Urea-induced transformation of native estrogen receptor and evidence for separate DNA-and ATP-binding sites

We have investigated the involvement of hydrophobic receptor domains during transformation of the native estrogen receptor to a form(s) with high affinity for immobilized DNA and ATP. In the presence of 6 M urea the intact estrogen-receptor complex was completely (greater than 90%, n = 12) transformed into a DNA-binding configuration but only partially (35-45%, n = 8) transformed into an ATP-binding state. Similar experiments performed with unliganded receptor preparations further distinguished the receptor's DNA and ATP binding properties. While the urea-induced increase in receptor affinity for DNA-agarose was estrogen-dependent, the urea-induced increase in affinity for ATP-agarose was steroid-independent. This is the first direct evidence that hydrophobic receptor domains may be involved in the steroid-dependent exposure of the DNA binding site. This event is partially reversible and suggests that electrostatic interactions alone may not be sufficient to accurately describe receptor recognition of specific DNA acceptor sites.     

Electrophysiological effects of ryanodine derivatives on the sheep cardiac sarcoplasmic reticulum calcium-release channel.

We have examined the effects of a number of derivatives of ryanodine on K+ conduction in the Ca2+ release channel purified from sheep cardiac sarcoplasmic reticulum (SR). In a fashion comparable to that of ryanodine, the addition of nanomolar to micromolar quantities to the cytoplasmic face (the exact amount depending on the derivative) causes the channel to enter a state of reduced conductance that has a high open probability. However, the amplitude of that reduced conductance state varies between the different derivatives. In symmetrical 210 mM K+, ryanodine leads to a conductance state with an amplitude of 56.8 +/- 0.5% of control, ryanodol leads to a level of 69.4 +/- 0.6%, ester A ryanodine modifies to one of 61.5 +/- 1.4%, 9,21-dehydroryanodine to one of 58.3 +/- 0.3%, 9 beta,21beta-epoxyryanodine to one of 56.8 +/- 0.8%, 9-hydroxy-21-azidoryanodine to one of 56.3 +/- 0.4%, 10-pyrroleryanodol to one of 52.2 +/- 1.0%, 3-epiryanodine to one of 42.9 +/- 0.7%, CBZ glycyl ryanodine to one of 29.4 +/- 1.0%, 21-p-nitrobenzoyl-amino-9-hydroxyryanodine to one of 26.1 +/- 0.5%, beta-alanyl ryanodine to one of 14.3 +/- 0.5%, and guanidino-propionyl ryanodine to one of 5.8 +/- 0.1% (chord conductance at +60 mV, +/- SEM). For the majority of the derivatives the effect is irreversible within the lifetime of a single-channel experiment (up to 1 h). However, for four of the derivatives, typified by ryanodol, the effect is reversible, with dwell times in the substate lasting tens of seconds to minutes. The effect caused by ryanodol is dependent on transmembrane voltage, with modification more likely to occur and lasting longer at +60 than at -60 mV holding potential. The addition of concentrations of ryanodol insufficient to cause modification does not lead to an increase in single-channel open probability, such as has been reported for ryanodine. At concentrations of > or = 500 mu M, ryanodine after initial rapid modification of the channel leads to irreversible closure, generally within a minute. In contrast, comparable concentrations of beta-alanyl ryanodine do not cause such a phenomenon after modification, even after prolonged periods of recording (>5 min). The implications of these results for the site(s) of interaction with the channel protein and mechanism of the action of ryanodine are discussed. Changes in the structure of ryanodine can lead to specific changes in the electrophysiological consequences of the interaction of the alkaloid with the sheep cardiac SR Ca2+ release channel.     

Muscarinic cholinergic receptor modulation of beta-adrenergic receptor affinity for catecholamines.

