The effect of a population bottleneck on the evolution of genetic variance/covariance structure.

It is well known that standard population genetic theory predicts decreased additive genetic variance (V(a) ) following a population bottleneck and that theoretical models including interallelic and intergenic interactions indicate such loss may be avoided. However, few empirical data from multicellular model systems are available, especially regarding variance/covariance (V/CV) relationships. Here, we compare the V/CV structure of seventeen traits related to body size and composition between control (60 mating pairs/generation) and bottlenecked (2 mating pairs/generation; average F = 0.39) strains of mice. Although results for individual traits vary considerably, multivariate analysis indicates that V(a) in the bottlenecked populations is greater than expected. Traits with patterns and amounts of epistasis predictive of enhanced V(a) also show the largest deviations from additive expectations. Finally, the correlation structure of weekly weights is not significantly different between control and experimental lines but correlations between necropsy traits do differ, especially those involving the heart, kidney and tail length.

Androgen regulation of rat renal angiotensinogen messenger RNA expression.

Renal angiotensinogen (ang-n) mRNA concentration in the male WKY rat increases significantly during puberty. Furthermore, renal angiotensinogen mRNA level in the adult female WKY rat is considerably lower than in the male. The present study investigates the role of androgen in differential renal ang-n mRNA expression. Northern and slot blot analyses with alpha-32P labeled ang-n cDNA (pRang 3) demonstrated that castration lowered ang-n mRNA levels in the male kidney by greater than or equal to 60% compared with control, suggesting that androgen may be involved with renal ang-n gene regulation. Moreover, male WKY rats castrated as weanlings and normal adult female WKY rats each implanted with testosterone displayed significant (P less than 0.05) increases in renal ang-n mRNA levels. Our observations, taken together with previous reports that androgen influences proximal tubule morphology and the tubular expression of transport proteins (e.g., Na+/H+ antiporter), may have important physiological implications for understanding the relationship between androgen and angiotensin in the regulation of tubular function.

Sodium regulation of angiotensinogen mRNA expression in rat kidney cortex and medulla.

Rat liver angiotensinogen cDNA (pRang 3) and mouse renin cDNA (pDD-1D2) were used to identify angiotensinogen and renin mRNA sequences in rat kidney cortex and medulla in rats on high and low salt diet. Angiotensinogen mRNA sequences were present in renal cortex and medulla in apparently equal proportions, whereas renin mRNA sequences were found primarily in renal cortex. Average relative signal of rat liver to whole kidney angiotensinogen mRNA was 100:3. Densitometric analysis of Northern blots demonstrated that renal cortical angiotensinogen mRNA concentrations increased 3.5-fold (P less than 0.001) and medulla, 1.5-fold (P less than 0.005) on low sodium compared with high sodium diet, whereas renal cortex renin mRNA levels increased 6.8-fold (P less than 0.0005). Dietary sodium did not significantly influence liver angiotensinogen mRNA levels. These findings provide evidence for sodium regulation of renal renin and angiotensinogen mRNA expressions, which supports potential existence of an intrarenally regulated RAS and suggest that different factors regulate renal and hepatic angiotensinogen.

In situ hybridization evidence for angiotensinogen messenger RNA in the rat proximal tubule. An hypothesis for the intrarenal renin angiotensin system.

We examined angiotensinogen gene expression in rat kidney by in situ hybridization histochemistry. Using a rat cRNA probe to angiotensinogen, we demonstrated angiotensinogen mRNA to be localized predominantly in the proximal renal tubule, with considerably lesser amounts in distal tubular segments and glomerular tufts. Previous studies have localized renin immunoreactivity to the juxtaglomerular cells, glomerular tufts, and proximal tubules. Such findings provide further evidence for a local tissue renin angiotensin system within the kidney which may influence regional function. Based on our data, we hypothesize that a major site of angiotensin production is the proximal tubule. We postulate that angiotensin synthesized in and/or around the proximal tubule may directly modulate tubular transport of sodium, bicarbonate, and water. In addition to the proximal tubule, the specific localization of the renin angiotensin components elsewhere in the kidney would also support the other proposed regional functions of the intrarenal system, including modulation of tubuloglomerular balance.

