Differential morphometric and ultrastructural remodelling in the left atrium and left ventricle in rapid ventricular pacing-induced heart failure.

BACKGROUND: Heart failure induced by rapid ventricular pacing (RVP) is associated with left atrial (LA) but not left ventricular (LV) hypertrophy. OBJECTIVE: To determine whether differences in wall tension correlate with the differential ultrastructural remodelling in the LA and LV chambers, changes in ultrastructure, systolic function and wall tension (an index of wall stress) were compared in dogs after RVP (n=7) and with no RVP (n=9). RESULTS: Compared with dogs with no RVP (controls), dogs with RVP had increased collagen volume fraction (5.3% versus 8.3%), myocyte cross-sectional area (245 versus 366 microm(2)) and hydroxyproline (222 versus 323 microg/mg protein) in the LA (all P<0.05), but not in the LV. The increase in systolic wall tension produced by RVP was greater in the LA (five versus 43 units, P<0.0004) than in the LV (227 versus 290 units, P<0.01) chambers and correlated closely with the collagen volume fraction (r=0.87), which in turn correlated with myocyte cross-sectional area (r=0.98). In the left atrium, wall tension correlated with wall stress (r=0.99). CONCLUSIONS: The results suggest that differential wall tension may provide the stimulus for differential ultrastructural remodelling (with more hypertrophy and collagen) between LA and LV chambers in RVP-induced cardiomyopathy.

Plakoglobin regulates the expression of the anti-apoptotic protein BCL-2.

Plakoglobin is a cytoplasmic protein and a homologue of beta-catenin and Armadillo of Drosophila with similar adhesive and signaling functions. These proteins interact with cadherins to mediate cell-cell adhesion and associate with transcription factors to induce changes in the expression of genes involved in cell fate determination and proliferation. Unlike the relatively well characterized role of beta-catenin in cell proliferation via activation of c-MYC and cyclin D1 gene expression, the signaling function of plakoglobin in regulation of cell growth is undefined. Here, we show that high levels of plakoglobin expression in plakoglobin-deficient human SCC9 cells leads to uncontrolled growth and foci formation. Concurrent with the change in growth characteristics we observe a pronounced inhibition of apoptosis. This correlates with an induction of expression of BCL-2, a prototypic member of apoptosis-regulating proteins. The BCL-2 expression coincides with decreased proteolytic processing and activation of caspase-3, an executor of programmed cell death. Our data suggest that the growth regulatory function of plakoglobin is independent of its role in mediating cell-cell adhesion. These observations clearly implicate plakoglobin in pathways regulating cell growth and provide initial evidence of its role as a pivotal molecular link between pathways regulating cell adherence and cell death.

Plakoglobin induces desmosome formation and epidermoid phenotype in N-cadherin-expressing squamous carcinoma cells deficient in plakoglobin and E-cadherin.

Pg is a homologue of beta-catenin and Armadillo, the product of the Drosophila segment polarity gene and has been shown to have both adhesive and signaling functions. It interacts with both classic and desmosomal cadherins. Pg interaction with the desmosomal cadherins is essential for desmosome assembly. Its precise role in the classic cadherin complexes is unclear, although Pg-E-cadherin interaction appears to be necessary for the formation of desmosomes. In addition to cadherins in adhesion complexes, Pg interacts with a number of proteins involved in regulation of cell differentiation and proliferation such as Lef-1/Tcf-1 transcription factors and the tumor suppressor protein APC. In this study, we have introduced Pg cDNA into SCC9 cells, a Pg- and E-cadherin-deficient squamous cell carcinoma line, which also lacks desmosomes. These cells have both alpha-catenin and beta-catenin, display unusual expression of N-cadherin, and have the typical fibroblastic phenotype of transformed cells. Pg-expressing SCC9 cells (SCC9P) formed desmosomes. Desmosome formation coincided with the appearance of an epidermoid phenotype, with increased adhesiveness and a contact-dependent decrease in growth. Biochemical characterization of SCC9P cells showed an increase in the expression and stability of N-cadherin and a decrease in level and stability of beta-catenin, without any apparent effects on alpha-catenin. These results show that, in the absence of E-cadherin, Pg can efficiently use N-cadherin to induce desmosome formation and epidermoid phenotype. They also suggest a role for Pg as one of the regulators of the intracellular beta-catenin levels and underscore the pivotal role of this protein in regulating cell adhesion and differentiation.

Spatial regulation of SpMTA metallothionein gene expression in sea urchin embryos by a regulatory cassette in intron 1.

