Calcified bicuspid aortic valve mass prolapsing into the left main coronary artery.

We report a case of a mobile calcific mass on the aortic valve that prolapsed into the left main coronary artery of a 51-year-old man. This case and a review of the literature suggest that calcific embolization to coronary arteries is a rare but possibly underrecognized complication of calcified degenerative or bicuspid aortic valves. This potentially catastrophic complication of calcified aortic valves needs to be suspected and recognized in clinical practice.

Validation of death certificate diagnosis of out-of-hospital coronary heart disease deaths in Olmsted County, Minnesota.

OBJECTIVE: To evaluate the validity of death certificate diagnosis of out-of-hospital (OOH) coronary heart disease (CHD) and sudden cardiac death (SCD) in Olmsted County, Minnesota, between 1981 and 1994. METHODS: In this review of the medical records, autopsy reports, and coroner's files, OOH deaths with heart disease as the underlying cause of death on the death certificate were classified into CHD (International Classification of Diseases, 9th Revision, Clinical Modification [ICD-9-CM] codes 410-414) and non-CHD (other ICD-9-CM heart disease codes) deaths. A 10% random sample (n = 174) of these death certificates was reviewed by physicians, and published validation criteria were applied to classify these deaths into validated CHD or non-CHD categories. Sudden cardiac death was defined as validated CHD that occurred at an OOH location with less than 24 hours between symptom onset and death. RESULTS: The death certificate definition of OOH CHD death (ICD-9-CM codes 410-414) had high sensitivity and positive predictive value of 91% and 96%, respectively. The specificity and the negative predictive value were slightly lower at 86% and 72%, respectively. The sensitivity of death certificate diagnosis of CHD for validated SCD was 89%, and the positive predictive value was 77%. Using a more restrictive definition of SCD, that is, less than 1 hour between the onset of symptoms and death, the positive predictive value of CHD codes for SCD was lower at 52%. CONCLUSIONS: In Olmsted County, the positive predictive values of death certificate diagnosis for OOH CHD and SCD are high. Relying on death certificate diagnoses results in about 5% underestimation of the true CHD rates, whereas their use as a surrogate for SCD yields a 16% overestimation of the true SCD rates.

Prognostic value of treadmill exercise testing in elderly persons.

BACKGROUND: Recent exercise testing guidelines recognized a gap in knowledge about the prognostic value of treadmill exercise testing in elderly persons. OBJECTIVE: To test the hypothesis that treadmill exercise testing has equal prognostic value among elderly (> or =65 years of age) and younger (<65 years of age) persons and to examine the incremental value of this testing over clinical data. DESIGN: Inception cohort with a median follow-up of 6 years. SETTING: Olmsted County, Minnesota. PATIENTS: All elderly (n = 514) and younger (n = 2593) residents of Olmsted County who underwent treadmill exercise testing between 1987 and 1989. MEASUREMENTS: Overall mortality and cardiac events (cardiac death, nonfatal myocardial infarction, and congestive heart failure). RESULTS: Compared with younger patients, elderly patients had more comorbid conditions, achieved a lower workload (6.0 and 10.7 metabolic equivalents; P < 0.001), and had a greater likelihood of a positive exercise electrocardiogram (28% and 9%; P < 0.001). With median follow-up of 6 years, overall survival (63% and 92%; P < 0.001) and cardiac event-free survival (66% and 95%; P < 0.001) were worse among elderly persons than among younger persons. Workload was the only treadmill exercise testing variable associated with all-cause mortality in both age groups, and the strength of association was similar. Workload and angina with exercise testing were associated with cardiac events in both age groups, whereas a positive exercise electrocardiogram was associated with cardiac events only in younger persons (P < 0.05 for all comparisons). After adjustment for clinical variables, workload was the only additional treadmill exercise testing variable that was predictive of death (P < 0.001) and cardiac events (P < 0.05); the strength of the association was similar in both age groups. Each 1-metabolic equivalent increase in exercise capacity was associated with a 14% and 18% reduction in cardiac events among younger and elderly persons, respectively. CONCLUSIONS: In elderly persons, treadmill exercise testing provided prognostic information that is incremental to clinical data. After adjustment for clinical factors, work-load was the only treadmill exercise testing variable that was strongly associated with outcome, and its prognostic effect was of the same magnitude in elderly and younger persons.

