Fitness Alters the Associations of BMI and Waist Circumference with Total and Abdominal Fat

Objective: We tested the following hypotheses in black and white men and women: 1) for a given BMI or waist circumference (WC), individuals with moderate cardiorespiratory fitness (CRF) have lower amounts of total fat mass and abdominal subcutaneous and visceral fat compared with individuals with low CRF; and 2) exercise training is associated with significant reductions in total adiposity and abdominal fat independent of changes in BMI or WC. Research Methods and Procedures: The sample included 366 sedentary male (111 blacks and 255 whites) and 462 sedentary female (203 blacks and 259 whites) participants in the HERITAGE Family Study. The relationships between BMI and WC with total fat mass (determined by underwater weighing) and abdominal subcutaneous and visceral fat (determined by computed tomography) were compared in subjects with low (lower 50%) and moderate (upper 50%) CRF. The effects of a 20‐week aerobic exercise training program on changes in these adiposity variables were examined in 86% of the subjects. Results: Individuals with moderate CRF had lower levels of total fat mass and abdominal subcutaneous and visceral fat than individuals with low CRF for a given BMI or WC value. The 20‐week aerobic exercise program was associated with significant reductions in total adiposity and abdominal fat, even after controlling for reductions in BMI and WC. With few exceptions, these observations were true for both men and women and blacks and whites. Discussion: These findings suggest that a reduction in total adiposity and abdominal fat may be a means by which CRF attenuates the health risk attributable to obesity as determined by BMI and WC.     

Structure determination and biochemical studies on Bacillus stearothermophilus E53Q serine hydroxymethyltransferase and its complexes provide insights on function and enzyme memory

Serine hydroxymethyltransferase (SHMT) belongs to the alpha-family of pyridoxal 5'-phosphate-dependent enzymes and catalyzes the reversible conversion of L-Ser and tetrahydrofolate to Gly and 5,10-methylene tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of one-carbon fragment in many biological processes. SHMT also catalyzes the tetrahydrofolate-independent conversion of L-allo-Thr to Gly and acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT (bsSHMT) suggested that E53 interacts with the substrate, L-Ser and tetrahydrofolate. To elucidate the role of E53, it was mutated to Q and structural and biochemical studies were carried out with the mutant enzyme. The internal aldimine structure of E53QbsSHMT was similar to that of the wild-type enzyme, except for significant changes at Q53, Y60 and Y61. The carboxyl of Gly and side chain of L-Ser were in two conformations in the respective external aldimine structures. The mutant enzyme was completely inactive for tetrahydrofolate-dependent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of tetrahydrofolate-independent reaction with L-allo-Thr. The results obtained from these studies suggest that E53 plays an essential role in tetrahydrofolate/5-formyl tetrahydrofolate binding and in the proper positioning of Cbeta of L-Ser for direct attack by N5 of tetrahydrofolate. Most interestingly, the structure of the complex obtained by cocrystallization of E53QbsSHMT with Gly and 5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal 5'-phosphate bound to Gly and active site Lys. However, density for 5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single conformation, whereas pyridoxal 5'-phosphate had two distinct conformations. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of 5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate is absent in the final structure. Spectral studies carried out with this mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gem-diamine form. This is the first report to provide direct evidence for enzyme memory based on the crystal structure of enzyme complexes.     

BMP4 promotes formation of primitive vascular networks in human embryonic stem cell-derived embryoid bodies

The vasculature develops primarily through two processes, vasculogenesis and angiogenesis. Although much work has been published on angiogenesis, less is known of the mechanisms regulating the de novo formation of the vasculature commonly called vasculogenesis. Human embryonic stem cells (hESC) have the capability to produce all of the cells of the body and have been used as in vitro models to study the molecular signals controlling differentiation and vessel assembly. One such regulatory molecule is bone morphogenetic protein-4 (BMP4), which is required for mesoderm formation and vascular/hematopoietic specification in several species. However, hESC grown in feeder-free conditions and treated with BMP4 differentiate into a cellular phenotype highly expressing a trophoblast gene profile. Therefore, it is unclear what role, if any, BMP4 plays in regulating vascular development in hESC. Here we show in two National Institutes of Health-registered hESC lines (BG02 and WA09) cultured on a 3D substrate of Matrigel in endothelial cell growth medium-2 that the addition of BMP4 (100 ng/ml) for 3 days significantly increases the formation and outgrowth of a network of cells reminiscent of capillary-like structures formed by mature endothelial cells (P<0.05). Analysis of the expression of 45 genes by quantitative real time-polymerase chain reaction on a low-density array of the entire culture indicates a rapid and significant downregulation of pluripotent and most ectodermal markers with a general upregulation of endoderm, mesoderm, and endothelial markers. Of the genes assayed, BMPR2 and RUNX1 were differentially affected by exposure to BMP4 in both cell lines. Immunocytochemistry indicates the morphological structures formed were negative for the mature endothelial markers CD31 and CD146 as well as the neural marker SOX2, yet positive for the early vascular markers of endothelium (KDR, NESTIN) and smooth muscle cells (alpha-smooth muscle actin [alpha SMA]). Together, these data suggest BMP4 can enhance the formation and outgrowth of an immature vascular system.     

Diagnosis of iron deficiency in groundnut,Arachis hypogaea L.

