Maternity validation using sire-only BovineSNP50 BeadChip data.

Based on pairwise identity-by-state (IBS) distances and whole-genome SNP data, kinship was investigated in the Israeli Holstein population. A total of 789 bulls, including most of the artificial insemination sires in service since 1987, were genotyped by the BovineSNP50 BeadChip. This sample included up to five generations. For each bull-by-bull combination, three states are possible for each marker: no match, a single match and both alleles match. Summing over all markers, the 932 598 IBS scores (three match frequencies*310 866 bull-by-bull combinations) were visualized using three-dimensional coordinates that corresponded to the frequencies of the three possible states. Results were reduced to two dimensions using the transformations x' = 0.7071(1 + freq1-freq2) and y' = 1.2247freq0. Bull-by-bull pairs were grouped according to their level of kinship, and canonical scores were calculated using discriminant analysis and the x' and y' features. Of the 474 pairs of recorded maternal grandsire-grandson with both individuals genotyped, the probability for 28 pairs to belong to this level of kinship was low (P < 0.05), suggesting an error rate of around 3% per generation in pedigree determination.

Signatures of contemporary selection in the Israeli Holstein dairy cattle.

Strong selection in the Israeli Holstein dairy cattle population over the last three decades should have left clear signatures of selection. Two experimental approaches were applied to detect evidence of contemporary selection based on the 54K BeadChip genotypes of ~1000 Israeli Holstein bulls: (i) the long-range haplotype test, which searches for structural evidence resulting from selective sweep, and (ii) direct analysis of the changes in haplotypes frequencies over time combined with linkage disequilibrium blocks haplotype-based association analysis. Ten traits were analyzed: the PD07 Israeli selection index, milk, milk fat, % fat, milk protein, % protein, somatic cell score, female fertility, milk production persistency and herd life. The long-range haplotype test detected ~15% of the 3288 haplotypes that showed significant positive frequency trends (P < 0.05) and was significantly correlated with the substitution effects of the haplotypes and the selection intensities for the different traits. Thirty signatures of recent selection, which correspond to both approaches and affect the Israeli PD07 selection index, were identified on 17 of the 29 autosomes. The second experimental approach also was used to estimate the selection intensity of the different traits. The correlation between the selection intensities for the traits analyzed, derived from changes in haplotype frequencies in the population of bulls, and those derived from trait-based analysis of the cow population was 0.93 over all traits. Thus, the changes in haplotypes frequencies in the bulls' population accurately estimate genetic trends in the general cow population and can be used to detect signatures of recent selection.

Differential expression of ruminant ZNF496 variants: Association with quantitative trait locus affecting bovine milk concentration and fertility.

A single nucleotide polymorphism in the intergenic region upstream of the ZNF496 gene on Bos taurus chromosome 7 displayed significant population-wide linkage disequilibrium with milk protein percentage in the Israeli Holstein population. The frequency of the allele associated with increased protein concentration was 10%. This single nucleotide polymorphism was located in the promoter region from which a 10-exon transcript of the bovine and the ovine ZNF496 genes are transcribed. The gene architecture was similar to the mouse ortholog Zkscan17. A 5-exon murine antisense transcript was complementary to the 5' untranslated Zkscan17 region that included a sequence domain conserved between mouse and ruminants, suggesting a regulatory function. In the bovine ZNF496 chromosomal region, segregation of a quantitative trait locus (QTL) for milk protein percentage was confirmed in a daughter design sire family. Concordance was not obtained between QTL status of bulls and any of the polymorphisms in the functional elements of ZNF496. This excludes these variations as the causative polymorphism under the assumption of no epigenetic effect for this locus. However, ZNF496 variants were differentially expressed in bovine ovaries, and only the paternal variant was expressed in liver and kidney in a sheep family with polymorphic ZNF496 sequence. Thus, the search for the mutation underlying the minor QTL allele, which is a top economically favorable allele in Israeli Holstein cattle, may be complicated by the presence of an imprinting center in this QTL confidence interval.

Characterization of the metabolic phenotype of chronically activated lymphocytes.

