleoid occlusion is not primarily responsible for mid-cell Z ring formation in the absence of thymine. We propose the existence of a specific structure at mid-cell that defines the Z ring nucleation site (NS). It is suggested that this NS is normally masked by the replisome upon initiation of replication or soon after entry into the elongation phase, and subsequently unmasked relatively late in the round. During spore outgrowth in the absence of thymine, this checkpoint control over mid-cell Z ring assembly breaks down prematurely.

Functional specificity of the replication fork-arrest complexes of Bacillus subtilis and Escherichia coli: significant specificity for Tus-Ter functioning in E. coli.

The Escherichia coli replication terminator TerB was inserted in its two alternate orientations into a Bacillus subtilis fork-arrest assay plasmid. After transferring these new plasmids into B. subtilis, which could overproduce the E. coli terminator protein Tus, it was shown that the E. coli Tus-TerB complex could cause polar replication fork arrest, albeit at a very low level, in B. subtilis. A new B. subtilis-E. coli shuttle plasmid was designed to allow the insertion of either the Terl (B. subtilis) or TerB (E. coli) terminator at the same site and in the active orientation in relation to the approaching replication fork generated in either organism. Fork-arrest assays for both terminator-containing plasmids replicating in both organisms which also produced saturating levels of either the B. subtilis terminator protein (RTP) or Tus were performed. The efficiency of the Tus-TerB complex in causing fork arrest was much higher in E. coli than in B. subtilis. The efficiency of the B. subtilis RTP-Terl complex was higher in B. subtilis than in E. coli, but the effect was significantly less. Evidently a specificity feature in E. coli operates to enhance appreciably the fork-arrest efficiency of a Tus-Ter complex. The specificity effect is of less significance for an RTP-Ter complex functioning in B. subtilis.

Septal localization of the membrane-bound division proteins of Bacillus subtilis DivIB and DivIC is codependent only at high temperatures and requires FtsZ.

Using immunofluorescence microscopy, we have examined the dependency of localization among three Bacillus subtilis division proteins, FtsZ, DivIB, and DivIC, to the division site. DivIC is required for DivIB localization. However, DivIC localization is dependent on DivIB only at high growth temperatures, at which DivIB is essential for division. FtsZ localization is required for septal recruitment of DivIB and DivIC, but FtsZ can be recruited independently of DivIB. These localization studies suggest a more specific role for DivIB in division, involving interaction with DivIC.

Utilization of subsidiary chromosomal replication terminators in Bacillus subtilis.

The Bacillus subtilis merodiploid strain GSY1127 contains a large nontandem duplication of a portion of its chromosome within its left (anticlockwise) replication segment. This causes displacement of the replication terminus region to a noticeably asymmetric location relative to oriC. The utilization of the subsidiary replication terminators, TerIII and TerV, in the merodiploid strain has been compared with that in B. subtilis 168. It is shown that TerIII is utilized to a significant extent in GSY1127 and that TerV is used only marginally at the most. Neither of these terminators is used to a measurable extent in the 168 strain. It is concluded that TerIII and TerV do indeed function as backups to the major terminator TerI, as has been generally thought. It is further concluded that, in the 168 strain, the vast majority of clockwise forks are arrested at the highly efficient TerI terminator, with fork fusion between the approaching forks occurring frequently while the clockwise fork is stationary at TerI.

Simultaneous bilateral tubeless percutaneous nephrolithotomy.

We present what is to our knowledge the first reported case of simultaneous bilateral tubeless (no nephrostomy tube) percutaneous nephrolithotomy. The 64-year-old man was rendered stone free with a single general anesthetic and discharged within 24 hours. The role, indications, and potential benefits of this novel technique are discussed.

Antibiotic resistance in Helicobacter pylori.

OBJECTIVE: To describe antibiotic resistance patterns in Helicobacter pylori. DESIGN: Culture and antibiotic sensitivity testing of antral and gastric body biopsy samples from patients having gastroscopy. PARTICIPANTS: Consecutive consenting patients aged 18 years or more presenting for gastroscopy from 1 July 1998 to 30 June 1999. SETTING: An open-access gastroscopy service at an urban university tertiary hospital. MAIN OUTCOME MEASURES: Number of H. pylori isolates showing resistance to antibiotics; correlates of such resistance with demographic and clinical information. RESULTS: Of 1580 patients undergoing endoscopy, 434 agreed to participate in the study. 108 (24.9%) had positive cultures for H. pylori, and 88 of these isolates (81%) were available for further testing. Resistance to metronidazole and clarithromycin was detected in 36% and 11%, respectively. No resistance was found to tetracycline or amoxycillin. Metronidazole resistance was commoner in younger patients (P = 0.0004) and macrolide resistance was commoner in those born outside Australia or New Zealand (P = 0.03). CONCLUSIONS: We found substantial resistance to metronidazole, and emerging clarithromycin resistance, but complete susceptibility to amoxycillin, tetracycline, gentamicin and cefaclor. These factors may influence the effectiveness of presently recommended eradication regimens.

Co-ordinating DNA replication with cell division in bacteria: a link between the early stages of a round of replication and mid-cell Z ring assembly.

Spores of a thymine-requiring strain of Bacillus subtilis 168, which is also temperature sensitive for the initiation of chromosome replication, were germinated and allowed to grow out at the permissive temperature in a minimal medium containing no added thymine. Under these conditions, there was no or very limited progression into the elongation phase of the first round of replication. In a significant proportion of the outgrown cells, a Z ring formed precisely at mid-cell and over the centrally positioned nucleoid, leading eventually to the formation of a mature division septum. When initiation of the first round of replication was blocked through a temperature shift and with thymine present, the Z ring was positioned acentrally. The central Z ring that formed in the absence of thymine was blocked by the presence of a DNA polymerase III inhibitor. It is concluded that the very early stages of a round of replication (initiation plus possibly limited progression into the elongation phase) play a key role in the precise positioning of the Z ring at mid-cell and between replicating daughter chromosomes.