Computational biomechanics of human brain with and without the inclusion of the body under different blast orientation

Three different human head models in a free space are exposed to blast waves coming from four different directions. The four head–neck–body models composed of model a, with the neck free in space; model b, with neck fixed at the bottom; and model c, with the neck attached to the body. The results show that the effect of the body can be ignored for the first milliseconds of the head–blast wave interactions. Also one can see that although most biomechanical responses of the brain have similar patterns in all models, the shear stresses are heavily increased after a few milliseconds in model b in which the head motion is obstructed by the fixed-neck boundary conditions. The free-floating head model results are closer to the attached-body model.     

Enhancement of Glucose transport in clone 9 cells by exposure to alkaline pH: Studies on potential mechanisms

Summary Incubation of a nontransformed rat liver cell line. Clone 9, at pH 8.5 resulted in an 16-fold stimulation of cytochalasin B-inhibitable 3-O-methylglucose (3-OMG) transport, an effect that was independent of the presence of serum. Exposure to 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated 3-OMG uptake, and the enhancement was not additive to that produced by incubation at pH 8.5. In cells depleted of protein kinase C activity by a 20-hr exposure to TPA, however, the stimulation of 3-OMG transport in response to incubation at alkaline pH was still fully demonstrable. In control and alkaline pH-exposed cells, the inhibition of 3-OMG uptake by cytochalasin B was consistent with a single-site ligand binding model (K ₁10^–7 m). Northern blot analysis demonstrated the presence of only the human erythrocyte/rat brain/HepG2 cell glucose transporter-mRNA isoform (EGT), and the abundance of this mRNA was unchanged following exposure to alkaline pH. Immunoblot analysis, using polyclonal antibodies directed against the carboxy-terminal dodecapeptide of EGT, demonstrated and 2.0-fold increase in the abundance of transporters in partially purified plasma membrane fractions following incubation at pH 8.5, while EGT abundance was unchanged in whole-cell extracts. It is concluded that the stimulation of glucose transport in response to incubation of Clone 9 cells at alkaline pH does not require the presence of serum or activation of protein kinase C, and that the response is at least in part mediated by an increase in the number of glucose transporters in the plasma membrane.     

Metabolic programming: fetal origins of obesity and metabolic syndrome in the adult

Exposure of the fetus to the intrauterine milieu can have profound effects on the health of the offspring in adulthood. Results of a series of studies demonstrate the powerful influence of the mother's metabolic state on whether the emerging adult develops obesity and hyperinsulinemia. Importantly, these attributes can be passed on to the next generation nongenetically and can be reversed and prevented.     

Control of Na+-K+-ATPase beta 1-subunit expression: role of 3'-untranslated region

Using in vitro translation and cell transfection assays, we previously demonstrated that the Na⁺-K⁺-ATPase ₁ mRNA species containing its longest 3'-untranslated region (UTR) exhibited the lowest translational efficiency. Here, employing deletions and in vivo expression assays, using direct injection of plasmids into rat ventricular myocardium, we identified a 143-nt segment located in the distal 3'-UTR of ₁ mRNA that was associated with decreased luciferase expression; interestingly, this segment contains three AUUUA motifs. Using RNA-protein binding assays and UV cross-linking of cRNA with cytosolic proteins of rat heart, we identified an 38-kDa protein that specifically bound to the cRNA encoding the 143-nt segment of ₁ mRNA 3'-UTR. Mutation of three nucleotides located in the middle region of the 143-nt segment, which was predicted to greatly disrupt a putative stem-loop structure of the cRNA in this region, was associated with reduced binding of the mutated cRNA to the protein migrating at 38 kDa. The cRNA encoding a segment of cyclooxygenase-2 mRNA 3'-UTR containing six AUUUA sequences did not bind the protein migrating at 38 kDa and did not compete with the binding of the wild-type 143-nt ₁ cRNA to the protein. The above results suggest that the 143-nt segment in the distal segment of the 3'-UTR of ₁ mRNA may play an important role in the control of ₁-subunit expression. RNA-protein binding; AUUUA sequence; plasmid expression in heart; direct myocardial injection; cardiac expression     

Thyroid hormone regulation of cardiac bioenergetics: role of intracellular creatine

The effect of thyroid hormone (T(3)) on the content of myocardial creatine (Cr), Cr phosphate (CrP), and high-energy adenine nucleotides and on cardiac function was examined. In the hearts of control and T(3)-treated rats perfused in vitro, while "low" and "high" contractile work was performed, T(3) treatment resulted in a approximately 50% reduction in CrP, Cr, total Cr content (Cr + CrP), and in the CrP-to-Cr ratio. In addition, there was a slight fall in myocardial content of ATP and a large rise in calculated free ADP (fADP), resulting in a significant decrease in the ATP-to-fADP ratio in the hearts of hyperthyroid compared with euthyroid rats. Moreover, there was a substantial decrease in the level of ATP in hearts of T(3)-treated rats under high work conditions. Importantly, the ratio of cardiac work to oxygen consumption was not altered by thyroid status. Treatment with T(3) also resulted in an almost threefold reduction in the content of Na(+)/Cr transporter mRNA in the ventricular myocardium and skeletal muscle but not in the brain. We conclude with the following: 1) changes in the expression of the Na(+)/Cr transporter mRNA correlate with Cr + CrP in the myocardium; 2) hearts of hyperthyroid rats contain lower levels of ATP and higher levels of fADP under both low and high work conditions but no reduction in efficiency of work output; and 3) the reduction in Cr and ATP in hearts of hyperthyroid rats may be the basis for the reduced maximal work capacity of the hyperthyroid heart.     