Activated lymphocytes proliferate, secrete cytokines, and can make antibodies. Normally activated B and T cells meet the bioenergetic demand for these processes by up-regulating aerobic glycolysis. In contrast, several lines of evidence suggest that pathogenic lymphocytes in autoimmune diseases like lupus meet ATP demands through oxidative phosphorylation. Using (13)C-glucose as a stable tracer, we found that splenocytes from mice with lupus derive the same fraction of lactate from glucose as control animals, suggesting comparable levels of glycolysis and non-oxidative ATP production. However, lupus splenocytes increase glucose oxidation by 40% over healthy control animals. The ratio between pentose phosphate cycle (PPC) activity and glycolysis is the same for each group, indicating that increased glucose oxidation is due to increased activity of the TCA cycle in lupus splenocytes. Repetitive stimulation of cultured human T cells was used to model chronic lymphocyte activation, a phenotype associated with lupus. Chronically activated T cells rely primarily on oxidative metabolism for ATP synthesis suggesting that chronic antigen stimulation may be the basis for the metabolic findings observed in lupus mice. Identification of disease-related bioenergetic phenotypes should contribute to new diagnostic and therapeutic strategies for immune diseases.

Paternity validation and estimation of genotyping error rate for the BovineSNP50 BeadChip.

Incorrect paternity assignment in cattle can have a major effect on rates of genetic gain. Of the 576 Israeli Holstein bulls genotyped by the BovineSNP50 BeadChip, there were 204 bulls for which the father was also genotyped. The results of 38 828 valid single nucleotide polymorphisms (SNPs) were used to validate paternity, determine the genotyping error rates and determine criteria enabling deletion of defective SNPs from further analysis. Based on the criterion of >2% conflicts between the genotype of the putative sire and son, paternity was rejected for seven bulls (3.5%). The remaining bulls had fewer conflicts by one or two orders of magnitude. Excluding these seven bulls, all other discrepancies between sire and son genotypes are assumed to be caused by genotyping mistakes. The frequency of discrepancies was >0.07 for nine SNPs, and >0.025 for 81 SNPs. The overall frequency of discrepancies was reduced from 0.00017 to 0.00010 after deletion of these 81 SNPs, and the total expected fraction of genotyping errors was estimated to be 0.05%. Paternity of bulls that are genotyped for genomic selection may be verified or traced against candidate sires at virtually no additional cost.

Importance of the carotid sinus baroreceptors in the regulation of myocardial performance.

The question of whether the carotid sinus baroreceptors modulate myocardial performance remains controversial. Several studies that have stressed their importance have been criticized because the possible role of cerebral ischemia and of other important variables was not eliminated. To reinvestigate this problem, we studied 21 dogs placed on total cardiopulmonary bypass. In each of these animals the carotid sinus regions were isolated and perfused with fully oxygenated blood at a constant flow rate; perfusion pressure was changed by varying the resistance to outflow from the isolated segments. Several indices of myocardial performance were assessed: right and left ventricular contractile force with Walton-Brodie strain gauge arches; the maximal rate of change in contractile force, dF/dt; the pressure developed within an isovolumic balloon inserted into the left ventricle; and the maximal rate of change of this pressure, dP/dt. When the pressure distending the carotid sinuses was raised from an average value of 34.1 +/-2.8 (SEM) mm Hg to 190.1 +/-4.7 mm Hg, right ventricular contractile force fell 14.9 +/-2.3% (P < 0.001); right ventricular dF/dt decreased 16.7 +/-3.0% (P < 0.01); left ventricular contractile force declined 14.9 +/-3.3% (P < 0.01); left ventricular dF/dt fell 19.3 +/-4.0% (P < 0.01); peak systolic pressure in the isovolumic balloon declined 18.2 +/-3.7% (P < 0.001); and dP/dt decreased 34.1 +/-4.0% (P < 0.01). Prior adrenalectomy and vagotomy and maintenance of heart rate at a constant level did not influence these results. The inverse relation between carotid sinus perfusion pressure and the indices of contractility that was observed in this investigation strongly suggests that the carotid sinus baroreceptors are an important regulatory mechanism in the control of myocardial performance.

Aspirin inhibits development of coronary atherosclerosis in cynomolgus monkeys (Macaca fascicularis) fed an atherogenic diet.

The effect of aspirin in the primary prevention of diet-induced atherogenesis in cynomolgus monkeys was studied. The diet consisted of 2% cholesterol and 10% butter by weight for 24 wk. Six monkeys received only the atherogenic diet and five monkeys received the diet plus aspirin, 81 mg/monkey per day. Aspirin did not affect plasma cholesterol levels or aortic atherosclerosis. Platelet aggregation to arachidonic acid was almost completely suppressed. Aspirin decreased significantly the number of coronary vessels with atherosclerotic involvement, and the number of coronary vessels narrowed by 20% or more. Thus, aspirin appears to exert a protective effect in the primary prevention of diet-induced coronary atherosclerosis in a primate model.