The SpMTA metallothionein (MT) gene of the sea urchin Strongylocentrotus purpuratus is restricted in its expression to the aboral ectoderm in gastrulae and pluteus larvae. The proximal 1.6 kb of the 5'-flanking region together with the 1.12-kb first intron of the SpMTA gene are sufficient for its correct cell-type specific expression in transgenic embryos. This restricted spatial expression is largely eliminated by deletion of an interior 405-bp region in the intron. Within this region is a 295-bp, genomically repetitive, transposon-like segment (Nemer et al., 1993), containing several sequence motifs highly homologous to posited regulatory elements in the promoters of other genes (Thiebaud et al., 1990). The P3A and P5 sites in this apparent regulatory cassette were shown through competition to bind with relatively high affinities the same nuclear factors, bound by their counterpart sites in the CyIIIa actin promoter.

Combinatorial regulation by promoter and intron 1 regions of the metallothionein SpMTA gene in the sea urchin embryo.

The SpMTA metallothionein gene of the sea urchin Strongylocentrotus purpuratus is regulated developmentally, histospecifically, and by heavy-metal induction. The sequenced 5' flank of the gene can be divided into proximal, middle, and distal regions, each containing a pair of metal response elements (MREs). Canonical 7-bp core sequences are present in all except the middle-region MREs c and d, which contain 1-bp mismatches. Metal-induced expression in transgenic blastulae was increased with each consecutive addition of the middle and distal regions to a chimeric reporter gene construct containing the proximal SpMTA promoter region. Reduced metal induction through point mutation of the distal MREs e and f indicated that the MREs themselves were largely responsible for the transcriptional increase. These activities were further enhanced by SpMTA intron 1, but not when a specific interior region of the intron had been deleted. The atypical MREs c and d did not support induction by themselves, i.e., when present alone with mutated proximal MREs a and b. However, in the presence of intron 1, they were able to substitute for the nullified MREs a and b in the promotion of metal-induced expression. This capability suggests, furthermore, that these atypical MREs, in addition to responding to an intron 1 region, participate cooperatively with the canonical proximal MREs.

Molecular size dependence of peritoneal transport.

The nature of the barrier between blood and peritoneal dialysate was studied by determining peritoneal solute transport rates as a function of molecular size using neutral dextrans, polymers with different molecular weights but uniform chemical composition. Transport rates for creatinine, p-aminohippurate (PAH), and neutral dextran (3,000 to 50,000 daltons) were measured during peritoneal dialysis in the awake rabbit using sequential isotonic and hypertonic dialysis solutions. The permeability-area product (PA) for the peritoneum was determined from the dependence of the dialysate concentration on time during the isotonic exchange. The dependence of PA on molecular size showed no additional hindrance by the peritoneum as molecules of larger size were considered. By accounting for convective solute transport during the hypertonic exchange, the solute reflection coefficient (sigma) and PA were simultaneously determined. The values of PA for creatinine and PAH were similar to those determined during the isotonic exchange, and sigma values for creatinine and PAH were 0.18 +/- 0.20 and 0.14 +/- 0.14, respectively. Dextran sigma values (3,000 to 22,000 daltons) were near unity (0.9 to 1.0) and relatively independent of molecular size, suggesting substantial hindrance to convective transport for this size range. This work demonstrates that the paradoxical transport properties of an "open" diffusive yet "tight" convective peritoneal barrier are primarily reflective of the unique structural properties of this biologic tissue and are not related to test solute heterogeneity.

Renal tubular acidosis in two horses: diagnostic studies.

An 11-year-old Quarter Horse mare and a 2-year-old Quarter Horse colt with clinical diagnoses of renal tubular acidosis (RTA) were donated to the University of California Veterinary Medical Teaching Hospital. A series of diagnostic tests was performed in an attempt to characterize the type and cause of RTA in these horses. Endogenous creatinine clearance and sodium sulfanilate clearance were within reference ranges; thus, no abnormality of glomerular function was detected. To assess renal tubular function in response to acid loading, each horse was given 0.1 g of NH4Cl/kg of body weight via nasogastric tube in 6 L of water. Urine acidification in response to the oral acid load was less than that observed in control horses. The urinary clearance ratio for sodium also was found to be greater for the principals than for the controls. These findings supported a diagnosis of RTA that closely resembled type 1 or distal RTA. In an attempt to determine the cause of RTA, renal ultrasonography, renal biopsy, and a mating study were performed. No abnormalities were identified, and the cause of RTA in these cases remained unknown.

Clinical features and treatment of renal tubular acidosis in two horses.

