Safety of pramlintide added to mealtime insulin in patients with type 1 or type 2 diabetes: a large observational study.

The objective of this Phase 4, open-label, multicentre, observational study was to fulfil food and drug administration (FDA) postapproval requirement to evaluate in healthcare practices the risk of insulin-induced severe hypoglycaemia following initiation of pramlintide therapy in N = 1297 patients with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) with inadequate glycaemic control. The duration of the study was approximately 6 months. During the adjustment period (0-3 months), the incidence and event rate of patient-ascertained severe hypoglycaemia (PASH) were 4.8% and 0.33 events/patient-year in patients with T1DM and 2.8% and 0.19 events/patient-year in patients with T2DM. During the maintenance period (>3-6 months), the incidence and event rate of PASH declined in patients with T1DM or T2DM. This study confirms that in healthcare practices, the risk of insulin-induced severe hypoglycaemia following the initiation of pramlintide is low in patients with T1DM or T2DM.

The sCYMV1 hairpin ribozyme: targeting rules and cleavage of heterologous RNA.

The catalytic center of the RNA from the negative strand of the satellite RNA of chicory yellow mottle virus type 1 (sCYMV1) is in the hairpin ribozyme family, has catalytic activity, and cleaves substrates before a preferred GUA sequence. This is different from that of the satellite RNA from the negative strand of tobacco ringspot virus (sTRSV) which prefers a GUC sequence at the site of cleavage. The sCYMV1 hairpin ribozyme has now been developed for cleaving heterologous RNA substrates. When helix 1 was extended from the native 5 bp to 6 bp with a newly added A:U base pair, catalytic activity increased three-fold. The preferred sequence for the substrate loop was the native A*GUA sequence where * is the site of cleavage. When each nucleotide in this sequence was changed to each of the other three nucleotides, catalytic activity decreased 66-100%. RNA targets, containing this A*GUA sequence, were located in both human papillomavirus and HIV-1. Ribozymes were developed which efficiently cleaved these targets in vitro. These results identify a new class of hairpin ribozymes capable of cleaving substrates before a preferred GUA sequence rather than the GUC preferred by the sTRSV hairpin ribozyme. This expands the repertoire of target sites available for gene therapy using the hairpin ribozyme.

Analysis of germline mutation spectra at the Huntington's disease locus supports a mitotic mutation mechanism.

Trinucleotide repeat disease alleles can undergo 'dynamic' mutations in which repeat number may change when a gene is transmitted from parent to offspring. By typing >3500 sperm, we determined the size distribution of Huntington's disease (HD) germline mutations produced by 26 individuals from the Venezuelan cohort with CAG/CTG repeat numbers ranging from 37 to 62. Both the mutation frequency and mean change in allele size increased with increasing somatic repeat number. The mutation frequencies averaged 82% and, for individuals with at least 50 repeats, 98%. The extraordinarily high mutation frequency levels are most consistent with a mutation process that occurs throughout germline mitotic divisions, rather than resulting from a single meiotic event. In several cases, the mean change in repeat number differed significantly among individuals with similar somatic allele sizes. This individual variation could not be attributed to age in a simple way or to ' cis ' sequences, suggesting the influence of genetic background or other factors. A familial effect is suggested in one family where both the father and son gave highly unusual spectra compared with other individuals matched for age and repeat number. A statistical model based on incomplete processing of Okazaki fragments during DNA replication was found to provide an excellent fit to the data but variation in parameter values among individuals suggests that the molecular mechanism might be more complex.

Cationic liposomes enhance adenovirus entry via a pathway independent of the fiber receptor and alpha(v)-integrins.

The ability of adenoviral vectors to mediate efficient gene delivery both in vitro and in vivo is limited by the availability of specific cell surface receptors and alpha(v)-containing integrins. We tested whether this limitation could be overcome by enhancing viral entry with cationic liposomes. In cultured vascular smooth muscle cells, delivery of adenoviral vectors in the presence of cationic liposomes increased vector-encoded transgene expression up to 20-fold. The increase in transgene expression was associated with the formation of adenovirus-lipid aggregates and an increase in the amount of vector DNA in the cells, suggesting that enhanced viral entry was responsible for the increase in gene expression. Treatment of the cells with an RGD-containing peptide or adenovirus type 5 fiber protein did not diminish liposome enhancement of transgene expression, indicating that liposomes increase viral entry via a pathway independent of the fiber receptor and of alpha(v) integrin-assisted endocytosis. Liposomes also significantly enhanced transgene expression from adenoviral vectors delivered to cells deficient in alpha(v)-containing integrins. The magnitude of liposome enhancement of transgene expression in cultured smooth muscle cells was greatest during brief periods of virus-cell contact and at low concentrations of virus. Despite these promising in vitro results, addition of liposomes did not improve in vivo adenoviral gene delivery into injured rat carotid arteries. Liposomes can improve adenoviral gene delivery in vitro; however, application of this observation to accomplish improved in vivo gene delivery remains a challenge.

