Threefold Excess Research Articles

Physicochemical Characterization and Purification of Cationic Lipoplexes

Cationic lipid-nucleic acid complexes (lipoplexes) consisting of dioleoyltrimethylammoniumpropane (DOTAP) liposomes and plasmid DNA were prepared at various charge ratios (cationic group to nucleotide phosphate), and the excess component was separated from the lipoplex. We measured the stoichiometry of the lipoplex, noted its colloidal properties, and observed its morphology and structure by electron microscopy. The colloidal properties of the lipoplexes were principally determined by the cationic lipid/DNA charge ratio and were independent of the lipid composition. In lipoplexes, the lipid membranes as observed in freeze-fracture electron microscopy were deformed into high-radius-of-curvature features whose characteristics depended on the lipid composition. Lipoplexes prepared at a threefold or greater excess of either DOTAP or DNA could be resolved into complexes with a defined stoichiometry and the excess component by sedimentation to equilibrium on sucrose gradients. The separated, positively charged complex retained high transfection activity and had reduced toxicity. The negatively charged lipoplex showed increased transfection activity compared to the starting mixture. In cryoelectron micrographs the positively charged complex was spherical and contained a condensed but indistinct interior structure. In contrast, the separated negatively charged lipoplexes had a prominent internal 5.9±0.1-nm periodic feature with material projecting as spikes from the spherical structure into the solution. It is likely that these two lipoplexes represent structures with different lipid and DNA packing.

In VitroAssembly Properties of Wild-Type and Cyclophilin-Binding Defective Human Immunodeficiency Virus Capsid Proteins in the Presence and Absence of Cyclophilin A

The cellular protein cyclophilin A (CypA) binds specifically to the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein and is incorporated into HIV-1 particles at a molar ratio of 1:10 (CypA/CA). Structural analysis of a CA–CypA complex suggested that CypA may destabilize interactions in the viral capsid and thus promote uncoating. We analyzed the influence of CypA on thein vitroassembly properties of wild-type (WT) CA and derivatives containing substitutions of Gly89 in the Cyp-binding loop. All variant proteins were significantly impaired in CypA binding. In the presence of CypA at a molar ratio of 1:10 (CypA/CA), WT CA assembled into hollow cylinders that were similar to those observed in the absence of CypA but slightly longer. Higher CypA concentrations inhibited cylinder formation. Variant CA proteins G89L and G89F yielded similar cylinders as the WT protein but were significantly more resistant to CypA. Cryoelectron microscopic analysis of WT cylinders assembled in the presence of CypA revealed direct binding of CypA to the outer surface. Electron diffraction patterns generated from these cylinders indicated that CypA causes local disorder. The addition of CypA to preassembled cylinders had little effect, however, and cylinders were only disrupted when incubated with a threefold molar excess of CypA for several hours. These results suggest that CypA does not efficiently destabilize CA interactions at the molar ratio observed in the virion and therefore is unlikely to serve as an uncoating factor.

Effect of a low-molecular-weight salt on colloidal dispersions of interpolyelectrolyte complexes

Colloidal dispersions of an interpolyelectrolyte complex were prepared by mixing dilute aqueous solutions of poly(dimethyldiallylammonium chloride) and the sodium salt of the alternating copolymer of maleic acid propene in amounts providing about a threefold excess of the charged groups of the cationic polyelectrolyte over those of the anionic polyelectrolyte. These dispersions were examined by means of analytical sedimentation, quasielastic light scattering, and laser Doppler microelectrophoresis. The experimental results obtained suggest that the particles of the interpolyelectrolyte complex are multicomplex aggregates bearing cationic charge. Such aggregates were assumed to consist of a hydrophobic core formed by coupled oppositely charged macromolecules and a hydrophilic shell formed by cationic macromolecules. Hydrodynamic and electrophoretic properties of these aggregates were found to be rather sensitive to variations in the ionic strength of the surrounding medium: with rising salt concentration, their sedimentation coefficient and hydrodynamic size increase, these increases becoming more strongly pronounced at higher salt concentrations, whereas their electrophoretic mobility gradually decreases. The salt effects revealed suggest that the aggregation level of the particles of the interpolyelectrolyte complex rises in response to an increase in the ionic strength of the surrounding medium. This phenomenon was associated with the salt-induced decrease of the stabilizing effect of the hydrophilic shells that protect such particles from progressive aggregation.

