Diabetic mitochondria are resistant to palmitoyl CoA inhibition of respiration, which is detrimental during ischemia.

The bioactive lipid intermediate palmitoyl CoA (PCoA) can inhibit mitochondrial ADP/ATP transport, though the physiological relevance of this regulation remains unclear. We questioned whether myocardial ischemia provides a pathological setting in which PCoA regulation of ADP/ATP transport would be beneficial, and secondly, whether the chronically elevated lipid content within the diabetic heart could make mitochondria less sensitive to the effects of PCoA. PCoA acutely decreased ADP-stimulated state 3 respiration and increased the apparent Km for ADP twofold. The half maximal inhibitory concentration (IC50 ) of PCoA in control mitochondria was 22 µM. This inhibitory effect of PCoA on respiration was blunted in diabetic mitochondria, with no significant difference in the Km for ADP in the presence of PCoA, and an increase in the IC50 to 32 µM PCoA. The competitive inhibition by PCoA was localised to the phosphorylation apparatus, particularly the ADP/ATP carrier (AAC). During ischemia, the AAC imports ATP into the mitochondria, where it is hydrolysed by reversal of the ATP synthase, regenerating the membrane potential. Addition of PCoA dose-dependently prevented this wasteful ATP hydrolysis for membrane repolarisation during ischemia, however, this beneficial effect was blunted in diabetic mitochondria. Finally, using 31 P-magnetic resonance spectroscopy we demonstrated that diabetic hearts lose ATP more rapidly during ischemia, with a threefold higher ATP decay rate compared with control hearts. In conclusion, PCoA plays a role in protecting mitochondrial energetics during ischemia, by preventing wasteful ATP hydrolysis. However, this beneficial effect is blunted in diabetes, contributing to the impaired energy metabolism seen during myocardial ischemia in the diabetic heart.

Mortality and cancer incidence following occupational radiation exposure: third analysis of the National Registry for Radiation Workers.

Mortality and cancer incidence were studied in the National Registry for Radiation Workers in, relative to earlier analyses, an enlarged cohort of 174 541 persons, with longer follow-up (to 2001) and, for the first time, cancer registration data. SMRs for all causes and all malignant neoplasms were 81 and 84 respectively, demonstrating a 'healthy worker effect'. Within the cohort, mortality and incidence from both leukaemia excluding CLL and the grouping of all malignant neoplasms excluding leukaemia increased to a statistically significant extent with increasing radiation dose. Estimates of the trend in risk with dose were similar to those for the Japanese A-bomb survivors, with 90% confidence intervals that excluded both risks more than 2-3 times greater than the A-bomb values and no raised risk. Some evidence of an increasing trend with dose in mortality from all circulatory diseases may, at least partly, be due to confounding by smoking. This analysis provides the most precise estimates to date of mortality and cancer risks following occupational radiation exposure and strengthens the evidence for raised risks from these exposures. The cancer risk estimates are consistent with values used to set radiation protection standards.

The functional purification of P-glycoprotein is dependent on maintenance of a lipid-protein interface.

P-Glycoprotein (P-gp) is a 180-kDa membrane-bound transporter which can confer the multi-drug resistance phenotype on tumor cells. We have examined the factors required to preserve activity of P-gp during its purification. The starting material for purification was plasma membranes from Chinese hamster ovary (CHrB30) cells, overexpressing P-glycoprotein. These membranes displayed drug stimulated ATPase activity (Vm = 897 +/- 55 nmol min(-1) mg(-1); Km = 1.8 +/- 0.4 mM) and high affinity binding of [3H]vinblastine (Kd = 36 +/- 5 nM; Bm = 161 +/- 11 pmol/mg). Several non-ionic detergents which readily solubilized P-glycoprotein significantly inhibited ATPase activity and drug binding at concentrations well below their respective CMC values. This inactivation was prevented by excess crude lipid mixtures, with the greatest protection afforded against dodecyl-maltoside. Furthermore, the significantly reduced binding affinity and capacity of solubilized P-gp was partly reversed by the addition of lipids. A combination of anion-exchange and hydroxyapatite chromatography were used to purify P-gp with high yield to greater than 90%. The purified, reconstituted P-gp displayed high ATPase activity (Vm = 2137 +/- 309; Km = 2.9 +/- 0.9 mM) which was stimulated by verapamil (EC50 = 3.8 +/- 0.6 microM) and inhibited by orthovanadate (3.1 +/- 0.8 microM). Pure P-gp also displayed high affinity vinblastine binding (Kd = 64 +/- 9 nM) with a capacity of 2320 +/- 192 pmol/mg. This purification scheme yields the highest P-gp activity reported to date, and indicates a dependence of function on maintaining a lipid-protein interface.