Intimal hyperplasia after vascular injury is inhibited by antisense cdk 2 kinase oligonucleotides.

The cell cycle regulatory enzyme, cdk (cyclin-dependent kinase) 2 kinase, is activated in the rat carotid artery after balloon angioplasty injury, and may mediate smooth muscle proliferation. To test the hypothesis that inhibition of the expression of this key enzyme can inhibit intimal hyperplasia, we studied the effect of antisense phosphorothioate oligodeoxynucleotides (ODN) against cdk 2 kinase administered by intraluminal delivery using hemagglutinating virus of Japan (HVJ)-liposome-mediated transfer. The specificity of antisense cdk 2 ODN was confirmed by the observation that mRNA level of cdk 2 kinase in injured vessels was markedly diminished by the antisense ODN treatment. At 2 wk after transfection, antisense cdk 2 ODN treatment (15 microM) resulted in a significant inhibition (60%) in neointima formation, compared with sense ODN-treated and untreated vessels. Since we have previously observed that cell division cycle 2 kinase mRNA was also activated after vascular injury, we administered the combination of antisense cdc 2 and cdk 2 ODN in this study. Antisense cdc 2 ODN alone (15 microM) only reduced intimal formation by 40%. Combined antisense treatment resulted in near complete inhibition of neointima formation. To understand the mechanism of the sustained effect of a single antisense ODN administration, we examined kinetics of ODN in the vessel wall. Using phosphorothioate FITC-labeled ODN, we transfected carotid artery using the HVJ-liposome method. Fluorescence localized immediately to the medial layer, and persisted up to 2 wk after transfection. These results demonstrate that a single intraluminal administration of antisense ODN directed to cell cycle regulatory genes (e.g., cdk 2 kinase) using the HVJ method can result in a sustained inhibition of neointima formation after balloon angioplasty in rat carotid injury model.

Evidence for direct local effect of angiotensin in vascular hypertrophy. In vivo gene transfer of angiotensin converting enzyme.

In vitro studies have demonstrated that angiotensin (Ang) II directly stimulates vascular smooth muscle cell (VSMC) growth. However, it is still unclear if Ang II exerts a direct effect on vascular hypertrophy in vivo independent of its effect on blood pressure. In vivo gene transfer provides the opportunity to assess the effects of increased activity of the vascular angiotensin system in the intact animal while avoiding an increase in circulating angiotensin or in blood pressure. Accordingly, we transfected the human angiotensin converting enzyme (ACE) vector into intact rat carotid arteries by the hemagglutinating virus of Japan-liposome method. 3 d after transfection, we detected increased ACE activity in the transfected artery. Immunohistochemistry localized immunoreactive ACE in the medial VSMC as well as in the intimal endothelial cells. The increase in vascular ACE activity was associated with a parallel increase in DNA synthesis as assessed by BrdU (bromo-deoxyuridine) index and vascular DNA content. This increase in DNA synthesis was abolished by the in vivo administration of an Ang II receptor-specific antagonist (DuP 753). Morphometry at 2 wk after transfection revealed an increase in the wall to lumen ratio of the ACE-transfected blood vessel as compared with control vector transfected vessels. This was accompanied by increases in protein and DNA contents without an increase in cell number. Local transfection of ACE vector did not result in systemic effects such as increased blood pressure, heart rate, or serum ACE activity. These morphological changes were abolished by the administration of the Ang II receptor antagonist. In this study, we used in vivo gene transfer to increase local expression of vascular angiotensin converting enzyme and provided proof that increased autocrine/paracrine angiotensin can directly cause vascular hypertrophy independent of systemic factors and hemodynamic effects. This approach has important potentials for defining the role of autocrine/paracrine substances in vascular biology and hypertension.

Entrainment mapping and radiofrequency catheter ablation of ventricular tachycardia in right ventricular dysplasia.