A mutation that improves soluble recombinant hemoglobin accumulation in Escherichia coli in heme excess.

High-level expression of soluble recombinant human hemoglobin (rHb) in Escherichia coli was obtained with several hemoglobin variants. Under identical conditions, two rHbs containing the Presbyterian mutation (Asn-108-->Lys) in beta-globin accumulated to approximately twofold less soluble globin than rHbs containing the corresponding wild-type beta-globin subunit accumulated. The beta-globin Providence(asp) mutation (Lys-82-->Asp) significantly improved soluble rHb accumulation compared to the wild-type beta-globin subunit and restored soluble accumulation of rHbs containing the Presbyterian mutation to wild-type levels. The Providenceasp substitution introduced a negatively charged residue into the normally cationic 2,3-bisphosphoglycerate binding pocket, potentially reducing the electrostatic repulsion in the absence of the polyanion. The average soluble globin accumulation when there was coexpression of di-alpha-globin and beta-Lys-82-->Asp-globin (rHb9.1) and heme was present in at least a threefold molar excess was 36% +/- 3% of the soluble cell protein in E. coli. The average total accumulation (soluble globin plus insoluble globin) was 56% +/- 7% of the soluble cell protein. Fermentations yielded 6.0 +/- 0. 3 g of soluble rHb9.1 per liter 16 h after induction and 6.4 +/- 0.2 g/liter 24 h after induction. The average total globin yield was 9.4 g/liter 16 h after induction. High-level accumulation of soluble rHb in E. coli depends on culture conditions, the protein sequence, and the molar ratio of the heme cofactor added.

A MULTICOPYc-mycTRANSGENE AS A NUCLEAR LABEL: OVERGROWTH OF Myctg50CELLS IN ALLOPHENIC MICE

To trace cell lineages and the origin and fate of cells in transplantation and embryo chimeras, a DNA/DNAin situhybridization cell labelling system was developed, based on a 50-copy murinec-myctransgene on mouse chromosome 8. Elevated levels of cMyc mRNA were found in Myc*tg50 (Myctg50/0 and Myctg50/Myctg50) transgenic tissues, but adult transgenic NMRI mice were anatomically and histologically indistinguishable from control NMRI mice and did not develop tumours on a wild-type or nude (nu/nu) background. The hybridization label detected transgenic nuclei with an efficiency of ∼80%. In muscle grafts, this transgene label was successfully applied to trace donor cells in a labelled host and to study the invasion of a graft by host cells. When the cMyc hybridization was used in allophenic mice of the control|acNMRI-Myctg50/? (nu/+ or +/+) type, an up to a three-fold excess of MYC*tg50 positive over control nuclei was found in all organs examined (ventricle, skeletal muscle, liver, small intestine). This overgrowth of MYC*tg50 cells is probably due to transgene expression. Four out of seven (C57BL/6×BALB/c) or (C57BL/6×NMRI)|acMYC*tg50 allophenic mice displayed anatomical abnormalities, e.g. an enlarged thymus and a tumour in the groin region. As these abnormalities were only observed in allophenic mice, they might be due to the imbalance of growth potential between MYC*tg50 transgenic and normal cells in the same individual.

Melanin-concentrating hormone: a functional melanocortin antagonist in the hypothalamus.

Melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) demonstrate opposite actions on skin coloration in teleost fish. Both peptides are present in the mammalian brain, although their specific physiological roles remain largely unknown. In this study, we examined the interactions between MCH and alpha-MSH after intracerebroventricular administration in rats. MCH increased food intake in a dose-dependent manner and lowered plasma glucocorticoid levels through a mechanism involving ACTH. In contrast, alpha-MSH decreased food intake and increased glucocorticoid levels. MCH, at a twofold molar excess, antagonized both actions of alpha-MSH. alpha-MSH, at a threefold molar excess, blocked the orexigenic properties of MCH. MCH did not block alpha-MSH binding or the ability of alpha-MSH to induce cAMP in cells expressing either the MC3 or MC4 receptor, the principal brain alpha-MSH receptor subtypes. These data suggest that MCH and alpha-MSH exert opposing and antagonistic influences on feeding behavior and the stress response and may function in a coordinate manner to regulate metabolism through a novel mechanism mediated in part by an MCH receptor.

