Dietary restriction increases variability in longevity.

Nutritional environments, particularly those experienced during early life, are hypothesized to affect longevity. A recent cross-taxa meta-analysis found that, depending upon circumstance, average longevity may be increased or decreased by early-life dietary restriction. Unstudied are the effects of diet during development on among-individual variance in longevity. Here, we address this issue using emerging methods for meta-analysis of variance. We found that, in general, standard deviation (s.d.) in longevity is around 8% higher under early-life dietary restriction than a standard diet. The effects became especially profound when dietary insults were experienced prenatally (s.d. increased by 29%) and/or extended into adulthood (s.d. increased by 36.6%). Early-life dietary restriction may generate variance in longevity as a result of increased variance in resource acquisition or allocation, but the mechanisms underlying these largely overlooked patterns clearly warrant elucidation.

Habitat-related specialization of lateral-line system morphology in a habitat-generalist and a habitat-specialist New Zealand eleotrid.

An investigation of intraspecific habitat-related patterns of variation in oculoscapular lateral-line superficial neuromasts (SN) identified a decrease in the ratio of total SNs to pores, and a trend towards decreased asymmetry in SNs in the habitat-generalist common bully Gobiomorphus cotidianus from fluvial habitats compared to lacustrine habitats, suggesting habitat-related phenotypic variability. A greater ratio of pores to SNs, as well as less variation in the total number and asymmetry of SNs observed in the fluvial habitat-specialist redfin bully Gobiomorphus huttoni may provide further evidence of variations in the oculoscapular lateral-line morphology of fluvial habitat G. cotidianus individuals serving as adaptations to more turbulent environments.

A quantitative review of MHC-based mating preference: the role of diversity and dissimilarity.

Sexual selection hypotheses stipulate that the major histocompatibility complex genes (MHC) constitute a key molecular underpinning for mate choice in vertebrates. The last four decades saw growing empirical literature on the role of MHC diversity and dissimilarity in mate choice for a wide range of vertebrate animals, but with mixed support for its significance in natural populations. Using formal phylogenetic meta-analysis and meta-regression techniques, we quantitatively review the existing literature on MHC-dependent mating preferences in nonhuman vertebrates with a focus on the role of MHC diversity and dissimilarity. Overall, we found small, statistically nonsignificant, average effect sizes for both diversity- and dissimilarity-based mate choice (r = 0.113 and 0.064, respectively). Importantly, however, meta-regression models revealed statistically significant support regarding female choice for diversity, and choice for dissimilarity (regardless of choosy sex) only when dissimilarity is characterized across multiple loci. Little difference was found among vertebrate taxa; however, the lack of statistical power meant statistically significant effects were limited to some taxa. We found little sign of publication bias; thus, our results are likely to be robust. In light of our quantitative assessment, methodological improvements and fruitful future avenues of research are highlighted.

Role of the Internet as an information resource for surgical patients: a survey of 877 patients.

BACKGROUND: This study was conducted to provide preliminary data regarding current Internet use practices for information about anaesthesia in patients undergoing elective surgical procedures at a major academic institution. METHODS: With IRB approval, 2936 patients coming for preanaesthetic evaluation at a tertiary academic hospital's preadmission testing (PAT) centre were invited to voluntarily participate in a 20-item questionnaire designed to obtain participants' characteristics and Internet use for information pertaining to their upcoming surgery. Data were analysed using statistical software SAS (Cary, NC, USA). Descriptive statistics were calculated for continuous variables using mean (sd), and for categorical data using n (%). Association analysis was performed using the Fisher's exact test. RESULTS: Eight hundred and seventy-seven patients (30%) responded. Of these, 356 (41%) looked for information about their medical condition, 321 (37%) for their surgery, 279 (32%) for surgeon, 163 (19%) for the hospital, and only 36 (4%) for information regarding anaesthesia. Of these 36 patients, 14 (39%) said the sites they used helped answer their questions regarding anaesthesia. Of the 831 patients who did not use the Internet for anaesthesia, 503 (57%) indicated that they would be receptive to being directed to specific websites for anaesthesia. CONCLUSIONS: Of the patients coming for elective surgery who responded (30%), the majority did not use the Internet to seek information regarding anaesthesia. Respondents indicated a high degree of interest in being directed to appropriate websites for further information. These results suggest that it may be beneficial to include information regarding reliable web-based resources to interested patients at preoperative visits.