The chromatin-associated protein H-NS.

H-NS is a major component of chromatin in enteric bacteria. H-NS plays a structural role in organising the chromosome, and influences DNA rearrangements as well as the expression of many genes. The biochemical and functional characteristics of H-NS are distinct from those of 'typical' DNA-binding proteins and much remains to be learned about the mechanism(s) by which H-NS acts. In this article we review our current understanding of the role of H-NS, and describe possible models by which H-NS might influence DNA structure and gene expression.

The multi-drug resistance reversal agent SR33557 and modulation of vinca alkaloid binding to P-glycoprotein by an allosteric interaction.

1. The interaction of the indolizin sulfone SR33557 with the multidrug resistance P-glycoprotein (P-gp), was used to explore the nature of drug binding site(s) on this transporter. The steady-state accumulation of [3H]-vinblastine in P-gp expressing CHrB30 cells was increased by SR33557 with greater potency than verapamil. Furthermore, SR33557 potentiated the affinity of verapamil to modulate vinblastine transport when added simultaneously. 2. Verapamil elicited a 1.5 to 2.5 fold stimulation of basal ATPase activity in CHrB30 membranes, whereas SR33557 and vinblastine inhibited activity, but only at relatively high concentrations. However, SR33557 and vinblastine decreased the Vmax but not the Km for verapamil stimulation of ATPase activity. This is indicative of a non-competitive interaction, most likely at distinct sites. 3. The specific [3H]-vinblastine binding to P-gp in CHrB30 cell membranes was displaced by SR33557 with an IC50 of 8.3 +/- 4.5 nM. Moreover, SR33557 caused a 3 fold increase in the dissociation rate of vinblastine binding to P-gp indicating a negative allosteric effect on the vinca alkaloid acceptor site. 4. These results demonstrate that SR33557 interacts with a site on P-gp which is distinct from, but allosterically linked to the vinca alkaloid site. The apparent broad substrate specificity displayed by P-gp may be explained by a multiple drug binding site model.

The molecular interaction of the high affinity reversal agent XR9576 with P-glycoprotein.

1 The kinetics and nature of equilibrium binding were used to characterize the molecular interaction of the anthranilic acid derivative [3H]-XR9576 with the multidrug resistance P-glycoprotein (P-gp). XR9576 displayed specific high-affinity binding to P-gp (Bmax = 275 pmol mg-1, Kd = 5.1 nM). The transport substrates [3H]-vinblastine and [3H]-paclitaxel displayed 4 fold and 20 fold lower affinity respectively for P-gp. The duration of action of XR9576 with P-gp was increased in comparison to that of vinblastine which displayed a slower rate of association and a faster dissociation rate. 2 The relative affinities of several modulators and transport substrates to interact with P-gp were determined from displacement drug equilibrium binding assays. Vinblastine and paclitaxel could only fractionally displace [3H]-XR9576 binding, displaying Ki values significantly different from their measured Kd values. This suggests a non-competitive interaction between XR9576 and the P-gp substrates vinblastine and paclitaxel. 3 XR9576 was shown to be a potent modulator of P-gp mediated [3H]-vinblastine and [3H]-paclitaxel transport as it increased the steady-state accumulation of these cytotoxics in CHrB30 cells to levels observed in non-P-gp-expressing AuxB1 cells (EC50 = 487+/-50 nM). This inhibition of drug transport is not mediated through competition for transport since [3H]-XR9576 accumulation was not influenced by P-gp expression or function. 4 These results demonstrate that the P-gp modulator XR9576 exhibits greater selectivity, duration of inhibition and potency of interaction with this transporter than any other reported modulators. Several lines of evidence suggest that XR9576 inhibits P-gp function by binding at a site which is distinct from the site of interaction of transport substrates. The two sites may be classified as serving modulatory or transport functions.

