Characterization of altered myocardial fatty acid metabolism in patients with inherited cardiomyopathy.

Inherited defects in myocardial long-chain fatty acid metabolism are increasingly recognized as a cause of cardiomyopathy and sudden death in children. To evaluate whether the phenotypic expression of these genetic diseases could be delineated using positron emission tomography (PET), 11 patients with inherited defects in fatty acid metabolism were evaluated and results were compared with those of 6 nonaffected siblings. Myocardial perfusion, myocardial oxygen consumption (MVO2), and long-chain fatty acid metabolism were determined noninvasively with PET using quantitative mathematical models. There were no differences in haemodynamics, perfusion, MVO2 or plasma substrate levels between groups. Patients with defects in enzymes of fatty acid beta-oxidation (acyl-CoA dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase deficiencies) (n = 5) had diminished myocardial palmitate oxidation compared with healthy siblings (3.2 +/- 3.0 vs. 13.0 +/- 5.6 nmol/g per min, p < 0.03) and a decrease in the percentage of MVO2 accounted for by palmitate (2% +/- 3% vs. 9% +/- 5%, p < 0.04). In these patients, extracted palmitate was shunted into a slow-turnover compartment (predominantly reflecting esterification to triglycerides) with expansion of palmitate in that pool (185 +/- 246 compared with 27 +/- 67 nmol/g in healthy siblings,p < 0.02). In contrast, myocardium of patients with carnitine deficiency (n = 6) (all on oral carnitine therapy) had normal palmitate extraction but expansion of the interstitial/cytosolic fatty acid pool (617 +/- 399 vs. 261 +/- 73 nmol/g in healthy siblings, p < 0.04), suggesting different mechanisms for handling upstream fatty acyl intermediates. Thus, PET can be used to noninvasively assess abnormal myocardial handling of fatty acids in patients with inherited defects of metabolism. This approach should be useful in the assessment of altered myocardial fatty acid metabolism associated with cardiomyopathy as well as for evaluating the efficacy of therapeutic interventions in affected patients.

Vitamin D deficiency rickets in children: prevalence and need for community education.

Nutritional rickets caused by a deficiency in vitamin D can be a crippling disease and is still prevalent today. Many feel that the vitamin D supplementation present in dairy products prevents any risk of developing vitamin D deficiency rickets. However, a combination of factors such as pigmented skin, inadequate sunlight, breastfeeding past 6 months with no supplementation, and insufficient calcium intake by the nursing mother and her baby often contribute to rickets diagnosed in children. Educating the medical community and those families at risk is critical to eradicate this preventable disease. Steps can be taken using stages of the Organizational Change Theory to involve school nurses, community health centers, and pediatricians in identifying these high-risk children. With prompt diagnosis and treatment, all symptoms can abate, with healthy bone growth resulting.

Genetic defects in acetylcholine signalling promote protein degradation in muscle cells of Caenorhabditis elegans.

A myosin-lacZ fusion, expressed in 103 muscle cells of Caenorhabditis elegans, reports on how proteolysis in muscle is controlled by neural and intramuscular signals. Upon acute starvation, the fusion protein is degraded in the posterior 63 cells of the body-wall muscle, but remains stable in 32 anterior body-wall muscles and 8 vulval muscle cells. This distinction correlates with differences in the innervation of these cells. Reporter protein in the head and vulval muscles becomes labile upon genetic 'denervation' in mutants that have blocks in pre-synaptic synthesis or release of acetylcholine (ACh) or post-synaptic reception at nicotinic ACh receptors (nAChR), whereas protein in all 103 muscles is stabilized by the nicotinic agonist levamisole in the absence of ACh production. Levamisole does not stabilize muscle protein in nAChR mutants that are behaviorally resistant to levamisole. Neural inputs thus exert negative control over the proteolytic process in muscle by stimulating muscle nicotinic ACh receptors.

Microtubule disassembly by ATP-dependent oligomerization of the AAA enzyme katanin.

Katanin, a member of the AAA adenosine triphosphatase (ATPase) superfamily, uses nucleotide hydrolysis energy to sever and disassemble microtubules. Many AAA enzymes disassemble stable protein-protein complexes, but their mechanisms are not well understood. A fluorescence resonance energy transfer assay demonstrated that the p60 subunit of katanin oligomerized in an adenosine triphosphate (ATP)- and microtubule-dependent manner. Oligomerization increased the affinity of katanin for microtubules and stimulated its ATPase activity. After hydrolysis of ATP, microtubule-bound katanin oligomers disassembled microtubules and then dissociated into free katanin monomers. Coupling a nucleotide-dependent oligomerization cycle to the disassembly of a target protein complex may be a general feature of ATP-hydrolyzing AAA domains.

Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit.

Microtubule disassembly at centrosomes is involved in mitotic spindle function. The microtubule-severing protein katanin, a heterodimer of 60 and 80 kDa subunits, was previously purified and shown to localize to centrosomes in vivo. Here we report the sequences and activities of the katanin subunits. p60 is a new member of the AAA family of ATPases, and we show that expressed p60 has microtubule-stimulated ATPase and microtubule-severing activities in the absence of p80. p80 is a novel protein containing WD40 repeats, which are frequently involved in protein-protein interactions. The p80 WD40 domain does not participate in p60 dimerization, but localizes to centrosomes in transfected mammalian cells. These results indicate katanin's activities are segregated into a subunit (p60) that possesses enzymatic activity and a subunit (p80) that targets the enzyme to the centrosome.

Transgene-coded chimeric proteins as reporters of intracellular proteolysis: starvation-induced catabolism of a lacZ fusion protein in muscle cells of Caenorhabditis elegans.

The product of an integrated transgene provides a convenient and cell-specific reporter of intracellular protein catabolism in 103 muscle cells of the nematode Caenorhabditis elegans. The transgene is an in-frame fusion of a 5'-region of the C. elegans unc-54 (muscle myosin heavy-chain) gene to the lacZ gene of Escherichia coli [Fire and Waterston (1989): EMBO J 8:3419-3428], encoding a 146-kDa fusion polypeptide that forms active beta-galactosidase tetramers. The protein is stable in vivo in well-fed animals, but upon removal of the food source it is inactivated exponentially (t1/2 = 17 h) following an initial lag of 8 h. The same rate constant (but no lag) is observed in animals starved in the presence of cycloheximide, implying that inactivation is catalyzed by pre-existing proteases. Both the 146-kDa fusion polypeptide (t1/2 = 13 h) and a major 116-kDa intermediate (t1/2 = 7 h) undergo exponential physical degradation after a lag of 8 h. Degradation is thus paradoxically faster than inactivation, and a number of characteristic immunoreactive degradation intermediates, some less than one-third the size of the parent polypeptide, are found in affinity-purified (active) protein. Some of these intermediates are conjugated to ubiquitin. We infer that the initial proteolytic cleavages occur in the cytosol, possibly by a ubiquitin-mediated proteolytic pathway and do not necessarily inactivate the fusion protein tetramer.

Detection of cardiac transplant rejection with 111In-labeled lymphocytes and gamma scintigraphy.

BACKGROUND: Cardiac transplantation is an increasingly important treatment for patients with end-stage heart failure. Rejection is one of the major limitations, and currently, serial endomyocardial biopsies are required to diagnose rejection. In the year after transplantation, patients routinely undergo 12, 14, or more biopsies. Infiltration of lymphocytes into the graft is a central feature of rejection. Previous studies from our laboratory have demonstrated the feasibility of detecting early rejection noninvasively with gamma scintigraphy after administration of autologous lymphocytes labeled with 111In. METHODS AND RESULTS: Eight patients were studied at the time of routine biopsy an average of 4.5 months after cardiac transplantation. Autologous lymphocytes were isolated and labeled with 111In. Forty-eight to 72 hours later, patients underwent planar scintigraphic imaging. Myocardial accumulation of labeled lymphocytes was quantified (indium excess, IE) with a previously described and validated technique. Animal studies have shown that an IE > or = 0.07 is associated with rejection. Two of four patients with biopsy grade 0 or 1A rejection had no excess accumulation of labeled lymphocytes. The other two patients with biopsy grade 0 or 1A had an average IE of 0.13 +/- 0.04 (SD), which may actually represent the higher sensitivity of the scintigraphic approach, since the whole myocardium is interrograted. All four patients with biopsy grade 1B rejection had increased accumulation of labeled lymphocytes (IE = 0.18 +/- 0.06, P = .06 compared with all patients with grade 0 or 1A biopsies). CONCLUSIONS: The development of a sensitive, specific, and noninvasive method of diagnosing cardiac allograft rejection in humans might obviate the need for endomyocardial biopsy as well as improve the accuracy of diagnosis. The results suggest that scintigraphic detection of labeled lymphocytes is a promising approach for the noninvasive detection of cardiac transplant rejection. In addition, the approach should permit the assessment of the efficacy of antirejection therapy.

Regional myocardial perfusion assessed with generator-produced copper-62-PTSM and PET.

