Role of indigenous arsenate and iron(III) respiring microorganisms in controlling the mobilization of arsenic in a contaminated soil sample.

In this study two different treatment options were investigated for the release of arsenic from a contaminated soil sample. The first option was based on the "bioaugmentation" principle and involved addition of a pure Fe(III)-reducing culture, i.e. Desulfuromonas palmitatis. The second option consisted in the "biostimulation" of indigenous bacteria and involved simple addition of nutrients. Due to the strong association of As with soil ferric oxides, the reductive dissolution of soil oxides by D. palmitatis lead to 45 % arsenic release in solution (2.15 mM). When only nutrients were supplied to the soil, the same amounts of Fe and As were dissolved with slower rates and most aqueous As was found to be in the trivalent state, indicating the presence of arsenate reducing species. The arsenate reducing microorganisms were enriched with successive cultures, using Na2HAsO4 as electron acceptor. The phylogenetic analysis revealed that the enriched microbial consortium contained Desulfosporosinus species, which are known arsenate reducers.

The pathogenesis of familial hypertrophic cardiomyopathy: early and evolving effects from an alpha-cardiac myosin heavy chain missense mutation.

Familial hypertrophic cardiomyopathy (FHC) is a genetic disorder resulting from mutations in genes encoding sarcomeric proteins. This typically induces hyperdynamic ejection, impaired relaxation, delayed early filling, myocyte disarray and fibrosis, and increased chamber end-systolic stiffness. To better understand the disease pathogenesis, early (primary) abnormalities must be distinguished from evolving responses to the genetic defect. We did in vivo analysis using a mouse model of FHC with an Arg403Gln alpha-cardiac myosin heavy chain missense mutation, and used newly developed methods for assessing in situ pressure-volume relations. Hearts of young mutant mice (6 weeks old), which show no chamber morphologic or gross histologic abnormalities, had altered contraction kinetics, with considerably delayed pressure relaxation and chamber filling, yet accelerated systolic pressure rise. Older mutant mice (20 weeks old), which develop fiber disarray and fibrosis, had diastolic and systolic kinetic changes similar to if not slightly less than those of younger mice. However, the hearts of older mutant mice also showed hyperdynamic contraction, with increased end-systolic chamber stiffness, outflow tract pressure gradients and a lower cardiac index due to reduced chamber filling; all 'hallmarks' of human disease. These data provide new insights into the temporal evolution of FHC. Such data may help direct new therapeutic strategies to diminish disease progression.

Stabilization of human urine doping control samples: II. microbial degradation of steroids.

The transportation of urine samples, collected for doping control analysis, does not always meet ideal conditions of storage and prompt delivery to the World Anti-Doping Agency (WADA) accredited laboratories. Because sample collection is not conducted under sterile conditions, microbial activity may cause changes to the endogenous steroid profiles of samples. In the current work, funded by WADA, a chemical mixture consisting of antibiotics, antimycotic substances and protease inhibitors was applied in urine aliquots fortified with conjugated and deuterated steroids and inoculated with nine representative microorganisms. Aliquots with and without the chemical mixture were incubated at 37 degrees C for 7 days to simulate the transportation period, whereas another series of aliquots was stored at -20 degrees C as reference. Microbial growth was assessed immediately after inoculation and at the end of the incubation period. Variations in pH and specific gravity values were recorded. Gas chromatography-mass spectrometry (GC-MS) analysis was performed for the detection of steroids in the free, glucuronide, and sulfate fractions. The addition of the chemical stabilization mixture to urine samples inhibited microorganism growth and prevented steroid degradation at 37 degrees C. On the other hand, four of the nine microorganisms induced alterations in the steroid profile of the unstabilized samples incubated at 37 degrees C.

Transgenic mouse model of stunned myocardium.