Male reproductive traits are differentially affected by dietary macronutrient balance but unrelated to adiposity.

Dietary factors influence male reproductive function in both experimental and epidemiological studies. However, there are currently no specific dietary guidelines for male preconception health. Here, we use the Nutritional Geometry framework to examine the effects of dietary macronutrient balance on reproductive traits in C57BL/6 J male mice. Dietary effects are observed in a range of morphological, testicular and spermatozoa traits, although the relative influence of protein, fat, carbohydrate, and their interactions differ depending on the trait being examined. Interestingly, dietary fat has a positive influence on sperm motility and antioxidant capacity, differing to typical high fat diet studies where calorie content is not controlled for. Moreover, body adiposity is not significantly correlated with any of the reproductive traits measured in this study. These results demonstrate the importance of macronutrient balance and calorie intake on reproductive function and support the need to develop specific, targeted, preconception dietary guidelines for males.

Genetic variation of macronutrient tolerance in Drosophila melanogaster.

Carbohydrates, proteins and lipids are essential nutrients to all animals; however, closely related species, populations, and individuals can display dramatic variation in diet. Here we explore the variation in macronutrient tolerance in Drosophila melanogaster using the Drosophila genetic reference panel, a collection of ~200 strains derived from a single natural population. Our study demonstrates that D. melanogaster, often considered a "dietary generalist", displays marked genetic variation in survival on different diets, notably on high-sugar diet. Our genetic analysis and functional validation identify several regulators of macronutrient tolerance, including CG10960/GLUT8, Pkn and Eip75B. We also demonstrate a role for the JNK pathway in sugar tolerance and de novo lipogenesis. Finally, we report a role for tailless, a conserved orphan nuclear hormone receptor, in regulating sugar metabolism via insulin-like peptide secretion and sugar-responsive CCHamide-2 expression. Our study provides support for the use of nutrigenomics in the development of personalized nutrition.

Controlled beat-wave Brillouin scattering in the ionosphere.

Stimulated Brillouin scattering experiments in the ionospheric plasma using a single electromagnetic pump wave have previously been observed to generate an electromagnetic sideband wave, emitted by the plasma, together with an ion- acoustic wave. Here we report results of a controlled, pump and probe beat-wave driven Brillouin scattering experiment, in which an ion-acoustic wave generated by the beating of electromagnetic pump and probe waves, results in electromagnetic sideband waves that are recorded on the ground. The experiment used the EISCAT facility in northern Norway, which has several high power electromagnetic wave transmitters and receivers in the radio frequency range. An electromagnetic pump consisting of large amplitude radio waves with ordinary (O) or extraordinary (X) mode polarization was injected into the overhead ionosphere, along with a less powerful probe wave, and radio sideband emissions observed on the ground clearly show stimulated Brillouin emissions at frequencies agreeing with, and changing with, the pump and probe frequencies. The experiment was simulated using a numerical full-scale model which clearly supports the interpretation of the experimental results. Such controlled beat-wave experiments demonstrate a way of remotely investigating the ionospheric plasma parameters.

Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina.

We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that the RL13 epitope occurs on the inner membrane's nucleoplasmic surface where the nuclear lamina is found. When nuclear envelopes are extracted with solutions containing nonionic detergent and high salt to solubilize nuclear membranes and pore complexes, most of these integral proteins remain associated with the insoluble lamina. Since the polypeptides recognized by RL13 are relatively abundant, they may function as lamina attachment sites in the inner nuclear membrane. Major cross-reacting antigens are found by immunoblotting and immunofluorescence microscopy in all rat cells examined. Therefore, these integral proteins are biochemical markers for the inner nuclear membrane and will be useful models for studying nuclear membrane biogenesis.

Monoclonal antibodies identify a group of nuclear pore complex glycoproteins.

Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an integral membrane protein. The eight antigens cofractionate with the nuclear pore complex under various conditions of ionic strength and detergent. It can be seen by immunofluorescence microscopy that the monoclonal antibodies reacting with these antigens stain the nuclear surface of interphase cells in a finely punctate pattern. When the nuclear envelope is disassembled and subsequently reformed during mitosis, the proteins are reversibly dispersed throughout the cytoplasm in the form of minute foci. By EM immunogold localization on isolated nuclear envelopes, the monoclonal antibodies label exclusively the nuclear pore complex, at both its nucleoplasmic and cytoplasmic margins. Considered together, our biochemical and localization data indicate that the eight nuclear envelope polypeptides are pore complex components. As shown in the accompanying paper (Holt, G. D., C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol., 104:1157-1164) these eight polypeptides contain a novel form of glycosylation, O-linked N-acetylglucosamine. The relative abundance and disposition of these O-linked glycoproteins in the pore complex are consistent with their having a role in nucleocytoplasmic transport.

Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine.

A novel form of protein-saccharide linkage consisting of single N-acetylglucosamine (GlcNAc) residues attached in O-linkages directly to the polypeptide backbone has been described (Holt, G. D., and G. W. Hart, 1986, J. Biol. Chem., 261:8049-8057). This modification was found on proteins distributed throughout the cell, although proteins bearing O-linked GlcNAc moieties were particularly abundant in the cytosolic and nuclear envelope fractions of rat liver. In the accompanying article (Snow, C. M., A. Senior, and L. Gerace, 1987, J. Cell. Biol., 104: 1143-1156), the authors describe monoclonal antibodies directed against eight proteins localized to the nuclear pore complex. These proteins occur on the cytoplasmic and nucleoplasmic (but not lumenal) sides of nuclear membranes. In this report, we demonstrate that all members of this group of pore complex proteins bear multiple O-linked GlcNAc residues. Further, we show that the O-linked GlcNAc moieties are linked via serine (and possibly threonine) side chains to these proteins. Perturbing the O-linked GlcNAc residues either by covalently attaching galactose to them or by releasing them with beta-N-acetylglucosaminidase strongly diminishes the immunoreactivity of the proteins with all of the monoclonal antibodies. However, the O-linked GlcNAc moieties are only part of the epitopes recognized, since O-GlcNAc-containing limit pronase fragments of nuclear pore complex proteins cannot be immunoprecipitated by these antibodies. These findings, taken together with those in the accompanying article, are a direct demonstration that proteins of the cytoplasm and nucleoplasm bear O-linked GlcNAc residues.

Prolactin binding to R3230AC mammary carcinoma and liver in hormone-treated and diabetic rats.

Specific 125I-labeled prolactin binding was measured in membrane particles prepared from R3230AC mammary carcinoma and liver of tumor-bearing Fischer rats after either prolactin, estrogen, or lergotrile mesylate treatment, or after the induction of diabetes by streptozotocin. Hormone binding to tumors was decreased by treatment with prolactin (0.5 or 1 mg/day) or estradiol valerate (7.5 mg/kg/week). In contrast, prolactin treatment did not affect prolactin binding to liver membrane particles, but estradiol valerate treatment resulted in a four-fold increase in prolactin binding to this tissue. Lergotrile mesylate, which lowers plasma prolactin levels, did not affect tumor growth or prolactin binding to either tumor or liver. Prolactin binding to both tumor and liver was significantly reduced in diabetic rats, suggesting that insulin may play an important role in controlling tissue sensitivity to prolactin. Specific binding of 125I-labeled prolactin to enzymatically dissociated cells from R3230AC tumors was demonstrated in vitro. The binding capacity of the cells was found to be of the same order of magnitude as the binding capacity in membrane preparations when appropriate corrections were applied for yields of cells and membranes. R3230AC tumor, which is responsive to prolactin appears therefore to be a useful model system for further study aimed at elucidation of growth and metabolic response to the hormone prolactin in breast cancer.

