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The genetic and epigenetic factors underlying the variable penetrance of homoplasmic mitochondrial DNA mutations are poorly understood. We investigated a 16-year-old patient with hypertrophic cardiomyopathy harboring a homoplasmic m.4277T>C mutation in the mt-tRNA(Ile) (MTTI) gene. Skeletal muscle showed multiple respiratory chain enzyme abnormalities and a decreased steady-state level of the mutated mt-tRNA(Ile). Transmitochondrial cybrids grown on galactose medium demonstrated a functional effect of this mutation on cell viability, confirming pathogenicity. These findings were reproduced in transmitochondrial cybrids, harboring a previously described homoplasmic m.4300A>G MTTI mutation. The pathogenic role of the m.4277T>C mutation may be ascribed to misfolding of the mt-tRNA molecule, as demonstrated by the altered electrophoretic migration of the mutated mt-tRNA. Indeed, structure and sequence analyses suggest that thymidine at position 4277 of mt-tRNA(Ile) is involved in a conserved tertiary interaction with thymidine at position 4306. Interestingly, the mutation showed variable penetrance within family members, with skeletal muscle from the patient's clinically unaffected mother demonstrating normal muscle respiratory chain activities and steady-state levels of mt-tRNA(Ile), while homoplasmic for the m.4277T>C mutation. Analysis of mitochondrial isoleucyl-tRNA synthetase revealed significantly higher expression levels in skeletal muscle and fibroblasts of the unaffected mother when compared with the proband, while the transient over-expression of the IARS2 gene in patient transmitochondrial cybrids improved cell viability. This is the first observation that constitutively high levels of aminoacyl-tRNA synthetases (aaRSs) in human tissues prevent the phenotypic expression of a homoplasmic mt-tRNA point mutation. These findings extend previous observations on aaRSs therapeutic effects in yeast and human.

The Pure Parsimony Haplotyping (PPH) problem is a NP-hard combinatorial optimization problem that consists of finding the minimum number of haplotypes necessary to explain a given set of genotypes. PPH has attracted more and more attention in recent years due to its importance in analysis of many fine-scale genetic data. Its application fields range from mapping complex disease genes to inferring population histories, passing through designing drugs, functional genomics and pharmacogenetics. In this article we investigate, for the first time, a recent version of PPH called the Pure Parsimony Haplotype problem under Uncertain Data (PPH-UD). This version mainly arises when the input genotypes are not accurate, i.e., when some single nucleotide polymorphisms are missing or affected by errors. We propose an exact approach to solution of PPH-UD based on an extended version of Catanzaro et al.[1] class representative model for PPH, currently the state-of-the-art integer programming model for PPH. The model is efficient, accurate, compact, polynomial-sized, easy to implement, solvable with any solver for mixed integer programming, and usable in all those cases for which the parsimony criterion is well suited for haplotype estimation.

We present an integer programming model for human leukocyte antigen (HLA) association studies based on the parsimony criterion. The model is simple, compact, easy to implement, and able to handle datasets containing up to 200 phenotypes. Computational experiments carried out on patients affected by psoriasis and severe alopecia areata show that the model is consistent with the experimental haplotype frequencies, showing, for the considered diseases at least, a high reliability of the predictions. These promising results provide perspective on computer-aided association studies and encourage the development of efficient exact computational approaches for haplotype estimation., (Copyright 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Background: Distance matrix methods constitute a major family of phylogenetic estimation methods, and the minimum evolution (ME) principle (aiming at recovering the phylogeny with shortest length) is one of the most commonly used optimality criteria for estimating phylogenetic trees. The major difficulty for its application is that the number of possible phylogenies grows exponentially with the number of taxa analyzed and the minimum evolution principle is known to belong to the NRho- hard class of problems., Results: In this paper, we introduce an Ant Colony Optimization (ACO) algorithm to estimate phylogenies under the minimum evolution principle. ACO is an optimization technique inspired from the foraging behavior of real ant colonies. This behavior is exploited in artificial ant colonies for the search of approximate solutions to discrete optimization problems., Conclusion: We show that the ACO algorithm is potentially competitive in comparison with state-of-the-art algorithms for the minimum evolution principle. This is the first application of an ACO algorithm to the phylogenetic estimation problem.

The General Time Reversible (GTR) model of nucleotide substitution is at the core of many distance-based and character-based phylogeny inference methods. The procedure described by Waddell and Steel (1997), for estimating distances and instantaneous substitution rate matrices, R, under the GTR model, is known to be inapplicable under some conditions, ie, it leads to the inapplicability of the GTR model. Here, we simulate the evolution of DNA sequences along 12 trees characterized by different combinations of tree length, (non-)homogeneity of the substitution rate matrix R, and sequence length. We then evaluate both the frequency of the GTR model inapplicability for estimating distances and the accuracy of inferred alignments. Our results indicate that, inapplicability of the Waddel and Steel's procedure can be considered a real practical issue, and illustrate that the probability of this inapplicability is a function of substitution rates and sequence length.We also discuss the implications of our results on the current implementations of maximum likelihood and Bayesian methods.

Motivation: The general-time-reversible (GTR) model is one of the most popular models of nucleotide substitution because it constitutes a good trade-off between mathematical tractability and biological reality. However, when it is applied for inferring evolutionary distances and/or instantaneous rate matrices, the GTR model seems more prone to inapplicability than more restrictive time-reversible models. Although it has been previously noted that the causes for intractability are caused by the impossibility of computing the logarithm of a matrix characterised by negative eigenvalues, the issue has not been investigated further., Results: Here, we formally characterize the mathematical conditions, and discuss their biological interpretation, which lead to the inapplicability of the GTR model. We investigate the relations between, on one hand, the occurrence of negative eigenvalues and, on the other hand, both sequence length and sequence divergence. We then propose a possible re-formulation of previous procedures in terms of a non-linear optimization problem. We analytically investigate the effect of our approach on the estimated evolutionary distances and transition probability matrix. Finally, we provide an analysis on the goodness of the solution we propose. A numerical example is discussed.