Inhibition of complement activity by humanized anti-C5 antibody and single-chain Fv

Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5G1.1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9. Humanized 5G1.1 antibody, Fab and scFv molecules have been produced by grafting the complementarity determining regions of 5G1.1 on to human framework regions. Competitive ELISA analysis indicated that no framework changes were required in the humanized variable regions for retention of high affinity binding to C5, even at framework positions predicted by computer modeling to influence CDR canonical structure. The humanized Fab and scFv molecules blocked complement-mediated lysis of chicken erythrocytes and porcine aortic endothelial cells in a dose-dependent fashion, with complete complement inhibition occurring at a three-fold molar excess, relative to the human C5 concentration. In contrast to a previously characterized anti-C5 scFv molecule, the humanized h5G1.1 scFv also effectively blocked C5a generation. Finally, an intact humanized h5G1.1 antibody blocked human complement lytic activity at concentrations identical to the original murine monoclonal antibody. These results demonstrate that humanized h5G1.1 and its recombinant derivatives retain both the affinity and blocking functions of the murine 5G1.1 antibody, and suggest that these molecules may serve as potent inhibitors of complement-mediated pathology in human inflammatory diseases.

Kaposi's sarcoma and non-Hodgkin's lymphomas in children and adolescents with AIDS.

To assess the proportion of, and the characteristics associated with, Kaposi's sarcoma (KS) and non-Hodgkin's lymphomas (NHL) in children (0-12 years) and adolescents (13-19 years) at AIDS diagnosis, in Europe and in the United States (US). Comparison of the proportions of KS and NHL at AIDS diagnosis, according to various characteristics, by means of the European Non-Aggregate AIDS Data Set (4400 children and 764 adolescents) and the US AIDS Public Information Data Set (4710 children and 1064 adolescents) updated to June 1993. Comparison of data was made using odds ratios (OR) and corresponding 95% confidence intervals. Between 0 and 19 years, the proportion of KS at AIDS diagnosis (0.5% in Europe and 0.9% in the US) increased with age, particularly in Europe, and was higher in males than in females. It decreased with time in the US but not in Europe. In the US, black children showed a nearly threefold excess of KS as compared with white children. Adolescents who reported homosexual intercourse with other males had significantly elevated risks of KS, both in Europe (OR, 6.3) and in the US (OR, 21.1). NHL was diagnosed in 0.9% of children in Europe and in 1.5% in the US. The proportion of NHL significantly increased with age (3.1% among adolescents in Europe and 3.0% in the US), and was higher in males than in females. In the US, NHL tended to decline in proportion with all AIDS cases over time and showed a nearly twofold higher frequency in white children. No association emerged between NHL and HIV transmission category. In agreement with incidence rates in the general paediatric population, but at variance with AIDS in adults, NHL turned out to be more common than KS in children and adolescents with AIDS. A strong excess of KS was already present in adolescents who reported homosexual intercourse.

An Anti-Interleukin 8 Monoclonal Antibody That Interferes with the Binding of Interleukin 8 to Cellular Receptors and the Activation of Human Blood Neutrophils

Interleukin 8 (IL-8) is a proinflammatory cytokine produced by a wide variety of cells. Interleukin 8 acts as a neutrophil activator and chemotactic factor. In the current studies, we examined the properties of a monoclonal antibody against human IL-8. The estimated affinity of the antibody was 1.74 x 10(7) liters/mol. The antibody interfered with the binding of radiolabeled recombinant human IL-8 (rhIL-8) to human blood neutrophils (IC50 = 3 x 10(-7) M, at an IL-8 concentration of 2.4 nM). Neutrophil degranulation elicited by 5 x 10(-6)-4 x 10(-8) M rhIL-8 was blocked by the antibody at three-fold molar excess. However, a higher concentration of anti-IL-8 antibody was needed to suppress the chemotactic activity of rhIL-8. The inhibition of neutrophil chemotaxis triggered by 2 x 10(-7)-2 x 10(-9) M rhIL-8 required 6 x 10(-5) M antibody. Similarly, a 300-fold molar excess of anti-IL-8 antibody [10(-5) M] was necessary to abrogate the increase in cytosolic free calcium in neutrophils stimulated with 4 x 10(-8) M rhIL-8. In addition, epitope analysis using synthetic peptides corresponding to different regions of the IL-8 molecule showed that peptide consisting of residues 44-72 (corresponding to the C-terminal of the IL-8 molecule) competed with the antibody for binding to rhIL-8. Because IL-8 is an important inflammatory mediator in several human diseases, anti-IL-8 antibodies may have pharmacological potential.