The effects of the muscarinic cholinergic agonist methacholine on affinity of beta-adrenergic receptors for isoproterenol and on isoproterenol-induced stimulation of adenylate cyclase activity were assessed in canine myocardium. GTP and guanyl-5'-yl imidoiphosphate both decreased the affinity of beta-adrenergic receptors for isoproterenol without altering the affinity of these receptors for propranolol. Methacholine (10 nM to 10 micronM) antagonized the guanine nucleotide-induced reduction in beta-adrenergic receptor affinity for isoproterenol. This effect of methacholine was reversed by atropine. The choline ester had no effect on the affinity of beta-adrenergic receptors for isoproterenol in the absence of guanine nucleotides. Likewise, methacholine had no effect on the affinity of beta-adrenergic receptors for propranolol, either in the presence or absence of guanine nucleotides. Methacholine also attenuated GTP-induced activation of adenylate cyclase or isoproterenol-induced activation of the enzyme in the presence of GTP. The effects of methacholine on myocardial adenylate cyclase activity were apparent only in the presence of GTP. These effects were also reversed by atropine. The choline ester had no effect on adenylate cyclase activity in the presence of guanyl-5'-yl imidodiphosphate or NaF. The results of the present study suggest that muscarinic cholinergic agonists can regulate both beta-adrenergic receptors and adenylate cyclase by modulating the effects of GTP.     

Isolation and characterization of adult rat hearts cells.

Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca²⁺ at concentrations greater than 10^?5m.     

Unmasking effect of alamethicin on the (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles

A mechanism for the activating effect of alamethicin on membrane enzymes was investigated, using a purified preparation of cardiac sarcolemmal vesicles. (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities were measured. alamethicin increased ouabain-sensitive (Na+,K+)-ATPase activity of sarcolemmal vesicles 5- to 7-fold and adenylate cyclase activity 2.5- to 4-fold. Adenylate cyclase retained its sensitivity to the beta-adrenergic agonist isoproterenol after membranes were treated with alamethicin. Alamethicin caused a 4- to 6-fold increase in the number of detectable (Na+,K+)-ATPase enzymic sites, but no increase ws noted for the number of muscarinic-cholinergic receptor-binding sites. Phosphorylation of endogenous proteins of sarcolemmal vesicles by an intrinsic cAMP-dependent protein kinase activity was stimulated 5- to 7-fold by alamethicin. The regulatory subunit of the membrane-bound cAMP-dependent protein kinase was labeled with the photoaffinity probe 8-azido-adenosine 3':5'[32P]monophosphate (8-N3-[32P]cAMP), and it migrated with an apparent molecular weight of 55,000 in sodium dodecyl sulfate polyacrylamide gels. Alamethicin stimulated autophosphorylation of the regulatory subunit by [gamma-32P]ATP 6-fold and incorporation of of 8-N3-[32P]cAMP into the subunit 2.6-fold. The results suggest that alamethicin disrupts membrane barriers of sarcolemmal vesicles, which are mostly right side out, giving substrates and activators access to enzymic sites in the interior of the vesicles, while preserving functional coupling of enzymes to their effectors.     

Studies on (pro)insulin biosynthesis and secretion of pancreatic islets of sand rats (Psammomys obesus) after different feeding conditions.

It was possible to induce different metabolic states in sand rats of our breeding colony or in newly caught Egyptian sand rats, respectively, by feeding a pellet diet or vegetable diet (green cabbage). Newly captured sand rats fed only on native food were used as reference group (group C). Plasma IRI-level and glucose in vivo and [3H]-leucine incorporation into proinsulin and insulin, insulin secretion and insulin content in vitro were investigated. Sand rats fed on pellet chow and ad libitum (group B) developed a hyperinsulinism and showed higher sensitivity of [3H]-leucine incorporation into proinsulin and insulin to glucose (maximal stimulation at 3 mM) and increased incorporation rates in vitro. Restriction of pellet food to 35-40 kcal/animal/day (group A) lead to changes of all parameters, which were investigated in the same direction as in group B, but to a much smaller extent. Newly captured sand rats, which were fed green cabbage for 4 to 6 weeks divided into two groups: One group (group D1) was comparable to the normal group (C) in IRI levels, glucose levels, glucose sensitivity and amount of [3H]-leucine incorporation. The other group (group D2) tended to group A. Marked changes in insulin content and insulin secretion of isolated pancreatic islets could not be found in any group.     

Efficacy of a Food Safety Comic Book on Knowledge and Self-Reported Behavior for Persons Living with AIDS

Introduction

Persons living with AIDS are highly vulnerable to foodborne enteric infections with the potential for substantial morbidity and mortality. Educational materials about foodborne enteric infections intended for this immunocompromised population have not been assessed for their efficacy in improving knowledge or encouraging behavior change.