PIP: Specific iodine-125-labeled prolactin binding was measured in membrane particles prepared from R3230AC mammary carcinoma and liver of tumor-bearing Fischer rats after either prolactin, estrogen, or lergotrile mesylate treatment, or after the induction of diabetes by streptozotocin. Hormone binding to tumors was decreased by treatment with prolactin (.5 or 1 mg/day) or estradiol valerate (7.5 mg/kg/week). In contrast, prolactin treatment was without affect on prolactin binding to liver membrane particles, but estradiol valerate treatment resulted in a 4-fold increase in prolactin binding to this tissue. Lergotrile mesylate, which lowers plasma prolactin levels, had no affect on tumor growth or prolactin binding to either tumor or liver. Prolactin binding to both tumor and liver was significantly reduced in diabetic rats, suggesting that insulin may play an important role in controlling tissue sensitivity to prolactin. Specific binding of iodine-labeled prolactin to enzymatically dissociated cells from R3230AC tumors was demonstrated in vitro. The binding capacity of the cells was found to be of the same order of magnitude as the binding capacity in membrane preparations when appropriate corrections were applied for yields of cells and membranes. R3230AC tumor, which is responsive to prolactin, appears therefore to be a useful model system for further study aimed at elucidation of growth and metabolic response to the hormone prolactin in breast cancer.

A comparative study of inner membrane enzymes and transport systems in mitochondria from R3230AC mammary tumor and normal rat mammary gland.

Mitochondria from a rat mammary tumor (R3230AC) have been compared with mitochondria from pregnant and lactating rat mammary glands, with particular attention paid to inner membrane enzymes and Transport proteins. In the tumor the mitochondrial adenosine triphosphatase was not activated by 2,4-dinitrophenol, in contrast to the mammary mitochondria from lactating or pregnant rats. Translocation of adenosine diphosphate across the inner membrane was found to be more rapid in the tumor by virtue of lovered Km adenosine diphosphate and raised Vmax. Transport of phosphate and dicarboxylic acids occurred at similar rates in all three types of mitochondria. The inner membrane proteins were also examined directly by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and some differences are noted. These results, although they indicate subtle differences between the inner mitochondrial membranes of tumor as compared with those of pregnant or lactating rat mammary glands, cannot form the basis of an explanation for enhanced glucose utilization and aerobic lactic acid production in this tumor.

Prolactin binding to 7,12-dimethylbenz(a)anthracene-induced mammary tumors and liver in diabetic rats.

Growth rates of 7,12-dimethylbenz(a)anthracene-induced mammary tumors and the specific 125I-labeled prolactin binding to membrane fractions prepared from livers and tumors were studied in rats made diabetic by streptozotocin injection. Growth was inhibited in a majority of tumors and prolactin binding was reduced in both tumors and livers from diabetic animals. Prolactin binding to individual tumors varied over a wide range in both intact and diabetic animals. Scatchard analysis of binding data revealed that the apparent affinity of prolactin binding to liver and tumor membranes was similar (Ka approximately 3.0 X 10(9) M-1) and was not affected by diabetes. We suggest that the reduction in prolactin binding to tumors may render these tissues less responsive to prolactin and thereby explain, at least in part, the observed inhibition of tumor growth in diabetic rats. However, some tumors in diabetic animals regressed despite relatively high levels of prolactin binding activity. Therefore, additional factors most certainly play important roles in the mechanism(s) by which the growth of 7,12-dimethylbenz(a)anthracene-induced tumors is impaired in the diabetic rat.

Prolactin binding to mammary gland, 7,12-dimethylbenz(a)-anthracene-induced mammary tumors, and liver in rats.

Specific binding of radioactively labeled prolactin was determined in membrane preparations from mammary glands and livers of rats during pregnancy and lactation. Prolactin binding to mammary gland increased throughout late pregnancy and early lactation, reached a maximum on Day 11 of lactation, and then declined. Maximum prolactin binding to liver membrane preparations was observed during late pregnancy and declined throughout lactation. Estradiol benzoate (20 mug/day), administered on Days 5 to 10 of lactation, reduced prolactin binding to mammary gland by 55%, increased binding to liver 2-fold, and reduced litter weight gain by 25%. Prolactin binding to 7,12-dimethylbenz(a)anthracene-induced mammary tumors was 3 times higher than that observed in lactating mammary gland. Administration of prolactin enhanced tumor growth but decreased specific prolactin binding to tumors. Lergotrile mesylate inhibited and estradiol benzoate (2 mug/day) enhanced tumor growth, but neither treatment affected prolactin binding to tumor membrane preparations. In contrast, higher doses of estradiol benzoate (20 mug/day) inhibited tumor growth and reduced prolactin binding. Prolactin binding varied widely within all groups of mammary tumors and was not clearly related to growth response or to altered circulating estrogen and/or prolactin levels. Hormone dependence in this animal tumor model is complex and may not be predicted on the basis of prolactin-binding capacity alone.