OBJECTIVES: The purpose of this study was to determine if entrainment mapping techniques and predictors of successful ablation sites previously tested in coronary artery disease can be applied to ventricular tachycardia (VT) in arrhythmogenic right ventricular dysplasia (ARVD). BACKGROUND: VT in ARVD has not been well characterized. Reentry circuits in areas of abnormal myocardium are the likely cause, but these circuits have not been well defined. METHODS: Mapping of 19 VTs in 5 patients with ARVD was performed. At 58 sites pacing entrained VT and radiofrequency current (RF) was applied to assess acute termination of VT. RESULTS: Sites classified as exits, central/proximal, inner loop, outer loop, remote bystander and adjacent bystander were identified by entrainment criteria. The reentrant circuit sites were clustered predominantly around the tricuspid annulus and in the right ventricular outflow tract (RVOT). RF ablation acutely terminated VT at 13 sites or 22% of the applications. Of the 19 VTs, eight were rendered noninducible and three were modified to a longer cycle length. In 2 patients ablation at a single site abolished two VTs. CONCLUSION: VT in ARVD shows many of the characteristics of VT due to myocardial infarction. Entrainment mapping techniques can be used to characterize reentry circuits in ARVD. The use of entrainment mapping to guide ablation is feasible.

The N + 1 difference: a new measure for entrainment mapping.

OBJECTIVES: The purpose of this study was to develop and test a new entrainment mapping measurement, the N + 1 difference. BACKGROUND: Entrainment mapping is useful for identifying re-entry circuit sites but is often limited by difficulty in assessing: 1) changes in QRS complexes or P-waves that indicate fusion, and 2) the postpacing interval (PPI) recorded directly from the stimulation site. METHODS: In computer simulations of re-entry circuits, the interval from a stimulus that reset tachycardia to a timing reference during the second beat after the stimulus was compared with the timing of local activation at the site during tachycardia to define an interval designated the N + 1 difference. The N + 1 difference was compared with the PPI-tachycardia cycle length (TCL) difference in simulations and at 65 sites in 10 consecutive patients with ventricular tachycardia (VT) after myocardial infarction and at 45 sites in 10 consecutive patients with atrial flutter. RESULTS: In simulations, the N + 1 difference was equal to the PPI-TCL difference. During mapping of VT and atrial flutter, the N + 1 difference correlated well with the PPI-TCL difference (r > or = 0.91, p < 0.0001), identifying re-entry circuit sites with sensitivity of > or = 86% and specificity of > or = 90%. Accuracy was similar using either the surface electrocardiogram or an intracardiac electrogram (Eg) as the timing reference. CONCLUSIONS: The N + 1 difference allows entrainment mapping to be used to identify re-entry circuit sites when it is difficult to evaluate Egs at the mapping site or fusion in the surface electrocardiogram.

Interruption of cytokine networks by poxviruses: lessons from myxoma virus.

Myxoma virus is an infectious poxvirus pathogen that induces a virulent systemic disease called myxomatosis in European rabbits. The disease is rapidly and uniformly fatal to susceptible rabbits and is characterized by generalized dysfunction of cellular immunity and multiple interruptions of the host cytokine network. A number of virus genes are classified as virulence factors because virus constructs bearing targeted gene disruptions induce attenuated disease symptoms. Many of these genes encode proteins that interact directly with effector elements of the host immune system. Included among these immunosubversive viral proteins are secreted mimics of host ligands or regulators (virokines) and homologues of cellular cytokine receptors (viroceptors). Five examples of these immune modulator proteins encoded by myxoma virus are reviewed: (1) myxoma growth factor, a member of the epidermal growth factor ligand superfamily; (2) SERP-1, a secreted serine proteinase inhibitor; (3) M11L, a receptor-like surface protein; (4) T2, a tumor necrosis factor receptor homologue; and (5) T7, an interferon-gamma receptor homologue. The origin of viral strategies designed to subvert immune regulation by host cytokines is considered in the context of the biology of myxoma virus within immunocompetent hosts.

Characterization of purified rabbit uterine renin: influence of pregnancy on uterine inactive renin.