High pH reduces DNA damage caused by bile from patients with familial adenomatous polyposis: antacids may attenuate duodenal polyposis.

Patients with familial adenomatous polyposis (FAP) develop periampullary duodenal tumours, suggesting that bile contributes to their formation. The hypothesis that bile contains carcinogens has been tested by looking for DNA adducts (markers of carcinogen exposure) in the duodenum of patients with or without FAP and by determining whether bile can produce DNA adducts in vitro. Using 32P-postlabelling to detect adducts, there was an excess (compared with unaffected patients) of DNA adducts in the duodenum of FAP patients and an excess of DNA adducts in the small bowel of rats treated with FAP bile, while bile from FAP patients formed significantly more DNA adducts in vitro than did bile from controls. In this study it is shown that the excess of adduct labelling produced by FAP bile in vitro depends on the pH of the incubate. While adduct labelling at pH 6-8 did not differ significantly between bile from six FAP patients and six controls, at pH 4-5 FAP bile, but not control bile, produced a near threefold excess of adduct labelling over that at pH 6-8. Therapy that increases duodenal pH may therefore alleviate duodenal polyposis.

Design, synthesis, and conformation of a model peptide of endothelin with cystine-stabilized alpha-helix motif.

A model 16-peptide of endothelin-1 (MET-1), which has the minimized sequence homology to the corresponding part of endothelin-1 (ET-1), was designed to confirm the cystine-stabilized alpha-helix motif. The model structure consists of an extended structure, a beta-turn part, and an alpha-helix structure that is stabilized by two disulfide bonds. The alpha-helix segment was designed to emphasize the amphiphilic nature. In order to combine the extended structure and the alpha-helix segment, a D-Ala-Pro sequence was selected to fix the beta-turn. The model endothelin 16-peptide amide was synthesized by solid-phase synthesis on a 4-methylbenzhydrylamine resin. Its conformation was examined by CD and two-dimensional (2D) 1H-nmr measurements. MET-1 showed similar CD patterns to ET-1 in both buffer and 50% aqueous trifluoroethanol solution. The 2D nmr experiments in 50% aqueous ethylene glycol revealed that MET-1 closely resembles the conformation of ET-1 with an extended structure, an alpha-helix, and a beta-turn unit in the same position of the sequence. Furthermore, model peptides without disulfide bond(s) could not assume a stable structure in aqueous solution, while they did have similar alpha-helical content in 50% trifluoroethanol with MET-1. When the two disulfide bridges were simultaneously formed, the peptide with the correct disulfide bonds (MET-1) was obtained in threefold excess to the isomer (apamin type, MET-2). These findings obtained by the modeling of ET-1 showed an important role for the stabilization of peptide conformation with disulfide bonds.

Physical Activity in Older Middle-aged Men and Reduced Risk of Stroke: The Honolulu Heart Program