Stunned myocardium is a syndrome of reversible contractile failure that frequently complicates coronary artery disease. Cardiac excitation is uncoupled from contraction at the level of the myofilaments. Selective proteolysis of the thin filament protein troponin I has been correlated with stunned myocardium. Here, transgenic mice expressing the major degradation product of troponin I (TnI1-193) in the heart were found to develop ventricular dilatation, diminished contractility, and reduced myofilament calcium responsiveness, recapitulating the phenotype of stunned myocardium. Proteolysis of troponin I also occurs in ischemic human cardiac muscle. Thus, troponin I proteolysis underlies the pathogenesis of a common acquired form of heart failure.

Impairment of endothelium-dependent dilation is an early event in children with familial hypercholesterolemia and is related to the lipoprotein(a) level.

Familial hypercholesterolemia is associated with premature atherosclerosis. Since endothelial dysfunction is an early event in atherogenesis, we used a noninvasive method to assess endothelial function in the systemic arteries of 30 children aged 7-17 yr (median 11) with familial hypercholesterolemia (2 homozygotes, 28 heterozygotes, total cholesterol 240-696 mg/dl) and 30 healthy age- and sex-matched controls. Using high resolution ultrasound, the diameter of the superficial femoral artery was measured at rest, in response to reactive hyperemia (with increased flow causing endothelium-dependent dilation), and after sublingual glyceryltrinitrate (causing endothelium-independent vasodilation). Flow-mediated dilation was present in the controls (7.5 +/- 0.7%) but was impaired or absent in the hypercholesterolemic children (1.2 +/- 0.4%, P < 0.0001). Total cholesterol was inversely correlated with flow-mediated dilation (r = -0.61, P < 0.0001). In the hypercholesterolemic children, flow-mediated dilation was inversely related to the lipoprotein(a) level (r = -0.61, P = 0.027) but not to other lipid fractions. Glyceryltrinitrate-induced dilation was present in all subjects but was lower in the hypercholesterolemia group (10.0 +/- 0.6% vs 12.4 +/- 0.8%, P = 0.023). Thus, impaired endothelium-dependent dilation is present in children with familial hypercholesterolemia as young as 7 yr of age and the degree of impairment is related to the lipoprotein(a) level.

beta(3)-adrenoceptor deficiency blocks nitric oxide-dependent inhibition of myocardial contractility.

The cardiac beta-adrenergic pathway potently stimulates myocardial performance, thereby providing a mechanism for myocardial contractile reserve. beta-Adrenergic activation also increases cardiac nitric oxide (NO) production, which attenuates positive inotropy, suggesting a possible negative feedback mechanism. Recently, in vitro studies suggest that stimulation of the beta(3)-adrenoceptor results in a negative inotropic effect through NO signaling. In this study, using mice with homozygous beta(3)-adrenoceptor deletion mutations, we tested the hypothesis that the beta(3)-adrenoceptor is responsible for beta-adrenergic activation of NO. Although resting indices of myocardial contraction were similar, beta-adrenergic-stimulated inotropy was increased in beta(3)(-/-) mice, and similar hyper-responsiveness was seen in mice lacking endothelial NO synthase (NOS3). NOS inhibition augmented isoproterenol-stimulated inotropy in wild-type (WT), but not in beta(3)(-/-) mice. Moreover, isoproterenol increased myocardial cGMP in WT, but not beta(3)(-/-), mice. NOS3 protein abundance was not changed in beta(3)(-/-) mice, and cardiac beta(3)-adrenoceptor mRNA was detected in both NOS3(-/-) and WT mice. These findings indicate that the beta(3)-adrenergic subtype participates in NO-mediated negative feedback over beta-adrenergic stimulation.

Interaction between neuronal nitric oxide synthase and inhibitory G protein activity in heart rate regulation in conscious mice.