Screening for important base identities in the hairpin ribozyme by in vitro selection for cleavage.

Random mutagenesis followed by an in vitro selection procedure was shown to be capable of identifying important bases of the hairpin ribozyme for cleavage of an RNA target sequence. The selection scheme enriched the RNA population for those molecules capable of efficient site-specific self-cleavage in the absence of ligation. Cleavable mutants were selected for all positions in loop 4 except for position A38, supporting the notion that A38 is an important base in the hairpin ribozyme. This has been confirmed by direct mutagenesis, validating the utility of this procedure. Thus, the method developed and reported here has utility for the selection of efficient hairpin ribozymes capable of highly efficient cleavage of a substrate RNA without a requirement for ribozyme-catalyzed ligation, conditions desired for many applications of catalytic RNA such as gene therapy.

Research on the effects of pharmacist-patient communication in institutions and ambulatory care sites, 1969-1994.

Research on the effects of pharmacist-patient communication that appeared in the pharmacy literature between 1969 and 1994 is reviewed. The terms patients education and patient counseling were used in identifying relevant research. Many authors used these terms interchangeably; also, the terms counseling and consultation often were not clearly defined. Studies of pharmacists' history-taking in institutional settings and of other communication with patients in ambulatory care settings were examined by decade. The research questions, theories, methods, results, and limitations were analyzed. More than 30 studies examined the effect of pharmacists' communication on patient outcomes, primarily knowledge and medication compliance; generally, the results suggested that pharmacists' communication led to increased knowledge and compliance. A few researchers raised concerns about patients' knowledge as an indicator of effective communication by pharmacists, and in the 1980s a few suggested that better medication compliance could be associated with the time and attention given to patients rather than the informational content of the interaction. Little was reported about the communication theories or models on which the studies were based, and there was little indication in most studies that patients' ideas about their therapy were considered. Often, the numbers of patients and pharmacists were small, and the pharmacists may have had training or motivation exceeding that of the average practitioner. In studies of pharmacists' versus physicians' history-taking, the physicians were not well described; their involvement and their approach may not have been comparable to those of the pharmacists. Before 1990, a few researchers had examined outcomes such as pulmonary function and control of diabetes. In the 1990s, more researchers looked at outcomes such as medication-related problems and use of health care resources. The research indicated that pharmacists can increase patients' knowledge of and compliance with their pharmacotherapy; however, additional research on other outcomes may be warranted.

Single sperm analysis of the trinucleotide repeats in the Huntington's disease gene: quantification of the mutation frequency spectrum.

The CAG triplet repeat region of the Huntington's disease gene was amplified in 923 single sperm from three affected and two normal individuals. Average-size alleles (15-18 repeats) showed only three contraction mutations among 475 sperm (0.6%). A 30 repeat normal allele showed an 11% mutation frequency. The mutation frequency of a 36 repeat intermediate allele was 53% with 8% of all gametes having expansions which brought the allele size into the HD disease range (> or = 38 repeats). Disease alleles (38-51 repeats) showed a very high mutation frequency (92-99%). As repeat number increased there was a marked elevation in the frequency of expansions, in the mean number of repeats added per expansion and the size of the largest observed expansion. Contraction frequencies also appeared to increase with allele size but decreased as repeat number exceeded 36. Our sperm typing data are of a discrete nature rather than consisting of smears of PCR product from pooled sperm. This allowed the observed mutation frequency spectra to be compared to the distribution calculated using discrete stochastic models based on current molecular ideas of the expansion process. An excellent fit was found when the model specified that a random number of repeats are added during the progression of the polymerase through the repeated region.

