Cardiac specific effects of thyroid hormone analogues.

There is significant interest in development of thyroid hormone analogues to harness specific properties as therapeutic agents for a variety of clinical indications including obesity, hypercholesterolemia, heart failure, and thyrotoxicosis. To date, most analogues have been designed to target liver specific effects, which can promote weight loss and lipid lowering through either tissue specific uptake or thyroid hormone receptor (TR) ß isoform selectivity at the same time minimizing the unwanted cardiac and bone effects. We have developed a molecular biomarker assay to study the induction of the transcription of the cardiac specific α-myosin heavy chain (MHC) gene as a more sensitive and specific measure of thyroid hormone action on cardiac myocytes. We tested 5 TRß and 1 TRα selective agonists as well as 2 putative TR antagonists in our α-MHC hnRNA assay. Using reverse transcription and polymerase chain reaction, we measured the induction of the α-MHC primary transcript in response to administration of drug. The TRα and only 2 of the TRß agonists were highly active, when compared to the effect of T3, at the level of the cardiac myocyte. In addition, our data suggests that the reason that the antagonist NH-3 is not able to block the T3-mediated induction of α-MHC is that it does not get transported into the cardiac myocyte. Our data suggest that this assay will be useful in preclinical studies of the potential cardiac specific effects of thyroid hormone analogues and that predictions of function based on structure are not necessarily accurate or complete.

Thyroid hormone and the cardiovascular system.

Thyroid hormone is an important regulator of cardiac function and cardiovascular hemodynamics. Triiodothyronine, (T(3)), the physiologically active form of thyroid hormone, binds to nuclear receptor proteins and mediates the expression of several important cardiac genes, inducing transcription of the positively regulated genes including alpha-myosin heavy chain (MHC) and the sarcoplasmic reticulum calcium ATPase. Negatively regulated genes include beta-MHC and phospholamban, which are down regulated in the presence of normal serum levels of thyroid hormone. T(3) mediated effects on the systemic vasculature include relaxation of vascular smooth muscle resulting in decreased arterial resistance and diastolic blood pressure. In hyperthyroidism, cardiac contractility and cardiac output are enhanced and systemic vascular resistance is decreased, while in hypothyroidism, the opposite is true. Patients with subclinical hypothyroidism manifest many of the same cardiovascular changes, but to a lesser degree than that which occurs in overt hypothyroidism. Cardiac disease states are sometimes associated with the low T(3) syndrome. The phenotype of the failing heart resembles that of the hypothyroid heart, both in cardiac physiology and in gene expression. Changes in serum T(3) levels in patients with chronic congestive heart failure are caused by alterations in thyroid hormone metabolism suggesting that patients may benefit from T(3) replacement in this setting.

Evaluation of two current generation enzyme immunoassays and an improved isolation-based assay for the rapid detection and isolation of rotavirus from stool.

BACKGROUND: Rapid and accurate rotavirus testing is important in decisions involving patient care and management. Quality assurance testing needs to be periodically performed, especially among widely used assays having a direct impact on patient care. OBJECTIVES: To evaluate the current generation Kallestad Pathfinder Direct antigen Detection system (PTH), and the widely used Rotaclone(R) Rotavirus EIA Diagnostic Kit (RTC), in comparison with an improved cell culture amplification-antigen detection (CCA-Ag) isolation-based assay. STUDY DESIGN: Two hundred stool specimens (specimen stored at > or =-75 degrees C), which had been previously tested by PTH, were tested by RTC and CCA-Ag. Discordant specimens were retested by PTH, blocking assay, polyacrylamide gel electrophoresis (PAGE), and/or electron microscopy (EM). RESULTS: Among 200 stool specimens, 197 were in accord by PTH, RTC and CCA-Ag. The sensitivity, specificity, positive and negative predictive values for RTC, PTH and CCA-Ag were, 100, 99, 99, 100, 100, 99, 99, 100; and 98, 100, 100, 98%, respectively. Among five initially discordant specimens, two required a period of 10 days to affect isolation. A non-cultivatable (CCA-Ag negative) but true positive specimen, was identified as rotavirus group A serotype G2 by RT-PCR. Four true positive but discordant specimens were blocking assay negative using one or both EIA kits. CONCLUSIONS: PTH and RTC are excellent rotavirus detection system. However, PTH is more expensive (ca. $3.50 vs. $2.00 per test), mandates a slightly longer turn-around time (ca. 1 vs. 1.5 h), and necessitates slightly more hands-on manipulative/preparative steps. Blocking assay was not a reliable confirmatory test for the resolution of specimen discordancy. A combination of CCA-Ag, PAGE, EM, and/or perhaps RT-PCR, is recommended as an appropriate test panel for the resolution of discordant results during assay evaluation. The newly modified and simplified 48-h rotavirus isolation-based assay may serve as a base line methodology in laboratory evalaution studies, as a laboratory support methodology during drug/vaccine efficacy trials, or for the testing of sources (e.g., biopsy/autopsy tissues) not approved for assay by commercial rotavirus kits.

Alterations in the Saccharomyces MAL-activator cause constitutivity but can be suppressed by intragenic mutations.

The Saccharomyces MAL-activator regulates the maltose-inducible expression of the MAL structural genes encoding maltose permease and maltase. Constitutive MAL-activator mutant alleles of two types were identified. The first were truncation mutations deleting C-terminal residues 283-470 and the second contained a large number of alterations compared to inducible alleles scattered throughout the C-terminal 200 residues. We used site-directed in vitro mutagenesis of the inducible MAL63 and MAL63/23 genes to identify the residues responsible for the negative regulatory function of the C-terminal domain. Intragenic suppressors that restored the inducible phenotype to the constitutive mutants were identified at closely linked and more distant sites within the MAL-activator protein. MAL63/mal64 fusions of the truncated mutants suggest that residues in the N-terminal 100 residues containing the DNA-binding domain also modulate basal expression. Moreover, a transcription activator protein consisting of LexA(1-87)-Gal4(768-881)-Mal63(200-470) allowed constitutive reporter gene expression, suggesting that the C-terminal regulatory domain is not sufficient for maltose-inducible control of this heterologous activation domain. These results suggest that complex and very specific intramolecular protein-protein interactions regulate the MAL-activator.

Constitutive mutations of the Saccharomyces cerevisiae MAL-activator genes MAL23, MAL43, MAL63, and mal64.

We report the sequence of several MAL-activator genes, including inducible, constitutive, and noninducible alleles of MAL23, MAL43, MAL63, and mal64. Constitutive alleles of MAL23 and MAL43 vary considerably from inducible alleles in their C-terminal domain, with many of the alterations clustered and common to both alleles. The 27 alterations from residues 238-461 of Mal43-C protein are sufficient for constitutivity, but the minimal number of alterations needed for the constitutive phenotype could not be determined. The sequence of mal64, a nonfunctional homologue of MAL63, revealed that Mal64p is 85% identical to Mal63p. Two mutations that activate mal64 and cause constitutivity are nonsense mutations resulting in truncated proteins of 306 and 282 residues. We conclude that the C-terminal region of the MAL-activator, from residues 283-470, contains a maltose-responsive negative regulatory domain, and that extensive mutation or deletion of the entire region causes loss of the negative regulatory function. Additionally, certain sequence elements in the region appear to be necessary for efficient induction of the full-length Mal63 activator protein. These studies highlight the role of ectopic recombination as an important mechanism of mutagenesis of the telomere-associated family of MAL loci.