Nitric oxide (NO) synthesized within mammalian sinoatrial cells has been shown to participate in cholinergic control of heart rate (HR). However, it is not known whether NO synthesized within neurons plays a role in HR regulation. HR dynamics were measured in 24 wild-type (WT) mice and 24 mice in which the gene for neuronal NO synthase (nNOS) was absent (nNOS-/- mice). Mean HR and HR variability were compared in subsets of these animals at baseline, after parasympathetic blockade with atropine (0.5 mg/kg i.p.), after beta-adrenergic blockade with propranolol (1 mg/kg i.p.), and after combined autonomic blockade. Other animals underwent pressor challenge with phenylephrine (3 mg/kg i.p.) after beta-adrenergic blockade to test for a baroreflex-mediated cardioinhibitory response. The latter experiments were then repeated after inactivation of inhibitory G proteins with pertussis toxin (PTX) (30 microgram/kg i.p.). At baseline, nNOS-/- mice had higher mean HR (711+/-8 vs. 650+/-8 bpm, P = 0.0004) and lower HR variance (424+/-70 vs. 1,112+/-174 bpm2, P = 0.001) compared with WT mice. In nNOS-/- mice, atropine administration led to a much smaller change in mean HR (-2+/-9 vs. 49+/-5 bpm, P = 0.0008) and in HR variance (64+/-24 vs. -903+/-295 bpm2, P = 0.02) than in WT mice. In contrast, propranolol administration and combined autonomic blockade led to similar changes in mean HR between the two groups. After beta-adrenergic blockade, phenylephrine injection elicited a fall in mean HR and rise in HR variance in WT mice that was partially attenuated after treatment with PTX. The response to pressor challenge in nNOS-/- mice before PTX administration was similar to that in WT mice. However, PTX-treated nNOS-/- mice had a dramatically attenuated response to phenylephrine. These findings suggest that the absence of nNOS activity leads to reduced baseline parasympathetic tone, but does not prevent baroreflex-mediated cardioinhibition unless inhibitory G proteins are also inactivated. Thus, neuronally derived NO and cardiac inhibitory G protein activity serve as parallel pathways to mediate autonomic slowing of heart rate in the mouse.

Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice.

To elucidate the role of cardiac myosin-binding protein-C (MyBP-C) in myocardial structure and function, we have produced mice expressing altered forms of this sarcomere protein. The engineered mutations encode truncated forms of MyBP-C in which the cardiac myosin heavy chain-binding and titin-binding domain has been replaced with novel amino acid residues. Analogous heterozygous defects in humans cause hypertrophic cardiomyopathy. Mice that are homozygous for the mutated MyBP-C alleles express less than 10% of truncated protein in M-bands of otherwise normal sarcomeres. Homozygous mice bearing mutated MyBP-C alleles are viable but exhibit neonatal onset of a progressive dilated cardiomyopathy with prominent histopathology of myocyte hypertrophy, myofibrillar disarray, fibrosis, and dystrophic calcification. Echocardiography of homozygous mutant mice showed left ventricular dilation and reduced contractile function at birth; myocardial hypertrophy increased as the animals matured. Left-ventricular pressure-volume analyses in adult homozygous mutant mice demonstrated depressed systolic contractility with diastolic dysfunction. These data revise our understanding of the role that MyBP-C plays in myofibrillogenesis during cardiac development and indicate the importance of this protein for long-term sarcomere function and normal cardiac morphology. We also propose that mice bearing homozygous familial hypertrophic cardiomyopathy-causing mutations may provide useful tools for predicting the severity of disease that these mutations will cause in humans.

Cardiomyopathy in Irx4-deficient mice is preceded by abnormal ventricular gene expression.

To define the role of Irx4, a member of the Iroquois family of homeobox transcription factors in mammalian heart development and function, we disrupted the murine Irx4 gene. Cardiac morphology in Irx4-deficient mice (designated Irx4(Delta ex2/Delta ex2)) was normal during embryogenesis and in early postnatal life. Adult Irx4(Delta ex2/Delta ex2) mice developed a cardiomyopathy characterized by cardiac hypertrophy and impaired contractile function. Prior to the development of cardiomyopathy, Irx4(Delta ex2/Delta ex2) hearts had abnormal ventricular gene expression: Irx4-deficient embryos exhibited reduced ventricular expression of the basic helix-loop-helix transcription factor eHand (Hand1), increased Irx2 expression, and ventricular induction of an atrial chamber-specific transgene. In neonatal hearts, ventricular expression of atrial natriuretic factor and alpha-skeletal actin was markedly increased. Several weeks subsequent to these changes in embryonic and neonatal gene expression, increased expression of hypertrophic markers BNP and beta-myosin heavy chain accompanied adult-onset cardiac hypertrophy. Cardiac expression of Irx1, Irx2, and Irx5 may partially compensate for loss of Irx4 function. We conclude that Irx4 is not sufficient for ventricular chamber formation but is required for the establishment of some components of a ventricle-specific gene expression program. In the absence of genes under the control of Irx4, ventricular function deteriorates and cardiomyopathy ensues.