Functional characterization of ribozymes expressed using U1 and T7 vectors for the intracellular cleavage of ANF mRNA.

Hammerhead ribozymes targeted to various GUC or GUA sites on rat atrial natriuretic factor (ANF) mRNA were developed. The catalytic activity of ribozymes to four of these sites, synthesized by transcription off synthetic oligodeoxynucleotide duplexes, was studied in detail. In vitro, ribozyme-mediated cleavage was highly Mg(2+)-dependent, and at concentrations approaching those found intracellularly, the rate but not the extent of cleavage was markedly reduced. To test for cellular activity, synthetic genes encoding the ribozymes were cloned between the initiation and termination sequences of the U1snRNA gene or between the T7RNA polymerase promoter and terminator sequences in pSP64. Both constructs had defined initiation and termination sequences to minimize transcript size and for message stability. In vitro the addition of T7 or U1 terminator sequences had variable effects on catalytic activity, presumably due to structural interactions between the ribozyme and the added sequence. The ribozyme-encoding plasmids were cotransfected with an expression plasmid containing a rat ANF cDNA into COS-1 cells using a liposome method, which provided high-level transfection efficiency. Quantitation of ANF mRNA by RNase protection showed marked decreases in ANF transcript levels with both the U1- and the T7-expressed ribozymes directed at three of the four sites on ANF mRNA. With all constructs, target accessibility, determined in vitro, was a more important determinant of intracellular ANF mRNA cleavage than catalytic activity per se. ANF mRNA cleavage was not merely due to an antisense effect, since a mutant construct that was catalytically inactive but could still bind produced less cleavage than the corresponding wild-type ribozyme construct. These findings indicate that both U1 and T7 vector systems provide efficient ribozyme expression for the intracellular cleavage of target mRNA.

Brefeldin A defines distinct pathways for atrial natriuretic factor secretion in neonatal rat atrial and ventricular myocytes.

The intracellular pathways for basal atrial natriuretic factor (ANF) secretion from the heart and their correlation with ANF processing to the active form were characterized in cultured neonatal rat atrial and ventricular myocytes. Brefeldin A, a fungal antimetabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was used to inhibit nascent protein trafficking. Thus, release of newly synthesized hormone was blocked, but release of stored hormone was unaffected. Whereas brefeldin A inhibited basal ventricular ANF release to 10% of the control value, basal ANF release from atrial cells was enhanced. Furthermore, basal atrial ANF secretion was inhibited by agents preventing myocyte depolarization, Ca2+ influx, release of Ca2+ from intracellular stores, or activation of protein kinase C, whereas ventricular ANF secretion was unaffected by these agents. Brefeldin A did not alter maturational processing of pro-ANF to ANF-(99-126) in either atrial or ventricular cultures. These findings indicate that (1) basal secretion of ANF from ventricular cells relies largely on newly synthesized hormone and is probably constitutive, (2) basal secretion of ANF from atrial cells is independent of transport of newly synthesized protein and occurs via a regulated pathway controlled at least in part by signaling changes associated with myocyte beating, and (3) processing of pro-ANF occurs either with constitutive or regulated secretion of hormone, which may indicate multiple cellular locations for the processing enzyme.

Cryptosporidium infections in a suburban community in Maracaibo, Venezuela.

A point prevalence survey for Cryptosporidium was conducted in 212 subjects two months to 70 years of age in a suburban area with a low socioeconomic status in Maracaibo City, Venezuela. Single stool specimens were collected and modified Ziehl-Neelsen carbol-fuchsin staining of 10% formalin-preserved stool was used to identify Cryptosporidium oocysts. Direct wet mounts, iron-hematoxylin-stained smears and formalin-ether concentrates were examined to determine the presence of other intestinal parasites. Cryptosporidium infections were identified in 21 subjects (9.9%), with a high percentage of asymptomatic carriers (15 of 21, 71.4%). Six children (28.5%) had gastrointestinal symptoms and four of them were infants. Cryptosporidium was the single detectable potential pathogenic parasite in only five (23.8%) of 21 patients. The infection rate with one or more parasites was high (82%) and multiple infections, including pathogenic helminths and protozoa, were observed in the majority of patients who passed oocysts. Our findings suggest that although Cryptosporidium is an important pathogen, the proportion of asymptomatic carriers may be high in areas of low socioeconomic status in developing countries.

