A novel, tissue-restricted zinc finger protein (HF-1b) binds to the cardiac regulatory element (HF-1b/MEF-2) in the rat myosin light-chain 2 gene.

The AT-rich element MEF-2 plays an important role in the maintenance of the muscle-specific expression of a number of cardiac and skeletal muscle genes. In the MLC-2 gene, an AT-rich element (HF-1b) which contains a consensus MEF-2 site is required for cardiac tissue-specific expression. The present study reports the isolation and characterization of a cDNA which encodes a novel C2H2 zinc finger (HF-1b) that binds in a sequence-specific manner to the HF-1b/MEF-2 site in the MLC-2 promoter. A number of independent criteria suggest that this HF-1b zinc finger protein is a component of the endogenous HF-1b/MEF-2 binding activity in cardiac muscle cells and that it can serve as a transcriptional activator of the MLC-2 promoter in transient assays. These studies suggest that, in addition to the previously reported RSRF proteins, structurally divergent transcriptional factors can bind to MEF-2-like sites in muscle promoters. These results underscore the complexity of the regulation of the muscle gene program via these AT-rich elements in cardiac and skeletal muscle.

Positive adaptations to weight-lifting training in the elderly.

Maximal weight-lifting performance, isometric strength, isokinetic torque, whole muscle and individual fiber cross-sectional areas, and muscle evoked contractile properties were assessed in 14 elderly males before and after 12 wk of weight-lifting training. Dynamic elbow flexion training of one arm resulted in a significant 48% mean increase in the maximal load that could be lifted once (1 RM) and a smaller improvement in isokinetic torque (8.8%) but no change in isometric strength. In the contralateral control arm, 1 RM and isokinetic torque increased by 12.7 and 6.5%, respectively, but isometric strength did not change. The interpolated twitch technique confirmed complete motor unit activation during a maximal isometric contraction of the elbow flexors before and after the training. Bilateral leg press training effected mean increases of 17 and 23% in isokinetic torque and dynamic lifting capacity, respectively. The mean maximal cross-sectional area of the elbow flexors (biceps brachii and brachialis) increased by 17.4% in the trained arm but did not change the control arm. The increase in the mean area of type II fibers in the biceps brachii muscle in the trained arm (30.2%) was greater than the corresponding change in the control arm (10.7%, P less than 0.05). The most significant change in the evoked contractile properties of the trained elbow flexors was the increase in twitch half-relaxation time. It is concluded that older individuals retain the potential for significant increases in strength performance and upper limb muscle hypertrophy in response to overload training.

Thyroid hormone receptor messenger ribonucleic acid in human granulosa and ovarian stromal cells.

OBJECTIVE: To examine whether mRNA for thyroid hormone receptors alpha and beta is present in human granulosa cells in nonstimulated ovaries. DESIGN: Paraffin-embedded sections of ovaries from normally cycling women were analyzed by in situ hybridization with oligonucleotide probes for thyroid hormone receptors alpha and beta. The sense strand oligonucleotide was used as a control for each of the probes. RESULTS: Granulosa cells from the preovulatory antral follicles examined showed positive staining for both the thyroid hormone receptor alpha and beta probes. Positive staining of ovarian stromal cells also was observed for both probes. CONCLUSION: Thyroid hormone receptor mRNAs are expressed in both granulosa cells and ovarian stromal cells found in nonstimulated ovaries. It is, therefore, conceivable that thyroid hormone may play a direct role in human ovarian physiology.

Pumping of water with ac electric fields applied to asymmetric pairs of microelectrodes.

Bulk fluid flow induced by an ac electric potential with a peak voltage below the ionization potential of water is described. The potential is applied to an ionic solution with a planar array of electrodes arranged in pairs so that one edge of a large electrode is close to an opposing narrow electrode. During half the cycle, the double layer on the surface of the electrodes charges as current flows between the electrodes. The electrodes charge in a nonuniform manner producing a gradient in potential parallel to the surface of the electrodes. This gradient drives the ions in the double layer across the surface of the electrode and this in turn drags the fluid across the electrode surface. The anisotropic nature of the pairs of electrodes is used to produce a net flow of fluid. The flow produced is approximately uniform at a distance from the electrodes that is greater than the periodicity of the electrode array. The potential and frequency dependence of this flow is reported and compared to a simple model. This method of producing fluid flow differs from electrical and thermal traveling-wave techniques as only a low voltage is required and the electrode construction is much simpler.

An unsaturated phosphonic acid analogue of phosphatidylethanolamine and its activity in blood-clotting systems.