Analysis of the negative transcriptional regulatory element in the angiotensin-converting enzyme gene.

We have characterized the sequence requirements and the protein binding properties of the previously identified transcriptional negative element present in the rabbit angiotensin-converting enzyme (ACE) gene. DNase footprinting experiments revealed that within the negative element (-715 to -610) several regions interact with proteins present in the nuclear extracts of ACE-expressing and -nonexpressing cell lines. Transfection analysis using the heterologous beta-actin promoter and mutated negative elements demonstrated that the SP1 site, the collagen-silencer-like sequence, and the inverted repeat elements are dispensable for their functioning. Deletion of the region between -692 to -668, however, completely eliminated the activity of the negative element, and mutation of the synapsin-silencer-like sequence present within this region vastly reduced it. This region (-692 to -668) by itself, when present in two copies, could effectively repress the activity of the beta-actin promoter. The same point mutations in the silencer element that destroyed its action on the beta-actin promoter greatly increased the transcriptional efficiency of the native ACE promoter. Electrophoretic mobility shift assay using the -692 to -668 ACE silencer sequence demonstrated the formation of a DNA/protein complex. UV cross-linking of the components of this complex revealed the presence of one prominent protein of approximately 21.5 kDa. This protein may be responsible for mediating the transcriptional-repressing activity of the ACE negative element. Homology between the ACE silencer and neuronal silencer consensus sequence, together with the promoter- and tissue-independent function of the the ACE silencer, suggests this element may bind a member of a large family of common negative regulatory transcription factors.

The cyclic AMP response elements of the genes for angiotensin converting enzyme and phosphoenolpyruvate carboxykinase (GTP) can mediate transcriptional activation by CREM tau and CREM alpha.

The potential of the CREM family of proteins to activate transcription of the genes encoding the testis-specific isozyme of angiotensin converting enzyme (ACET) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (EC 4.1.1.32) were investigated. Both CREM tau and CREM alpha bind efficiently to the putative cyclic AMP response element (CRE) present in the ACET gene (CRET) and to the CRE in the PEPCK gene. In HepG2 cells, the CRE was required for the strong stimulation by CREM tau of the expression of a chimeric PEPCK (-210 to +73)-chloramphenicol acetyl transferase (CAT) gene. The CRE could be mutated to the CRET sequence without losing the stimulatory effects of CREM tau. However, a similar chimeric gene driven by the regulatory region of the ACET gene, which contains the CRET site, could only be stimulated by CREM tau when its imperfect TATA element was mutated to an authentic TATA. Surprisingly, CREM alpha, an alleged inhibitor of CRE-mediated transcription, stimulated the expression of both PEPCK-CAT and ACET-CAT genes in HepG2 cells, a process which required the presence of the CRE and the CRET sites, respectively. In contrast, when the same CRE elements were used to drive the transcription of a chimeric gene containing the thymidine kinase promoter linked to the CAT structural gene, CREM alpha inhibited its expression in HepG2 and JEG3 cells. The expression of the same chimeric gene, however, was stimulated by CREM alpha in F9 embryonal carcinoma cells. These results demonstrated that the nature of the transcriptional effects of CREM isoforms on CRE-mediated transcription depends on the specific gene, the specific cell type and the promoter context of the CRE site.

Signal transduction alterations in peripheral nerves from streptozotocin-induced diabetic rats.