The extent to which the clinical effects of angiotensin receptor blockers (ARB) are related to ambient renin system activity remains poorly defined. Therefore, we measured blood pressure (BP), large (C1) and small (C2) arterial compliance, systemic vascular resistance (SVR), plasma renin activity (PRA), and the 24-h urinary excretion of sodium (UNaV) and aldosterone before and 1, 2, 4, and 24 h after administration of single doses of placebo, and 5, 20, and 40 mg of the ARB olmesartan medoximil to 12 unmedicated normotensive subjects. In the basal state, SVR was inversely related to UNaV (r = -0.3, P =.04); the greater the UNaV, the more vasodilated the subject. Indices of arterial compliance, both C1 (r = -0.32, P =.03) and C2 (r = -0.35, P =.02) were inversely related to the basal PRA. Renin also predicted olmesartan-induced changes in C1 (r = 0.43, P =.004) and C2 (r = 0.33, P =.04). The greater the basal PRA, the less the arterial compliance, and the more compliance improved after olmesartan. Both systolic (P =.003) and diastolic (P <.0001) BP fell significantly on olmesartan compared with placebo (MANOVA with time), and relations were observed between the basal PRA and olmesartan-induced changes in pressure (systolic BP: r = -0.414, P =.012; diastolic BP: r = -0.561 P <.0001)-the greater the initial PRA, the more olmesartan lowered BP. Furthermore, the more pressure fell, the more PRA rose reciprocally (r = -0.44, P =.007). Finally, aldosterone excretion fell (sig = 0.05) on each dose of olmesartan compared with placebo. We conclude that 1) the inverse relation of UNaV and SVR illustrates the reciprocal role of volume versus constrictor factors in maintaining normal BP; and 2) PRA is a physiologic determinant of arterial compliance in normal individuals and of the response to the ARB olmesartan. Measurement of PRA may help to predict clinical ARB responses in individual subjects.

Delayed graft function (DGF), defined as persistent renal failure that requires dialysis within the first week after kidney transplantation, occurs commonly after cadaveric renal transplantation (CRT). This has important implications for long-term outcome because the 1-year allograft survival rate is significantly reduced when DGF occurs. The mechanisms contributing to the development of DGF are not well established. However, several lines of evidence indicate that excess renin system activity, in both the cadaver kidney donor and recipient, contributes importantly to the pathogenesis of DGF. If this hypothesis can be verified in clinical studies, then pharmacologic agents that interrupt the renin-angiotensin system (eg, type 1 angiotensin II receptor blockade, angiotensin converting enzyme inhibition, and beta-adrenergic blockade) in the donor and recipient might significantly improve the outcome of cadaveric renal transplants.

Background: Inhibition of the renin-angiotensin system is known to raise serum potassium [K(+)] levels in patients with renal insufficiency or diabetes. No study has evaluated the comparative effects of an angiotensin-converting enzyme (ACE) inhibitor versus an angiotensin receptor blocker (ARB) on the changes in serum [K(+)] in people with renal insufficiency., Methods: The study was a multicenter, randomized, double crossover design, with each period lasting one month. A total of 35 people (21 males and 14 females, 19 African Americans and 16 Caucasian) participated, with the mean age being 56 +/- 2 years. Mean baseline serum [K(+)] was 4.4 +/- 0.1 mEq/L. The glomerular filtration rate (GFR) was 65 +/- 5 mL/min/1.73 m(2), and blood pressure was 150 +/- 2/88 +/- 1 mm Hg. The main outcome measure was the difference from baseline in the level of serum [K+], plasma aldosterone, and GFR following the initial and crossover periods., Results: For the total group, serum [K(+)] changes were not significantly different between the lisinopril or valsartan treatments. The subgroup with GFR values of < or = 60 mL/min/1.73 m(2) who received lisinopril demonstrated significant increases in serum [K(+)] of 0.28 mEq/L above the mean baseline of 4.6 mEq/L (P = 0.04). This increase in serum [K(+)] was also accompanied by a decrease in plasma aldosterone (P = 0.003). Relative to the total group, the change in serum [K(+)] from baseline to post-treatment in the lisinopril group was higher among those with GFR values of < or = 60 mL/min/1.73 m(2). The lower GFR group taking valsartan, however, demonstrated a smaller rise in serum [K(+)], 0.12 mEq/L above baseline (P = 0.1), a 43% lower value when compared with the change in those who received lisinopril. This blunted rise in [K(+)] in people taking valsartan was not associated with a significant decrease in plasma aldosterone (P = 0.14)., Conclusions: In the presence of renal insufficiency, the ARB valsartan did not raise serum [K(+)] to the same degree as the ACE inhibitor lisinopril. This differential effect on serum [K(+)] is related to a relatively smaller reduction in plasma aldosterone by the ARB and is not related to changes in GFR. This study provides evidence that increases in serum [K(+)] are less likely with ARB therapy compared with ACE inhibitor therapy in people with renal insufficiency.

Previous research indicates a role of pheromones in novel male-induced early pregnancy disruptions. Although some reports suggest that urine alone is sufficient to produce this effect, others raise procedural concerns and fail to replicate such effects. On Days 1 to 5 after insemination, female CF-1 mice had their nasal regions repeatedly painted with water, urine from males housed in isolation, or urine from males housed in proximity to females. Almost all (87.5%) of the control females delivered litters. There was a small nonsignificant reduction in proportion parturient (78.5%) among females exposed to urine of males housed without social contact. The proportion of females parturient (57.1%) after treatment with urine from males housed in proximity to females was significantly different from controls. The magnitude of the effect of socially stimulated male urine is substantially less than that recorded when males are housed directly above inseminated females separated by a wire-mesh grid. These data suggest that production of pregnancy-disrupting male pheromones is stimulated by contact with conspecifics.