Reflex cardiovascular depression produced by stimulation of pulmonary stretch receptors in the dog.

To study the possible reflex effects of stimulation of pulmonary stretch receptors on the cardiovascular system, experiments were designed that would allow separate assessment of the responses of the heart, the total peripheral vascular resistance, and the resistance of the innervated hindlimb that was perfused at a constant flow rate. In every experiment, inflation of the lungs to a positive pressure of 20 mm Hg produced significant negative inotropic and chronotropic effects. Heart rate fell an average of 22.3+/-3.8% (SEM) (P < 0.01), pressure recorded from within an isovolumic balloon in animals on total cardiopulmonary bypass fell an average of 14.3+/-4.6% (P < 0.05), dp/dt recorded from within the balloon declined an average of 31.4 +/- 6.0% (P < 0.01), and contractile force measured with a Walton-Brodie strain gauge arch fell an average of 18.6 +/-2.2% (P < 0.01). Similarly, a depressor response to inflation of the lungs was noted in the periphery as manifested by an average decrease in total peripheral vascular resistance of 21.9+/-2.5% in the animals on total cardiopulmonary bypass (P < 0.01), and by an average decrease in perfusion pressure in the isolated hindlimb of 26.0 +/-3.8% (P < 0.01). After bilateral cervical vagotomy, the cardiovascular responses to inflation of the lungs were either abolished or markedly lessened. Thus, sudden expansion of the lungs activates the afferent arm of a depressor reflex, which produces negative inotropic and chronotropic responses, in addition to arterial vasodilation. The receptors are sensitive to stretch and the afferent pathway runs predominantly in the vagus nerves.

Mechanisms of reflex vasodilation: assessment of the role of neural reuptake of norepinephrine and release of histamine.

The mechanisms of reflex vasodilation were studied in an innervated canine hindlimb preparation which was perfused at a constant rate. Reflex vasodilation was produced by suddenly increasing the pressure in the trunk by the intravenous injection of norepinephrine, with consequent stimulation of the baroreceptors. When the basal vasoconstrictor tone exerted by the sympathetic nervous system on the systemic arterial bed was minimized, either by pretreatment with the alpha adrenergic blocking agent phenoxybenzamine or with reserpine, which depletes endogenous catecholamine stores, reflex vasodilation was virtually abolished. Administration of cocaine, a drug which blocks reuptake of norepinephrine by the nerve terminals, significantly reduced reflex vasodilation, the response after cocaine averaging 47% of the vasodilator response in the control period. Cocaine also potentiated the vasoconstriction caused by intra-arterially administered norepinephrine but attenuated the vasoconstriction induced by tyramine. The antihistamine, tripelennamine, had effects similar to those of cocaine. It is suggested, therefore, that reflex vasodilation results from a sudden decrease in the level of norepinephrine at the neuroeffector junction, which is a consequence of the cessation of norepinephrine secretion, together with continued and possibly augmented uptake. When the uptake mechanism is impaired, either by the administration of cocaine or tripelennamine, the magnitude of reflex vasodilation is diminished. It does not appear necessary to postulate active secretion of a vasodilator substance to account for reflex vasodilation.

Ligand recognition by murine anti-DNA autoantibodies. II. Genetic analysis and pathogenicity.

Although anti-DNA autoantibodies are an important hallmark of lupus, the relationships among anti-DNA structure, reactivity, and pathogenicity have not been fully elucidated. To further investigate these relationships, we compare the variable genes and primary structure of eight anti-DNA mAbs previously obtained from an MRL/MpJ-lpr/lpr mouse along with the ability of three representative mAbs to induce nephritis in nonautoimmune mice using established adoptive transfer protocols. One monospecific anti-single-stranded (ss) DNA (11F8) induces severe diffuse proliferative glomerulonephritis in nonautoimmune mice whereas another anti-ssDNA with apparently similar in vitro binding properties (9F11) and an anti-double-stranded DNA (4B2) are essentially benign. These results establish a murine model of anti-DNA-induced glomerular injury resembling the severe nephritis seen in lupus patients and provide direct evidence that anti-ssDNA can be more pathogenic than anti-double-stranded DNA. In vitro binding experiments using both protein-DNA complexes and naive kidney tissue indicate that glomerular localization of 11F8 may occur by recognition of a planted antigen in vivo. Binding to this antigen is DNase sensitive which suggests that DNA or a DNA-containing molecule is being recognized.