From 1965 to 1968, the Honolulu Heart Program began following 8,006 men in a prospective study of cardiovascular disease. At the time of study enrollment, an estimate of current 24-hour habitual physical activity was collected from each subject. On the basis of a calculated physical activity index, subjects were classified as being inactive, partially active, or active. This report examines the relation between the levels of physical activity and stroke that occurred among 7,530 of the men over 22 years of follow-up. Risk of stroke was examined separately in younger (45-54 years) middle-aged men and older (55-68 years) middle-aged men. Among the older men, those who were inactive or partially active experienced a three- to fourfold excess incidence of hemorrhagic stroke as compared with active men (p < 0.01). There was a two- to threefold excess of intracerebral hemorrhage in men who were inactive or partially active as compared with those who were active (p < 0.05). An excess of subarachnoid hemorrhage was observed in inactive older men, with only one event occurring in those who were active (p < 0.05). After exclusion of subjects with hypertension, diabetes mellitus, and left ventricular hypertrophy, the relative risk of hemorrhagic stroke for inactive men versus active men was 3.7 (95% confidence interval (CI) 1.3-10.4). In older men who did not smoke cigarettes, the relative risk of thromboembolic stroke among inactive men versus active men was 2.8 (95% CI 1.2-6.7), and when partially active older men were compared with those who were active, the relative risk was 2.4 (95% CI 1.0-5.7). These findings persisted after control for the residual effects of systolic blood pressure and other risk factors for stroke. Benefits of physical activity in reducing the risk of thromboembolic stroke were not observed in men who smoked cigarettes. The authors conclude that physical activity may be important in reducing the risk of stroke, particularly among nonsmoking men in older middle age.

The mRNA Poly(A)-Binding Protein: Localization, Abundance, and RNA-Binding Specificity

The poly(A)-binding protein (PABP) binds to the messenger (mRNA) 3′-poly(A) tail found on most eukaryotic mRNAs and together with the poly(A) tail has been implicated in governing the stability and the translation of mRNA. In order to further understand the role of the PABP in these processes, we have undertaken a detailed analysis of the cellular localization, the abundance, and the RNA-binding properties of the human PABP (hPABP). We raised monoclonal antibodies against the 70-kDa hPABP and confocal immunofluorescence microscopy with these antibodies reveals that it is localized exclusively to the cytoplasm. The hPABP exhibits a very low turnover rate in these cells and quantitative immunoblotting experiments demonstrated that growing HeLa cells contain a surprisingly high number of approximately 8 × 106 PABP molecules per cell, which corresponds to an intracellular concentration of about 4 μM. In an in vitro selection/amplification assay from random sequence oligonucleotide pools the hPABP selects oligo(rA)-rich sequences and it binds oligo(rA)25 with an apparent Kd of 7 nM. The hPABP binds to unrelated RNA sequences with an about 100-fold lower affinity (Kd ⩾ 0.5 μM). The abundance of the hPABP indicates that there is an approximately three-fold excess of the protein over binding sites on cytoplasmic poly(A). This excess and the high concentration of the hPABP, which is three orders of magnitude above its Kd for oligo(rA)25, suggest that the hPABP may bind to additional, lower affinity binding sites in vivo.

Ethnic differences in ECG amplitudes in North American white, black, and Hispanic men and women: Effect of obesity and age

General trends were investigated in the age-evolution of electrocardiogram (ECG) patterns using ECG data of North American white, black, and Hispanic men and women aged 25–74 years, derived from the Second National Health and Nutrition Examination Survey and the Hispanic Health and Nutrition Examination Survey. The data were stratified by race, sex, age, and obesity. There were substantial racial differences in ECG amplitudes. In general, ECG amplitudes and amplitude combinations used in left ventricular hypertrophy (LVH) criteria were larger in blacks than in whites. RV 5 was smaller in all age groups of Hispanic women and in younger men, and RaVL was larger in Hispanics than in whites. There was, however, no significant difference in RaVL + SV 3 amplitude between Hispanics and whites except in the youngest age group. The QRS frontal plane axis increased in men and women in all three racial groups by about 8° per decade of age. Overweight was associated with 14° more horizontal axis. RaVL and RaVL + SV 3 amplitudes increased systematically with age and overweight, but there was an opposite tendency in RV 5 amplitude. Multivariate logistic regression analyses indicated that age and the QRS axis had a relatively minor influence on LVH likelihood. Being black was associated with a more than fourfold excess of LVH by the Minnesota code criteria and a nearly threefold excess of LVH by the Cornell voltage criteria. Being overweight did not influence the odds ratio for LVH by the Cornell voltage criteria except in Hispanics, but the odds ratio for LVH was reduced by about 25% according to the Minnesota code criteria. The odds ratio of having LVH differed substantially for men and women by the Minnesota code and Cornell voltage criteria. Whereas there was a more than twofold excess likelihood of LVH in men by the Minnesota code criteria, there was a more than threefold excess of LVH likelihood in women by the Cornell voltage criteria when age, obesity, QRS axis, and race were simultaneously included as covariates in the logistic regression model.