Prorenin is expressed in certain extrarenal tissues, but normally only the kidneys process prorenin to renin and secrete renin into the circulation. Although transgenic animal lines containing the human renin (hREN) structural gene with either 0.9-kb or 3-kb 5'-flanking DNA express the transgene appropriately in renal juxtaglomerular cells and secrete hREN into the circulation, the source of the circulating renin is not known. In the present study, we observed that 13-kb hREN transgenic mice that contain the structural gene and 0.9-kb 5'-flanking DNA express hREN mRNA in many unusual tissues. We also observed that circulating hREN levels in 13-kb hREN mice increased after bilateral nephrectomy. These results suggested that the hREN gene is expressed at inappropriate locations where prorenin might be processed to renin. To determine if more distal sequences flanking the hREN gene might contribute to cell and tissue specificity, we used a 45-kb hREN genomic fragment that contained the structural gene and about 25-kb 5'- and 8-kb 3'-flanking DNA sequences to generate 3 separate transgenic lines that contained the intact transgene sequences. Ribonuclease protection assays revealed a much narrower tissue distribution of hREN expression than in the 13-kb hREN transgenic mice. In each 45-kb hREN line, hREN mRNA was present only in the kidney, adrenal, lung, eye, ovary, and brain. Moreover, 24 hours after nephrectomy, human plasma renin fell to very low levels, indistinguishable from those of nontransgenic littermates, indicating that their circulating hREN is of renal origin. These studies suggest that sequences flanking the structural gene, missing from previous hREN transgenic lines, suppress renin gene expression at inappropriate extrarenal sites where cellular proteases, to which prorenin is not normally exposed, could convert prorenin to renin, resulting in abnormal secretion of renin into the plasma.

To create physiological models of the human renin-angiotensin system in transgenic animals, the component genes should be expressed in the correct tissues and cells and respond appropriately to physiological stimuli. We recently showed that mice carrying a 45-kb human renin genomic fragment, containing approximately 25 kb 5'-flanking DNA and 6 kb 3'-flanking DNA, express the transgene in a highly cell- and tissue-specific pattern. More importantly, in contrast to previous models, human renin in the circulating plasma of these mice is derived exclusively from the kidneys. In the present study, we tested the responses of both human and mouse renal renin expression and secretion of the 45-kb hREN transgenic mice to a variety of physiological and pharmacological stimuli. A sodium-deficient diet, angiotensin-converting enzyme inhibition, and beta1-adrenergic stimulation each increased both human and mouse plasma renin concentration significantly, whereas elevated blood pressure and/or increased plasma angiotensin II levels suppressed them. Human and mouse renal renin mRNA levels changed similarly but to a lesser degree. These studies demonstrate that human renin synthesis and secretion respond appropriately in 45-kb hREN mice to physiological stimuli. This most likely results from appropriate cell-specific expression of the transgene conferred by the extended transgene flanking sequences.

Despite the strong conservation of proximal 5'-flanking DNA sequences, cell transfection and transgenic animal studies have failed to provide a unifying hypothesis to explain the expression of both mouse and human renin genes. Recently, sequences contained in the mouse Ren-1c gene 5'-flanking DNA (-2866 to -2625) were shown to contain an enhancer-like element that stimulates Ren-1c promoter activity in renin-expressing As4.1 cells approximately 80-fold. Earlier studies using transgenic mice had suggested that this same region is required for the cell-specific expression of mouse renin genes. Since existing human renin genomic clones lack sequences homologous to the mouse renin enhancer, we isolated several human P1 and P1 artificial chromosome genomic clones that contain > 80 kb spanning the human renin gene. Analysis of these clones by Southern blot hybridization and long-rang polymerase chain reaction showed that they contain sequences homologous to the mouse enhancer at approximately 12 kb upstream of the transcription start site. Mouse and human sequences were 59% identical over a 650-bp region that contained the minimal enhancer from the mouse Ren-1c gene. However, a 1-kb fragment containing the entire human enhancer homology failed to stimulate human renin promoter activity in transiently transfected As4.1 cells. Further deletional analysis showed that a 220-bp region of the human sequence highly conserved in the mouse Ren-1c gene exhibited up to 47-fold transcriptional stimulation, although this was lower than the maximal effect exhibited by the minimal mouse enhancer (223-fold). Taken together, these observations suggest that sequences surrounding the conserved enhancer core stimulate enhancer activity in the mouse gene but suppress activity in the human gene. The high transcriptional activity of the mouse enhancer may have evolved to support the exceptionally high plasma renin concentrations found in mice. However, the enhancer core and surrounding conserved sequences may play an additional role in directing cell specificity.

We measured plasma prorenin and renin levels, renal renin mRNA, renal anti-renin and anti-prorenin-prosequence immunoreactivity, and blood pressure in maturing Brookhaven Dahl salt-sensitive (Dahl S) and salt-resistant (Dahl R) rats during 14 days of low (0%), medium (0.4%), or high 4%) NaCl diets. Blood pressure was higher in Dahl S rats and did not increase with high NaCl. Seven-week-old Dahl R rats had twofold and sixfold higher levels of plasma prorenin and renal prosequence immunoreactivity, respectively, which by 9 weeks were the same as in Dahl S rats. The anti-renin antiserum, BR1-5, was found to detect prorenin better than renin; Dahl S rats had suppressed renal anti-renin immunoreactivity relative to Dahl-R rats. Dahl R rats were unresponsive to high NaCl, whereas in Dahl S rats, plasma renin and renal prosequence immunoreactivity fell by 90% (P < .01), renal anti-renin immunoreactivity and renal renin MRNA fell by 35% (P < .05 for both), and plasma prorenin fell by 30% (P = NS). NaCl depletion increased prorenin/renin parameters similarly in both strains. There were direct relationships among all of the prorenin/renin parameters. Between low and high salt diets in Dahl S rats, plasma renin increased 20-fold, plasma total renin (renin plus prorenin) and renal renin mRNA both increased threefold, and plasma prorenin increased twofold. The results indicate that under steady-state conditions, plasma and renal renin/prorenin parameters change concordantly and that plasma total renin (renin plus prorenin) reflects changes in renal renin mRNA. The lower blood pressure of Dahl R rats is associated with later maturation-related declines in plasma and renal prorenin. Suppression of plasma renin may delay the salt-induced blood pressure rise in Dahl S rats. Finally, the renin system and blood pressure of Dahl R rats have remarkable disregard for a high salt diet.

