Enhanced PKC beta II translocation and PKC beta II-RACK1 interactions in PKC epsilon-induced heart failure: a role for RACK1.

Recent investigations have established a role for the beta II-isoform of protein kinase C (PKC beta II) in the induction of cardiac hypertrophy and failure. Although receptors for activated C kinase (RACKs) have been shown to direct PKC signal transduction, the mechanism through which RACK1, a selective PKC beta II RACK, participates in PKC beta II-mediated cardiac hypertrophy and failure remains undefined. We have previously reported that PKC epsilon activation modulates the expression of RACKs, and that altered epsilon-isoform of PKC (PKC epsilon)-RACK interactions may facilitate the genesis of cardiac phenotypes in mice. Here, we present evidence that high levels of PKC epsilon activity are commensurate with impaired left ventricular function (dP/dt = 6,074 +/- 248 mmHg/s in control vs. 3,784 +/- 269 mmHg/s in transgenic) and significant myocardial hypertrophy. More importantly, we demonstrate that high levels of PKC epsilon activation induce a significant colocalization of PKC beta II with RACK1 (154 +/- 7% of control) and a marked redistribution of PKC beta II to the particulate fraction (17 +/- 2% of total PKC beta II in control mice vs. 49 +/- 5% of total PKC beta II in hypertrophied mice), without compensatory changes of the other eight PKC isoforms present in the mouse heart. This enhanced PKC beta II activation is coupled with increased RACK1 expression and PKC beta II-RACK1 interactions, demonstrating PKC epsilon-induced PKC beta II signaling via a RACK1-dependent mechanism. Taken together with our previous findings regarding enhanced RACK1 expression and PKC epsilon-RACK1 interactions in the setting of cardiac hypertrophy and failure, these results suggest that RACK1 serves as a nexus for at least two isoforms of PKC, the epsilon-isoform and the beta II-isoform, thus coordinating PKC-mediated hypertrophic signaling.

Women living long, living well: community driven women's health priorities.

The Office on Women's Health of the Department of Health and Human Services led an effort to seek feedback on women's health issues. The response showed that women in communities nationwide focused less on individual organ-specific health issues than on broader strategies they thought were critical to improving and sustaining women's health programs. This article summarizes the result of those discussions as expressed in the "Women Living Long, Living Well" framework.

Cardiac-specific abrogation of NF- kappa B activation in mice by transdominant expression of a mutant I kappa B alpha.

Nuclear factor-kappaB (NF-kappa B) is a pleiotropic oxidant-sensitive transcription factor that is present in the cytosol in an inactive form complexed to an inhibitory kappaB (I kappa B) monomer. Various stimuli, including ischemia, hypoxia, free radicals, cytokines, and lipopolysaccharide (LPS), activate NF-kappa B by inducing phosphorylation of I kappa B. Phosphorylation of serine residues at positions 32 and 36 is critical for ubiquitination and degradation of I kappa B alpha with consequent migration of NF-kappa B to the nucleus. Although NF-kappa B is thought to contribute to numerous pathophysiologic processes, definitive assessment of its role has been hindered by the inability to achieve specific inhibition in vivo. Pharmacologic inhibitors of NF-kappa B are available, but their utility for in vivo studies is limited by their relative lack of specificity. Targeted ablation of genes encoding NF-kappa B subunits has not been productive in this regard because of fetal lethality in the case of p65 and functional redundancy in the Rel family of proteins. To overcome these limitations, we have created a viable transgenic mouse that expresses a phosphorylation-resistant mutant of I kappa B alpha (I kappa B alpha(S32A,S36A)) under the direction of a cardiac-specific promoter. Several transgenic lines were obtained with copy numbers ranging from one to seven. The mice exhibit normal cardiac morphology and histology. Total myocardial I kappa B alpha protein level is elevated 3.5- to 6.5-fold with a concomitant 50-60% decrease in the level of I kappa B beta. Importantly, expression of I kappa B(S32A,S36A) results in complete abrogation of myocardial NF-kappa B activation in response to tumor necrosis factor- alpha (TNF-alpha) and LPS stimulation. Thus, novel transgenic mice have been created that make it possible to achieve cardiac-specific and selective inhibition of NF-kappa B in vivo. These transgenic mice should be useful in studies of various cardiac pathophysiological phenomena that involve NF-kappa B activation, including ischemic preconditioning, heart failure, septic shock, acute coronary syndromes, cardiac allograft rejection, and apoptosis.