Using specific antibody raised against renal renin, we have documented that the majority of the uterine renin-like activity in gravid and nongravid uteri is immunoreactive renin. To characterize its physiochemical properties, we obtained highly purified uterine renin by two affinity chromatographic steps, pepstatin and antirenin. Uterine renin has a pH optimum of 6, an apparent mol wt of 38K, and a Km of 1.7 microM for homologous substrate. These properties are identical to those of renal renin and are not influenced by the pregnant state. In the basal state, an inactive form of the uterine enzyme constitute 55 +/- 10% of the total uterine renin. During pregnancy, active renin increased 40-fold as inactive renin fell to 4 +/- 3% of the total renin concentration. The renal renin concentration fell as plasma renin increased during pregnancy. These data suggest that the increased uterine renin concentrations during pregnancy are probably due to increased local production and conversion of renin precursor to the active enzyme. This stimulation of the uterine renin level appears to be independent of renal renin.

A comparative study of the distributions of renin and angiotensinogen messenger ribonucleic acids in rat and mouse tissues.

Previous studies have reported the presence of renin mRNAs in several mouse tissues and angiotensinogen mRNAs in various rat tissues. Clarification as to whether renin and angiotensinogen mRNAs are coexpressed in the same tissues of the same animal species is important for understanding the biology of the tissue renin-angiotensin system. We employed mouse renin cDNA and rat angiotensinogen cDNA to compare tissue distributions of renin and angiotensinogen in RNAs of the rat and mouse. Both cDNA probes readily cross-hybridize with the corresponding mRNA of the other species. Our results demonstrate several patterns of distribution. Renin and angiotensinogen mRNAs are readily detected in kidney and adrenals of both species. In brain and heart, angiotensinogen mRNAs are present in concentrations that far exceed renin mRNA levels in these organs in both species. In mouse and rat livers, angiotensinogen, but not renin, mRNA is demonstrated. In rat testis, only renin mRNA can be detected, whereas in mouse testes both renin and angiotensinogen mRNA are present. In CD-1 male mouse submandibular gland, renin mRNA exists in high concentrations, whereas angiotensinogen mRNA is present in low levels. In contrast, neither renin nor angiotensinogen mRNA could be detected in rat salivary gland. In summary, our study demonstrates the widespread codistribution of renin and angiotensinogen mRNAs in many tissues of both species, allowing for the possibility of local angiotensin production. However, tissue and species differences in these gene expressions also exist. Understanding differential tissue expressions of these genes will provide additional important insight into the biology of the renin-angiotensin system.

Identification of renin and angiotensinogen messenger RNA sequences in mouse and rat brains.

Components of the renin angiotensin system have been demonstrated in mouse and rat brains. However, local synthesis of renin has not been documented. In this study, we employed mouse submandibular gland renin complementary DNA (pDD-1D2) and rat liver angiotensinogen complementary DNA (pRang3) to examine whether renin and angiotensinogen RNA sequences exist in mouse and rat brain. Angiotensinogen messenger RNA sequences were readily demonstrable in whole rat and mouse brain using Northern blot hybridization analysis. Using large quantities (greater than 100 micrograms) of brain total RNA and the sensitive complementary RNA probe, we were able to detect low levels of renin RNA sequences in the brains of both species. The relatively low concentration of brain renin messenger RNA and high concentration of angiotensinogen messenger RNA raises several interesting questions about the distribution of these two proteins and their relative contribution to activity of the brain renin-angiotensin system. In summary, our data demonstrate the expression of both renin and angiotensinogen genes in mouse and rat brains and provide definitive evidence for an independent endogenous brain renin angiotensin system.

Tissue-specific regulation of renin expression in the mouse.

