A Crystal Structure of the Bifunctional Antibiotic Simocyclinone D8, Bound to DNA Gyrase

Simocyclinones are bifunctional antibiotics that inhibit bacterial DNA gyrase by preventing DNA binding to the enzyme. We report the crystal structure of the complex formed between the N-terminal domain of the Escherichia coli gyrase A subunit and simocyclinone D8, revealing two binding pockets that separately accommodate the aminocoumarin and polyketide moieties of the antibiotic. These are close to, but distinct from, the quinolone-binding site, consistent with our observations that several mutations in this region confer resistance to both agents. Biochemical studies show that the individual moieties of simocyclinone D8 are comparatively weak inhibitors of gyrase relative to the parent compound, but their combination generates a more potent inhibitor. Our results should facilitate the design of drug molecules that target these unexploited binding pockets

Phosphorylation and Transport in the Na-K-2Cl Cotransporters, NKCC1 and NKCC2A, Compared in HEK-293 Cells

Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 is only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. NKCC2 regulation is poorly understood because of its restricted distribution and difficulties with its expression in mammalian cell cultures. Here we compare phosphorylation of the N-termini of the cotransporters, measured with phospho-specific antibodies, with bumetanide-sensitive transport of K+ (86Rb+) (activity) in HEK-293 cells stably expressing fNKCC1 or fNKCC2A which were cloned from ferret kidney. Activities of transfected transporters were distinguished from those of endogenous ones by working at 37°C. fNKCC1 and fNKCC2A activities were highest after pre-incubation of cells in hypotonic low-[Cl−] media to reduce cell [Cl−] and volume during flux measurement. Phosphorylation of both transporters more than doubled. Pre-incubation with ouabain also strongly stimulated fNKCC1 and fNKCC2A and substantially increased phosphorylation, whereas pre-incubation in Na+-free media maximally stimulated fNKCC1 and doubled its phosphorylation, but inhibited fNKCC2A, with a small increase in its phosphorylation. Kinase inhibitors halved phosphorylation and activity of both transporters whereas inhibition of phosphatases with calyculin A strongly increased phosphorylation of both transporters but only slightly stimulated fNKCC1 and inhibited fNCCC2A. Thus kinase inhibition reduced phosphorylation and transport, and transport stimulation was only seen when phosphorylation increased, but transport did not always increase with phosphorylation. This suggests phosphorylation of the N-termini determines the transporters' potential capacity to move ions, but final activity also depends on other factors. Transport cannot be reliably inferred solely using phospho-specific antibodies on whole-cell lysates

Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia.

The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected]

Effects of quality improvement in health facilities and community mobilization through women's groups on maternal, neonatal and perinatal mortality in three districts of Malawi: MaiKhanda, a cluster randomized controlled effectiveness trial

BACKGROUND: Maternal, perinatal and neonatal mortality remains high in low-income countries. We evaluated community and facility-based interventions to reduce deaths in three districts of Malawi. METHODS: We evaluated a rural participatory women's group community intervention (CI) and a quality improvement intervention at health centres (FI) via a two-by-two factorial cluster randomized controlled trial. Consenting pregnant women were followed-up to 2 months after birth using key informants. Primary outcomes were maternal, perinatal and neonatal mortality. Clusters were health centre catchment areas assigned using stratified computer-generated randomization. Following exclusions, including non-birthing facilities, 61 clusters were analysed: control (17 clusters, 4912 births), FI (15, 5335), CI (15, 5080) and FI + CI (14, 5249). This trial was registered as International Standard Randomised Controlled Trial [ISRCTN18073903]. Outcomes for 14 576 and 20 576 births were recorded during baseline (June 2007–September 2008) and intervention (October 2008–December 2010) periods. RESULTS: For control, FI, CI and FI + CI clusters neonatal mortality rates were 34.0, 28.3, 29.9 and 27.0 neonatal deaths per 1000 live births and perinatal mortality rates were 56.2, 55.1, 48.0 and 48.4 per 1000 births, during the intervention period. Adjusting for clustering and stratification, the neonatal mortality rate was 22% lower in FI + CI than control clusters (OR = 0.78, 95% CI 0.60–1.01), and the perinatal mortality rate was 16% lower in CI clusters (OR = 0.84, 95% CI 0.72–0.97). We did not observe any intervention effects on maternal mortality. CONCLUSIONS: Despite implementation problems, a combined community and facility approach using participatory women's groups and quality improvement at health centres reduced newborn mortality in rural Malawi

Maternal and Newborn-care Practices During Pregnancy, Childbirth, and the Postnatal Period: A Comparison in Three Rural Districts in Bangladesh

The aim of this study was to examine the prevalence of maternal and newborn-care practices among women reporting a birth in the previous year in three districts in different divisions of Bangladesh. In 2003, 6,785 women, who had delivered a newborn infant in the previous year, across three districts in Bangladesh, were interviewed. Overall, less than half of the women received any antenatal care, and 11% received a minimum of four check-ups. Only 18% took iron tablets for at least four months during pregnancy. Over 90% of the 6,785 deliveries took place at home, and only 11% were attended either by a doctor or by a nurse. The mothers reported three key hygienic practices in 54% of deliveries: at\uadtendants washing their hands with soap and boiling cord-tie and blade for cutting the cord. Forty-four percent of the 6,785 infants were bathed immediately after delivery, and 42% were given colostrum as their first food. The results suggest that maternal and newborn-care remains a cause of concern in rural Bangladesh. Short-term policies to promote healthy behaviour in the home are needed, in addition to the long-term goal of skilled birth attendance

Na+,K+-pump stimulation improves contractility in isolated muscles of mice with hyperkalemic periodic paralysis