Synergism between cAMP and ATP in signal transduction in cardiac myocytes.

ATP transiently increases the intracellular Ca2+ concentration in cardiac myocyte suspensions. Pretreatment with norepinephrine (NE) greatly potentiates the ATP response. We performed experiments on adult rat myocyte suspensions loaded with fura-2 to investigate the mechanism of NE potentiation. We found that forskolin (an activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase), and permeative adenosine 3',5'-cyclic monophosphate (cAMP) analogues potentiate the increase in cytosolic Ca2+ concentration induced by ATP. NE, forskolin, and 8-(4-chlorophenylthio)-cAMP all increase Vmax of the Ca2+ response curve of ATP. Measurement of cAMP by radioimmunoassay confirmed that the changes in the ATP response were accompanied by an increase in cAMP. These results suggest that the noradrenergic potentiation of the ATP-induced Ca2+ mobilization involves cAMP as a second messenger. Patch-clamp studies of isolated myocytes showed that neither NE nor forskolin alters the inward current elicited by ATP, but rather they increase the duration of secondary slow action potentials elicited by ATP. NE also increases the Ca2+ current through L-type Ca2+ channels in the myocytes. We conclude that NE potentiates the ATP-induced Ca2+ transient by increasing cAMP levels and that one of the early events is the increase of the inward Ca2+ current during the action potential.

Extracellular ATP activates coordinated Na+, Pi, and Ca2+ transport in cardiac myocytes.

Activation of an ATP receptor has previously been shown to induce cytosolic [Ca2+] transients in rat ventricular myocytes. A slower but larger [Ca2+] increase which can cause cell hypercontraction follows the transient when extracellular Pi is increased. This second phase of the [Ca2+] response is stimulated by ATP or adenosine 5'-(gamma-thio)triphosphate in a medium containing 11.2 mM Pi, but not by high concentrations of 2-methylthio-ATP, which stimulate only the initial [Ca2+] transient. Replacing medium Na+ with N-methyl-D-glucamine suppresses this Pi-dependent [Ca2+] increase following ATP addition, suggesting a causal relationship between Na+ transport and Ca2+ influx. Blocking voltage-sensitive Na+ channels, Na(+)-H+ exchange, or Na(+)-K(+)-Cl- cotransport did not reduce ATP-induced cell hypercontraction in 11.2 mM Pi medium, suggesting that these transporters are not involved. ATP stimulation of Na(+)-Pi cotransport was investigated with isotopic methods. The results were consistent with the hypothesis that extracellular ATP stimulates Na(+)-Pi cotransport, which activates Na(+)-Ca2+ exchange. A novel Pi-dependent ATP receptor-effector system has been demonstrated in cardiac cells, and it may have significant effects on cellular transport, contractility, and bioenergetics.

ATP receptor-induced Ca2+ transients in cardiac myocytes: sources of mobilized Ca2+.

Addition of micromolar concentrations of extracellular ATP to adult rat cardiac ventricular myocytes increases cytosolic Ca2+ concentration ([Ca2+]). Experiments were performed on fura-2-loaded myocytes to determine whether the [Ca2+] rise was due to Ca2+ influx, release of Ca2+ from the sarcoplasmic reticulum (SR), or a combination of both. BAY K 8644 and nifedipine affected ATP-induced [Ca2+] transients, indicating involvement of voltage-sensitive Ca2+ channels. Addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or Ca2+ channel blockers significantly reduced cytosolic [Ca2+] changes due to addition of ATP or KCl without depleting Ca2+ stores (shown by ionomycin treatment in a Ca2+-free medium), demonstrating that these responses require Ca2+ influx. Depletion of intracellular Ca2+ stores by caffeine or ryanodine also diminished cytosolic [Ca2+] responses, indicating that a portion of the increased cytosolic [Ca2+] is due to Ca2+ release from SR. Norepinephrine potentiates the ATP-Ca2+ response, and this effect was not inhibited by depletion of intracellular stores. Although the data show that there are two Ca2+ sources in the cytosolic Ca2+ response to ATP, the pattern is also consistent with the hypothesis of Ca2+-induced Ca2+ release from cardiac SR.

The indignant page: techniques of neutralization in the publications of pedophile organizations.