Increasing biochemical evidence suggests that the renin-angiotensin system may be present in may extrarenal tissues. We have employed the mouse submandibular gland renin complementary DNA (pDD-1D2) and the rat liver angiotensinogen complementary DNA (pRang 3) to demonstrate that renin and angiotensinogen messenger RNAs are expressed in the mouse kidney, submandibular gland, heart, adrenal, brain, and testis. To elucidate the factors that influence local tissue renin-angiotensin expressions, we studied tissue renin messenger RNA and enzymatic levels of male mice in response to sodium depletion and castration. Sodium depletion resulted in increased renin expression in the kidney, heart, and adrenal, but not in the submandibular gland and testis. Castration lowered renin levels in all extrarenal tissues but appeared to increase renin level in the kidney. Taken together, the above data demonstrate tissue-specific regulation of renin expression and imply different functions for the sodium responsive and nonresponsive systems.

Ablation therapy for cardiac arrhythmias.

Ablation has become an important and, in some cases, the first-line therapy for a number of tachyarrhythmias. The feasibility of treating arrhythmias with ablation was initially demonstrated with surgical ablation techniques. Recently, catheter ablation techniques have replaced the surgical approach in nearly all cases. Catheter ablation is highly effective for the Wolff-Parkinson-White syndrome, atrioventricular nodal reentry, and atrial ectopic tachycardia. It is effective for atrial flutter, although approximately one quarter of patients treated with catheter ablation continue to require therapy for concomitant atrial fibrillation. The surgical maze procedure has proved to be feasible for preventing atrial fibrillation. The risks and long-term efficacy of catheter ablation maze procedures for atrial fibrillation need to be defined. The efficacy of ablation for ventricular tachycardia varies with the type of tachycardia. Catheter ablation is very effective for the rare idiopathic ventricular tachycardias that occur in structurally normal hearts and for bundle-branch reentry ventricular tachycardia, which occurs most frequently in patients with dilated cardiomyopathy. When performed at an experienced center, surgical ablation is an excellent option for selected patients with ventricular tachycardia due to prior myocardial infarction who have a discrete aneurysm but otherwise well-preserved ventricular function. Catheter ablation shows promise for this arrhythmia, but it can be offered only to those patients who have relatively slow tachycardias that allow catheter mapping. Substantial advances in mapping and ablation technology will continue to occur, allowing nonpharmacologic control of cardiac arrhythmias to be achieved in an ever greater number of patients.

Isolation of a yeast artificial chromosome contig spanning the X chromosomal translocation breakpoint in a patient with Rett syndrome.

Rett syndrome is a neurodevelopmental disorder observed exclusively in females. A de novo X;3 translocation was detected in a patient (TH) with Rett syndrome. The X chromosomal breakpoint maps to Xp21.3 between the distal end of the Duchenne muscular dystrophy (DMD) gene and the DXS28 (C7) locus. To determine if this translocation caused the Rett syndrome in this patient, our efforts focused on mapping and cloning of the X chromosomal breakpoint in this patient. Toward these goals, we generated a set of radiation-reduced hybrid cell lines for the short arm of the X chromosome to use as a source for region-specific markers. Using Alu-PCR, 13 new DNA markers were isolated from a radiation-reduced hybrid, which retained both DMD and DXS28. These markers were localized within Xp21 using DNA from males with various interstitial deletions in this region. Two new markers, K23-2p and K23b-1, were found to be closer flanking markers to the X chromosomal breakpoint than DMD and DXS28. Long range restriction mapping using K23-2p and K23b-1 determined that the maximum distance between them was 800 kb. Several of the new markers were developed into sequence tagged-sites and were used to isolate yeast artificial chromosome (YAC) clones. A total of 22 YAC clones was isolated and characterized; these YACs were then developed into 3 large contigs in the Xp21.3 region. This effort resulted in the cloning of the region containing the X chromosomal translocation breakpoint of the Rett syndrome patient in a 170-kb YAC clone.

Implantable cardioverter defibrillators in heart failure.

Implantable cardioverter defibrillators provide effective and reliable treatment of spontaneous VT and VF. These devices can be expected to decrease the risk for arrhythmic death in patients with heart failure but do not improve overall survival when death from severe pump dysfunction is imminent. Appropriate patient selection is a major aspect of arrhythmia management. Future devices will incorporate features that have the potential to reduce atrial arrhythmias, improve ventricular function, monitor hemodynamics, and prevent sudden arrhythmic death.