Renin can be detected in cardiovascular and other tissues but it disappears after bilateral nephrectomy indicating that tissues can take up or bind renal renin from the circulation. If renin uptake is the result of specific binding, plasma prorenin may be a natural antagonist of tissue directed renin-angiotensin systems. To investigate if specific prorenin/renin uptake occurs in rat tissues, binding studies were performed, with rat microsomal membrane preparations using recombinant rat prorenin metabolically labeled with 35S-methionine as a probe. A high affinity binding site for both renin and prorenin was identified. Affinities for prorenin and renin were approximately 200 and 900 pmol/L, respectively. Binding was reversible, saturable, and pH and temperature dependent. The relative binding capacities of membranes from various rat tissues were as follows (fmol/mg): renal cortex (55), liver (54), testis (63), lung (31), brain (18), renal medulla (15), adrenal (17), aorta (7), heart (4), and skeletal muscle (1). Bound prorenin was displaced by rat and human renin or prorenin but not by the prosequence of rat prorenin, angiotensin I or II, rat or human angiotensinogen, the renin inhibitor SQ30697, atrial natriuretic factor, amylase, insulin, bovine serum albumin, hemoglobin, heparin, lysozyme, ovalbumin, cytochrome C, pepsin, pepsinogen, ribonuclease A, mannose-6-phosphate, alpha-methyl mannoside, gonadotropin releasing hormone, or an antibody to hog renin binding protein. these results demonstrate specific binding of prorenin to a site in rat tissues, herein named ProBP, that also binds renin. It is possible that differences in prorenin/renin binding capacity determine the activity of tissue-directed renin-angiotensin systems and that prorenin is a natural antagonist. Alternatively, a prorenin/renin receptor may have been identified that may function by transducing an intracellular signal.

Inseminated female CF-1 mice (Mus musculus) were exposed on days 1 to 5 of pregnancy to unfamiliar outbred males. In the first experiment, inseminated females were each housed directly with the sire, a preputialectomized male, or an intact male. Both types of novel male attempted to mate with the female during this period, unlike the sire. Reinsemination occurred in a significant proportion of the females that were exposed to novel males; this effect was equivalent for preputialectomized and intact males. In two subsequent experiments, we refined a paradigm of indirect exposure to novel males through a wire-mesh grid, which prevents mating and reinsemination. Two or three males housed directly above each female through a grid disrupt pregnancy in most cases, but housing the males below the females is much less likely to do so. In a final experiment, each inseminated female was housed below two males that were either preputialectomized or sham-preputialectomized. Whereas 29 of 33 undisturbed controls were parturient, only eight of 32 females exposed to sham-preputialectomized males and six of 32 exposed to preputialectomized males were parturient. These results suggest that nonvolatile pheromones are involved in novel-male-induced pregnancy disruptions, but that preputial gland emissions are not necessary for such disruptions.

Background: The most common form of idiopathic ventricular tachycardia (VT) is repetitive monomorphic VT (RMVT), which is characterized by frequent ventricular ectopy and salvos of nonsustained VT with intervening sinus rhythm. Unlike most other forms of idiopathic VT, this tachycardia typically occurs at rest and is nonsustained. The mechanism of RMVT is undefined. Because of a common site of origin, the right ventricular outflow tract (RVOT), we hypothesized that RMVT is mechanistically related to paroxysmal sustained, exercise-induced VT, which has been shown to be consistent with cAMP-mediated triggered activity. Therefore, in this study, we sought to identify (1) the mechanism of RMVT at the cellular level by using electropharmacological probes known to activate either stimulatory or inhibitory G proteins and thereby modify intracellular cAMP levels, (2) potential autonomic triggers of RMVT through analysis of heart rate variability, and (3) whether well-characterized somatic activating mutations in the stimulatory G protein, G alpha s, underlie RMVT., Methods and Results: Twelve patients with RMVT underwent electrophysiological study. Sustained monomorphic VT was reproducibly initiated and terminated with programmed stimulation and/or isoproterenol infusion in 11 of the 12 patients (the other patient had incessant RMVT). Induction of VT demonstrated cycle length dependence and was facilitated by rapid atrial or ventricular pacing. Termination of VT occurred in response to interventions that either lowered stimulated levels of intracellular cAMP (and thus decreased intracellular Ca2+)--ie, adenosine (12 of 12), vagal maneuvers or edrophonium (8 of 9), and beta-blockade (3 of 5)--or directly decreased the slow-inward calcium current--ie, verapamil (10 of 12). Analysis of heart rate variability during 24-hour ambulatory monitoring in 7 patients showed that the sinus heart rate is increased and accelerates before nonsustained VT (P < .05), whereas high-frequency heart rate variability is unchanged. These findings are consistent with transient increases in sympathetic tone preceding nonsustained VT. Finally, myocardial biopsy samples were obtained from the site of origin of the VT (typically the RVOT) and from the right ventricular apex from 9 patients. Genomic DNA was extracted from each biopsy sample, and three exons of G alpha s in which activating mutations have previously been described were amplified by polymerase chain reaction. All sequences from these regions were found to be identical to that of control., Conclusions: Although the arrhythmia occurs at rest, the constellation of findings in idiopathic VT that is characterized by RMVT is consistent with the mechanism of cAMP-mediated triggered activity. Therefore, the spectrum of VT resulting from this mechanism includes not only paroxysmal exercise-induced VT but also RMVT.

It is well-established that novel males can disrupt early pregnancy in house mice. Inseminated female C57BL mice were either left undisturbed or each exposed indirectly to a novel HS male through a wire-mesh grid during days 1-6 of pregnancy. Varied dosages of antibodies to 17 beta-estradiol were administered to females exposed to males. Vehicle-treated females exposed to novel males showed fewer litters than did nonexposed controls. Male-exposed females given 1 ml daily of the antibody showed rates of pregnancy comparable to those observed in controls. These data suggest that estrogen levels might play a role in strange-male-induced pregnancy disruptions, converging with evidence implicating estrogens in stress-induced pregnancy blocks.

Previous evidence suggests that androgen activity is necessary for strange males to disrupt early pregnancy in mice. Inseminated females were housed below castrated males, separated by a wire-mesh grid. Castrated males did not disrupt pregnancy, whereas those given daily injections of 27 or 81 micrograms of 17 beta-estradiol did so. In conjunction with previous evidence, these data suggest a similarity between the hormones involved in the capacity of males to disrupt pregnancy and the hormones directly implicated in the females' vulnerability to pregnancy disruption.

