Effects of kappa-opioid receptor activation on myocardium.

Kappa-opioid receptor stimulation of the heart transiently increases twitch amplitude and decreases Ca2+-dependent actomyosin Mg2+-ATPase activity through an undetermined mechanism. One purpose of the present study was to determine if the increase in twitch amplitude is due to changes in myofilament Ca2+ sensitivity. We also wanted to determine if kappa-opioid receptor activation alters maximum actin-myosin ATPase activity and Ca2+ sensitivity of tension in a way consistent with protein kinase A or protein kinase C (PKC) action. Rat hearts were treated with U50,488H (a kappa-opioid receptor agonist), phenylephrine plus propranolol (alpha-adrenergic receptor stimulation), isoproterenol (a beta-adrenergic receptor agonist), or phorbol 12-myristate 13-acetate (PMA, receptor independent activator of PKC) or were untreated (control), and myofibrils were isolated. U50,488H, phenylephrine plus propranolol, and PMA all decreased maximum Ca2+-dependent actomyosin Mg2+-ATPase activity, whereas isoproterenol treatment increased maximum Ca2+-dependent actomyosin Mg2+- ATPase activity. Untreated myofibrils exposed to exogenous PKC-epsilon, but not PKC-delta, decreased maximum actomyosin Mg2+-ATPase activity. Langendorff-perfused hearts treated with U50,488H, phenylephrine plus propranolol, or isoproterenol had significantly higher ventricular ATP levels compared with control hearts. PKC inhibitors abolished the effects of U50,488H on Ca2+-dependent actomyosin Mg2+-ATPase activity and myocardial ATP levels. U50,488H and PMA treatment of isolated ventricular myocytes increased Ca2+ sensitivity of isometric tension compared with control myocytes at pH 7.0. The U50,488H-dependent increase in Ca2+ sensitivity of tension was retained at pH 6.6. Together, these findings are consistent with the hypotheses that 1) the positive inotropy associated with kappa-opioid receptor activation may be due in part to a PKC-mediated increase in myofilament Ca2+-sensitivity of tension and 2) the kappa-opioid receptor-PKC pathway is a modulator of myocardial energy status through reduction of actomyosin ATP consumption.

Superinhibition of sarcoplasmic reticulum function by phospholamban induces cardiac contractile failure.

To determine whether selective impairment of cardiac sarcoplasmic reticulum (SR) Ca(2+) transport may drive the progressive functional deterioration leading to heart failure, transgenic mice, overexpressing a phospholamban Val(49) --> Gly mutant (2-fold), which is a superinhibitor of SR Ca(2+)-ATPase affinity for Ca(2+), were generated, and their cardiac phenotype was examined longitudinally. At 3 months of age, the increased EC(50) level of SR Ca(2+) uptake for Ca(2+) (0.67 +/- 0.09 microm) resulted in significantly higher depression of cardiomyocyte rates of shortening (57%), relengthening (31%), and prolongation of the Ca(2+) signal decay time (165%) than overexpression (2-fold) of wild type phospholamban (68%, 64%, and 125%, respectively), compared with controls (100%). Echocardiography also revealed significantly depressed function and impaired beta-adrenergic responses in mutant hearts. The depressed contractile parameters were associated with left ventricular remodeling, recapitulation of fetal gene expression, and hypertrophy, which progressed to dilated cardiomyopathy with interstitial tissue fibrosis and death by 6 months in males. Females also had ventricular hypertrophy at 3 months but exhibited normal systolic function up to 12 months of age. These results suggest a causal relationship between defective SR Ca(2+) cycling and cardiac remodeling leading to heart failure, with a gender-dependent influence on the time course of these alterations.

Role for PKC in the adenosine-induced decrease in shortening velocity of rat ventricular myocytes.

We previously demonstrated that both adenosine receptor activation and direct activation of protein kinase C (PKC) decrease unloaded shortening velocity (V(max)) of rat ventricular myocytes. The goal of this study was to further investigate a possible link among adenosine receptors, phosphoinositide-PKC signaling, and V(max) in rat ventricular myocytes. We determined that the adenosine receptor agonist R-phenylisopropyladenosine (R-PIA, 100 microM) and the alpha-adrenergic receptor agonist phenylephrine (Phe, 10 microM) increased turnover of inositol phosphates. PKC translocation from the cytosol to the sarcolemma was used as an indicator of PKC activation. Western blot analysis demonstrated an increased PKC-epsilon translocation after exposure to R-PIA, Phe, and the PKC activators dioctanoylglycerol (50 microM) and phorbol myristate acetate (1 microM). PKC-alpha, PKC-delta, and PKC-zeta did not translocate to the membrane after R-PIA exposure. Finally, PKC inhibitors blocked R-PIA-induced decreases in V(max) as well as Ca(2+)-dependent actomyosin ATPase in rat ventricular myocytes. These results support the conclusions that adenosine receptors activate phosphoinositide-PKC signaling and that adenosine receptor-induced PKC activation mediates a decrease in V(max) in ventricular myocytes.