UNLABELLED: We have previously demonstrated that myocardial perfusion can be estimated accurately in experimental animals with the generator-produced positron-emitting tracer, 62Cu-pyruvaldehyde bis (N4-methylthio- semicarbazone)(62Cu-PTSM) and PET. This study evaluated the feasibility of quantifying regional myocardial blood flow using 62Cu-PTSM and PET in human subjects. METHODS: Regional perfusion was estimated using a previously described and validated two-compartment model from dynamic PET scans obtained after an intravenous bolus of 62Cu-PTSM in 10 healthy volunteers and in 6 patients with coronary artery disease at rest: and in 9 of the volunteers and 4 of the patients after administration of dipyridamole intravenously. Flow estimates were compared with those obtained using H2(15)O. RESULTS: Contrast was high between myocardium and blood or lung with 62Cu-PTSM, resulting in high-quality myocardial images. Liver uptake was also high. At flows of up to 1.5 ml/g/min, flow estimated with 62Cu-PTSM correlated closely with estimates obtained with H2(15)O (y = 0.71x .21, n = 169 regional comparisons, r = 0.66, p < 0.55), but this relationship was not maintained at higher flows. CONCLUSION: The results demonstrate that quantification of myocardial perfusion with 62Cu-PTSM is feasible in human subjects but cannot be used to estimate hyperemic flows due most likely to the strong binding of the tracer to human serum albumin. Copper-62-PTSM congeners with less avidity for human albumin may prove more suitable for evaluation of hyperemic flows.

Effects of time discrepancies between input and myocardial time-activity curves on estimates of regional myocardial perfusion with PET.

UNLABELLED: Estimates of myocardial perfusion with PET using kinetic models require faithful recording of radioactivity content in blood and myocardium. Typically the arterial time-activity curve is obtained by placing a region of interest (ROIs) within the left atrial or left ventricular cavity. However, curves generated from these regions appear earlier in time than tissue time-activity curves obtained from ROIs within the myocardial tissue, and such time discrepancies can lead to errors in flow estimates. METHODS: The magnitude of these time discrepancies and their effect on estimates of regional myocardial perfusion using oxygen-15-water were measured in 30 normal subjects evaluated at rest and again after administration of dipyridamole. RESULTS: Under baseline conditions, the left atrial curve appeared 0.97 +/- 0.67 (s.d.) before the ascending aorta input curve (p < 0.05) and estimated perfusion decreased from 1.28 +/- 0.28 ml/g/min using the left atrial curve uncorrected for time to 0.98 +/- 0.27 ml/g/min after correction (p < 0.05). After dipyridamole, the left atrial curve appeared 0.68 +/- 0.72 sec before the ascending aorta curve (p < 0.05) and estimated perfusion decreased from 3.60 +/- 1.40 ml/g/min using the left atrial curve uncorrected for time to 3.24 +/- 1.26 ml/g/min using the time-corrected curve (p < 0.05). Because the magnitude of time discrepancies between the left ventricular and ascending aortic curves was less (0.25 +/- 0.34 and 0.19 +/- 0.23 sec at rest and after dipyridamole, respectively), effects on flow estimates were more modest. CONCLUSIONS: The results of this study demonstrate that time discrepancies between input and tissue time-activity curves can affect estimates of myocardial flow. Correction for this potential source of error is proposed.

Research on lesbianism: selected effects of time, geographic location, and data collection technique.

It is the intention of this article both to be descriptive of elements of the lesbian life style that appear to be consistent over time and to examine the results of using widely different data collection techniques attempting to differentiate such behaviors. In addition, the study from which the data are derived examined areas of change and social movement among selected areas of personal commitment or interaction. Research in the area of covert behavior is extremely difficult. Certain types of covert behavior preclude traditional survey and sampling procedures, making parameter estimates for the general population, as well as precluding the use of inferential statistics for data analysis. The masking of the deviant self is perhaps most pronounced where the covert activity in question is illegal (Klockars, 1974). Given this, a comparison of the impact of different research techniques on the quality of data generated in the study of deviant behavior would appear to be important. The data were collected from three separate groups involving three data collection times spread over a 10-year period, involving three geographic locations, and involving two different data collection techniques. A total of 394 lesbians were interviewed or responded to a questionnaire distributed with the cooperation of a large, well-known homophile organization. An analysis was made, and both significant and nonsignificant differences in sample types are discussed. It should be noted that these data represent a small segment of the data generated by the study. The parts of them presented were chosen because they address pertinent theoretical and methodological questions in the area of researching covert behavior.