One of the principal aims of our research is to determine the mechanisms which direct renin gene expression to different sites. We recently demonstrated that human renin (hRen) 5'-flanking DNA sequences -148 +/- 11 can drive the transient expression of a linked luciferase reporter gene transfected into pituitary GC cells. This activity was found to be dependent on the binding of Pit-1 to a site approximately 70 bp upstream from the transcription start site. Pit-1 is a pituitary-specific transcription factor which is involved in directing the cell-specific expression of growth hormone (GH) and prolactin (PRL) gene expression to somatotrope and lactotrope cells of the anterior pituitary. Thus, Pit-1 may be play a role in directing the expression of renin to primate lactotrope cells. Renin promoter-driven luciferase or CAT hybrid genes were found to be expressed following transfection into primary, or early passage cell cultures of placental chorionic membranes, and the renin-secreting renal tumor cell line As4.1. As with GC cells, deletion or mutagenesis of the Pit-1 site reduced activity several-fold in both placental and renal cells. These results suggest that members of the POU family of transcription factors, or some other closely related group such as the Hox proteins, participate in directing renin gene expression to placental and juxtaglomerular cells.

Developmental stage- and tissue-specific expression of the rat growth hormone (rGH) gene is conferred by DNA sequences within 237 base pairs of the transcription start site. Although binding of a number of transcription factors including Pit-1, Sp1, GHF3, and thyroid hormone receptor (T3R) stimulates rGH expression, several studies have suggested that interactions between these factors are important in determining cell specificity and responsiveness to extracellular signals. We have directly tested this hypothesis by creating a set of nested insertional mutations at two positions in the rGH promoter. Sequences were inserted at either position -148, separating GHF-3 and T3R binding sites from the downstream Pit-1 and Sp 1 binding sites, or at -51, separating the above elements from the TATA box. All insertions were made in the context of the rGH gene -237/+8 5'-flanking DNA, linked to a chloramphenicol acetyltransferase reporter gene and tested for activity by transient transfection in GC pituitary tumor cells. Insertions at both -148 and -51 caused sharp distance-dependent reductions in serum-stimulated expression such that insertions of 23 base pairs at -51 or 44 base pairs at -148 were sufficient to isolate the effects of sequences upstream of the insertion point. Insertions at -148 reduced T3 responsiveness severalfold but had little or no effect on stimulation by forskolin, whereas insertions at -51 reduced both T3 and forskolin responsiveness. Our results are consistent with the hypothesis that expression and regulation of the rGH gene is dependent on short-range protein-protein interactions, which are more critically dependent on spacing than the relative orientation of the transcription factor binding sites.

Although the renal juxtaglomerular cell is the source of circulating renin, the renin gene is also expressed at a number of extrarenal sites including lactotrope cells of human and ovine pituitaries. In the present study, we demonstrate that GC cells, a pituitary lactotrope precursor cell line, efficiently express transiently transfected hybrid genes containing human renin 5'-flanking DNA sequences -148/+11. Gel mobility shift competition analyses show that a highly conserved sequence in human and rodent renin 5'-flanking DNAs (human coordinates: -80/-58) binds a nuclear factor from GC cells, most likely the pituitary-specific factor Pit-1. Deletional and mutational analyses demonstrate that this site is a major determinant of renin promoter activity in GC cells. Transfection of a Pit-1 expression construct into HeLa cells, where activity of the human renin promoter is low, stimulates expression of cotransfected renin-luciferase constructs. Moreover, activation of the human renin promoter by co-expression of Pit-1 is dependent on an intact Pit-1 site. Taken together, these data strongly suggest that Pit-1 activates pituitary renin gene expression. This finding raises the possibility that member(s) of the POU family of transcription factors, of which Pit-1 is an archetypal member, may direct renin expression in other tissues, including the kidney.

Transcriptional regulation by thyroid and steroid hormone receptors requires their recognition and binding of specific DNA sequences. However, little is known about the mechanisms whereby DNA bound receptors regulate transcription. In the present study, we examined the effects of thyroid hormone receptor (TR) binding on DNA conformation using various TR recognition sites contained within sets of circularly permuted flanking sequences. We show that under conditions where TR binds predominantly as monomer, the conformation of a number of binding sites is changed in a manner consistent with receptor induced bending. Despite similar affinities for receptor binding, not all binding sites tested showed evidence for receptor-induced bending. Notably, the conformation of a sequence from the frog vitellogenin 2 gene, which confers a positive transcriptional response when bound by estrogen receptor (ER), but a negative response when bound by TR, appeared to be unaffected by binding of either TR or ER. The observations suggest that the ability of the receptor to alter DNA architecture is strongly dependent on sequence characteristics other than those required for receptor binding. While both partly purified TR from rat liver and TR translated in vitro were able to induce DNA bending, the bend centers and bend angles produced by these different sources of receptor differed. However, addition of a receptor-depleted fraction from the rat liver TR preparation to in vitro translated receptor stimulated TR binding and appeared to form heterodimers with TR. This resulted in changes in both bend centers and bend angles to resemble more closely those produced by native receptor. Together, these results suggest that receptor-induced DNA bending may be specific to TRs and that the position and degree of bending is further modulated by the formation of heterodimers between TRs and accessory protein(s).