Inhibition of Succinate Dehydrogenase by Malonic Acid Produces an “Excitotoxic” Lesion in Rat Striatum

Excitotoxicity and defects in neuronal energy metabolism have both been implicated in the pathogenesis of neurodegenerative disease. These two mechanisms may be linked through the NMDA receptor, activation of which is dependent on neuronal membrane potential. Because the ability to maintain membrane potential is dependent on neuronal energy metabolism, bioenergetic defects may affect NMDA receptor-mediated excitotoxicity. We now report that reversible inhibition of succinate dehydrogenase (SDH), an enzyme central to both the tricarboxylic acid cycle and the electron transport chain, produces an "excitotoxic" lesion in rat striatum that can be blocked by the NMDA antagonist MK-801. Male Sprague-Dawley rats received intrastriatal stereotaxic injections of the SDH inhibitor malonic acid (1 or 2 mumol) in combination with intraperitoneal injections of vehicle or MK-801 (5 mg/kg) 30 min before and 210 min after malonic acid. Animals were killed 72 h after surgery, and brains were processed for histology, cytochrome oxidase activity, and [3H]MK-801 and [3H]AMPA autoradiography. The higher dose of malonic acid (2 mumol) produced large lesions that were markedly attenuated by treatment with MK-801 (28.1 +/- 3.6 vs. 4.7 +/- 2.6 mm3; p < 0.001). [3H]MK-801 and [3H]AMPA binding were reduced in the lesions by 60 and 63%, respectively. One micromole of malonic acid produced smaller lesions that were almost completely blocked by MK-801 treatment (9.6 +/- 1.3 vs. 0.06 +/- 0.04 mm3; p < 0.0001). The toxic effects of malonic acid were due specifically to inhibition of SDH inasmuch as coinjection of a threefold excess of succinate with the malonic acid blocked the striatal lesions (p < 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)

Antioxidant effect of coenzyme Q on hydrogen peroxide-activated myoglobin.

In recent years increased attention has been focused on the reduced forms of coenzyme Q as antioxidant compounds inhibiting lipid peroxidation in model systems and in biological membranes, but in spite of extensive experimental evidences the molecular mechanisms responsible for the antioxidant activity of ubiquinones are still debated. Ferrylmyoglobin and/or its free radical form are regarded as powerful oxidizing agents capable of promoting oxidation of essential cellular constituents, particularly cell membranes. Therefore, we investigated the effects of ubiquinol on the formation and survival of ferryl species of myoglobin and on metmyoglobin itself. The addition of a threefold molar excess of hydrogen peroxide to a solution of metmyoglobin induces the rapid formation of a compound with the spectral characteristics of ferrylmyoglobin. The reaction is complete within 4 min, producing up to 76% of ferrylmyoglobin, which remains stable for at least 30 min. The addition of ubiquinol-1 to the same solution provokes a rapid and progressive reduction of ferrylmyoglobin to metmyoglobin and oxymyoglobin. Ubiquinol-1, furthermore, is also capable of protecting metmyoglobin against oxidation when added in the solution before hydrogen peroxide. Ubiquinol-1, indeed, is effective at both limiting the maximal ferrylmyoglobin level attained (59% inhibition) and accomplishing complete removal of the ferryl form (in about 15 min). The results demonstrate that ubiquinol is capable of reducing both ferrylmyoglobin and metmyoglobin to oxymyoglobin, providing a novel antioxidant mechanism for coenzyme Q.