PIP: Experiments designed to study the changes in prolactin (PRL) binding to rat mammary tissue and liver during pregnancy and lactation, to study the relationship between PRL binding and growth in 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors and to measure PRL binding in DMBA tumors, lactating mammary gland and liver following administration of pharmacological doses of estrogen are described. Specific binding of radioactive PRL binding to mammary gland increased throughout late pregnancy and early lactation, reached a maximum on Day 11 of lactation and then declined. Maximum binding to liver membrane preparations was observed during late pregnancy and declined throughout lactation. The administration of 20 mcg estradiol benzoate (EB/day on Days 5-10 of lactation, reduced PRL binding to mammary gland by 55%, increased binding to liver 2-fold and reduced litter weight gain by 25%. PRL binding to DMBA-induced mammary tumor was 3 times higher than that observed in lactating mammary gland. PRL administration enhanced tumor growth but decresed specific PRL binding to tumors. Lergotrile mesylate inhibited and 2 mcg EB enhanced tumor growth, but neither treatment affected PRL binding to tumor membrane preparations. However, 20 mcg EB inhibited tumor growth and reduced PRL binding. PRL binding varied widely within all groups of mammary tumors and was not clearly related to growth response or to altered circulating estrogen and/or PRL levels. It is concluded that hormone dependence in the rat tumor model is complex and may not be predicted on the basis of PRL-binding capacity alone.

Digital Imaging Capability for Caries Detection: A Meta-analysis.

The objective of this study was to identify the diagnostic capability of photostimulable phosphor plates (PSPs) and direct digital sensors (DDSs) in the detection of interproximal caries. Studies were identified that evaluated the diagnostic capability of PSPs and DDSs in detecting interproximal caries in human teeth, in both dentin and enamel. Histologic sections were the gold standard. This systematic review searched several electronic databases. In addition, Google Scholar and reference lists of the finally included studies were screened. QUADAS-2 was applied to evaluate the risk of bias among included studies. Six studies were finally included; 4 of which were considered homogeneous enough to conduct a meta-analysis. The meta-analysis evaluated 668 interproximal human tooth surfaces. All studies used extracted human teeth ranging from no caries present to caries into dentin. Each tooth was radiographed by both PSP and DDS technologies and then submitted for histologic analysis as the gold standard. Meta-analysis showed that intraoral digital imaging is of high specificity but low sensitivity in the detection of interproximal caries. The sensitivity and specificity for different studies with PSPs varied substantially from 15% to 54% and from 84% to 100%, respectively. Direct sensor analysis sensitivity and specificity ranged from 16% to 56% and from 90% to 100%, respectively. Newer PSP and DDS technologies had statistically significant higher sensitivities, yet the differences in diagnostic capabilities between the older and newer technologies were clinically insignificant. Both digital systems were excellent in identifying surfaces without caries (specificity) but were not sensitive enough to reliably identify interproximal surfaces with caries. Clinicians must therefore remain vigilant in performing a careful clinical examination and other diagnostic tests rather than relying solely on radiographic imaging to diagnose interproximal caries. Knowledge Transfer Statement: This study will help clinicians make an evidence-based decision when deciding which digital radiography system to use when evaluating interproximal caries. Time, patient radiation safety, cost, and image quality are factors to be considered. The performance of the different available digital imaging systems was compared with the current gold standard-a histologic analysis-via meta-analysis.

Membrane adenosine triphosphatase activities in rat pancreas.