Although both promoter and enhancer sequences of the PRL gene 5'-flanking DNA are required for cell-specific, high level expression in transgenic animals, reports of the relative contributions of these elements determined in transient transfection experiments have varied. In this study we examined the transcriptional activities of proximal promoter (-422/+33) and distal enhancer (-1956/-1530) sequences of the rat (r) PRL gene by transient transfection of hybrid genes containing these sequences into two rat pituitary cell lines, GC and 235-1. These cell lines exhibit characteristics either of mammosomatotropes, which express both PRL and the evolutionarily related GH gene (GC), or lactotropes, which express only PRL (235-1). As lactotropes are thought to differentiate from a mammosomatotrope precursor cell, comparisons between these cell lines provide the opportunity to examine the mechanisms that activate PRL and GH genes during development. We show that the relative contributions of promoter and enhancer elements differ between GC and 235-1 cells. Although maximal enhancer-driven activity was similar between these cell lines, promoter sequences were more active in GC (5-10% maximal) than 235-1 cells (1-2% maximal). However, no apparent differences in factor binding to the rPRL promoter region could be correlated with differences in activity, suggesting that differential factor modification, rather than different factors, is involved. As the rGH promoter exhibited a similar pattern of activity in these cell lines, these observations suggest that promoter as well as enhancer elements contribute to the cell specificity of PRL and GH gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of hGH-1 expression is mediated by the pituitary-specific transcription factor GHF-I/Pit-I which binds the 5'-flanking DNA at two sites: I (-96/-70) and II (-134/-106). Although the factor(s) which direct the placental-specific expression of hCS-1 are not known, hCS-1 sequences are transcriptionally active in pituitary cells. In the present study we examined the effects of sequence differences between hGH-1 and hCS-1 5'-flanking DNAs in determining their basal and thyroid hormone-regulated promoter activities. We showed that Sp1 is a major determinant of both hGH-1 and hCS-1 promoter activities and that in hGH-1, binding and activation by Sp1 are modulated by interference from GHF-I/Pit-1 binding at the adjacent site II sequence. A single base which differed in site II of hCS-1 greatly reduced GHF-1/Pit-1 binding and thus facilitated binding and activation by Sp1. Further differences in promoter activity of hGH-1 and hCS-1 sequences were accounted for by a thyroid hormone-responsive element between -62/-48 in the hCS-1 gene. However, induction by T3 was independent of either Sp1 or GH-1/Pit-1 binding in the site II region. These data demonstrate that a small number of base changes between hGH and hCS promoter sequences subserve a number of mechanisms which may differentially modulate the expression of hGH and hCS genes.

In one experiment, female C57- and HS-stain mice were inseminated according to standard procedures and randomly assigned to conditions involving administration of various adrenal and ovarian steroids in the first trimester of pregnancy. The pattern of effects was similar in the two strains, although generally C57 females produced fewer and smaller litters than did HS females. Oestradiol benzoate injections completely blocked pregnancy at doses far less than those effective for other steroids. Corticosterone injections did not produce any significant pregnancy block. Androstenedione injections produced a pregnancy block at 500 micrograms per day. Dehydroepiandrosterone produced a mild pregnancy block at both 500 micrograms and 100 micrograms per day. In a second experiment, the dose-response curve for oestradiol was examined. Daily doses of 0.333 microgram and greater completely blocked pregnancy, a dose of 0.111 microgram did so in the majority of females, and smaller doses had little effect. These results, taken together with other data, suggest that oestrogenic action may mediate the stress-induced block of pregnancy.

It is established that diverse psychological stressors administered in the first trimester of pregnancy can cause the pregnancy to fail. The physiological mediators of this phenomenon are unknown. In Experiment 1, two strains of female mice were inseminated and randomly assigned to either first trimester physical restraint, restraint plus progesterone, or control conditions. Restrained females produced fewer pregnancies than did controls, and a reversal of this block through the daily administration of 500 micrograms progesterone was seen in HS but not C57 mice. Experiment 2 demonstrated that exposure to a predator will also block pregnancy in the first trimester in C57 mice, and that this can also be counteracted with progesterone administration. Experiment 3 showed that metyrapone, a drug which blocks the conversion of progesterone to corticosterone, also partially reverses a restraint-induced pregnancy block. These results suggest that lowered progesterone levels may be involved in pregnancy blocks induced by psychological stress.

The transcriptional activity of renin genes in the mouse kidney and submandibular gland (SMG) was examined by measurement of renin messenger RNA (mRNA) (nanograms per g tissue) and compared with renin activity (micromoles angiotensin I per h/g tissue). In control adult mice renin mRNA and renin activity (in parentheses) were 1.8 +/- 0.24 (11 +/- 1.1) in male kidney, 3.6 +/- 0.66 (18 +/- 2.8) in female kidney, 230 +/- 34 (903 +/- 59) in male SMG, and 31 +/- 6 (188 +/- 47) in female SMG (mean +/- SE, n = 6). The ratio of renin mRNA among these four tissues was similar to that of renin activity (1:2:100:17, respectively). Although the values in male kidney were one one-hundredth those in SMG, 1000 to 10000 times more SMG cells are involved per gram of tissue so that, per renin-synthesizing cell, kidney values would be 10 to 100 times SMG values. Expression of renin gene(s) in a renal juxtaglomerular cell may thus be higher than in a SMG granular duct cell. Values in adult male SMG were a consequence of a 40-fold rise at puberty, were decreased to 16% (+/- 3.8) by castration, but were not significantly influenced by treatment with testosterone, T4, propylthiouracil, sodium depletion, or spironolactone. Renin mRNA in adult female SMG was 18-fold higher than juvenile values, was increased 10-fold (+/- 1.6) by testosterone (to adult male levels) and 5.5-fold (+/- 0.81) by T4 (P less than 0.005), but was decreased to 42% (+/- 29) of normal by propylthiouracil (P less than 0.05). Propylthiouracil caused a small but significant decrease in testosterone-treated female values. In the kidney renin mRNA was increased 3.0-fold (+/- 0.30) by captopril, 2.3-fold (+/- 0.23) by a low sodium diet, and 1.7-fold (+/- 0.13) by spironolactone after 1 week, whereas T4, testosterone, or propylthiouracil had little or no effect. Sialoadenectomy increased renin mRNA and renin in male but not in female kidney, suggesting that a SMG factor, possibly renin, may have a role in suppression of renin synthesis in male kidney. In conclusion, measurement of renin mRNA suggests that testosterone regulates renin gene expression by a direct effect in male mouse SMG, whereas in the female regulation is by thyroid hormone. In the kidney conditions which increase renin content are accompanied by parallel (5-fold higher) increases in renin mRNA, suggesting enhanced expression of renin gene(s) in renal juxtaglomerular cells in chronic low sodium states.