An unsaturated phosphonolipid analogous to phosphatidylethanolamine,rac-dioleoylglyceryl(2-aminoethyl)phosphonate, was synthesized by a general method introduced by Baer for similar saturated substances. An improvement was made in the preparation of the phthalimidoethyl-phosphonic acid precursor.The phosphonolipid was purified by DEAE cellulose and silicic acid chromatography. It was tested by comparison with synthetic phosphatidyl (dioleoyl) ethanolamine and phosphatidyl(dilinoleoyl) ethanolamine in the Hicks-Pitney test and in a test for prothrombin conversion by using purified blood coagulation factors. In both tests it had more acceleratory activity than the synthetic phosphatidylethanolamines.

The Bridge Program evaluation process.

The Bridge Program was designed to enrich the undergraduate nursing experience of students from underrepresented groups at three schools of nursing in the Baltimore area. An evaluation of the Program was conducted in the Spring of 1996 using data from a variety of sources. Data were collected using student, faculty and mentor surveys, and student interviews. The evaluation demonstrated that students were enthusiastic about the Bridge Program despite the heavy work load. Students identified the connection with the faculty mentor as the Program's greatest benefit. Faculty and mentors generally viewed the Program as satisfactory. The strengths of the Program as seen by faculty and mentors were in the areas of students' personal and professional growth, faculty-student relationships, learning resources, and preparation for graduate study.

Inverted conversions: reading the Bible and writing the lesbian subject in Oranges are not the only fruit.

The prominence of the Bible as intertext in Oranges Are Not the Only Fruit, both a coming-of-age and a coming-out story, has puzzled readers. This paper argues that Winterson's articulation of a lesbian subject is actually inseparable from her revisionary engagement of the Bible. By repeatedly turning and re-turning several types of narrative about the origins of identity and story-making. Winterson reconstructs both some biblical texts and a hallmark of the gay and lesbian literary tradition as precursors for the prophetic voice of the main character.

Tales from the grave: Opposing autopsy reports from a body exhumed.

We report an autopsy case of a 42-year-old woman who, when discovered, had been dead in her apartment for approximately 1 week under circumstances involving treachery, assault and possible drug overdose. This case is unique as it involved two autopsies of the deceased by two different medical examiners who reached opposing conclusions. The first autopsy was performed about 10 days after death. The second autopsy was performed after an exhumation approximately 2 years after burial. Evidence collected at the crime scene included blood samples from which DNA was extracted and analysed, fingerprints and clothing containing dried body fluids. The conclusion of the first autopsy was accidental death due to cocaine toxicity; the conclusion of the second autopsy was death due to homicide given the totality of evidence. Suspects 1 and 2 were linked to the death of the victim by physical evidence and suspect 3 was linked by testimony. Suspect 1 received life in prison, and suspects 2 and 3 received 45 and 20 years in prison, respectively. This case indicates that cocaine toxicity is difficult to determine in putrefied tissue and that exhumations can be important in collecting forensic information. It further reveals that the combined findings of medical examiners, even though contradictory, are useful in determining the circumstances leading to death in criminal justice. Thus, this report demonstrates that such criminal circumstances require comparative forensic review and, in such cases, scientific conclusions can be difficult.

Acute regulation of the epidermal growth factor receptor in response to nerve growth factor.

PC12 cells possess specific receptors for both nerve growth factor and epidermal growth factor, and by an unknown mechanism, nerve growth factor is able to attenuate the propagation of a mitogenic response to epidermal growth factor. The differentiation response of PC12 cells to nerve growth factor, therefore, predominates over the proliferative response to epidermal growth factor. We have observed that the addition of nerve growth factor to PC12 cells rapidly produces a decrease in surface 125I-epidermal growth factor binding capacity. Unlike previously described nerve growth factor effects on 125I-epidermal growth factor binding capacity, which required several days of nerve growth factor exposure, the decreases we report occur within minutes of nerve growth factor addition: A 50% decrease in 125I-epidermal growth factor binding capacity is evident at 10 min. This rapid nerve growth factor response is concentration dependent; inhibition of 125I-epidermal growth factor binding is detectable at nerve growth factor levels as low as 0.2 ng/ml and is maximal at approximately 50 ng/ml, consistent with known ranges of biological activity. No demonstrable differences in the rate of epidermal growth factor receptor synthesis or degradation were observed in cells acutely exposed to nerve growth factor. Scatchard analysis revealed that acute nerve growth factor treatment decreased the number of both high- and low-affinity 125I-epidermal growth factor binding sites, while the receptor affinity remained unchanged. We have also investigated the involvement of various potential intracellular mediators of nerve growth factor action and of known intracellular modulatory systems of the epidermal growth factor receptor for their capacity to participate in this nerve growth factor activity.

Stimulation of tyrosine-specific protein phosphorylation and phosphatidylinositol phosphorylation by orthovanadate in rat liver plasma membrane.