Methods/Results

AIDS patients in four healthcare facilities in Chicago, New Orleans, and Puerto Rico were recruited using fliers and word of mouth to healthcare providers. Those who contacted research staff were interviewed to determine food safety knowledge gaps and risky behaviors. A food safety educational comic book that targeted knowledge gaps was created, piloted, and provided to these patients who were instructed to read it and return at least 2 weeks later for a follow-up interview. The overall food safety score was determined by the number of the 26 knowledge/belief/behavior questions from the survey answered correctly. Among 150 patients who participated in both the baseline and follow-up questionnaire, the intervention resulted in a substantial increase in the food safety score (baseline 59%, post-intervention 81%, p<0.001). The intervention produced a significant increase in all the food safety knowledge, belief, and behavior items that comprised the food safety score. Many of these increases were from baseline knowledge below 80 percent to well above 90%. Most (85%) of the patients stated they made a change to their behavior since receiving the educational booklet.

Conclusion

This comic book format intervention to educate persons living with AIDS was highly effective. Future studies should examine to what extent long-term behavioral changes result.     

Artificial vision with wirelessly powered subretinal electronic implant alpha-IMS

This study aims at substituting the essential functions of photoreceptors in patients who are blind owing to untreatable forms of hereditary retinal degenerations. A microelectronic neuroprosthetic device, powered via transdermal inductive transmission, carrying 1500 independent microphotodiode-amplifier-electrode elements on a 9 mm² chip, was subretinally implanted in nine blind patients. Light perception (8/9), light localization (7/9), motion detection (5/9, angular speed up to 35 deg s⁻¹), grating acuity measurement (6/9, up to 3.3 cycles per degree) and visual acuity measurement with Landolt C-rings (2/9) up to Snellen visual acuity of 20/546 (corresponding to decimal 0.037 or corresponding to 1.43 logMAR (minimum angle of resolution)) were restored via the subretinal implant. Additionally, the identification, localization and discrimination of objects improved significantly (n = 8; p < 0.05 for each subtest) in repeated tests over a nine-month period. Three subjects were able to read letters spontaneously and one subject was able to read letters after training in an alternative-force choice test. Five subjects reported implant-mediated visual perceptions in daily life within a field of 15° of visual angle. Control tests were performed each time with the implant''s power source switched off. These data show that subretinal implants can restore visual functions that are useful for daily life.     

Characterization and quantification of triple helix formation in chromosomal DNA

DNA-binding molecules that recognize specific sequences offer a high potential for the understanding of chromatin structure and associated biological processes in addition to their therapeutic potential, e.g. as positioning agents for validated anticancer drugs. A prerequisite for the development of DNA-binding molecules is the availability of appropriate methods to assess their binding properties quantitatively at the desired target sequence in the human genome. We have further developed a capture assay to assess triplex-forming oligonucleotide (TFO) binding efficiency quantitatively. This assay is based on bifunctional, psoralen and biotin-conjugated, TFOs and real-time PCR analysis. We have applied this novel quantification method to address two issues that are relevant for DNA-binding molecules. First, we have compared directly the extent of TFO-binding in three experimental settings with increasing similarity to the situation in vivo, i.e. naked genomic DNA, isolated cell nuclei, or whole cells. This comparison allows us to characterize factors that influence genomic triplex formation, e.g. chromosomal DNA organization or intracellular milieu. In isolated nuclei, the binding was threefold lower compared to naked DNA, consistent with a decreased target accessibility int he nucleosomal environment. Binding was detected in whole cells, indicating that the TFO enters the nucleus and binds to its target in intact cells in vivo, but the efficiency was decreased (tenfold) compared to nuclei. Secondly, we applied the method to characterize the binding properties of two different TFOs targeting the same sequence. We found that an antiparallel-binding GT-containing TFO bound more efficiently, but with less target sequence selectivity compared to a parallel-binding CU-containing TFO. Collectively, a sensitive method to characterize genomic triplex formation was described. This may be useful for the determination of factors driving TFO binding efficiency and, thus, may improve the usefulness of triplex-mediated gene targeting for studies of chromatin structure as well as for therapeutic antigene strategies.