A single site (Ser16) phosphorylation in phospholamban is sufficient in mediating its maximal cardiac responses to beta -agonists.

Phospholamban (PLB) can be phosphorylated at Ser(16) by cyclic AMP-dependent protein kinase and at Thr(17) by Ca(2+)-calmodulin-dependent protein kinase during beta-agonist stimulation. A previous study indicated that mutation of S16A in PLB resulted in lack of Thr(17) phosphorylation and attenuation of the beta-agonist stimulatory effects in perfused mouse hearts. To further delineate the functional interplay between dual-site PLB phosphorylation, we generated transgenic mice expressing the T17A mutant PLB in the cardiac compartment of the null background. Lines expressing similar levels of T17A mutant, S16A mutant, or wild-type PLB in the null background were characterized in parallel. Cardiac myocyte basal mechanics and Ca(2+) kinetics were similar among the three groups. Isoproterenol stimulation was associated with phosphorylation of both Ser(16) and Thr(17) in wild-type PLB and Ser(16) phosphorylation in T17A mutant PLB, whereas there was no detectable phosphorylation of S16A mutant PLB. Phosphorylation of Ser(16) alone in T17A mutant PLB resulted in responses of the mechanical and Ca(2+) kinetic parameters to isoproterenol similar to those in wild-type myocytes, which exhibited dual-site PLB phosphorylation. However, those parameters were significantly attenuated in the S16A mutant myocytes. Thus, Ser(16) in PLB can be phosphorylated independently of Thr(17) in vivo, and phosphorylation of Ser(16) is sufficient for mediating the maximal cardiac responses to beta-adrenergic stimulation.

The transgenic expression of highly inhibitory monomeric forms of phospholamban in mouse heart impairs cardiac contractility.

Transgenic mice were generated with cardiac-specific overexpression of the monomeric, dominant-acting, superinhibitory L37A and I40A mutant forms of phospholamban (PLN), and their phenotypes were compared with wild-type (wt) mice or 2-fold overexpressors of wt PLN (wtOE). The level of PLN monomer in cardiac microsomes was increased 11-13-fold, and the apparent affinity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase for Ca(2+) was decreased from pCa 6.22 in wt or 6.12 in wtOE to 5.81 in L37A and 5.72 in I40A. Basal physiological parameters, measured in isolated myocytes, indicated a significant reduction in the rates of shortening (+dL/dt) and relengthening (-dL/dt). Hemodynamic measurements indicated that peak systolic pressure was unaffected but that pressure changes (+dP/dt and -dP/dt) were lowered significantly in both mutant lines, and relaxation time (tau) was also lengthened significantly. Echocardiography for both mutants showed depressed systolic function and an increase in left ventricular mass of over 1.4-fold. Significant decreases in left ventricular shortening fraction and velocity of circumferential shortening and increases in ejection time were corrected by isoproterenol. The use of antibodies specific against Ser(16)- and Thr(17)-PLN peptides showed that phosphorylation of both pentameric and monomeric PLN were increased between 1.2- and 2.4-fold in both the L37A and I40A lines but not in the wtOE line. These observations show that overexpression of superinhibitory mutant forms of PLN causes depression of contractile parameters with induction of cardiac hypertrophy, as assessed with echocardiography.

Effects of adenosine and protein kinase C stimulation on mechanical properties of rat cardiac myocytes.

Exposure of the heart to adenosine decreases heart rate and left ventricular developed pressure. However, little is known regarding the influence of adenosine on mechanical properties of isolated ventricular myocytes and the intracellular mechanism(s) by which adenosine acts. Therefore, in the present study we compared the effects of the adenosine receptor agonist R-phenylisopropyladenosine (R-PIA) and protein kinase C (PKC) activator dioctanoylglycerol (DOG) on Ca2+ sensitivity of tension, maximum isometric tension, and velocity of unloaded shortening (Vmax) in enzymatically isolated, drug-treated, and subsequently skinned ventricular myocytes. Neither R-PIA (100 microM) nor DOG (50 microM) affected Ca2+ sensitivity of tension or maximum isometric tension compared with controls. However, both R-PIA and DOG treatment caused approximately 25% decrease in Vmax during maximum activation compared with controls. This suggests adenosine and PKC decrease actin-myosin interaction through an alteration of myofilament proteins. The observed similarity of response after R-PIA and DOG treatment is consistent with the hypothesis that effects of adenosine are mediated by activation of the PKC pathway in isolated ventricular myocytes.