Awareness, perception, and knowledge of heart disease risk and prevention among women in the United States. American Heart Association Women's Heart Disease and Stroke Campaign Task Force.

CONTEXT: One of 2 women in the United States dies of heart disease or stroke, yet women are underdiagnosed and undertreated for these diseases and their risk factors. Informed decisions to prevent heart disease and stroke depend on awareness of risk factors and knowledge of behaviors to prevent or detect these diseases. OBJECTIVE: Assess (1) knowledge of risks of heart disease and stroke and (2) perceptions of heart disease and its prevention among women in the United States. DESIGN AND SETTING: Telephone survey conducted in 1997 of US households, including an oversample of African American and Hispanic women. PARTICIPANTS: One thousand respondents 25 years or older; 65.8% white, 13.0% African American, and 12.6% Hispanic. MAIN OUTCOME MEASURES: Knowledge of heart disease and stroke risks, perceptions of heart disease, and knowledge of symptoms and preventive measures. RESULTS: Only 8% of the respondents identified heart disease and stroke as their greatest health concerns; less than 33% identified heart disease as the leading cause of death. More women aged 25 to 44 years identified breast cancer as the leading cause of death than women 65 years or older. Women aged 25 to 44 years indicated they were not well informed about heart disease and stroke. Although 90% of the women reported that they would like to discuss heart disease or risk reduction with their physicians, more than 70% reported that they had not. CONCLUSIONS: Most women do not perceive that heart disease is a substantial health concern and report that they are not well informed about their risk. Age influenced knowledge to a greater extent than ethnicity. Programs directed at young women that address the effects of lifestyle behaviors on long-term health are needed. Better communication between physicians and patients is also warranted.

The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene.

The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the late phase of ischemic preconditioning (PC) in vivo. A total of 321 mice were used. Wild-type mice preconditioned 24 h earlier with six cycles of 4-min coronary occlusion/4-min reperfusion exhibited a significant (P < 0.05) increase in myocardial iNOS protein content, iNOS activity (assessed as calcium-independent L-citrulline formation), and nitrite + nitrate tissue levels. In contrast, endothelial NOS protein content and calcium-dependent NOS activity remained unchanged. No immunoreactive neuronal NOS was detected. When wild-type mice were preconditioned 24 h earlier with six 4-min occlusion/4-min reperfusion cycles, the size of the infarcts produced by a 30-min coronary occlusion followed by 24 h of reperfusion was reduced markedly (by 67%; P < 0.05) compared with sham-preconditioned controls, indicating a late PC effect. In contrast, when mice homozygous for a null iNOS allele were preconditioned 24 h earlier with the same protocol, infarct size was not reduced. Disruption of the iNOS gene had no effect on early PC or on infarct size in the absence of PC. These results demonstrate that (i) the late phase of ischemic PC is associated with selective up-regulation of iNOS, and (ii) targeted disruption of the iNOS gene completely abrogates the infarct-sparing effect of late PC (but not of early PC), providing unequivocal molecular genetic evidence for an obligatory role of iNOS in the cardioprotection afforded by the late phase of ischemic PC. Thus, this study identifies a specific protein that mediates late PC in vivo.

Ischemic preconditioning increases iNOS transcript levels in conscious rabbits via a nitric oxide-dependent mechanism.

Recent studies implicate iNOS as the mediator of the late phase of ischemic preconditioning (PC). However, it is unknown whether induction of iNOS activity is mediated by transcriptional, post-transcriptional, translational, or post-translational mechanisms. To address this issue, we isolated and sequenced a partial iNOS cDNA expressed in preconditioned rabbit myocardium. Using a rabbit-specific probe generated from this sequence, we measured the steady state levels of the iNOS transcript after ischemic PC [six cycles of 4-min occlusion/4-min reperfusion (O/R)]. Three hours after ischemic PC, the iNOS mRNA levels in the ischemic/reperfused region were increased approximately three-fold relative to samples from the non-ischemic region and from control rabbits. This increase in mRNA levels was completely abolished by pretreatment with the NOS inhibitor Nomega -nitro- L-arginine. Conversely, administration of the NO donor nitroglycerin induced an increase in iNOS mRNA levels similar to that induced by ischemic PC. We conclude that in the conscious rabbit, ischemic PC induces an increase in iNOS mRNA levels, and that this induction is triggered by increased generation of NO during the PC stimulus. These results provide direct evidence that upregulation of iNOS is a natural response of the heart to a brief ischemic stress and that NO itself, in the absence of ischemia, upregulates myocardial iNOS transcript levels, a finding that may have implications for nitrate therapy. This previously unrecognized NO-dependent upregulation of iNOS mRNA is likely to play an important role in the development of late PC as well as in many other pathophysiological conditions in which NO is implicated.