The involvement of the pituitary-adrenocortical axis in the control of the lordosis reflex was investigated; In Experiment 1, estrogen-primed ovariectomized (ovx) and adrenalectomized-ovariectomized (adx-ovx) females were treated chronically with dexamethasone, a compound blocking ACTH release from the pituitary. Dexamethasone inhibited lordosis, effectively blocking an adrenalectomy-induced facilitation of the reflex. In Experiment 2, corticosterone was similarly administered chronically; this compound also inhibited lordosis in adx-ovx females. In Experiment 3, acute peripheral administration of synthetic ACTH caused a marked increase in lordosis in ovx females. The results suggest that in the adrenally intact animal, ACTH may exert its effect through adrenal steroids. An acute elevation of adrenal steroids may increase lordosis, whereas a chronic elevation may decrease it.

The biosynthesis of renin as a higher molecular weight 'prorenin' was demonstrated by in vitro incorporation of [35S] methionine into nascent polypeptides of submandibular gland tissue from adult male mice. Immunoprecipitation with anti-renin and electrophoresis identified a Mr 44,500, pI 6.4 prorenin which normally represented 5% of renin-immunoreactive protein in tissue extracts and which was rapidly converted during in vitro labeling into a Mr 40,000, pI 6.2 species. The latter was subsequently processed slowly to forms of Mr 35,500, pI 5.6 and Mr 34,000, pI 5.4. The influence of processing enzymes was then eliminated by synthesizing renin in a cell-free translation system containing rabbit reticulocyte lysate and mRNA isolated from submandibular glands. This yielded an even larger species of Mr 46,000 likely to be 'preprorenin'. A clone bank of mouse submandibular gland cDNA was prepared. This consisted of E. coli RRI transformed with plasmid pBR322 in which cDNA had been inserted at the PstI site so that the bacteria could express the encoded polypeptide. Renin-immunoreactive colonies were identified suggesting the expression of renin-like proteins by a prokaryote. The cDNA which was 700-1000 base pairs big was excised for sequencing.

A three-dimensional model of human renin has been used to define amino acid residues involved in interaction with substrate. The structure revealed proteolytic cleavage sites on the surface of the molecule that may help explain numerous experimental observations concerning prorenin and its activation and forms of renin differing in size.

The beta and gamma subunits of nerve growth factor were isolated and tested for inactive-renin-activating activity using human amniotic fluid as a convenient source of inactive renin. Only the gamma subunit, which is an arginine esteropeptidase, could activate inactive renin. The role of this enzyme in sites containing both it and inactive renin warrants investigation.

Ovariectomized mice were given replacement estrogen and progesterone, and tested for sexual receptivity in the presence of mounting males after various pharmacological manipulations of adrenocortical hormone activity. In Experiment I, females received a chronic regimen of varied dosages of metyrapone, which blocks adrenal conversion of desoxycorticosterone to corticosterone. In each of three repeated measures, females given an intermediate dosage (800 micrograms/animal/injection) showed substantially higher levels of receptivity than those given vehicle injections or other dosages. In Experiment 2, corticosterone administration reversed the facilitatory action of metyrapone on receptivity. In Experiment 3, chronic administration of either desoxycorticosterone or progesterone failed to elevate receptivity. These findings suggest that corticosterone titer may play a role in modulating female receptivity in sexually inexperienced mice.

A DNA coding for mouse submandibular gland (MSG) renin was used in studies of the biosynthesis and processing of MSG renin and precursors and as a probe for identification of the human renin gene. A 2 kilodalton signal peptide was demonstrated by cleavage of preprorenin (45 kilodaltons) in the presence of microsomal membranes from dog pancreas. Prior selection of renin mRNA by hybridization with its cDNA obviated the possibility that a protease translated from total MSG mRNA could be activated by the microsomal membranes and then act on the primary translation product. In vitro labelling experiments with female MSG demonstrated that prorenin (43 kilodaltons) is rapidly converted to renin (38 kilodaltons) and that testosterone stimulated synthesis by increasing transcription of renin mRNA. Electrophoresis under non-reducing conditions demonstrated 38 kilodalton renin which when reduced ran as two bands of 33 and 5 kilodaltons. Thus, native MSG renin has two chains linked by disulphide bonding. Hydrolysis of the 38 kilodalton single chain occurred only slowly during in vitro labelling. Prorenin bound only weakly to a pepstatin affinity column and could be activated by adding trypsin to column fractions. Both 38 kilodalton single and two chain renin bound strongly however, suggesting that both are active. Base sequencing of MSG renin cDNA indicated an Arg-Arg residue additional to the published amino acid sequence of the heavy chain. This may account for the two forms seen on isoelectric focusing. Mouse renin cDNA was used as a hybridization probe in screening a human genomic library in order to identify the human renin gene. The DNA in positive colonies had overlapping restriction maps and the coding region was found.

In four experiments, different preparations and modes of prenatal administration of ACTH all failed to produce any substantial effects upon male sexual behavior in mice. In Experiment 1, CD-1 females implanted during the third trimester of pregnancy with osmotic pumps releasing varied dosages of ACTH1-24 produced male offspring with essentially normal copulatory behavior. In Experiment 2, prenatal injections of high doses of ACTH1-24 had no effect upon male sexual activity. In Experiment 3, osmotic pumps releasing ACTH1-39 during the third trimester of pregnancy had no effect upon sexual behavior of offspring. However, aggressive behavior was significantly reduced, relative to untreated controls, in offspring of all females implanted with pumps, including those releasing only saline. In Experiment 4, third-trimester injections of ACTH1-39 in long-acting gel form had no effect on the sexual behavior or aggression of offspring of C57 strain females. In most of these experiments, ACTH treatment significantly reduced body weight. These results do not confirm previous suggestions that pituitary-adrenal hormones influence the perinatal differentiation of sexually dimorphic behavior.

1. The existence of a biosynthetic precursor of renin (prorenin) has been inferred, but never established experimentally. The aim of the present study was to provide this evidence. 2. In a continuous labelling experiment using submandibular glands of male Quackenbush mice (an extraordinarily rich source of renin) a molecule of Mr 44 500 was immunoprecipitated with antiserum to renin. 3. The Mr 44 500 protein was converted to Mr 40 000 renin in a time-dependent fashion during incubation of tissue at 37 degrees C: 15 min after addition of [35S] methionine only 16% of immunoprecipitable radioactivity was renin, whereas by 4 h this had increased to 86%. The Mr 44 500 protein would therefore appear to be a 'pro' form of renin. 4. Submandibular gland mRNA was translated in a cell-free system derived from rabbit reticulocyte lysate and a molecule of Mr 46 000 was immunoprecipitated with antirenin. This was preprorenin and the Mr of the 'pre' region would thus be 1500, which is consistent with the size generally found for the 'pre' region of secretory proteins. 5. The present study has therefore demonstrated for the first time the biosynthesis of a 'pro' form of renin (prorenin) and its conversion to renin intracellularly.