Comparison of two murine models of familial hypertrophic cardiomyopathy.

Although sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), individuals bearing a mutant cardiac myosin binding protein C (MyBP-C) gene usually have a better prognosis than individuals bearing beta-cardiac myosin heavy chain (MHC) gene mutations. Heterozygous mice bearing a cardiac MHC missense mutation (alphaMHC(403/+) or a cardiac MyBP-C mutation (MyBP-C(t/+)) were constructed as murine FHC models using homologous recombination in embryonic stem cells. We have compared cardiac structure and function of these mouse strains by several methods to further define mechanisms that determine the severity of FHC. Both strains demonstrated progressive left ventricular (LV) hypertrophy; however, by age 30 weeks, alphaMHC(403/+) mice demonstrated considerably more LV hypertrophy than MyBP-C(t/+) mice. In older heterozygous mice, hypertrophy continued to be more severe in the alphaMHC(403/+) mice than in the MyBP-C(t/+) mice. Consistent with this finding, hearts from 50-week-old alphaMHC(403/+) mice demonstrated increased expression of molecular markers of cardiac hypertrophy, but MyBP-C(t/+) hearts did not demonstrate expression of these molecular markers until the mice were >125 weeks old. Electrophysiological evaluation indicated that MyBP-C(t/+) mice are not as likely to have inducible ventricular tachycardia as alphaMHC(403/+) mice. In addition, cardiac function of alphaMHC(403/+) mice is significantly impaired before the development of LV hypertrophy, whereas cardiac function of MyBP-C(t/+) mice is not impaired even after the development of cardiac hypertrophy. Because these murine FHC models mimic their human counterparts, we propose that similar murine models will be useful for predicting the clinical consequences of other FHC-causing mutations. These data suggest that both electrophysiological and cardiac function studies may enable more definitive risk stratification in FHC patients.

Aging is associated with endothelial dysfunction in healthy men years before the age-related decline in women.

OBJECTIVES: This study assessed whether aging is associated with progressive endothelial dysfunction, whether the pattern of any age-related decline in vascular health is different in men and women and whether any gender difference is consistent with known changes in hormonal status. BACKGROUND: Coronary and cerebrovascular disease are much less common in young and middle-aged women compared with men, although the gender difference in death from atherosclerosis is less marked after the menopause. Endothelial dysfunction is an early event in atherogenesis and is important in dynamic plaque stenosis in later life. The effect of aging on endothelial function in men and women, however, is not well known. METHODS: We used high resolution ultrasound to study endothelium-dependent and endothelium-independent vascular responses. Brachial artery physiology was investigated in 238 subjects (103 men, 135 women; mean [+/- SD] age 38 +/- 17 years, range 15 to 72) with no known risk factors for atherosclerosis. The responses to reactive hyperemia (flow-mediated dilation, which is endothelium dependent) and to glyceryl trinitrate (an endothelium-independent dilator) were assessed for all the subjects and then for men and women separately. RESULTS: On multivariate analysis for the whole group, reduced flow-mediated dilation was related to older age (r = -0.34, p < 0.0001). In men, flow-mediated dilation was preserved in subjects aged < or = 40 years but declined thereafter at 0.21%/year. In women, flow-mediated dilation was stable until the early 50s, after which it declined at 0.49%/year (p = 0.002 compared with men). In contrast, there was no significant change in the glyceryl trinitrate response with aging in either gender. CONCLUSIONS: Aging is associated with progressive endothelial dysfunction in normal humans, and this appears to occur earlier in men than in women. In women, however, a steep decline commences at around the time of the menopause. This is consistent with a protective effect of estrogens on the arterial wall.