Two horses were admitted separately for evaluation and treatment of profound hyperchloremic metabolic acidosis without azotemia. One, an 11-year-old Quarter Horse mare, had been depressed and ataxic for 2 days. The other, a 2-year-old Quarter Horse colt, had a 6-week history of depression, anorexia, and weight loss. Both horses responded to fluid and electrolyte therapy, but required daily oral administration of sodium bicarbonate for maintenance. In each case, the diagnosis was renal tubular acidosis.

Effects of intravenous infusion of doxorubicin on blood chemistry, blood pressure and heart rate in rabbits.

The effects of a 1 h continuous infusion of doxorubicin (12.5 mg kg-1, 200 mg M-2) on blood chemistry was examined in rabbits over a 6-h period. Plasma glucose levels remained unchanged while insulin levels were significantly decreased to 39, 45 and 61% of the zero time value (12.8 +/- 2.9 ng ml-1) at 30, 60 and 120 min, respectively, after starting the drug infusion. Plasma cortisol levels were increased to 141, 140 and 131% of the initial zero time value (12.3 +/- 2.2 ng ml-1) at 120, 240 and 360 min, respectively. Doxorubicin had no effect on plasma electrolytes, osmolality and urea nitrogen but significantly increased plasma creatinine over the corresponding control value (2.2 +/- 0.8 micrograms ml-1 to 4.9 +/- 0.7 micrograms ml-1) at 120 min and the level remained elevated for the remaining period of the study. Systolic and diastolic pressure, and heart rate were also depressed at 240 and 360 min. The data collected in the present study indicate that the doxorubicin infusion might have a direct effect on beta cells in the pancreas as well as muscle tissue. Changes in cortisol, blood pressure and heart rate appear to be secondary to other effects produced by doxorubicin.

Gene regulatory divergence among species estimated by altered developmental patterns in interspecific hybrids.

Disturbances in the schedules of gene expression in developing interspecific fish hybrids have been used to draw inferences about the extent of gene regulatory divergence between species and about the degree to which this gene regulatory divergence is correlated with structural gene divergence, as estimated by genetic distance. Sperm from each of 10 different species representing six genera within the family Centrarchidae was used to fertilize eggs of the Florida largemouth bass (Micropterus salmoides floridanus). The genetic distances (D; Nei 1978) between the parental species used to form the hybrids ranged from 0.133 to 0.974. The developmental success and temporal patterns of gene expression of each of the hybrids were compared with those of the Florida largemouth bass. As the genetic distance between the paternal species and the Florida largemouth bass increased, there was a general decline in developmental success in the hybrid embryos as demonstrated by the observed reductions in the percentage of hatching and by progressively earlier and more extensive morphological abnormalities. Concomitantly, progressively more marked alterations in developmental schedules of expression of 15 enzyme loci occurred in the hybrids as the genetic distance between parental species increased. However, observed deviations from this trend for a few species may represent an uncoupling of the rates and modes of evolution of structural genes from those for genes regulating developmental processes.

Relative developmental success of interspecific Lepomis hybrids as an estimate of gene regulatory divergence between species.

The developmental success of interspecific Lepomis hybrids is used as an index of gene regulatory divergence between the green sunfish, L. cyanellus, and each of three other parental species, longear sunfish, L. megalotis, warmouth, L. gulosus, and bluegill, L. macrochirus. This gene regulatory divergence is compared to the degree of structural gene divergence among these four species (genetic distance [Nei, '78], D, ranged from 0.206 to 0.586). The developmental success of the hybrid embryos at the level of morphogenesis was higher than expected from the genetic distance between the parental species. The rates of morphogenesis of the hybrid embryos were the same as that for the green sunfish embryos. The percentage of embryos that hatched was relatively high in all crosses. However, two of the hybrid crosses resulted in enhanced percentages of hatched embryos. Slight increases in the extent of morphological abnormalities were observed in hybrids from crosses between more distantly related parental species. The schedules and levels of enzyme locus expression of the hybrids, assessed spectrophotometrically and electrophoretically for nine enzyme systems (encoded in a total of 14 loci), were different from each other and from those of the green sunfish embryos. Alterations in the time of first enzyme appearance and in the time of first increase in enzyme activity in the developing hybrid embryos were not correlated with genetic distance between parental species. However, the extents of alteration of enzyme activities over the entire period of hybrid embryogenesis were correlated with the genetic distance. We attribute the morphological and molecular anomalies observed in the hybrids to gene regulatory incompatibilities between species. Although the exact number of mutational differences and their relative developmental impacts are not known, some inferences can be drawn about the degree of divergence in gene regulation between species. It appears that an uncoupling of the rates of structural and regulatory gene evolution can occur between species of some taxa, an observation that has implications for the roles of gene regulatory differences in organismic evolution.