In Experiment 1, adult male C57 mice were castrated, housed individually or in groups of four, and repeatedly injected with either of two doses of testosterone. Control mice were sham-castrated, individually or group housed, and injected with oil vehicle. In repeated tests of sexual behavior with receptive females, isolated males in all surgery-dose combinations showed significantly more mounts and intromissions than did their group-housed counterparts. Ejaculations were fully restored by testosterone in castrated grouped males but not in castrated isolated males. In Experiment 2, administration of either of two doses of testosterone failed to elevate the sexual behavior of intact group-housed males. These experiments show that housing with other males depresses all major measures of sexual behavior, and suggest that this is probably independent of testicular hormones.

Novel male house mice (Mus musculus) can disrupt early pregnancy in females. Previous research focused on pheromonal rather than behavioral mediation of this phenomenon. In Experiment 1, novel males were housed with females shortly after insemination. Litter production was negatively correlated with the males' sexual activity. Experiment 2 replicated this finding with a larger sample. In Experiment 3, females were exposed to castrated males. Testosterone-treated males completely blocked pregnancy, whereas untreated males did not. In Experiment 4, castrated testosterone-treated males were presented at intervals after insemination. Pregnancy was totally blocked at Days 3 and 4 and mostly blocked at Days 1 and 2 but was less affected at Days 5 and 6. In Experiment 5, females were exposed through a wire-mesh grid to castrated males. Pregnancies occurred in all conditions, even with testosterone-treated males. These data suggest a role for sexual activity in male-induced pregnancy blocks.

Corticosterone was administered through various modes into several brain regions of estrogen-treated adrenalectomized-ovariectomized female rats. Daily administration of 20 micrograms corticosterone dissolved in propylene glycol through intracerebral cannulae effectively inhibited female sexual receptivity at each of four sites: third ventricle, ventro-medial hypothalamus, preoptic area, and septum. Similar administration of lesser daily doses failed to inhibit receptivity. Implantation of pure crystalline corticosterone also had no effect on receptivity at any site. Beeswax pellets chronically releasing low doses of corticosterone significantly inhibited receptivity when implanted in the medial hypothalamus and preoptic area, with similar nonsignificant trends in the lateral septum and medial forebrain bundle, but had no effect in the dorsal hippocampus or the amygdala. The effective sites are similar to those in which estrogen activity is known to induce receptivity and those in which serotonergic activity is believed to inhibit receptivity, but do not entirely correspond to sites in the brain showing the greatest uptake of corticosterone.

In Experiment 1, when previously isolated male mice were paired and given a female, they fought before beginning to mount, and the more aggressive male ejaculated somewhat more frequently. Males housed together for several days showed little aggression when jointly given a female, but those that were more aggressive in the home cage clearly ejaculated more frequently. In Experiment 2, males were paired for 4 days after a period of isolation. More aggressive males showed more ejaculations when subsequently tested individually with females, but not when pair members conjointly encountered females. In Experiment 3, males were paired for several weeks before encountering females. In cases in which home cage dominance was constant, the more aggressive males ejaculated more frequently both when tested individually and when tested as pairs.

The presence of renin mRNA in a tissue is a manifestation of the expression of the renin gene by that tissue. In the present study 32P-labeled, single-stranded renin complementary DNA was used as a specific probe for the detection of renin mRNA in tissues by hybridization of their complementary base sequences. With use of the technique of hybridization histochemistry, renin cDNA probe bound strongly to sections of submandibular gland from male Quackenbush mice, a tissue which displays high androgen-dependent renin synthesis. Less was present in the female gland. In the kidney the probe localized in the cortex. A dot hybridization assay was developed to measure renin mRNA in serially diluted total RNA spotted onto nitrocellulose filters along with renin cDNA standards. From the concentration the ratio of renin mRNA between male and female submandibular gland and kidney was calculated as approx. 100:8:1:1, respectively, which is similar to the relative proportion of renin concentrations between these tissues. The present study thus provides the basis for investigations of the expression of the renin gene by tissues, particularly those with high renin biosynthetic activity.

A 14 kilo-base pair DNA clone (lambda HCMHC8) was isolated from a human genomic library by hybridization with a complementary DNA coding for a rabbit cardiac myosin heavy chain. lambda HCMHC8 hybridized to RNA isolated from cardiac but not skeletal muscle and formed heteroduplexes with a genomic clone coding for the fast type of rabbit cardiac myosin heavy chain. lambda HCMHC8 represented at least the 3' half of the gene and contained over 11 exons which together spanned 4 kb of the coding region estimated to be 6 kb. Probes made from lambda HCMHC8 were used to rescreen the library in order to isolate overlapping clones and so extend the sequence (estimated to be approximately 25 kb for the whole gene, including introns). In addition, a clone having a different restriction map was isolated suggesting that man, like rat and rabbit, has two cardiac myosin heavy chain genes. These may code for proteins having different ATPase activity and be expressed in different proportions in different cardiac states, including hypertension.

Previously it has been established that adrenalectomy facilitates lordosis in estrogen-primed ovariectomized female rats and that corticosterone administration restores lordosis to preadrenalectomy levels. The present study examined the effects of an inhibitor of the synthesis of corticosterone, metyrapone, upon lordosis in ovariectomized females. In Experiment 1, chronic administration of moderate doses of metyrapone was found to facilitate lordosis. In Experiment 2, a single metyrapone administration at various doses and time intervals before testing had a mild facilitatory effect on lordosis. Experiment 3 compared the effects of metyrapone on ovariectomized and adrenalectomized-ovariectomized females. The absence of a facilitatory effect in adrenalectomized females suggests that the drug's effect on lordosis is mediated by its established inhibitory effects on 11 beta-hydroxylation in the adrenal. These data are consistent with indications that corticosterone titer modulates female receptivity.