Late ventricular arrhythmia is rare after early repair of tetralogy of Fallot.

OBJECTIVES: This study was conducted to describe the incidence of ventricular arrhythmia during prospective long-term follow-up in a group of patients who had repair of tetralogy of Fallot during early childhood. BACKGROUND: Ventricular arrhythmia has been a common finding in patients who have undergone repair of tetralogy of Fallot in late childhood or as adults. Whether earlier repair lowers the incidence of late ventricular arrhythmia or late sudden death is unknown. METHODS: Twenty-nine asymptomatic patients who underwent repair at age 1.2 to 7.7 years (mean [+/- SD] age 4 +/- 1.4 years) between 1979 and 1984 were studied. Twenty-one patients had simple repair (Group A), and eight had complex or multiple operations (Group B). All had ambulatory electrocardiographic monitoring preoperatively, postoperatively, at early follow-up (after 4.2 +/- 1.3 years) and again at late follow-up (after 11.8 +/- 1.3 years). At late follow-up, 28 subjects also underwent echocardiography, and 26 had an exercise test. RESULTS: No patient had significant ventricular arrhythmia (> or = modified Lown grade 2) before or immediately after repair. There was no significant increase in the incidence of arrhythmia at early and late follow-up (14% to 28%), but at each of these periods the incidence of ventricular arrhythmia was higher in Group B patients (3 [43%] of 7 vs. 1 [5%] of 22 with early repair, p = 0.03; 6 [75%] of 8 vs. 2 [10%] of 21 with late repair, p = 0.001). No patient had symptoms of arrhythmia, and there were no sudden deaths. Late ventricular arrhythmia did not correlate with estimated right ventricular systolic pressure, outflow tract gradient or degree of pulmonary incompetence or right ventricular dilation. On exercise, 5 (19%) of 26 patients had ventricular premature complexes at low levels of exercise that were suppressed at maximal exercise in all patients. CONCLUSIONS: Late ventricular arrhythmia is rare in patients with successful early correction of tetralogy of Fallot, unless complex or multiple operations are performed.

Percutaneous balloon dilatation of the atrial septum: immediate and midterm results.

OBJECTIVES: To assess the effectiveness of atrial septostomy by percutaneous balloon dilatation in patients with congenital heart defects or primary pulmonary hypertension. PATIENTS AND DESIGN: Twenty three patients (15 boys, eight girls; aged 10 days to 10 years; 17 with congenital heart defects and six with primary pulmonary hypertension), all haemodynamically unstable under optimal medical treatment, underwent atrial septostomy by percutaneous balloon dilatation. INTERVENTIONS: The balloon catheter entered the left atrium through a patent foramen ovale (n = 14) or via transseptal puncture in cases with an intact atrial septum (n = 9). The size of the balloons used ranged from 13 to 18 mm. RESULTS: There were no complications. The interatrial communication (mm) increased (P < 0.05) after dilatation and remained unchanged (P = NS) during a 16.6 (13.8) month follow up (2 (1.7) v 8.8 (1.4) v 8.2 (1.1), respectively). Transatrial gradient (mm Hg) fell and arterial oxygenation (%) improved both in patients with transposition (6.3 (0.8) v 0.8 (1) (P = 0.0001) and 40.6 (4.2) v 76.5 (4.8) (P = 0.0001), respectively) and in those with mitral atresia (13.4 (1.9) v 2 (1.4) (P = 0.0001) and 77.1 (3.9) v 81.5 (4.2) (P = 0.008), respectively). There were two failures, one early and one late, both in the group of patients with mitral atresia or stenosis. A decrease in arterial oxygenation (94.8 (1.5) v 83 (2.4), P = 0.004) and an increase in left atrial pressure (6.8 (0.9) v 8.3 (1.2), P = 0.02) and cardiac index (2.3 (0.2) v 3.1 (0.2) l/min/m2, P = 0.002) was observed in patients with primary pulmonary hypertension. CONCLUSIONS: Percutaneous balloon dilatation is an effective and safe procedure for creating an adequate interatrial communication that can be used as an alternative to blade septostomy.