A deep band I-band selected galaxy sample - Implications for galaxy evolution

New spectroscopic observations of a statistically complete sample of 50 very faint I-band selected galaxies with I AB obs ∼0.5±0.1. This is the same as simple no-evolution predictions, despite a roughly threefold excess in the number-magnitude counts relative to a no-evolution q 0 =0.5 model. Within this cosmological framework, this supports the idea that the faint field galaxy population is dominated by a rapidly evolving population of numerous and presumably relatively low-mass galaxies with the spectral energy distributions of Irr galaxies and rest-frame V-band luminosities around M V ∼−20.0, i.e., about 1 mag below the present-day L *

Exercise and pacing tests for the diagnosis of single coronary artery disease

General trends were investigated in the age-evolution of electrocardiogram (ECG) patterns using ECG data of North American white, black, and Hispanic men and women aged 25–74 years, derived from the Second National Health and Nutrition Examination Survey and the Hispanic Health and Nutrition Examination Survey. The data were stratified by race, sex, age, and obesity. There were substantial racial differences in ECG amplitudes. In general, ECG amplitudes and amplitude combinations used in left ventricular hypertrophy (LVH) criteria were larger in blacks than in whites. RV5 was smaller in all age groups of Hispanic women and in younger men, and RaVL was larger in Hispanics than in whites. There was, however, no significant difference in RaVL + SV3 amplitude between Hispanics and whites except in the youngest age group. The QRS frontal plane axis increased in men and women in all three racial groups by about 8° per decade of age. Overweight was associated with 14° more horizontal axis. RaVL and RaVL + SV3 amplitudes increased systematically with age and overweight, but there was an opposite tendency in RV5 amplitude. Multivariate logistic regression analyses indicated that age and the QRS axis had a relatively minor influence on LVH likelihood. Being black was associated with a more than fourfold excess of LVH by the Minnesota code criteria and a nearly threefold excess of LVH by the Cornell voltage criteria. Being overweight did not influence the odds ratio for LVH by the Cornell voltage criteria except in Hispanics, but the odds ratio for LVH was reduced by about 25% according to the Minnesota code criteria. The odds ratio of having LVH differed substantially for men and women by the Minnesota code and Cornell voltage criteria. Whereas there was a more than twofold excess likelihood of LVH in men by the Minnesota code criteria, there was a more than threefold excess of LVH likelihood in women by the Cornell voltage criteria when age, obesity, QRS axis, and race were simultaneously included as covariates in the logistic regression model.

Na(+)-K(+)-ATPase alpha 1- and beta 1-subunit degradation: evidence for multiple subunit specific rates.

Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein consisting of an alpha-catalytic and a beta-glycoprotein subunit. Because these two subunits are derived from two separate genes, they may not be synthesized with stoichiometric equivalence. The aim of this study was to estimate relative rates of synthesis and degradation of nascent and mature Na(+)-K(+)-ATPase alpha- and beta-subunits to determine whether either of the nascent subunits accumulates in excess and, if so, the fate of the excess subunits. We studied a pig kidney cell line (LLC-PK1/Cl4) that expresses only alpha 1- and beta 1-subunits. Relative synthesis and degradation rates of nascent subunits were first estimated by pulsing cells for 10 min with [35S]methionine followed by chase periods of up to 120 min and by immunoprecipitation. We found that directly after labeling, beta-subunits were present in threefold excess over alpha-subunits and that nearly 50% of this beta-subunit pool was degraded by 60 min. Nascent alpha-subunits were not degraded during the chase period. In a second strategy to examine relative rates of nascent alpha- vs. beta-subunit accumulation, cells were pulsed for 5-60 min and immunoprecipitated directly (without chase). The rate of accumulation of labeled alpha was greater than that of beta between 5 and 60 min, consistent with the results of the pulse-chase strategy, demonstrating a significant component of degradation of beta during this period. Despite the very different degradation rates of newly synthesized alpha- vs. beta-subunits, the degradation rates of alpha- and beta-subunits beyond 4 h after synthesis were indistinguishable (t0.5 = 10-12 h).(ABSTRACT TRUNCATED AT 250 WORDS)

A One-Step Synthesis of 2,3-Diydro-1,4-benzothiazines and Phenothiazines from 1,3-Thiazolidine Derivatives of Cyclohexanones

N-Acetyl-1,3-thiazolidine derivatives of cyclohexanones, when treated with a threefold excess N-bromosuccinimide in anhydrous chloroform at room temperature, smoothly afford N-acetyl-2,3-dihydro-1,4-benzothiazines. This represents the first general route to such heterocyclic system in one step from aliphatic precursors. The synthesis of optically active N-acetyl-2,3-dihydro-1,4-benzothiazines is also reported