Electromyographic and urethral pressure profilometry: assessment of urethral function before and after perineal urethrostomy in cats.

Urethral pressure and electromyographic profiles were recorded in 10 healthy adult male cats before and after perineal urethrostomy. There was a significant decrease (P = 0 at 1 week after surgery, P = 0.002 at 3 weeks after surgery) in the mean maximum urethral pressure profile with associated loss of periurethral striated muscle activity.

Metabolism and renal handling of sodium arsenate in dogs.

Renal handling of sodium arsenate was studied in 5 dogs. Using a low dose (0.73 mg/kg of body weight) of sodium arsenate given IV, variable arsenite concentrations were detected in plasma and urine. Using a medium dose (7.33 mg/kg), the renal tubule cells were determined to be the probable sites of reabsorption of arsenate, reduction of arsenate to arsenite, and secretion or diffusion of the latter into urine. However, using a high dose (14.66 mg/kg), despite a similar pattern of reduction of arsenate to arsenite, marked reabsorption of arsenite into plasma took place instead of secretion or diffusion into urine. Because of reabsorption, the amount of arsenite in plasma (18.4 +/- 3.5% of the total As) was about 3 times higher than that measured during the medium dose experiment (6.0 +/- 1.0%). During the clearance experiments which lasted 110 minutes, only 40% to 45% of the arsenate infused was excreted in urine, and a minimal amount of dimethylarsinic acid (DMAA) was detected. In contrast, by the next day, DMAA was the major metabolite excreted in urine. This excretion of As as DMAA was partly due to delayed excretion of 55% to 60% As that was stored in the body and was subsequently metabolized.

Nephrotoxicity of sodium arsenate in dogs.

Nephrotoxicity of sodium arsenate was evaluated in dogs to determine the pathophysiologic basis for renal lesions caused by this heavy metal. Examination of biopsy specimens indicated that the low dose of the As salt (0.73 mg/kg of body weight) produced histologic changes consisting of mild degeneration and vacuolation of renal tubular epithelium. Vacuolation involved mainly the ascending thick portion of the nephron. Clinical pathologic changes were not demonstrable at this dosage level according to glomerular filtration rate (creatinine clearance), fractional reabsorption of sodium, potassium, and chloride; plasma osmolar and free water clearance; and urinalysis. The medium dose (7.33 mg/kg) resulted in alterations determined by urinalysis, but did not markedly affect other clinical pathologic measurements. Histopathologic changes were equal to or greater than those seen with the low dose. Tubular necrosis was observed in the cortical portion of the nephron and the ascending thick limb. The high dose (14.66 mg/kg) consistently produced marked changes in all parameters evaluated. Clinical pathologic alterations were compatible with acute tubular necrosis involving all segments of the nephron. Histologically, moderate glomerular sclerosis and severe tubular necrosis were observed. During recovery from the high dose of As, a gradual compensatory healing process was observed that was evident in all clinical pathologic parameters and was confirmed from sequential renal biopsy specimens.

Carotid artery exteriorization for percutaneous catheterization in sheep and dogs.

Summary and reliable physiologic or pharmacologic investigation in animal models often requires arterial catheterization in awake animal subjects. This can be facilitated by surgical exteriorization of the carotid artery for subsequent percutaneous catheterization. A simplified surgical procedure is described that has been demonstrated to have limited morbidity and excellent applicability for multiple use, as well as to ensure high blood flow rates when needed. The surgical technique involves transfer of the carotid artery from a deep paratracheal position to a ventrolateral neck subcutaneous position, protected from the underlying mattress sutures by the sternomastoid muscle.

Continuous low flow hemofiltration with sorbent regeneration of ultrafiltrate.

In summary, a series of experiments have culminated in the maintenance of 2 anephric sheep for 48 and 72 hrs with CLF-HF treatment. Ewe A died of hemorrhage secondary to systemic heparinization and recent surgery. Ewe B was terminated electively. The technique of CLF-HF proved satisfactory in the control of urea, creatinine and water. Sodium and hydrogen ion balance required substantial therapeutic manipulations in addition to CLF-HF to maintain satisfactory balance with the present system. Technical and functional performance of the ultrafilter was better than anticipated. Cartridge performance was sufficiently satisfactory to demonstrate that the procedure will work. The unphysiologic linking of urea, hydrogen and sodium sorption resulted in difficulty in maintaining satisfactory sodium and hydrogen balance. Coupling a disposable reprocessing system, which lacks the deficiencies mentioned above, with continuous hemofiltration can provide a truly wearable artificial kidney.