Racial Differences in Metabolic Predictors of Obesity among Postmenopausal Women

NICKLAS, BARBARA S., DORA M. BERMAN, DAWN C. DAVIS, C. LYNNE DOBROVOLNY, AND KAREN E. DENNIS. Racial differences in metabolic predictors of obesity among postmenopausal women. Ober Res. Objective: This study determined whether there are racial differences in resting metabolic rate (RMR), fat oxidation, and maximal oxygen consumption (VO,max) in obese [body mass index (BMI = 34±2 kg/m²)], postmenopausal (58±2 years) women. Research Methods and Procedures: Twenty black and 20 white women were matched for fat mass and lean mass (LM), as determined by dual energy X-ray absorptiometry. RMR and fat oxidation were measured by indirect calorimetry in the early morning after a 12-hour fast using the ventilated hood technique. VO₂max was measured on a treadmill during a progressive exercise test to voluntary exhaustion. Results: RMR, adjusted for differences in LM, was 5% higher in white than black women (1566±27 and 1490±26 kcal/day, respectively; p<0. 05); and fat oxidation rate was 17% higher in white than black women (87±4 and 72±3 g/day, respectively; p<0. 01). VO₂max (L/minute) was 150 mL per minute (8%) higher (p<0. 05) in white than black women. VO₂max correlated with LM in black (r = 0. 44, p = 0. 05) and white (r=0. 53, p<0. 05) women, but the intercept of the regression line was higher in white than black women (p<0. 05), with no significant difference in slopes. In a multiple regression model including race, body weight, LM, and age, LM was the only independent predictor of RMR (r² = 0. 46, p<0. 0001), whereas race was the only independent predictor of fat oxidation (r² = 0. 18,p<0. 05). The best predictors of VO,max were LM (r² = 0. 22, p<0. 05) and race (cumulative r² = 0. 30, p<0. 05). Discussion: These results show there are racial differences in metabolic predictors of obesity. Determination of whether these ethnic differences lead to, or are an effect of, obesity status or other lifestyle factors requires further study.     

Role of Oxidative Stress and the Glutathione System in Loss of Dopamine Neurons Due to Impairment of Energy Metabolism

Abstract: Alterations in the glutathione system and impairment in energy metabolism have both been implicated in the loss of dopamine neurons in Parkinson's disease. This study examined the importance of cellular glutathione and the involvement of oxidative stress in the loss of mesencephalic dopamine and GABA neurons due to inhibition of energy metabolism with malonate, the reversible, competitive inhibitor of succinate dehydrogenase. Consistent with previous findings, exposure to malonate for 24 h followed by 48 h of recovery caused a dose-dependent loss of the dopamine population with little effect on the GABA population. Toxicity was assessed by simultaneous measurement of the high-affinity uptake of [³H]dopamine and [¹⁴C]GABA. Total glutathione content in rat mesencephalic cultures was decreased by 65% with a 24-h pretreatment with 10 µM buthionine sulfoxamine. This reduction in glutathione level greatly potentiated damage to both the dopamine and GABA populations and removed the differential susceptibility between the two populations in response to malonate. These findings point to a role for oxidative stress occurring during energy impairment by malonate. Consistent with this, several spin-trapping agents, α-phenyl-tert-butyl nitrone and two cyclic nitrones, MDL 101,002 and MDL 102,832, completely prevented malonate-induced damage to the dopamine neurons in the absence of buthionine sulfoxamine. The spin-trapping agents also completely prevented toxicity to both the dopamine and GABA populations when cultures were exposed to malonate after pretreatment with buthionine sulfoxamine to reduce glutathione levels. Counts of tyrosine hydroxylase-positive neurons verified enhancement of cell loss by buthionine sulfoxamine plus malonate and protection against cell loss by the spin-trapping agents. NMDA receptors have also been shown to play a role in malonate-induced dopamine cell loss and are associated with the generation of free radicals. Consistent with this, toxicity to the dopamine neurons due to a 1-h exposure to 50 µM glutamate was attenuated by the nitrone spin traps. These findings provide evidence for an oxidative challenge occurring during inhibition of energy metabolism by malonate and show that glutathione is an important neuroprotectant for midbrain neurons during situations when energy metabolism is impaired.     