Induction of GLUT1 mRNA in response to azide and inhibition of protein synthesis

The biochemical behaviour of four commercial strains of Saccharomyces cerevisiae was studied in the presence of calcium ions, acrylamide and bisacrylamide. Calcium ions at a concentration of 300 µM induced an increase of NAD(P)⁺ reduction in commercial Turkish and American strains, while in Chilean and Brazilian commercial strains, it diminished NAD(P)⁺ reduction. On the other hand, polyacrylamide monomers (acrylamide and bisacrylamide) induced a decrease of NAD(P)⁺ reduction in all strains studied in this paper. When membrane potential () and oxygen consumption were measured in the presence of polyacrylamide monomers, a decrease of both was observed in all strains studied.     

Aromatic Anchor at an Invariant Hormone-Receptor Interface: FUNCTION OF INSULIN RESIDUE B24 WITH APPLICATION TO PROTEIN DESIGN*

Crystallographic studies of insulin bound to fragments of the insulin receptor have recently defined the topography of the primary hormone-receptor interface. Here, we have investigated the role of Phe^B24, an invariant aromatic anchor at this interface and site of a human mutation causing diabetes mellitus. An extensive set of B24 substitutions has been constructed and tested for effects on receptor binding. Although aromaticity has long been considered a key requirement at this position, Met^B24 was found to confer essentially native affinity and bioactivity. Molecular modeling suggests that this linear side chain can serve as an alternative hydrophobic anchor at the hormone-receptor interface. These findings motivated further substitution of Phe^B24 by cyclohexanylalanine (Cha), which contains a nonplanar aliphatic ring. Contrary to expectations, [Cha^B24]insulin likewise exhibited high activity. Furthermore, its resistance to fibrillation and the rapid rate of hexamer disassembly, properties of potential therapeutic advantage, were enhanced. The crystal structure of the Cha^B24 analog, determined as an R₆ zinc-stabilized hexamer at a resolution of 1.5 Å, closely resembles that of wild-type insulin. The nonplanar aliphatic ring exhibits two chair conformations with partial occupancies, each recapitulating the role of Phe^B24 at the dimer interface. Together, these studies have defined structural requirements of an anchor residue within the B24-binding pocket of the insulin receptor; similar molecular principles are likely to pertain to insulin-related growth factors. Our results highlight in particular the utility of nonaromatic side chains as probes of the B24 pocket and suggest that the nonstandard Cha side chain may have therapeutic utility.     

Detection of Low-Level Mixed-Population Drug Resistance in Mycobacterium tuberculosis Using High Fidelity Amplicon Sequencing

Undetected and untreated, low-levels of drug resistant (DR) subpopulations in clinical Mycobacterium tuberculosis (Mtb) infections may lead to development of DR-tuberculosis, potentially resulting in treatment failure. Current phenotypic DR susceptibility testing has a theoretical potential for 1% sensitivity, is not quantitative, and requires several weeks to complete. The use of “single molecule-overlapping reads” (SMOR) analysis with next generation DNA sequencing for determination of ultra-rare target alleles in complex mixtures provides increased sensitivity over standard DNA sequencing. Ligation free amplicon sequencing with SMOR analysis enables the detection of resistant allele subpopulations at ≥0.1% of the total Mtb population in near real-time analysis. We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay’s performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples. SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations. This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.     

Molecular Dynamics Simulation Study of the Interaction of Piscidin 1 with DPPC Bilayers: Structure-Activity Relationship

Abstract

To study structure-activity relationship of antimicrobial peptides and to design novel antimicrobial peptides with selectivity for bacterial cells, we have performed molecular dynamics simulations of the interaction of Piscidin (Pis1) and its two analogues (Pis1-AA and Pis1-PG) with dipalmitoylphosphatidylcholine (DPPC) bilayer through 45 ns. Our results inform us of the detailed location and orientation of the peptide with respect to the bilayer as well as provide about hydrogen-bond-formation patterns and electrostatics interactions. Simulations show that Pis1 and Pis-AA form the most hydrogen bonds and Pis-PG forms the fewest hydrogen bonds with lipid. Thus, Pis1 and Pis-AA should have stronger interactions with the lipid head group when compared to Pis-PG. Experimental studies have shown that Pis1 and Pis1-AA have a high antimicrobial and hemolytic activities, and Pis1-PG has low hemolytic activity while keeps potent antimicrobial activity. Our results complement the previous experimental studies. According to our MD results and previous experimental studies, Pis1 and Pis1-AA are more effective at the zwitterionic bilayer comparing Pis1-PG. These properties of Pis1-PG could be accordance with its low hemolytic activities.