Drug binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide hydrolysis.

P-glycoprotein (P-gp) confers multiple drug resistance on cancer cells by acting as a plasma membrane localized ATP-dependent drug efflux pump. Currently, there is little information on the nature of the communication between the energy-providing nucleotide binding domains (NBDs) and the drug binding sites of P-gp to generate transport of substrate. Many substrates and modulators cause alterations in ATP hydrolysis, but what effect do the various stages of the catalytic cycle have on drug interaction with P-gp? Vanadate trapping of Mg.ADP caused a reversible decrease in the binding capacity of the transported substrate [(3)H]-vinblastine and the nontransported modulator [(3)H]XR9576 to P-gp in CH(r)B30 cell membranes. The non-hydrolyzable nucleotide analogue ATP-gamma-S also caused a reduction in the binding capacity of [(3)H]-vinblastine but not for the modulator [(3)H]XR9576. This indicates that signaling to the NBDs following binding of a nontransported modulator is different to that transmitted upon interaction of a transported substrate. Second, it appears that the binding of nucleotide, rather than its hydrolysis, causes the initial conformational shift in the drug-binding site during a transport cycle.

Communication between multiple drug binding sites on P-glycoprotein.

P-glycoprotein, a member of the ATP-binding cassette transporter family, is able to confer resistance on tumors against a large number of functionally and chemically distinct cytotoxic compounds. Several recent investigations suggest that P-glycoprotein contains multiple drug binding sites rather than a single site of broad substrate specificity. In the present study, radioligand-binding techniques were used to directly characterize drug interaction sites on P-glycoprotein and how these multiple sites interact. The drugs used were classified as either 1) substrates, which are known to be transported by P-glycoprotein (e.g., vinblastine) or 2) modulators, which alter P-glycoprotein function but are not themselves transported by the protein (e.g., XR9576). Drug interactions with P-glycoprotein were either competitive, at a common site, or noncompetitive, and therefore at distinct sites. Based on these data, we can assign a minimum of four drug binding sites on P-glycoprotein. These sites fall into two categories: transport, at which translocation of drug across the membrane can occur, and regulatory sites, which modify P-glycoprotein function. Intriguingly, however, some modulators interact with P-glycoprotein at a transport site rather than a regulatory site. The pharmacological data also demonstrate that both transport and regulatory sites are able to switch between high- and low-affinity conformations. The multiple sites on P-glycoprotein display complex allosteric interactions through which interaction of drug at one site switches other sites between high- or low-affinity conformations. The data are discussed in terms of a model for the mechanism of transport by P-glycoprotein.

A randomised controlled trial of care of the perineum during second stage of normal labour.

OBJECTIVE: To compare the effect of two methods of perineal management used during spontaneous vaginal delivery on the prevalence of perineal pain reported at 10 days after birth. DESIGN: Randomised controlled trial. SETTING: Two English maternity care units. SAMPLE: 5471 women who gave birth between December 1994 and December 1996. METHODS: At the end of the second stage of labour women were allocated to either the 'hands on' method, in which the midwife's hands put pressure on the baby's head and support ('guard') the perineum; lateral flexion is then used to facilitate delivery of the shoulders, or the 'hands poised' method, in which the midwife keeps her hands poised, not touching the head or perineum, allowing spontaneous delivery of the shoulders. MAIN OUTCOME MEASURE: Perineal pain in the previous 24 hours reported by women in self-administered questionnaire 10 days after birth. RESULTS: Questionnaires were completed by 97% of women at 10 days after birth. 910 (34.1%) women in the 'hands poised' group reported pain in the previous 24 hours compared with 823 (31.1%) in the 'hands on' group (RR 1.10, 95% CI 1.01 to 1.18: absolute difference 3%, 0.5% to 5%, P = 0.02). The rate of episiotomy was significantly lower in the 'hands poised' group (RR 0.79, 99% CI 0.65 to 0.96, P = 0.008) but the rate of manual removal of placenta was significantly higher (RR 1.69, 99% CI 1.02 to 2.78; P = 0.008). There were no other statistically significant differences detected between the two methods. CONCLUSION: The reduction in pain observed in the 'hands on' group was statistically significant and the difference detected potentially affects a substantial number of women. These results provide evidence to enable individual women and health professionals to decide which perineal management is preferable.