Vital Genes That Flank Sex-Lethal, an X-Linked Sex-Determining Gene of DROSOPHILA MELANOGASTER

The X-chromosome:autosome balance in D. melanogaster appears to control both sex determination and dosage compensation through effects on a maternally influenced sex-linked gene called Sex-lethal (Sxl; 1-19.2). To facilitate molecular and genetic analysis of Sxl, we attempted to determine the locations of all ethyl methanesulfonate (EMS)-mutable genes vital to both sexes in the region between 6E1 and 7B1. This area includes approximately 1 cM of the genetic map on each side of Sxl and was reported by C. B. Bridges to contain 26 salivary gland polytene chromosome bands. The region appears rather sparsely populated with genes vital to both sexes, since the 122 recessive lethal mutations we recovered fell into only nine complementation groups. From one to 38 alleles of each gene were recovered. There was a preponderance of embryonic lethals in this area, although the lethal periods of loss-of-function mutations included larval, pupal and adult stages as well. Since the screen required that mutations be recessive and lethal to males, our failure to recover new Sxl alleles was the result expected for a gene with a female-specific function. An attempt was made to identify recessive male-specific lethals in this region, but none were found. Precise map positions were determined for eight of the nine vital genes. An interesting feature of the map is the location of Sxl in the middle of a 0.6- to 0.7-cM interval that appears to be devoid of genes vital to both sexes. The genetic location was determined of breakpoints near Sxl for all available chromosome rearrangements. Sxl is most likely located just to the left of band 7A1. We determined the relationship of our EMS-induced mutations in these nine genes to alleles induced by others. From this we conclude that the various genes appear to differ significantly from each other in their relative sensitivity to mutation by EMS vs. X rays.     

V(D)J recombinase-mediated deletion of the hprt gene in T-lymphocytes from adult humans.

The hprt T-cell cloning assay allows the detection of mutations occurring in vivo in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of T-lymphocytes. We have shown previously that the illegitimate activity of V(D)J recombinase accounts for about 40% of the hprt mutations in T-lymphocytes of human newborns as measured with umbilical cord blood samples (Fuscoe et al., 1991). This mechanism results in deletion of hprt exons 2 + 3. In this report, we examined a collection of 314 HPRT-deficient clones derived from adult humans for evidence that the mutations were caused by this mechanism by analyzing exons 2 + 3 deletion mutations. DNA sequence analysis of deletion breakpoint junctions showed that 8 of the mutations were the result of V(D)J recombinase activity. The frequency of the recombinase-mediated mutations was similar in the adults and newborns (2-4 x 10(-7). However, since the hprt mutant frequency is about 10-fold higher in the adult than in the newborn, the recombinase-mediated mutations account for only a few percent of the adult mutations. These mutations are likely to have occurred during early development and persist into adulthood. Unregulated expression of V(D)J recombinase activity may be an important mechanism for genomic rearrangements in the genesis of cancer.     

Chromosome micromanipulation

Two types of unusual motion within the spindle have heen studied in a grasshopper (Melanoplus differentialis) spermatocyte. The first is the motion of granules placed by micromanipulation within the normally granule-free spindle. The most specific motions are poleward, approximate the speed of the chromosomes in anaphase, and occur in the area between the kinetochores and the nearer pole during both metaphase and anaphase. Exactly the same transport properties were earlier observed by Bajer inHaemanthus endosperm spindles. The absence of significant motion in the interzone between the separating chromosomes at anaphase has been unequivocally demonstrated inMelanoplus spermatocytes. Thus very specific motion of non-kinetochoric materials is probably a general spindle capability which would much restrict admissible models of mitotic force production,if the same forces move both granules and chromosomes. The second unusual motion is seen following chromosome detachment from the spindle by micromanipulation during anaphase. These tend to move toNearer pole rather than to the pole the chromosome's kinetochoresFace. The latter preference was earlier demonstrated after detachment during prometaphase or metaphase and has been confirmed without exception in the present studies. The apparent preference for motion to the nearer pole in anaphase provides the first evidence for poleward forces within each half-spindle which cannot be entirely specified by the chromosomal spindle fibers. Almost certainly these would be the usual forces responsible for chromosome motion since they act specifically at the kinetochores of detached chromosomes. This evidence requires interpretation, however because additional factors influence chromosome motion following detachment at anaphase. On thesimplest interpretation, certain current models of mitosis clearly are not satisfactory and others are favored.     

Inhibition of NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine

1-methyl-4-phenylpyridine (MPP+), a major metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) inhibited the ADP-stimulated and uncoupled oxidation of NADH-linked substrates by brain mitochondrial preparations. MPTP itself was ineffective. The apparent Ki's for MPP+ inhibition of pyruvate or glutamate oxidation by purified rat brain mitochondria were approximately 300 and 400 microM, respectively; with mouse brain mitochondria the values were lower, 60 and 150 microM, respectively. Succinate oxidation was unaffected by either compound. Compromise of mitochondrial oxidative capacity by MPP+ could be an important factor in mechanisms underlying the toxicity of MPTP.     