The nitric oxide hypothesis of late preconditioning.

Ischemic preconditioning (PC) occurs in two phases: an early phase, which lasts 2-3 h, and a late phase, which begins 12-24 h later and lasts 3-4 days. The mechanism for the late phase of PC has been the focus of intense investigation. We have recently proposed the "NO hypothesis of late PC", which postulates that NO plays a prominent role both in initiating and in mediating this cardioprotective response. The purpose of this essay is to review the evidence supporting the NO hypothesis of late PC and to discuss its implications. We propose that, on day 1, a brief ischemic stress causes increased production of NO (probably via eNOS) and .O2-, which then react to form ONOO-, ONOO-, in turn, activates the epsilon isoform of protein kinase C (PKC), either directly or via its reactive byproducts such as .OH. Both NO and secondary species derived from .O2- could also stimulate PKC epsilon independently. PKC epsilon activation triggers a complex signaling cascade that involves tyrosine kinases (among which Src and Lck appear to be involved) and probably other kinases, the transcription factor NF-kappa B, and most likely other as yet unknown components, resulting in increased transcription of the iNOS gene and increased iNOS activity on day 2, which is responsible for the protection during the second ischemic challenge. Tyrosine kinases also appear to be involved on day 2, possibly by modulating iNOS activity. According to this paradigm, NO plays two completely different roles in late PC: on day 1, it initiates the development of this response, whereas on day 2, it protects against myocardial ischemia. We propose that two different NOS isoforms are sequentially involved in late PC, with eNOS generating the NO that initiates the development of the PC response on day 1 and iNOS then generating the NO that protects against recurrent ischemia on day 2. The NO hypothesis of late PC puts forth a comprehensive paradigm that can explain both the initiation and the mediation of this complex phenomenon. Besides its pathophysiological implications, this hypothesis has potential clinical reverberations, since NO donors (i.e., nitrates) are widely used clinically and could be used to protect the ischemic myocardium in patients.

Osteogenic protein-1 mRNA expression is selectively modulated after acute ischemic renal injury.

Osteogenic protein-1 (OP-1) is a morphogenetic factor highly expressed in the kidney and involved in tissue repair and development. Homozygous OP-1-deficient mice die shortly after birth due mainly to arrest of renal growth and differentiation. Because postischemic injury involves several repair mechanisms, this study examined whether kidney OP-1 mRNA expression is modulated after ischemia. Acute ischemic renal injury was achieved in rats by unilateral clamping of the renal pedicle followed by reperfusion. Rats were killed at 3, 6, 12, 24, and 48 h and 7 d after reperfusion, and kidneys were microdissected and analyzed by histology and Northern and Western blots. Changes in OP-1 mRNA were determined by measuring the ratio of OP-1/glyceraldehyde 3-phosphate dehydrogenase signals for each OP-1 transcript (4.0 and 2.4 kb) from ischemic, opposite, and sham-operated rats. The OP-1 mRNA content for transcript 4.0 kb was fivefold lower in the whole ischemic kidney compared with that in sham animals 24 h after reperfusion. In the ischemic medulla, OP-1 mRNA was strikingly downregulated 20-fold when compared with the ischemic cortex. Results for transcript 2.4 kb and for the other time points were comparable. OP-1 mRNA expression was also affected in the opposite medulla compared with the sham medulla. However, only in the ischemic medulla was the relative OP-1 content significantly lower at all time points. Similar results were obtained when analyzing OP-1 protein by Western blot at 24 h after reperfusion. Seven days after reperfusion, the levels of OP-1 mRNA returned to baseline. In conclusion, kidney OP-1 mRNA and protein are selectively downregulated in the medulla after acute ischemic renal injury. OP-1 modulation may be a key element for kidney repair.

Female genital mutilation. Female circumcision. Who is at risk in the U.S.?

