Relationship of body composition to somatotype.

The purpose of this study is to determine the relationship in college-aged women between somatotype using both Sheldon's ('69) and Heath and Carter's ('67) procedures, and body composition, as measured by whole-body 40K counting and body density. Sheldon's endomorphy is closely associated with height and weight; Heath and Carter's first component is significantly related to weight and body fatness. Lean body mass (LBM) as a weight or as a percent is not closely related to Sheldon's mesomorphy or Heath and Carter's second component. However, when LBM and height are used as independent variables to estimate somatotype, both variables are significantly related to Heath and Carter's second component, accounting for 61% of the variance. Thus Heath and Carter's second component is significantly associated with LBM for a given body height. Most of the variation in Sheldon's ectomorphy and Heath and Carter's third component can be accounted for by weight and height. Sheldon's somatotype for all three components is not as closely related to body composition as Heath-Carter's. Body composition, as measured by either 40K counting or body density, is found to be important in accounting for variation in Heath and Carter's first and second components.     

Na+ effects on transition of DNA and polynucleotides of variable linear charge density.

A range of linear charge densities of the ordered and disordered forms of DNA or polynucleotides can be obtained experimentally by acid or alkaline titration, or by the investigation of unusual complexes involving protonated bases or three-stranded helices. The variation of melting temperatures with Na⁺ concentration for various of these systems is known and in some cases is complemented by structural and thermodynamic information. We have extended the condensation–screening theory of Manning [Biopolymers, 11 , 937–955 (1972)] to these systems. The stabilizing and destabilizing effects of Na⁺ (condensation and screening, respectively) and be independently varied, and the theory is successful in predicting the qualitative (in some cases, quanittative) behaviour that is observed. Comparison of theory and experiment indicates that the axial phosphate distance b for single-stranded polynucleotides increases with increasing pH. Values of the critical parameter ξ are obtained for the various polynucleotide structures. These values are essential for an understanding of ionic effects on charged ligand–polynucleotide interactions.     

Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold “tetramer”. Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance.     

The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site.     

Pre-storage centrifugation conditions have significant impact on measured microRNA levels in biobanked EDTA plasma samples

BackgroundIn the past few years, an increasing number of studies have reported the potential use of microRNAs (miRNA) as circulating biomarkers for diagnosis or prognosis of a wide variety of diseases. There is, however, a lack of reproducibility between studies. Due to the high miRNA content in platelets this may partly be explained by residual platelets in the plasma samples used. When collecting fresh plasma samples, it is possible to produce cell-free/platelet-poor plasma by centrifugation. In this study, we systematically investigated whether biobanked EDTA plasma samples could be processed to be suitable for miRNA analysis.Materials and methodsBlood samples were collected from ten healthy volunteers and centrifuged to produce platelet-poor-plasma (PPP) and standard biobank plasma. After one week at ?80 °C the biobanked EDTA plasma was re-centrifuged by different steps to remove residual platelets. Using RT-qPCR the levels of 14 miRNAs in the different plasma preparations were compared to that of PPP.ResultsWe were able to remove residual platelets from biobanked EDTA plasma by re-centrifugation of the thawed samples. Nevertheless, for most of the investigated miRNAs, the miRNA level was significantly higher in the re-centrifuged biobanked plasma compared to PPP, even when the platelet count was reduced to 0–1×10⁹/L.ConclusionWe found, that pre-storage centrifugation conditions have a significant impact on the measured EDTA plasma level of miRNAs known to be present in platelets. Even for the miRNAs found to be less effected, we showed that a 1.5–3 fold change in plasma levels may possible be caused by or easily overseen due to sample preparation and/or storage.     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.