A hyphal-specific chitin synthase gene (CHS2) is not essential for growth, dimorphism, or virulence of Candida albicans.

In the dimorphic fungus Candida albicans, the CHS2 gene encodes a chitin synthase that is expressed preferentially in the hyphal form. Gene disruption of CHS2 in this diploid asexual fungus was achieved by the "ura-blaster" protocol described for Saccharomyces [Alani, E., Cao, L. & Kleckner, N. (1987) Genetics 116, 541-545]. This involves the sequential disruption of multiple alleles by integrative transformation with URA3 as a single selectable marker. After disrupting the first CHS2 allele, the Ura- phenotype was recovered through cis recombination between repeated hisG sequences that flanked the URA3 marker in the disruption cassette, which was then used again to disrupt further CHS2 alleles. This method of gene disruption is well suited to the mutational analysis of this genetically recalcitrant human pathogen. Three rounds of disruption were required, suggesting that the strain SGY243 is triploid for the CHS2 locus. The resulting homozygous delta chs2::hisG null mutants were viable and made germ tubes with a normal morphology. The germ tubes were formed more slowly than parental strains in serum-containing medium and the germinating cells had a 40% reduction in their chitin content compared to germ tubes of the parent strain. The chitin content of the yeast form was not affected. A prototrophic strain of the chs2 null mutant was not attenuated significantly in its virulence when tested in normal and immunosuppressed mice.

Influence of family functioning on maternal-fetal attachment.

Although the development of a positive relationship between the mother and infant most likely begins during pregnancy, the correlates of maternal-fetal attachment are poorly understood. This study examined the influence of family functioning on maternal-fetal attachment in a sample of varied ethnic and socioeconomic backgrounds. The authors surveyed 339 pregnant women in their last trimesters with the Family Adaptability and Cohesion Scales-III (FACES-III), Maternal-Fetal Attachment Scale (MFA), and a demographic interview. On the basis of correlational and regression analyses, the demographic variables of parity, ethnicity, age, education, and occupation of primary wage earner correlated significantly with maternal-fetal attachment. When entered in a multiple regression analysis, parity, ethnicity, and occupation explained 12% of the variance in the MFA scores. The FACES-III total score and the subscale scores of adaptability and cohesion also correlated significantly with scores on the MFA and explained an additional 3% of the variance in the MFA beyond that explained by the demographics. Support of positive family dynamics during pregnancy by health care workers may potentially increase the quality of maternal-fetal attachment.

Getting the story straight: evaluating the test-retest reliability of a university health history questionnaire.

This study was designed to establish the reliability of a health history questionnaire used as a screening tool for incoming university students. The authors used a test-retest design, with a test interval of 6 months, on a sample of medical and nursing students. The analysis focused on overall reliability of the questionnaire and reproducibility of specific items, based on question format. Questionnaire items of specific interest were those with dichotomous yes/no response options versus open-ended format questions, those using the words frequently or recently, or those that asked multiple questions. Demographic characteristics of the subjects were considered in the evaluation of reliability. Overall reliability of the questionnaire (93.6%) was above the anticipated level of 90%, and subject sex or program of study did not show any significant differences in reproducibility of responses. Although wording of questions did not affect item reliability, dichotomous format questions demonstrated a higher degree of reliability (96.4%) than the overall reliability of the questionnaire. Recommendations for enhancing the reliability of the questionnaire are based on item analysis and information gathered from interviews with subjects.

Herpes encephalitis: MR monitoring of response to acyclovir therapy.

Viral encephalitis secondary to herpes simplex virus type I is thought to be the single most important cause of fatal, sporadic encephalitis in the United States. Magnetic resonance not only improves early diagnosis of herpes encephalitis but also is effective in documenting response to antiviral therapy.

Epidemiologic characteristics of cytomegalovirus infection in mothers and their infants.

In this longitudinal study of cytomegalovirus in 4578 pregnant women of middle/upper socioeconomic status in Houston, 52% had cytomegalovirus antibody when enrolled, and 48% were serologically susceptible. Studies were completed on 3899 mothers and their infants; 2.2% of these women experienced primary cytomegalovirus during pregnancy and 24% of those with primary infection transmitted cytomegalovirus to their infants. Of 22 cytomegalovirus-infected infants, 2 had disease at birth and 20 were asymptomatic. One symptomatic infant (primary maternal infection) has developmental delay. The other (immunocompromised mother with cytomegalovirus antibody before pregnancy) had hepatitis but has no symptoms at 1 year of age. On follow-up, 4 of 16 infants asymptomatic at birth have sequelae (hearing loss in 3, developmental delay in 1). All four were born to mothers with primary cytomegalovirus infection. Infant outcome was not related to trimester of maternal infection.

