Changes in ionic currents and beta-adrenergic receptor signaling in hypertrophied myocytes overexpressing G alpha(q).

Transgenic overexpression of G alpha(q) causes cardiac hypertrophy and depressed contractile responses to beta-adrenergic receptor agonists. The electrophysiological basis of the altered myocardial function was examined in left ventricular myocytes isolated from transgenic (G alpha(q)) mice. Action potential duration was significantly prolonged in G alpha(q) compared with nontransgenic (NTG) myocytes. The densities of inward rectifier K(+) currents, transient outward K(+) currents (I(to)), and Na(+)/Ca(2+) exchange currents were reduced in G alpha(q) myocytes. Consistent with functional measurements, Na(+)/Ca(2+) exchanger gene expression was reduced in G alpha(q) hearts. Kinetics or sensitivity of I(to) to 4-aminopyridine was unchanged, but 4-aminopyridine prolonged the action potential more in G alpha(q) myocytes. Isoproterenol increased L-type Ca(2+) currents (I(Ca)) in both groups, with a similar EC(50), but the maximal response in G alpha(q) myocytes was approximately 24% of that in NTG myocytes. In NTG myocytes, the maximal increase of I(Ca) with isoproterenol or forskolin was similar. In G alpha(q) myocytes, forskolin was more effective and enhanced I(Ca) up to approximately 55% of that in NTG myocytes. These results indicate that the changes in ionic currents and multiple defects in the beta-adrenergic receptor/Ca(2+) channel signaling pathway contribute to altered ventricular function in this model of cardiac hypertrophy.

The expression of SR calcium transport ATPase and the Na(+)/Ca(2+)Exchanger are antithetically regulated during mouse cardiac development and in Hypo/hyperthyroidism.

The mouse has been used extensively for generating transgenic animal models to study cardiovascular disease. Recently, a number of transgenic mouse models have been created to investigate the importance of sarcoplasmic reticulum (SR) Ca(2+)transport proteins in cardiac pathophysiology. However, the expression and regulation of cardiac SR Ca(2+)ATPase and other Ca(2+)transport proteins have not been studied in detail in the mouse. In this study, we used multiplex RNase mapping analysis to determine SERCA2, phospholamban (PLB), and Na(+)/Ca(2+)-exchanger (NCX-1) gene expression throughout mouse heart development and in hypo/hyperthyroid animals. Our results demonstrate that the expression of SERCA2 and PLB mRNA increase eight-fold from fetal to adult stages, indicating that SR function increases with heart development. In contrast, the expression of the Na(+)/Ca(2+)-exchanger gene is two-fold higher in fetal heart compared to adult. Our study also makes the important observation that in hypothyroidic hearts the NCX-1 mRNA and protein levels were upregulated, whereas the SERCA2 mRNA/protein levels were downregulated. In hyperthyroidic hearts, however, an opposite response was identified. These findings are important and point out that the expression of NCX-1 is regulated antithetically to that of SERCA2 during heart development and in response to alterations in thyroid hormone levels.

Reproduction of Globodera tabacum solanacearum in Seven Flue-Cured Tobacco-Producing Soils.

The tobacco cyst nematode (Globodera tabacum solanacearum) continues to pose a serious threat to flue-cured tobacco production in Virginia and nearby states. Soils were sampled from five uninfested and two infested flue-cured tobacco-producing locations. Twenty-three edaphic factors were characterized to determine if any were correlated with G. t. solanacearum reproduction. Comparisons were also made between pasteurized and natural soils to determine if biological suppression of G. t. solanacearum reproduction might be occurring in currently uninfested areas. Differences in G. t. solanacearum reproduction were noted among the soils, but results were inconsistent across the three trials conducted in this study. Only soil pH correlated significantly with nematode reproduction, and then only in one of three trials. Globodera tabacum solanacearum reproduced with similar efficiency in natural and pasteurized soils.

Impaired cardiac performance in heterozygous mice with a null mutation in the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene.

The sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene encodes both SERCA2a, the cardiac sarcoplasmic reticulum Ca2+ pump, and SERCA2b, which is expressed in all tissues. To gain a better understanding of the physiological functions of SERCA2, we used gene targeting to develop a mouse in which the promoter and 5' end of the gene were eliminated. Mating of heterozygous mutant mice yielded wild-type and heterozygous offspring; homozygous mutants were not observed. RNase protection, Western blotting, and biochemical analysis of heart samples showed that SERCA2 mRNA was reduced by approximately 45% in heterozygous mutant hearts and that SERCA2 protein and maximal velocity of Ca2+ uptake into the sarcoplasmic reticulum were reduced by approximately 35%. Measurements of cardiovascular performance via transducers in the left ventricle and right femoral artery of the anesthetized mouse revealed reductions in mean arterial pressure, systolic ventricular pressure, and the absolute values of both positive and negative dP/dt in heterozygous mutants. These results demonstrate that two functional copies of the SERCA2 gene are required to maintain normal levels of SERCA2 mRNA, protein, and Ca2+ sequestering activity, and that the deficit in Ca2+ sequestering activity due to the loss of one copy of the SERCA2 gene impairs cardiac contractility and relaxation.

Effects of Tobacco Cyst Nematode on Growth of Flue-cured Tobacco.