The membrane ATPase activities present in rat pancreas were studied to investigate the possible role of ATPase enzymes in HCO3(-) secretion in the pancreas. It was found that all the HCO3(-)-sensitive (anion-sensitive) ATPase activity was accountable as pancreatic mitochondrial ATPase, thus supporting the view that a distinct plasma membrane 'bicarbonate-ATPase' is not involved in HCO3(-) secretion in pancreas. A remarkably high Mg+- and CA2+-requiring ATPase activity (30 mumol ATP hydrolysed/min per mg) was found in the plasma membrane fraction (rho = 1.10-1.13). This activity has been characterized in some detail. It is inhibited by p-fluorosulfonylbenzoyladenosine, an affinity label analogue of ATP and the analogue appears to label covalently a protein of Mr approximately 35 000. The (Ca2+ + Mg2+)-ATPase activity did not form a 'phosphorylated-intermediate' and was vanadate-insensitive. These and other tests have served to demonstrate that the (Ca2+ + Mg2+)-ATPase activity is different in properties from (Na+ + K+)-ATPase, Ca2+-ATPase, (H+ + K+)-ATPase or mitochondrial H+-ATPase. Apart from the (Ca2+ + Mg2+)-ATPase of plasma membrane and mitochondrial ATPase, the only other membrane ATPase activities noted were (Na+ + K+)-ATPase, which occurred in the same fractions as the (Ca2+ + Mg2+)-AtPase at rho = 1.10-1.13 and was of surprisingly low activity, and an ATPase activity in light membrane fractions (rho - 1.08-1.09) derived from zymogen granule membranes. At this time, therefore, there is no obvious candidate for an ATPase activity at the luminal surface of pancreatic cells which is directly involved in ion transport, but the results presented here direct attention to the high activity (Ca2+ + Mg2+)-ATPase in the plasma membrane fraction.

Catalytic mechanism of F1-ATPase.

The structure of the core catalytic unit of ATP synthase, alpha 3 beta 3 gamma, has been determined by X-ray crystallography, revealing a roughly symmetrical arrangement of alternating alpha and beta subunits around a central cavity in which helical portions of gamma are found. A low-resolution structural model of F0, based on electron spectroscopic imaging, locates subunit a and the two copies of subunit b outside of a subunit c oligomer. The structures of individual subunits epsilon and c (largely) have been solved by NMR spectroscopy, but the oligomeric structure of c is still unknown. The structures of subunits a and delta remain undefined, that of b has not yet been defined but biochemical evidence indicates a credible model. Subunits gamma, epsilon, b, and delta are at the interface between F1 and F0; gamma epsilon complex forms one element of the stalk, interacting with c at the base and alpha and beta at the top. The locations of b and delta are less clear. Elucidation of the structure F0, of the stalk, and of the entire F1F0 remains a challenging goal.

Effects of cardiovascular drugs on ATPase activity of P-glycoprotein in plasma membranes and in purified reconstituted form.

Drug interactions with P-glycoprotein (Pgp) were quantitatively assessed using ATPase assay. Two experimental systems were used, (i) plasma membranes isolated from a multidrug-resistant cell line, which contained 30% Pgp as fraction of total membrane protein, and (ii) purified reconstituted Pgp. The cardioactive drugs verapamil, quinidine, diltiazem, nifedipine, and a series of digitalis analogs, interacted directly with Pgp as shown on ATPase in both systems. Apparent affinities of drug binding were calculated. Direct competition was shown between digitoxin and verapamil. Drug-drug interaction in vivo at the level of Pgp is expected from the results. This approach seems well-suited for empirical determination of drug interactions with Pgp, and prediction of drug-drug interactions.

ATP synthase: what we know about ATP hydrolysis and what we do not know about ATP synthesis.

In ATP synthase, X-ray structures, demonstration of ATP-driven gamma-subunit rotation, and tryptophan fluorescence techniques to determine catalytic site occupancy and nucleotide binding affinities have resulted in pronounced progress in understanding ATP hydrolysis, for which a mechanism is presented here. In contrast, ATP synthesis remains enigmatic. The molecular mechanism by which ADP is bound in presence of a high ATP/ADP concentration ratio is a fundamental unknown; similarly P(i) binding is not understood. Techniques to measure catalytic site occupancy and ligand binding affinity changes during net ATP synthesis are much needed. Relation of these parameters to gamma-rotation is a further goal. A speculative model for ATP synthesis is offered.