Female genital mutilation/female circumcision (FGM/FC) refers to a group of traditional practices that involve partial or total removal of the external female genitalia or other injury to the female genital organs for cultural, religious, or other non-therapeutic reasons. These practices are usually performed by a nonmedical practitioner in the home or other nonclinical setting. Complications occurring immediately after the practice as well as those encountered months and years afterward can result in disability or premature death. In 1996 Congress directed the Department of Health and Human Services to develop estimates of the prevalence of women and girls with or at risk for FGM/FC in the United States. This paper reports those estimates, as derived by the Centers for Disease Control and Prevention, which showed that in 1990 there were an estimated 168,000 girls and women living in the United States with or at risk for FGM/FC.

On-site, rapid HIV testing with same-day results and counseling.

BACKGROUND: New rapid HIV antibody tests have allowed provision of results and result-specific counseling on the day on initial visit, and have the potential to increase the efficiency of HIV counseling and testing. METHODS: To evaluate the use of rapid testing with same-day results in public clinics, the Single Use Diagnostic System HIV-1 rapid assay was used for a 3-month period at an anonymous testing clinic and a sexually transmitted disease (STD) clinic in Dallas, Texas. Non-reactive rapid test results were reported as HIV-negative. Reactive results were reported as 'preliminary positive'. These procedures were compared with standard testing during a baseline period, with respect to number of clients receiving results and post-test counseling, client satisfaction, counselor acceptance, cost and effectiveness at reducing HIV risk. RESULTS: Rapid testing resulted in an increase in the number of persons learning their serostatus: a 4% increase for uninfected and a 16% increase for infected clients at the Anonymous Testing Clinic; a 210% increase for uninfected patients and a 23% increase for infected patients at the STD clinic. Rapid testing resulted in a cost saving of US$ 11 per test in both the anonymous and STD clinics. Of those previously tested, 88% responded that they preferred the rapid test. In the year following initial HIV test, clients tested with rapid and standard procedures were equally likely to return to the clinic with a new STD (odds ratio, 0.97; 95% confidence interval, 0.7-1.4). CONCLUSIONS: Rapid, on-site HIV testing was feasible, preferred by clients, and, resulted in significant improvement in the number of persons learning their serostatus, without increasing the costs or decreasing the effectiveness of counseling and testing.

Managed care--issues and opportunities: a view from the CDC.

Major changes are occurring in the health system in the United States. Currently the term managed care is most synonymous with this era of change. The Centers for Disease Control, as the nation's federal agency responsible for prevention of morbidity and premature mortality, offers its perspective on the issues and opportunities for prevention and community health promotion presented in managed care, describes some of its collaborative projects with public health agencies and managed care organizations, and discusses some of the prevention aspects of HEDIS 3.0.

Ablation of the murine alpha myosin heavy chain gene leads to dosage effects and functional deficits in the heart.

The alpha-myosin heavy chain (alpha-MyHC) is the major contractile protein expressed in the myocardium of adult mice. We have produced mice carrying a null mutation of alpha-MyHC by homologous recombination in murine ES cells. Homozygous null animals die between 11 and 12 d in utero of gross heart defects, while alpha-MyHC+/- heterozygotes survive and appear externally normal. The presence of a single functional alpha-MyHC+ allele in heterozygous animals results in reduced levels of the transcript and protein as well as fibrosis and alterations in sarcomeric structure. Examination of heart function using a working heart preparation revealed severe impairment of both contractility and relaxation in a subset of the alpha-MyHC+/- animals. Thus, two alpha-MyHC+ alleles are necessary for normal cardiac development, and hemizygosity for the normal allele can result in altered cardiac function.

Construction of an ultrasound biopsy phantom.

The technique for construction of an agar-based ultrasound biopsy phantom is described. Features include tissue equivalent reflectivity, long life and non-shadowing targets. The phantom is useful for learning the necessary co-ordination between needle and probe for ultrasound needle guidance. This skill should initially be practised in vitro, on a device such as this.

Counseling and testing for HIV prevention: costs, effects, and cost-effectiveness of more rapid screening tests.