Anti-DNA autoantibodies and systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that affects most of the organs and tissues of the body, causing glomerulonephritis, arthritis, and cerebritis. SLE can be fatal with nephritis, in particular, predicting a poor outcome for patients. In this review, we highlight what has been learned about SLE from the study of mouse models, and pay particular attention to anti-DNA autoantibodies, both as pathological agents of lupus nephritis and as DNA-binding proteins. We summarize the current approaches used to treat SLE and discuss the targeting of anti-DNA autoantibodies as a new treatment for lupus nephritis.

The hairpin ribozyme substrate binding-domain: a highly constrained D-shaped conformation.

The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order to form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crosslinking and structural approaches in conjunction with molecular modeling (constraint-satisfaction program MC-SYM), we have investigated the conformation of the substrate-binding domain in the context of the active docked ribozyme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked state of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-binding strand to cleave a circular substrate, and provides essential information concerning the organization of the substrate in the active conformation. The internal geometry of the substrate-binding strand places G8 of the substrate-binding strand near the cleavage site, which has allowed us to predict the crucial role played by this nucleotide in the reaction chemistry.

Echocardiographic detection of cardiac involvement in patients with chronic renal failure.

Echocardiography was performed in 50 patients in chronic renal failure with symptoms suggestive of possible cardiac involvement. Pericardial effusions were detected in 33 and pericardial thickening in 22. In five patients solid material, probably fibrinous, was seen adherent to the parietal or visceral pericardium. The left ventricle was dilated in 18 patients and showed definitely impaired contractility in 16. The left ventricular (LV) posterior wall was abnormally thick in 19 patients. In two, the ventricular septum was more than 1.3 times as thick as the LV posterior wall. Other echocardiographic findings included calcification in the mitral annulus region, mitral valve vegetations, and thickened chordae tendineae. Echocardiography has proved to be of great help in the assessment of symptomatic patients with chronic renal failure not only in diagnosing the presence and extent of pericardial effusion and thickening, but also in detecting impaired myocardial contractility, calcification in the posterior mitral annulus region, and vegetations of bacterial endocarditis.

Conformational studies of hairpin sequences from the ColE1 cruciform.

Inverted repeat sequences derived from the ColE1 cruciform were investigated by nuclear magnetic resonance (NMR) and UV spectroscopy. It was shown that 15 different sequences exist as stable hairpin structures over a range of buffer conditions and DNA concentrations. Experiments with six oligomers (1-6) containing the native stem sequence and five base loops, found that the two hairpins with the wild-type loops (1-2) served as upper and lower bounds for the thermodynamic stability of all the other sequences. NMR experiments, including rotational correlation time measurements and NOESY spectra, were then performed on 1, the most stable hairpin sequence to begin to uncover a structural basis of its stability.

Effects of propranolol, minoxidil, and clofibrate on cholesterol-induced atherosclerosis in stumptail macaques (Macaca arctoides).

Propanolol, minoxidil, and clofibrate, three different classes of pharmacological agents used clinically in various conditions related to atherosclerosis, were shown not to have any intrinsic potentiating effects on the development of atherosclerosis in stumptail macaques fed an atherogenic diet. We did obtain, however, some results that suggest that clofibrate and propranolol may exert some beneficial actions.

Diet-induced atherosclerosis and experimental hypertension in stumptail macaques (Macaca arctoides). Effects of antihypertensive drugs and a non-atherogenic diet in the evolution of lesions.

This study was carried out to determine the evolution of atherosclerotic lesions during a therapeutic period during which regression might be appreciated. We produced aortic and coronary atherosclerosis in 27 young adult stumptail macaques (Macaca arctoides) by feeding a diet supplemented with 2% cholesterol and 25% fat. Hypertension was produced by bilateral or unilateral narrowing of the renal artery. After six months of this regimen, four monkeys were killed (group 1) and 23 monkeys were divided into three groups: group 2 received unsupplemented diet; group 3 received the same diet as group 2 and drug treatment for hypertension; group 4 was continued on the atherogenic diet and received antihypertensive drug treatment. The results indicate that deleting the atherogenic diet leads to a decrease in the lipid content of the lesions and a transformation of the lipid laden atherosclerotic plaques into lipid-poor, fibro-collagenous lesions, with a decrease in the amount of coronary luminal narrowing. Partial control of systolic hypertension by antihypertensive drugs did not accelerate the involution of the atherosclerotic lesions over the relatively short period of this study. No statistically significant correlation by regression analysis was observed between the level of blood pressure elevation, the plasma renin activity, or the degree of the drug response, and the severity and extent of the atherosclerotic lesions. Furthermore, severe arterial hypertension without an atherogenic diet (group 5) produced arteriosclerosis of the aorta, and intensified branch cushions in the coronary arteries, without inducing lipid deposition in either vascular bed.