Summary Investigations into iron deficiency have been hindered by the lack of a satisfactory diagnostic tissue test, which in turn results from the total iron content of plant tissue commonly being an unreliable index of the iron status. Our measurements of chlorotic and normal leaves of field grown groundnut (Arachis hypogaea L.) showed that total iron was unsatisfactory as the measure of iron status of plant tissue. It was found that iron status was better assessed from an estimate of the ferrous iron content of fresh leaf materials obtained by extraction with o-phenanthroline. Extractable iron content increased with leaf age. Chlorotic buds or the first fully opened leaf always contained less than 6μg extractable-Fe/g fresh tissue. Approved for publication as ICRISAT Journal Article No. 307.     

Mutations Altering a Structurally Conserved Loop-Helix-Loop Region of a Viral Packaging Motor Change DNA Translocation Velocity and Processivity

Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage λ gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.     

Identification of C4 responsive genes in the facultative C4 plant Hydrilla verticillata

The aquatic monocot Hydrilla verticillata (L.f.) Royle is a well-documented facultative C₄ NADP-malic enzyme species in which the C₄ and Calvin cycles operate in the same cell with the specific carboxylases confined to the cytosol and chloroplast, respectively. Several key components had already been characterized at the molecular level, thus the purpose of this study was to begin to identify other, less obvious, elements that may be necessary for a functional single-cell C₄ system. Using differential display, mRNA populations from C₃ and C₄ H. verticillata leaves were screened and expression profiles compared. From this study, 65 clones were isolated and subjected to a customized macroarray analysis; 25 clones were found to be upregulated in C₄ leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C₄ upregulated genes were identified. Among these one encoded a previously recognized C₄ phosphoenolpyruvate carboxylase, and two encoded distinct pyruvate orthophosphate dikinase isoforms, new findings for H. verticillata. Genes that encode a transporter, an aminotransferase and two chaperonins were also upregulated. Twelve false positives, mostly housekeeping genes, were determined from the Northern/semi-quantitative RT-PCR analyses. Sequence data obtained in this study are listed in the dbEST database (DV216698 to DV216767). As a single-cell C₄ system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C₄ system in a C₃ crop species.Srinath K. Rao and Hiroshi Fukayama contributed equally to this study.     

Intraabdominal hydatid cyst: a case report

BACKGROUND: Hydatid disease is caused by Echinococcus granulosus, endemic in cattle and sheep-raising regions of the world such as Central Europe, South America, Australia, New Zealand and South Africa. Although hydatid disease is more common in liver and lung, it also affects brain, kidney, spleen and muscle. We present a case of intraabdominal hydatid cyst, diagnosed by fine needle aspiration cytology, producing an indentation of the liver, which is uncommon. CASE: A male patient presented with right side abdominal pain. On ultrasonography an intraabdominal solid mass (right hypochondrial) was revealed, and subsequently FNA was done. Smears were diagnostic of hydatid cyst. CONCLUSION: FNAC is a sensitive and rapid technique in diagnosis of hydatid cysts. The present case is unusual, owing to its presentation as a solid abdominal mass seeding over the liver and mimicking malignancy radiologically.     

Characterization of a glutathione conjugate of the 1,4-benzosemiquinone-free radical formed in rat hepatocytes

Rat hepatocytes treated with 1,4-benzoquinone formed 1,4-benzosemiquinone and 2-S-glutathionyl-1,4-benzosemiquinone radicals as detected by ESR spectroscopy. The 2-S-glutathionyl-1,4-benzosemiquinone radical was first obtained from the reaction of 1,4-benzoquinone with glutathione. Glutathione both reduced benzoquinone to form benzosemiquinone and conjugated benzoquinone to form 2-S-glutathionyl-1,4-benzosemiquinone radical. The ratio of these two radicals depended upon the ratio of 1,4-benzoquinone to glutathione. At near equimolar ratios, the 2-S-glutathionyl-1,4-benzosemiquinone radical was predominantly formed. This radical was characterized by computer simulation of the experimental spectra and identified by comparison of its hyperfine coupling constants with those of chemical analogues. The 2-S-glutathionyl-1,4-benzosemiquinone radicals formed inside hepatocytes, and then crossed the plasma membrane into the media.     

Sex-specific effects of <Emphasis Type="Italic">ACE</Emphasis> I/D and <Emphasis Type="Italic">AGT</Emphasis>-M235T on pulse pressure: the HyperGEN Study

Evidence shows that an elevated pulse pressure (PP) may lead to an increased risk of cardiovascular morbidity and mortality. There is also evidence that PP is a sexually dimorphic trait, and that genetic factors influence inter-individual variation in PP. The aim of this project was to assess the genotype-by-sex interaction on PP in a sample of mostly hypertensive African American and White participants using candidate genes involved in the renin–angiotensin–aldosterone system. Subjects were participants in the HyperGEN Study, including men (43%) and women (57%) over the age of 55 years (mean age = 65). Candidate gene polymorphisms used were ACE insertion/deletion (1,789 subjects genotyped) and AGT-M235T (1,800 subjects genotyped). We employed linear regression methods to assess the genotype-by-sex interaction. For ACE, genotype-by-sex interaction on PP was detected (P = 0.04): the “D/D” genotype predicted a 2.2 mmHg higher pulse pressure among women, but a 1.2 mmHg lower PP among men, compared to those with an “I” allele, after adjusting for age, weight, height, ethnicity, and antihypertension medication use. A similar interaction was found for systolic blood pressure. The genotype-by-sex interaction was consistent across ethnicity. The interaction was evident among those on antihypertensive medications (P = 0.05), but not among those not taking such medications (P = 0.55). In our analysis of AGT, no evidence of a genotype-by-sex interaction affecting PP, SBP, or DBP was detected. This evidence for a genotype-by-sex interaction helps our understanding of the complex genetic underpinnings of blood pressure phenotypes.