Factors predictive of cytomegalovirus immune status in pregnant women.

We performed serological tests for antibody to cytomegalovirus on 1989 pregnant women of middle-to-upper socioeconomic status at the time of their first obstetric visit. Fifty percent of the women had antibody to cytomegalovirus. Analysis with a stepwise logistic regression model revealed that seropositivity was independently correlated with nonwhite race, less than 16 years of education, being breast-fed as an infant, the presence of children five to 18 years of age in the home, and maternal age greater than or equal to 30 years. Conversely, women not possessing these risk factors were more likely to be seronegative; 69% of the women without any of the five factors lacked antibody to cytomegalovirus. Serological screening for antibody would be more useful in obstetric practices where the majority of patients lack these risk factors.

Colon carcinoma metastatic to the thyroid gland.

Metastatic carcinoma to the thyroid gland rarely is encountered in clinical practice; however, autopsy series have shown that it is not a rare occurrence. A case of adenocarcinoma of the colon with metastases to the thyroid is reported. A review of the literature reveals that melanoma, breast, renal, and lung carcinomas are the most frequent tumors to metastasize to the thyroid. Metastatic disease must be considered in the differential diagnosis of cold nodules on radionuclide thyroid scans, particularly in patients with a known primary.

Human smooth muscle cells cultured from atherosclerotic plaques and uninvolved vessel wall.

Smooth muscle cells (SMC) were cultured from atherosclerotic plaques and uninvolved arteries to determine if differences exist between growth characteristics or ultrastructure of the cultured cells. Eighteen aortic punch biopsies provided the uninvolved tissue, and 58 carotid plaques provided the atherosclerotic tissue. Eighty percent of the samples yielded viable cultured cells, which reached a maximum population doubling time during log phase growth of 72 h (seeding density = 1.0 x 10(4) cells/cm2, 2nd passage). Growth characteristics of both normal and plaque-derived cells were the same in vitro. Growth rate declined with time in culture, and cell division ceased by the 5th or 6th passage. In culture, spindle shaped cells formed the "hill and valley" configuration typical of SMC. Plaque-derived SMC were ultrastructurally similar to SMC from uninvolved vessel wall. Proliferative potential did not vary with age or sex, with method of culture, or with whether the cells were plaque derived or not.

Gestalt closure and the human visual evoked cortical potential.

Previous research examined the relationship between perceptual psychological content and the human visual evoked cortical potential (VECP). Each study suffered a common methodological problem. The stimuli's properties covaried with their perceptual-psychological content. To circumvent this problem, VECP's were recorded before and after Ss (14 men and women) "closed" Gestalt figures. A small, consistent increase in amplitude was observed after figures were organized.

The core course in medical microbiology at Baylor College of Medicine.

During the past five years at Baylor College of Medicine student performance in microbiology, as measured by scores on the examination of the National Board of Medical Examiners, has improved from less (mean of 78 percent) than the national average of 80+ percent to considerably greater than the national average (mean of 85 percent). Only about one-half the time usually given to microbiology is allotted to the course at Baylor (107 hours). Principal features of the course are annually revised lecture handouts, medically oriented laboratory sessions with a manual written especially for the course, and clinical demonstrations of infectious disease. The pattern of performance in the microbiology course did not occur in two other basic science courses at Baylor. The improvement in performance appeared to be related to the course format, increased teaching proficiency, and the allocation of hours to the various subdisciplines.

Voice predicts affect during psychotherapy.

This study examined the relationship between emotions and the frequency and power characteristics of the voice in psychotherapy. The intensity of fear, anger, depression, and total affect in each of four interviews with one patient was rated every 20 seconds on a nine-point scale. Significant agreement among judges was achieved. Voice samples from each epoch in which there was sufficient speech were subjected to spectral analysis of the frequencies between 0 and 1000 HZ. These spectra were scored for nine frequency and power parameters. Multiple linear regression equations were then developed from two interviews, using the nine voice spectral variables as predictors and the mean ratings for each affect as the criterion variables. Significant multiple correlations were achieved between every rated affect and various combinations of voice variables. The beta weights and constants from these equations were then employed in the successful prediction of levels of anger, fear, depression, and total affect in one interview, and the levels of depression and total affect in another interview. In addition, epochs of conflict differed from "pure" affect epochs, and pure epochs of anger, fear, and depression differed from each other in various frequency and power characteristics of the voice. Voice spectral measures may be an objective means of identifying and quantifying affect in psychotherapy.