Microscopic polyangiitis and Kawasaki disease without overt clinical cardiovascular manifestations and with abnormal cardiovascular magnetic resonance findings.

Abnormal cardiovascular magnetic resonance findings were found in a patient with microscopic polyangiitis and a patient with Kawasaki disease that presented without overt clinical cardiovascular manifestations.

Stabilization of human urine doping control samples: III. Recombinant human erythropoietin.

BACKGROUND: The tampering of athlete's urine samples by the addition of proteolytic enzymes during the doping control sampling procedure was reported recently. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in urine samples to chemically inactivate proteolytic enzymes and improve the electrophoteric signal of erythropoietin (EPO) in human urine. METHODS: The stabilization mixture applied was a combination of antibiotics, antimycotic substances and protease inhibitors. A series of incubation experiments were conducted under controlled conditions in the presence and absence of the stabilization mixture in urine aliquots spiked with six proteases. Two different analytical techniques were applied for the qualitative and quantitative EPO measurement: isoelectric focusing (IEF) and chemiluminescent immunoassay respectively. RESULTS: The addition of the chemical stabilization mixture into urine aliquots substantially improved EPO detection in the presence of proteolytic enzymes following incubation at 37 degrees C or storage at -20 degrees C. CONCLUSIONS: The results of this study indicated that the stabilization of urine prior to the sample collection procedure with the proposed chemical mixture might prove to be a useful tool for the preservation of anti-doping samples.

Nonlinear isochrones in murine left ventricular pressure-volume loops: how well does the time-varying elastance concept hold?

The linear time-varying elastance theory is frequently used to describe the change in ventricular stiffness during the cardiac cycle. The concept assumes that all isochrones (i.e., curves that connect pressure-volume data occurring at the same time) are linear and have a common volume intercept. Of specific interest is the steepest isochrone, the end-systolic pressure-volume relationship (ESPVR), of which the slope serves as an index for cardiac contractile function. Pressure-volume measurements, achieved with a combined pressure-conductance catheter in the left ventricle of 13 open-chest anesthetized mice, showed a marked curvilinearity of the isochrones. We therefore analyzed the shape of the isochrones by using six regression algorithms (two linear, two quadratic, and two logarithmic, each with a fixed or time-varying intercept) and discussed the consequences for the elastance concept. Our main observations were 1) the volume intercept varies considerably with time; 2) isochrones are equally well described by using quadratic or logarithmic regression; 3) linear regression with a fixed intercept shows poor correlation (R(2) < 0.75) during isovolumic relaxation and early filling; and 4) logarithmic regression is superior in estimating the fixed volume intercept of the ESPVR. In conclusion, the linear time-varying elastance fails to provide a sufficiently robust model to account for changes in pressure and volume during the cardiac cycle in the mouse ventricle. A new framework accounting for the nonlinear shape of the isochrones needs to be developed.

Minimal force-frequency modulation of inotropy and relaxation of in situ murine heart.