Construction of a shuttle vector containing a single O6-methylguanine: a probe for mutagenesis in mammalian cells.

A shuttle vector, pKE15, was constructed for investigating the mechanisms by which single carcinogen-DNA adducts induce mutations in mammalian cells. pKE15 contains the SV40 origin of replication, the neomycin resistance gene, SV40 polyadenylation sequences and the pML2 origin of replication. Transfection of pKE15 into CHO cells established the G418-resistant phenotype; the frequency of G418-resistant clones was approximately 10(-4), a value that is similar to those obtained with other SV40-based vectors expressing the neomycin resistance gene. A tetranucleotide containing O6-methylguanine, a DNA adduct formed by carcinogenic alkylating agents, was incorporated into a 4-base gap positioned in the center of a PstI site. The tetranucleotide containing the adduct was physically mapped to a 14-base-pair region of the shuttle vector that included the ligation target, the PstI site. It was incorporated approximately equally into either of the complementary strands of the shuttle vector. The ligation efficiency of the tetranucleotide into the gapped genome was approximately 100% and was independent of the concentration of tetranucleotide used at concentrations ranging over one order of magnitude. The potential applications of the site-specifically modified genome for establishing the mutagenic fate of O6-methylguanine in repair-proficient and -deficient CHO cells are discussed.

Caring for the person receiving ventilatory support at home: care givers' needs and involvement.

Little is known of the needs of family members who provide home care to persons receiving ventilatory support (VS). In a structured interview of 44 care givers of 29 persons receiving VS, finances, provisions for emergencies, information, family relationships, and continuity of care were ranked as the most important needs. Needs for support services were ranked highest in importance to the care givers of persons receiving continuous VS, care givers who did not live in, and children of persons receiving VS. Parents of persons receiving VS ranked educational needs and attention to other family members of highest importance. Handling emergencies was most important to less experienced care givers, whereas financial and respite needs were more important to experienced and full-time care givers. Findings suggest the need for early participation of community health care professionals in care giver preparation, negotiations with third-party payers, 24-hour support services, information networks, and long-term, comprehensive coverage of services.

Specialization in adult health/medical surgical nursing: what does it mean?

In this descriptive study, all NLN-accredited master's programs in the United States were surveyed to describe the content and form of the Adult Health/Medical Surgical (AH/MS) Nursing component of the curricula. An 82% response rate was achieved. The study confirmed a high degree of diversity in the AH/MS Nursing component of master's degree programs. It revealed a lack of consensus on the amount and kind of content necessary for specialization in AH/MS Nursing. Few curricula were based on an identified nursing model/theoretical framework, although students in most programs were required to utilize a nursing model as a framework for advanced or specialty practice. The structure of class and clinical learning activities varied markedly. Ideas about graduate faculty's role in clinical instruction were dissimilar. Findings from the study could guide decision making for the AH/MS Nursing component of master's-level curricula. Curriculum revision seems to be inherent in the nurse-faculty role, yet curriculum decision making can be an onerous task. This is especially true in today's master's nursing programs where clinical specialty areas are not well defined and diversity and ambiguity abound. As faculty members in a master's nursing program where numerous specialty areas had been developed, we became increasingly frustrated in our attempts to identify specific knowledge and skills that should be acquired by students specializing in adult health nursing. Traditionally, this rather broad specialty area has been a "catch-all" for the study of a wide variety of age groups and health alterations.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of learning needs of nurse educators: continuing education implications.

Because of current structure, content, and outcomes of master's nursing curricula, administrators responsible for hiring nurse educators often have no choice but to employ clinical experts as teachers. This article reports on the results of an investigation designed to ascertain the self-reported learning needs of nurse educators in a southern state. This investigation confirmed that nurse educators have numerous learning needs specific to fulfillment of the nurse educator role. Findings suggest that learning needs differed according to the educator's level of academic preparation and type of employment setting. The results provide useful insights for individuals planning continuing educational offerings for nurse educators with varied backgrounds.