We have previously determined the presence of muscarinic receptors and the expression of several G proteins in homogenates and myelin fractions from rat sciatic nerves. In the present study we investigated whether changes in several signal transduction pathways in peripheral nerves might be responsible for some of the biochemical abnormalities (e.g., phosphoinositide metabolism) present in sciatic nerves from streptozotocin-induced diabetic rats. Sciatic nerves from 5 week diabetic rats that were prelabelled with [3H]-myo-inositol displayed a significant increase in the basal release of inositol mono- and bis-phosphate, while carbamylcholine-stimulated release was significantly smaller. Basal- and forskolin-stimulated adenylyl cyclase activity was significantly decreased in sciatic nerve homogenates from diabetic animals. However, we were unable to detect any significant differences in the levels of cAMP in intact nerves or in nerve segments that were incubated in the presence or absence of forskolin. ADP-ribosylation experiments showed that in sciatic nerves from experimentally diabetic rats there was a significant increase in the ADP-ribosylation catalyzed by cholera and pertussis toxins. Measurements of the levels of alpha-subunits of G proteins revealed that the expression of Gq/11 alpha, Gs alpha, and Gi-3 alpha was increased by 30 to 50%. These results indicate that during the course of experimental diabetes, peripheral nerves exhibit an abnormal production of inositol phosphates and cAMP, together with an abnormal expression and/or function of G proteins. One of the consequences of such alterations is the diminished release of inositol phosphates triggered by muscarinic agonists in diabetic sciatic nerves.

Identification of positive and negative transcriptional regulatory elements of the rabbit angiotensin-converting enzyme gene.

The two tissue-specific mRNAs encoding the isozymes of rabbit angiotensin-converting enzyme (ACE) are generated from the same gene by alternative choice of two transcription initiation sites 5.7 kb apart. In the current study, we have characterized the regulatory sites controlling the transcription of the larger pulmonary isozyme mRNA. For this purpose, reporter genes driven by varying lengths of upstream region of the ACE gene were transfected into ACE-producing cells. Our results demonstrated that the transcription of this gene is primarily driven by positive elements within the first 274 bp DNA upstream of the transcription initiation site. The reporter gene driven by this region was expressed in two ACE-producing cells but not in two ACE-non-producing cells thereby establishing its tissue specificity. Our experiments also revealed the existence of a strong negative element located between -692 and -610 positions. This element suppressed the expression of the reporter gene in a dose-dependent and position and orientation-independent fashion thus suggesting that it is a true silencer element. It could also repress the expression of a reporter gene driven by the heterologous strong promoter of the beta-actin gene. The repressing effects of the negative element could be partially overcome by cotransfecting the isolated negative element along with the reporter gene containing the negative element. This result was possibly due to the functional removal of a limiting trans-acting factor which binds to this element. Electrophoretic mobility shift assays revealed that the negative element can form several complexes with proteins present in the nuclear extract of an ACE-producing cell line. At least part of the negative element is strongly conserved in the upstream regions of the human and mouse ACE genes.

Identification of G protein subtypes in peripheral nerve and cultured Schwann cells.

In this study, we investigated the expression of various G proteins in whole sciatic nerves, in myelin and nonmyelin fractions from these nerves, and in membranes of immortalized Schwann cells. In myelin, nonmyelin, and Schwann cell membranes we detected two 39-40-kDa pertussis toxin substrates that were resolved on separation on urea-gradient gels. Two cholera toxin substrates with apparent molecular masses of 42 and 47 kDa were present in nerve and brain myelin and in Schwann cell membranes. In these membranes, a third 45-kDa cholera toxin substrate, which displayed the highest labeling, was also present. Immunoblotting with specific antisera allowed the identification of G(o) alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Gq/G11 alpha, and the two isoforms of Gs alpha in nerve homogenates, nerve, and brain myelin fractions. In Schwann cell membranes we identified G(o) alpha, Gi2 alpha, Gi3 alpha, and proteins from the Gq family, but no immunoreactivity toward anti-Gi1 alpha antiserum was detected. In these membranes, anti-Gs alpha antibody recognized the three cholera toxin substrates mentioned above, with the 45-kDa band displaying the highest immunoreactivity. Relative to sciatic nerve myelin, the Schwann cell membranes revealed a significantly higher expression of Gi3 alpha and the absence of Gi1 alpha. The different distribution of G proteins among the different nerve compartments might reflect the very specialized function of Schwann cells and myelin within the nerve.