Conformation in solution of hemoglobin Osler (alpha 2 A beta 2 145 Tyr replaced by Asp).

Computer simulations of Gelin and Karplus ((1977) Proc. Natl. Acad. Sci. U.S.A. 74, 801-805) suggest that in hemoglobin upon ligation the penultimate tyrosyl residues of the subunits are not expelled from the hydrophobic pockets described in the crystals between the helices E and F (Perutz, M.F. (1970) Nature 228, 726-737). This implies that both the liganded and unliganded conformations of hemoglobin may be affected by mutations involving such residues. Investigation of the conformational behavior of liganded and unliganded hemoglobin Osler was conducted measuring the functional properties, the subunits dissociation, the CD and electronic spectra, the protons absorption upon interaction with polyanions, and the reactivity of the -SH groups of the protein. The results suggest that both the liganded and unliganded conformations of the system are affected by the mutation, confirming the anticipations of Gelin and Karplus on the relevance of tyrosine at beta 145 for both allosteric states of hemoglobin.     

THE COMPLEX BINDING OF γ-AMINOBUTYRIC ACID TO A RAT BRAIN SYNAPTIC MEMBRANE PREPARATION

The association of γ-aminobutyric acid (GABA) with rat brain synaptic membranes is complex and involves more than one set of binding sites. This fact is suggested by the form of experimentally determined binding curves and confirmed by the destruction of one set of homogenous sites by covalent modifications with iodine. The difference in binding of GABA by two sets of membranes which have been exhaustively iodinated in the presence and absence of an excess of GABA corresponds to 1/6 of the binding expressed by uniodinated membranes after equilibration with 0.25 μm -GABA. This difference appears to be the result of the expression of a homogeneous set of independent sites, each of which contains an iodinatable'residue that is protected by bound GABA. Examination of the factors leading to the retention of radioactive ligand by a synaptic membrane preparation is suggestive of a method for determining the maximum number of binding sites for a given ligand and for evaluating solution space without displacing the bound species or adding a presumably inert tracer. Three additional measurements of retention of high ligand concentration are shown to be sufficient for the determination. Employment of the method facilitates the investigation of the interaction of the membrane preparations with individual agonists and antagonists.     

Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands

The antigen I/II (AgI/II) family polypeptides, ranging from 1310 to 1653 amino acid (aa) residues, are cell wall anchored adhesins expressed by most indigenous species of oral streptococci. The polypeptides interact with a wide range of host molecules, in particular salivary agglutinin glycoprotein (SAG or gp340), and with ligands on other oral bacteria. To determine the receptor recognition properties of six different AgI/II family polypeptides from strains of Streptococcus gordonii, Streptococcus intermedius and Streptococcus mutans, the genes were cloned and expressed on the surface of the surrogate host Lactococcus lactis. The S. gordonii SspA and SspB polypeptides mediated higher binding levels of L. lactis cells to surface immobilized gp340 than did S. intermedius Pas protein, or S. mutans SpaP or PAc proteins. However, the AgI/II proteins were all similar in their abilities to mediate aggregation of lactococci by fluid phase gp340. The SpaP(I) polypeptide from S. mutans Ingbritt, which was C-terminally truncated by approximately 400 aa residues, did not bind gp340. Lactococci expressing AgI/II proteins, including SpaP(I), were aggregated by a synthetic 16 aa residue peptide SRCRP2 derived from the aa repeat block sequences within gp340. In coaggregation assays, SspB from S. gordonii was unique in mediating coaggregation with only group A and group E strains of Actinomyces naeslundii. All the other AgI/II polypeptides mediated coaggregation with group C and group D strains of A. naeslundii. Analysis of chimeric protein constructs revealed that coaggregation specificity was determined by sequences within the N-terminal half of AgI/II protein. A synthetic peptide (20 aa residues), which defines a putative adhesion epitope within the C-terminal region of polypeptide, inhibited AgI/II-mediated aggregation by gp340 but did not affect coaggregation with A. naeslundii. These results suggest that different mechanisms operate in interactions of AgI/II family polypeptides with native gp340, gp340 SRCR domain peptide, and A. naeslundii. Specificity of these interactions appears to be determined by discontinuous but interacting regions of the polypeptides, thus providing flexibility in receptor recognition for streptococcal colonization of the human host.