There is considerable consensus in this and other societies that adult sexual behavior with children is exploitative and victimizing in nature. The publications of three United States pedophile organizations that advocate adult sexual behavior with children and that are politically and socially active in attempts to decriminalize that behavior--the Rene Guyon Society, the Childhood Sensuality Circle, and the North American Man/Boy Love Association--are analyzed as to how they justify, normalize, or rationalize their practices and philosophy in light of that consensus. The framework for that analysis is the sociological concept of techniques of neutralization.

Extracellular ATP induces Ca2+ transients in cardiac myocytes which are potentiated by norepinephrine.

Isolated rat ventricular cardiac myocytes loaded with the fluorescent calcium indicator fura2 showed significant changes in intracellular calcium concentrations upon exposure to greater than 1 microM ATP (EC50 = 7.4 +/- 1.3 microM, n = 4, SE), suggesting that extracellular ATP may have an important influence on myocardial contractility. The response was found to be highly ATP specific and required extracellular calcium. Furthermore, 30 s pretreatment of the cells with 0.2-1 microM norepinephrine decreased the concentration of ATP required for the Ca2+ transient, shifting the EC50 for ATP to 1.7 +/- 0.1 microM (n = 3, SE). beta-Propranolol (a beta 1-receptor antagonist) prevented potentiation, whereas phentolamine (an alpha 1-receptor antagonist) did not, indicating that regulation is through the beta 1-adrenergic receptor. ATP and norepinephrine released locally from sympathetic neurons may act in concert through the ATP and beta 1-adrenergic receptors to regulate myocardial calcium homeostasis.

Measurement of the internal pH of mast cell granules using microvolumetric fluorescence and isotopic techniques.

The intragranular pH of isolated mast cell granules was measured. Because of the minute amounts of isolated granules available, two techniques were developed by modifying aminoacridine fluorescence and [14C]methylamine accumulation techniques to permit measurements with microliter sample volumes. Granule purity was demonstrated by electron microscopy, ruthenium red exclusion, and biochemical (histamine, mast cell granule protease) analysis. The internal pH was determined to be 5.55 +/- 0.06, indicating that the pH environment within mast cell granules is not significantly different from that of previously studied granule types (i.e., chromaffin, platelet, pancreatic islet, and pituitary granules). Collapse of the pH gradient by NH+4 was demonstrated with both techniques. No evidence of Cl-/OH- or specific cation/H+ transport was found, and major chloride permeability could not be unequivocably demonstrated. Ca2+ and Cl- at concentrations normally present extracellularly destabilized granules in the presence of NH+4, but this phenomenon does not necessarily indicate a role for these ions in the exocytotic release of granule contents from intact cells. The pH measurement techniques developed for investigating the properties of granules in mast cells may be useful for studying other granules that can be obtained only in limited quantities.

Intracellular Ca2+ mobilization activated by extracellular ATP in Ehrlich ascites tumor cells.

Addition of 0.4-25 microM extracellular ATP results in transient, dose-dependent increases in cytosolic free calcium measured in Ehrlich ascites tumor cells. In cells incubated with 1 mM extracellular Ca2+, ATP induces a triphasic Ca2+ transient: an initial rapid increase (2-3 s), a second, slower phase of increase (60-90 s), and, finally, a gradual return to near resting [Ca2+]i (4-5 min). Several findings demonstrate that the initial, rapid phase of Ca2+ transient results from a mobilization of Ca2+ from a non-mitochondrial intracellular store, while the second, slow phase of increase is produced by enhanced influx of Ca2+ across the plasma membrane. Successive additions of extracellular ATP can elicit repetitive Ca2+ transients if the initially added ATP is removed either through the action of native ecto-ATPase activity or exogenous hexokinase. Other adenine nucleotides, including non-hydrolyzable ATP analogs, neither alter cytosolic [Ca2+] nor antagonize the ATP-induced effects. Conversely, other nucleotide triphosphates (ITP, UTP, and GTP) induce Ca2+ transients which are identical to those produced by ATP. A variety of experimental results indicate that these actions of ATP and other nucleotide triphosphates are not due to a generalized increase in plasma membrane permeability. The results suggest that, in these transformed cells, ATP may act in a manner similar to other Ca2+ mobilizing hormones and growth factors.

Counterphobic behavior in multiply molested children.

That multiply molested children are somehow responsible for their victimization is a conclusion unacceptable to the author, who examines one possible cause for the paradoxical behavior of these children.