In patients with hyperkalemic periodic paralysis (HyperKPP), attacks of muscle weakness or paralysis are triggered by K+ ingestion or rest after exercise. Force can be restored by muscle work or treatment with β2-adrenoceptor agonists. A missense substitution corresponding to a mutation in the skeletal muscle voltage-gated Na+ channel (Nav1.4, Met1592Val) causing human HyperKPP was targeted into the mouse SCN4A gene (mutants). In soleus muscles prepared from these mutant mice, twitch, tetanic force, and endurance were markedly reduced compared with soleus from wild type (WT), reflecting impaired excitability. In mutant soleus, contractility was considerably more sensitive than WT soleus to inhibition by elevated [K+]o. In resting mutant soleus, tetrodotoxin (TTX)-suppressible 22Na uptake and [Na+]i were increased by 470 and 58%, respectively, and membrane potential was depolarized (by 16 mV, P < 0.0001) and repolarized by TTX. Na+,K+ pump–mediated 86Rb uptake was 83% larger than in WT. Salbutamol stimulated 86Rb uptake and reduced [Na+]i both in mutant and WT soleus. Stimulating Na+,K+ pumps with salbutamol restored force in mutant soleus and extensor digitorum longus (EDL). Increasing [Na+]i with monensin also restored force in soleus. In soleus, EDL, and tibialis anterior muscles of mutant mice, the content of Na+,K+ pumps was 28, 62, and 33% higher than in WT, respectively, possibly reflecting the stimulating effect of elevated [Na+]i on the synthesis of Na+,K+ pumps. The results confirm that the functional disorders of skeletal muscles in HyperKPP are secondary to increased Na+ influx and show that contractility can be restored by acute stimulation of the Na+,K+ pumps. Calcitonin gene-related peptide (CGRP) restored force in mutant soleus but caused no detectable increase in 86Rb uptake. Repeated excitation and capsaicin also restored contractility, possibly because of the release of endogenous CGRP from nerve endings in the isolated muscles. These observations may explain how mild exercise helps locally to prevent severe weakness during an attack of HyperKPP

Activation of Thiazide-Sensitive Co-Transport by Angiotensin II in the cyp1a1-Ren2 Hypertensive Rat

Transgenic rats with inducible expression of the mouse Ren2 gene were used to elucidate mechanisms leading to the development of hypertension and renal injury. Ren2 transgene activation was induced by administration of a naturally occurring aryl hydrocarbon, indole-3-carbinol (100 mg/kg/day by gastric gavage). Blood pressure and renal parameters were recorded in both conscious and anesthetized (butabarbital sodium; 120 mg/kg IP) rats at selected time-points during the development of hypertension. Hypertension was evident by the second day of treatment, being preceded by reduced renal sodium excretion due to activation of the thiazide-sensitive sodium-chloride co-transporter. Renal injury was evident after the first day of transgene induction, being initially limited to the pre-glomerular vasculature. Mircoalbuminuria and tubuloinsterstitial injury developed once hypertension was established. Chronic treatment with either hydrochlorothiazide or an AT1 receptor antagonist normalized sodium reabsorption, significantly blunted hypertension and prevented renal injury. Urinary aldosterone excretion was increased ∼20 fold, but chronic mineralocorticoid receptor antagonism with spironolactone neither restored natriuretic capacity nor prevented hypertension. Spironolactone nevertheless ameliorated vascular damage and prevented albuminuria. This study finds activation of sodium-chloride co-transport to be a key mechanism in angiotensin II-dependent hypertension. Furthermore, renal vascular injury in this setting reflects both barotrauma and pressure-independent pathways associated with direct detrimental effects of angiotensin II and aldosterone

Regulation of Alr1 Mg Transporter Activity by Intracellular Magnesium

Mg homeostasis is critical to eukaryotic cells, but the contribution of Mg transporter activity to homeostasis is not fully understood. In yeast, Mg uptake is primarily mediated by the Alr1 transporter, which also allows low affinity uptake of other divalent cations such as Ni2+, Mn2+, Zn2+ and Co2+. Using Ni2+ uptake to assay Alr1 activity, we observed approximately nine-fold more activity under Mg-deficient conditions. The mnr2 mutation, which is thought to block release of vacuolar Mg stores, was associated with increased Alr1 activity, suggesting Alr1 was regulated by intracellular Mg supply. Consistent with a previous report of the regulation of Alr1 expression by Mg supply, Mg deficiency and the mnr2 mutation both increased the accumulation of a carboxy-terminal epitope-tagged version of the Alr1 protein (Alr1-HA). However, Mg supply had little effect on ALR1 promoter activity or mRNA levels. In addition, while Mg deficiency caused a seven-fold increase in Alr1-HA accumulation, the N-terminally tagged and untagged Alr1 proteins increased less than two-fold. These observations argue that the Mg-dependent accumulation of the C-terminal epitope-tagged protein was primarily an artifact of its modification. Plasma membrane localization of YFP-tagged Alr1 was also unaffected by Mg supply, indicating that a change in Alr1 location did not explain the increased activity we observed. We conclude that variation in Alr1 protein accumulation or location does not make a substantial contribution to its regulation by Mg supply, suggesting Alr1 activity is directly regulated via as yet unknown mechanisms

Exploiting bacterial DNA gyrase as a drug target: current state and perspectives

DNA gyrase is a type II topoisomerase that can introduce negative supercoils into DNA at the expense of ATP hydrolysis. It is essential in all bacteria but absent from higher eukaryotes, making it an attractive target for antibacterials. The fluoroquinolones are examples of very successful gyrase-targeted drugs, but the rise in bacterial resistance to these agents means that we not only need to seek new compounds, but also new modes of inhibition of this enzyme. We review known gyrase-specific drugs and toxins and assess the prospects for developing new antibacterials targeted to this enzyme