Orthovanadate stimulated the incorporation of 32P from [gamma-32P]ATP by Triton X-100-solubilized rat liver plasma membrane into endogenous, trichloroacetic acid-precipitable materials as well as added (Glu4:Tyr1) copolymers. Extraction of incubation mixture with chloroform-methanol-HCl revealed that the increase in 32P incorporation by vanadate was predominantly into endogenous phospholipids. [32P]Phosphatidylinositol 4-phosphate (PtdIns-4-P) was identified by thin-layer chromatography as the major phosphorylated product of vanadate stimulation, which also resulted in elevated 32P, predominantly in P-Tyr in endogenous membrane proteins. Vanadate effects on protein tyrosine and phosphatidylinositol phosphorylation were concomitant and exhibited similar sensitivity. These effects of vanadate were enhanced by the presence of either dithiothreitol or NAD(P)H. Phosphatidylinositol phosphorylation could also be stimulated by a substrate of and inhibited by a synthetic inhibitory copolymer of tyrosine kinase. These results suggest that vanadate, an oxygen radical producer, stimulates a tyrosine kinase-PtdIns kinase coupled system much like those described for a number of growth factors and oncogene encoded products.

Activation of a membrane-associated phosphatidylinositol kinase through tyrosine-protein phosphorylation by naphthoquinones and orthovanadate.

We have previously reported that several naphthoquinones stimulated tyrosine-specific protein phosphorylation in isolated rat liver membranes. Our more recent study demonstrated a similar effect by orthovanadate, which concomitantly stimulated phosphorylation of protein-tyrosine and phosphatidylinositol (Ptd-Ins). Results presented here show a simultaneous increase in PtdIns phosphorylation along with stimulation of tyrosine-protein phosphorylation by naphthoquinones. This PtdIns kinase resembles the type I PtdIns kinase in that it was insensitive to adenosine inhibition. The product, nevertheless, comigrated with a PtdIns-4-phosphate standard in TLC using three different solvent systems. Stimulation of PtdIns phosphorylation by vanadate or naphthoquinones could be achieved in the following preparations: intact rat liver membranes, Triton X-100-solubilized membranes, solubilized membranes partially purified by Sephacryl chromatography, solubilized membranes purified by wheat germ agglutinin chromatography. The naphthoquinone or vanadate-activated PtdIns kinase activity could be isolated by antiphosphotyrosine antibody-agarose affinity chromatography. The relative potencies of a series of ring-substituted naphthoquinones in the stimulation of tyrosine-protein phosphorylation, PtdIns kinase activity, dithiothreitol-dependent oxygen consumption, and cytochrome c reduction were highly correlated. We conclude that oxidant(s) produced by redox cycling of naphthoquinones stimulated an adenosine-insensitive PtdIns kinase through tyrosine phosphorylation of the enzyme.

Atrial natriuretic factor inhibits vasopressin secretion from rat posterior pituitary.

The effects of synthetic atrial natriuretic factor (ANF) were studied in superfused rat posterior pituitary gland. ANF (10(-6)M, 10(-10)M) significantly inhibited basal as well as KC1 (50 mM) or angiotensin II-stimulated immunoreactive arginine vasopressin secretion. The magnitude of inhibition was greater at 10(-6)M than at 10(-10)M. ANF also decreased cAMP secretion and increased cGMP secretion from the posterior pituitary. These results suggest that ANF directly acts on the posterior pituitary to inhibit arginine vasopressin secretion and that this effect is, at least, partly mediated by the changes in cyclic nucleotide production.

Feedback regulation of phospholipase C-beta by protein kinase C.

Treatment of a variety of cells and tissues with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC) results in the inhibition of receptor-coupled inositol phospholipid-specific phospholipase C (PLC) activity. To determine whether or not the targets of TPA-activated PKC include one or more isozymes of PLC, studies were carried out with PC12, C6Bu1, and NIH 3T3 cells, which contain at least three PLC isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of the cells with TPA stimulated the phosphorylation of serine residues in PLC-beta, but the phosphorylation state of PLC-gamma and PLC-delta was not changed significantly. Phosphorylation of bovine brain PLC-beta by PKC in vitro resulted in a stoichiometric incorporation of phosphate at serine 887, without any concomitant effect on PLC-beta activity. We propose, therefore, that rather than having a direct effect on enzyme activity, the phosphorylation of PLC-beta by PKC may alter its interaction with a putative guanine nucleotide-binding regulatory protein and thereby prevent its activation.

A novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

HF-1 b, an SP1 -related transcription factor, is preferentially expressed in the cardiac conduction system and ventricular myocytes in the heart. Mice deficient for HF-1 b survive to term and exhibit normal cardiac structure and function but display sudden cardiac death and a complete penetrance of conduction system defects, including spontaneous ventricular tachycardia and a high incidence of AV block. Continuous electrocardiographic recordings clearly documented cardiac arrhythmogenesis as the cause of death. Single-cell analysis revealed an anatomic substrate for arrhythmogenesis, including a decrease and mislocalization of connexins and a marked increase in action potential heterogeneity. Two independent markers reveal defects in the formation of ventricular Purkinje fibers. These studies identify a novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.