Application of the gel shift assay to study the affinity and specificity of anti-DNA autoantibodies.

We have demonstrated that the gel shift assay, a powerful method to study protein.DNA interactions under equilibrium conditions, is both an accurate and precise method to measure the affinity of anti-DNA.DNA immune complexes. One difficulty in performing gel shift assays is disruption of protein.DNA equilibria during the time needed for complexes to enter the gel matrix. However, we have found that highly cross-linked polyacrylamide gels which are known to form non-restrictive matrices, do not perturb anti-DNA.DNA complexes. Using anti-ssDNA BV04-01 as a model antibody, we find good agreement between the dissociation constants (Kd) measured in the gel shift assay using a 5.4% polyacrylamide gel cross-linked with 0.6% (bis)acrylamide, and those obtained previously by fluorescence quenching. Because gel shift assays require only nanogram quantities of analyte and can be performed in several hours, it is well suited for a range of anti-DNA binding studies.

Successful treatment of drug-resistant atrial tachycardia and intractable congestive heart failure with permanent coupled atrial pacing.

Temporary coupled atrial stimulation slowed the ventricular rate by nearly 50% in an adolescent patient with intractable congestive heart failure and focal repetitive atrial tachycardia that was resistant to drug treatment. Because of the success with the temporary pacemaker, a specially designed permanent pacemaker was implanted to provide coupled atrial stimulation. The necessary electrophysiologic conditions for ventricular slowing by coupled atrial pacing are: (1) an atrial effective refractory period shorter than that of the atrioventricular junction, and (2) depolarization of the ectopic atrial pacemaker by the responses to coupled atrial stimulation. During a 4 year follow-up period the treatment resulted in elimination of the tachycardia, followed by return of the heart size to normal and complete clinical recovery. Coupled atrial stimulation can provide effective treatment in selected patients with disabling drug-resistant atrial tachycardia in whom this mode of therapy is shown to be effective by careful electrophysiologic studies.

Submitral calcification or sclerosis in elderly patients: M mode and two dimensional echocardiography in "mitral anulus calcification".

Submitral calcification or sclerosis was visualized with M mode echocardiography in 84 elderly patients, 35 of whom were also studied with two dimensional echocardiography. Posterior submitral calcification, commonly referred to as "mitral anulus calcification," was present in 82 patients and was located in the angle between the posterior mitral leaflet and left ventricular posterior wall, rather than in the mitral anulus proper. M mode scans from the left ventricle to the left atrium showed that posterior submitral calcification ended abruptly in 66 cases, and in these it became contiguous with the posterior atrioventricular junction (true mitral anulus) in only 14 instances, in 16 patients the posterior submitral calcification sloped anteriorly to merge with the posterior aortic root. Anterior submitral calcification was visualized in 12 patients, 10 of whom also had posterior submitral calcification. Anterior submitral calcification was usually located immediately anterior to the base of the anterior mitral cusp. In two cases, if appeared to arise in the region between the aortic and mitral rings; in one instance, it was located in the mid left ventricle, in the mitral chordal region. We suggest that the terms anterior and posterior submitral calcification are more appropriate than "mitral anulus calcification" because in most cases such calcific deposits do not appear to be located in or to arise from the true mitral anulus.

Echocardiographic features of second degree atrioventricular block.

Echocardiographic studies demonstrated abnormalities of motion of the pulmonary valve, the aortic root and valve, the mitral and tricuspid valves, the left ventricle, and the left atrium in two patients with second-degree atrioventricular block. During Wenckebach 3:2 atrioventricular conduction, ventricular beats exhibited alternately long and short periods of systolic opening of the pulmonary and aortic valves and alternately large and small left ventricular stroke volumes. During 4:3 and 3:2 Wenckebach atrioventricular conduction, the left ventricular stroke volume was directly proportional to the preceding end-diastolic volume. During 2:1 atrioventricular conduction, the blocked atrial contractions may produce movements of the left atrial wall, thereby revealing the true atrial rate when the blocked P waves are obscured in the electrocardiogram by their superimposition on preceding T waves.