1. The normal influence of heart rate (HR) on cardiac contraction and relaxation in the mouse remains uncertain despite its importance in interpreting many genetically engineered models. Prior in vivo data have repeatedly shown positive effects only at subphysiological heart rates, yet depressed basal conditions and use of load-dependent parameters probably have an impact on these results. 2. Open-chest mice of various strains (n = 16, etomidate/urethane anaesthesia) were instrumented with a miniaturized pressure-volume catheter employing absolute left ventricular (LV) volume calibration. HR was slowed (< 400 beats min(-1)) using ULFS-49, and atrial or ventricular pacing was achieved via an intra-oesophageal catheter. Pressure-volume data yielded cardiac-specific contractile indexes minimally altered by vascular load. 3. At a resting HR of 600 beats min(-1), peak pressure-rise rate (dP/dt(max)) was 16 871 +/- 2941 mmHg s(-1) (mean +/- S.D.) and the relaxation time constant was 3.9 +/- 0.8 ms, similar to values in conscious animals. Within the broad physiological range (500-850 beats min(-1)), load-insensitive contractile indexes and relaxation rate varied minimally, whereas dP/dt(max) peaked at 600 +/- 25 beats min(-1) and decreased at higher rates due to preload sensitivity. Contraction and relaxation were enhanced modestly (13-15 %) at HRs of between 400 and 500 beats min(-1). 4. The minimal force-frequency dependence was explained by rapid calcium cycling kinetics, with a mechanical restitution time constant of 9 +/- 2.7 ms, and by dominant sarcoplasmic reticular buffering (recirculation fraction of 93 +/- 1 %). 5. The mouse normally has a very limited force-frequency reserve at physiological HRs, unlike larger mammals and man. This is important to consider when studying disease evolution and survival of genetic models that alter calcium homeostasis and SR function.

Epiphytic populations of Pseudomonas syringae on barley.

The epiphytic populations of Pseudomonas syringae were monitored on 23 barley entries planted in the field in four replications during the summer of 1986, and on six selected entries during the summer of 1987, from the second-leaf stage until senescence. Populations were initially low (0-3 log colony-forming units (cfu) per leaf) in all but one entry; they generally increased throughout the season, and at the end they reached 3-7 log cfu/leaf. Significant differences among the average epiphytic populations were found in the 1986 trial; only one entry, however, had a significantly different average population in the 1987 trial. The slopes of population increase were also compared: significant differences were observed in 1986 but not in 1987. In addition to epiphytic population counts, the percentage of ice nucleation active bacteria was determined in the population isolated from each leaf sample, and averaged throughout the season for each entry. Significant differences were observed in 1986 and in 1987. When the entries were ranked according to their average epiphytic population and compared between the two experiments, they were found to be very similar. The same was not true for the other parameters studied in the experiment.

Estimation of parallel conductance by dual-frequency conductance catheter in mice.

The conductance catheter method has substantially enhanced the characterization of in vivo cardiovascular function in mice. Absolute volume determination requires assessment of parallel conductance (V(p)) offset because of conductivity of structures external to the blood pool. Although such a determination is achievable by hypertonic saline bolus injection, this method poses potential risks to mice because of volume loading and/or contractility changes. We tested another method based on differences between blood and muscle conductances at various catheter excitation frequencies (20 vs. 2 kHz) in 33 open-chest mice. The ratio of mean frequency-dependent signal difference to V(p) derived by hypertonic saline injection was consistent [0.095 +/- 0.01 (SD), n = 11], and both methods were strongly correlated (r(2) = 0.97, P < 0.0001). This correlation persisted when the ratio was prospectively applied to a separate group of animals (n = 12), with a combined regression relation of V(p(DF)) = 1.1 * V(p(Sal)) - 2.5 [where V(p(DF)) is V(p) derived by the dual-frequency method and V(p(Sal)) is V(p) derived by hypertonic saline bolus injection], r(2) = 0.95, standard error of the estimate = 1.1 microl, and mean difference = 0.6 +/- 1.4 microl. Varying V(p(Sal)) in a given animal resulted in parallel changes in V(p(DF)) (multiple regression r(2) = 0.92, P < 0.00001). The dominant source of V(p) in mice was found to be the left ventricular wall itself, since surrounding the heart in the chest with physiological saline or markedly varying right ventricular volumes had a minimal effect on the left ventricular volume signal. On the basis of V(p) and flow probe-derived cardiac output, end-diastolic volume and ejection fraction in normal mice were 28 +/- 3 microl and 81 +/- 6%, respectively, at a heart rate of 622 +/- 28 min(-1). Thus the dual-frequency method and independent flow signal can be used to provide absolute volumes in mice.