The importance of cholesterol in maintenance of P-glycoprotein activity and its membrane perturbing influence.

In tumour cell lines that display multidrug resistance, expression of P-glycoprotein (P-gp) alters many aspects of biomembrane organization in addition to its well-characterized drug transport activity. We have developed a reconstitution system to directly investigate the effect of purified P-gp on the biophysical properties of lipid bilayers. Using a mixed detergent system it was possible to efficiently reconstitute P-gp at lipid:protein ratios as low as 2.5 (w/w) by removal of detergent using adsorption to SM-2 BioBeads. P-gp was able to alter many biophysical parameters associated with lipid organization within bilayers. For example, the changes in overall fluidity and excimer formation by lipid analogues indicate modified packing organization of bilayer constituents. Surprisingly, given its role in conferring drug resistance, P-gp insertion into bilayers also caused significantly increased permeability to aqueous compounds, also reflecting a modified phospholipid environment. Translocation of various phospholipid species between leaflets of the bilayer was increased in the presence of P-gp; however, the effect was not dependent on ATP hydrolysis by the protein. Physiological concentrations of cholesterol modified P-gp function and the degree to which it perturbed bilayer organization. The basal ATPase activity of P-gp was increased in a dose-dependent fashion by the incorporation of cholesterol in PC:PE liposomes. In addition, the degree to which the modulator verapamil was able to stimulate this basal ATPase activity was reduced by the presence of cholesterol in proteoliposomes. However, the potency of verapamil was unaltered, suggesting a specific effect, not simply caused by lower drug penetration into the cholesterol containing bilayers. In summary, P-gp is able to cause perturbation in the organization of bilayer constituents. Cholesterol imparted "stability" to this perturbation of bilayer organization by P-gp and moreover this led to altered protein function.

Epidemiological studies of UK test veterans: II. Mortality and cancer incidence.

An epidemiological study was set up in the 1980s of UK participants in the UK atmospheric nuclear weapons testing programme. A large cohort of test participants was established along with a closely matched comparison or control group. Three analyses of mortality and cancer incidence have been carried out. This review describes the development of the evidence on possible effects on test participants with especial emphasis on the most recent analysis. Other sources of evidence, particularly from studies of other groups of test participants, are also considered. It was concluded that overall levels of mortality and cancer incidence in UK nuclear weapons test participants were similar to those in a matched control group, and overall mortality was lower than expected from national rates. There was no evidence of an increased raised risk of multiple myeloma among test participants in recent years, and the suggestion in the first analysis of this cohort of a raised myeloma risk relative to controls is likely to have been a chance finding. There was some evidence of a raised risk of leukaemia other than chronic lymphatic leukaemia among test participants relative to controls, particularly in the early years after the tests. Whilst this could be a chance finding, the possibility that test participation caused a small absolute risk of leukaemia other than chronic lymphatic leukaemia cannot be ruled out.

Follow up of mortality and incidence of cancer 1952-98 in men from the UK who participated in the UK's atmospheric nuclear weapon tests and experimental programmes.