The effects of infection by tobacco cyst nematode (Globodera tabacum solanacearum) on growth of flue-cured tobacco cultivars NC 567 (resistant) and K 326 (susceptible) were evaluated in the field in 1993 and 1994. Infection by G. t. solanacearum suppressed number of leaves, plant height, and fresh weight of leaves and feeder roots. Correlations between weekly egg densities of G. t. solanacearum collected from soil and host growth during 11 weeks after transplanting (WAT) were often inconsistent between cultivars and years. However, consistent correlations were obtained between root weight and egg densities collected 9 WAT, as well as between leaf weight from susceptible K 326 and nematode egg densities 6 WAT. Leaf and feeder root weights were significantly correlated with the area under the curve for all nematodes per gram of feeder root for K 326 in 1993 and for both cultivars in 1994. Reduction in feeder root weight by G. t. solanacearum was similar for the resistant and susceptible cultivars. Reduction in fresh leaf weight by G. t. solanacearum was twice as great (P

Effect of aging on EGF-stimulated replication of specific genes in rat hepatocytes.

EGF-stimulated replication of specific genes was examined in primary hepatocyte cultures from mature (6 months) and senescent (24 months) rats. Basal and EGF-stimulated [3H]thymidine incorporation and DNA polymerase alpha activities, as well as total cellular DNA, were also assessed. The genes examined were dihydrofolate reductase (DHFR) and c-myc, as well as total mitochondrial DNA (mt DNA). Although [3H]thymidine incorporation, DNA polymerase alpha activity, total cellular DNA, DHFR, and c-myc gene specific DNA replication stimulated by EGF are reduced with age, mt DNA replication is not affected by either EGF or age. Chromosomal DNA replication is mediated mainly by DNA polymerase alpha while mt DNA replication is mediated by its own DNA polymerase gamma. Thus, the age-related decline in stimulated DNA replication appears to be associated mainly with the DNA polymerase alpha activation pathway.

Chimeric papillomavirus-like particles.

We have constructed chimeric papillomavirus-like particles (CVLPs) by replacing the 34-carboxy-terminal amino acids of the HPV 16 L1 protein with various parts of the HPV 16 E7 protein. Chimeric proteins were expressed by recombinant baculoviruses and analyzed by electron microscopy for their ability to assemble into virus capsids. We were able to produce CVLPs in high efficiencies with inserts of up to 60 amino acids. CVLPs are able to induce a neutralizing antibody response, assayed by inhibition of hemagglutination of mouse erythrocytes. CVLPs are interacting with the putative receptor for papillomaviruses as they were shown to hemagglutinate mouse red blood cells and bind to and penetrate cells in vitro. As CVLPs follow a similar intracellular pathway as observed earlier for BPV VLPs, we speculate that CVLPs can be used to deliver peptides into mammalian cells in vitro and in vivo, possibly reaching the pathway for MHC class I presentation.

Characterization of transcriptional regulation of the kdp operon of Salmonella typhimurium.

The transcriptional control of the kdpFABC (K+ transport) operon of Salmonella typhimurium was characterized with a lacZ fusion. The kdpFABC operon of this organism was induced by K+ limitation and high osmolality, and osmotic induction was antagonized by a high concentration of K+. In the trkA (sapG) kdp+ mutant background, high concentrations of K+ inhibited growth, along with repressing the kdp operon. This result, which has not been reported for Escherichia coli, is inconsistent with the model in which the signal for the induction of the kdp operon is turgor loss.

Aging reduces the numbers of hepatocytes synthesizing DNA in response to EGF and epinephrine.

Primary cultures of hepatocytes were prepared from young (6 month) and old (24 month) Wistar rats and exposed to epinephrine or epidermal growth factor. Incorporation of [3H]thymidine into DNA was determined both radiochemically and autoradiographically. The numbers of responding cells and degree of response per cell were determined and the results confirmed by FACScan analysis. Such analyses clearly demonstrate a reduced number of hepatocytes capable of responding to the above stimuli in cultures obtained from old rats. Thus, changes in numbers of responding cells may be an important mechanism involved in reduced responsiveness of the aged liver to agents which stimulate DNA synthesis and cell division.

Effect of aging on regulation of sdi-1 in rat hepatocytes.

We examined basal and EGF stimulated DNA synthesis as well as sdi-1 mRNA and protein in primary hepatocyte cultures, and basal levels of sdi-1 mRNA and protein in whole liver homogenates from 6 and 24 month old rats. Since EGF stimulated DNA synthesis decreases with age, it was hypothesized that basal and EGF stimulated levels of sdi-1 mRNA and protein, an inhibitor of DNA synthesis, might increase. Surprisingly, however both sdi-1 mRNA and protein actually decreased both in cells and homogenates of old rats. These results indicate that the age-related impairment in EGF stimulated DNA synthesis in hepatocytes appears to occur prior to or parallel with sdi-1 expression and cannot be explained on the basis of increased inhibition due to elevated levels of this protein.

Effect of aging on EGF stimulated DNA synthesis and EGF receptor levels in primary cultured rat hepatocytes.

Epidermal growth factor (EGF) stimulated DNA synthesis and EGF receptor levels were examined in primary cultured hepatocytes from 6, 12 and 24 month old rats. EGF stimulated DNA synthesis began after 12h and reached a peak at 48 h. Although no age difference was seen in the time course of DNA synthesis, the magnitude of synthesis at the peak time in 12 and 24 month old rat hepatocytes was reduced approximately 50 and 70%, as compared to that at 6 months. Hepatocyte EGF receptors exhibited no age difference in the equilibrium dissociation constant (Kd) or the density (Bmax). These results indicate that EGF stimulated DNA synthesis in rat hepatocytes declines with age, and that this reduction is not due to decreased receptor density or specific binding affinity.