New rapid human immunodeficiency virus (HIV) antibody tests permit many individuals to receive test results and appropriate counseling at one clinic visit. Because currently used tests require significant time for processing, all individuals must return for a second visit for test results and counseling. Since return rates for the second visit are low, the more rapid tests present an opportunity to improve the efficiency of HIV counseling and testing. The authors compared the costs and effectiveness of the currently used counseling and testing procedure and a streamlined procedure made possible by the new, more rapid screening tests. When test-positive clients are given preliminary screening test results, the rapid procedure is more cost-effective than the current procedure. Since over 90% of the clients in most clinics will test negative, the rapid counseling and testing procedure allows the vast majority of clients to be counseled and tested and to receive their results and posttest counseling in one visit. However, in the case where the goal of HIV counseling and testing is to focus only on infected individuals, if information regarding a positive result from the rapid screening test is not given to clients at the initial visit before a confirmatory test is performed, then the rapid counseling and testing procedure is not more cost-effective than the current procedure.

Performance of a rapid, on-site human immunodeficiency virus antibody assay in a public health setting.

Rapid, on-site human immunodeficiency virus (HIV) testing has the potential to improve the delivery of prevention services in publicly funded counseling and testing sites. The Single Use Diagnostic System (SUDS) HIV-1 is the only rapid enzyme immunoassay (EIA) approved for diagnostic use in the United States. To evaluate the feasibility of using SUDS in public clinics and to validate the test's performance in a public health laboratory, we conducted blinded SUDS testing on plasma sent for HIV testing. From 19 March through 30 June 1993, 1,923 consecutive samples from a sexually transmitted diseases clinic and an HIV counseling and testing clinic were tested on site with SUDS. Tests done in the first two weeks with a malfunctioning centrifuge n = 402) and those done when there were excessively high temperatures in the laboratory (n = 53) were analyzed separately. Of 1,466 tests, 39 were positive by both SUDS and EIA (with Western blot [immunoblot] confirmation) and 7 were SUDS positive and EIA negative. Western blotting was used as the "gold standard" to adjudicate these discrepancies. There were no SUDS-negative and EIA-positive tests. Compared with that of EIA (with Western blot confirmation), the sensitivity of SUDS was 100% (95% confidence interval, 88.8 to 100%) and the specificity was 99.5% (95% confidence interval, 98.9 to 99.8%). The positive predictive value of SUDS was 88% in the STD clinic and 81% in the HIV counseling and testing clinic. There was a 7.7-fold increase in false positives, from 0.48 to 3.7%, when there was inadequate centrifugation and when the temperature exceeded the manufacturer's recommendations. Rapid, on-site HIV testing by the SUDS assay is feasible and practical in public health settings. The test can be performed accurately, at reasonable cost, and within the time frame of a typical clinic visit. Caution should be used, however, as two conditions adversely affected the accuracy of this test: inadequate specimen preparation and elevated temperature.

Differential phospholamban gene expression in murine cardiac compartments. Molecular and physiological analyses.

Phospholamban, the regulator of the Ca2+ pump in cardiac sarcoplasmic reticulum, is differentially expressed between murine atrial and ventricular muscles. Quantitative analyses of RNA isolated from atrial flaps and ventricular apices indicated that the phospholamban gene transcript copy number is 2.5-fold higher in the ventricle compared with the atrium of the FVB/N mouse and 6-fold higher in the ventricle compared with the atrium of the B6D2/F1 mouse strain. These findings were corroborated by in situ hybridization studies of cardiopulmonary sections from both murine strains, and phospholamban transcripts were also observed in pulmonary myocardia of both strains. Analyses of phospholamban transcript levels relative to alpha-myosin heavy chain (alpha-MHC) revealed a 3-fold higher phospholamban abundance in the ventricle compared with the atrium of the FVB/N murine strain. However, the relative mRNA level of Ca(2+)-ATPase (ratio of sarcoplasmic reticulum Ca(2+)-ATPase [SERCA2] to alpha-MHC) in the ventricle was 80% of that in the atrium. Consequently, the relative ratio of phospholamban to SERCA2 mRNA was 4.2-fold lower in the atrium than in the ventricle. The lower transcript ratio of phospholamban to SERCA2 in the atrium was associated with significantly shortened times to half-relaxation (17.40 +/- 0.71 milliseconds for atrium versus 30.58 +/- 2.04 milliseconds for ventricle), assessed in isolated superfused cardiac tissue preparations recorded at maximum length tension. Contraction times, measured as times to peak tension, were also significantly shortened in atrial muscle (27.36 +/- 0.82 milliseconds) compared with ventricular muscle (44.60 +/- 2.55 milliseconds), assessed in the same tissue preparations. These findings suggest that phospholamban gene expression is differentially regulated in murine atrial and ventricular muscles and that this differential expression may be associated with differences in the contractile parameters of these cardiac compartments.