AIMS: To extend and analyse follow up of mortality and cancer incidence among men who took part in the UK's atmospheric nuclear weapon tests and experimental programmes 40-50 years ago, with particular reference to multiple myeloma and leukaemia. METHODS: A total of 21,357 servicemen and male civilians from the UK who participated in the tests and a control group of 22,333 male controls were followed over the period 1952-98. Analyses were conducted of mortality from various causes, and of mortality and incidence for 27 types of cancer. RESULTS: Rates of mortality from all causes continued to be similar among test participants and controls with the longer follow up, with standardised mortality ratios (SMRs) of 89 and 88 respectively over the full follow up period. For all cancers, the corresponding SMRs were 93 for participants and 92 for controls. Mortality from multiple myeloma was consistent with national rates both for participants and controls, and the relative risk (RR) of myeloma incidence among participants relative to controls was 1.14 (90% CI 0.74 to 1.74) over the full follow up period and 0.79 (90% CI 0.45 to 1.38) during the extended period of follow up (1991-98). Over the full follow up period, leukaemia mortality among participants was consistent with national rates, while rates among controls were significantly lower, and there was a suggestion of a raised risk among test participants relative to controls (RR 1.45, 90% CI 0.96 to 2.17); the corresponding RR for leukaemia incidence was 1.33 (90% CI 0.97 to 1.84). After excluding chronic lymphatic leukaemia (CLL), which is not thought to be radiation inducible, the RR of leukaemia mortality increased to 1.83 (90% CI 1.15 to 2.93), while that for incidence was little changed. Analysis of subgroups of participants with greater potential for exposure provided little evidence of increased risks, although the numbers of men involved were smaller and the statistical power was therefore less. Among other types of cancer, only for liver cancer incidence was there evidence of differences in rates between participants and controls in both the earlier and in the additional period of follow up. Mortality rates among test participants from causes other than cancer were generally similar to those among the controls. CONCLUSIONS: Overall levels of mortality and cancer incidence in UK nuclear weapons test participants have continued to be similar to those in a matched control group, and overall mortality has remained lower than expected from national rates. There was no evidence of an increased raised risk of multiple myeloma among test participants in recent years, and the suggestion in the first analysis of this study of a raised myeloma risk is likely to have been a chance finding. There was some evidence of a raised risk of leukaemia other than CLL among test participants relative to controls, particularly in the early years after the tests, although a small risk may have persisted more recently. This could be a chance finding, in view of low rates among the controls and the generally small radiation doses recorded for test participants. However, the possibility that test participation caused a small absolute risk of leukaemia other than CLL cannot be ruled out.

Occupational radiation exposure and mortality: second analysis of the National Registry for Radiation Workers.

The National Registry for Radiation Workers (NRRW) is the largest epidemiological study of UK radiation workers. Following the first analysis published in 1992, a second analysis has been conducted using an enlarged cohort of 124,743 workers, updated dosimetry and personal data for some workers, and a longer follow-up. Overall levels of mortality were found to be less than those expected from national rates; the standardised mortality ratio for all causes was 82, increasing to 89 after adjusting for social class. This 'healthy worker effect' was particularly strong for lung cancer and for some smoking-related non-malignant diseases. Analysis of potential radiation effects involved testing for any trend in mortality risk with external dose, after adjusting for likely confounding factors. For leukaemia, excluding chronic lymphatic leukaemia (CLL), the central estimate of excess relative risk (ERR) per Sv was similar to that estimated for the Japanese atomic bomb survivors at low doses (without the incorporation of a dose-rate correction factor); the corresponding 90% confidence limits for this trend were tighter than in the first analysis, ranging from just under four times the risk estimated at low doses from the Japanese atomic bomb survivors to about zero. For the grouping of all malignancies other than leukaemia, the central estimate of the trend in risk with dose was closer to zero than in the first analysis; also, the 90% confidence limits were tighter than before and included zero. Since results for lung cancer and non-malignant smoking-related diseases suggested the possibility of confounding by smoking, an examination was made, as in the first analysis, of all malignancies other than leukaemia and lung cancer. In this instance the central estimate of the ERR per Sv was similar to that from the A-bomb data (without the incorporation of a dose-rate correction factor), with a 90% confidence interval ranging from about four times the A-bomb value to less than zero. For multiple myeloma there was an indication of an increasing trend in risk with external dose (p = 0.06), although the evidence for this trend disappeared after omitting workers monitored for exposure to internal emitters. The second NRRW analysis provides stronger inferences than the first on occupational radiation exposure and cancer mortality; the 90% confidence intervals for the risk per unit dose are tighter than before, and now exclude values which are greater than four times those seen among the Japanese A-bomb survivors, although they are also generally consistent with an observation of no raised risk. Furthermore, there is evidence, of borderline statistical significance, of an increasing risk for leukaemia excluding CLL, and, as with solid cancers, the data are consistent with the A-bomb findings.

Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle.

P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.