Characterization of affinity-purified juvenile hormone esterase from the plasma of the tobacco hornworm, Manduca sexta

Juvenile hormone (JH) esterase found primarily in the hemolymph and tissues of insects is a low abundance protein involved in the ester hydrolysis of insect juvenile hormones, JHs. The enzyme was purified from the larval plasma of wild-type Manduca sexta using an affinity column prepared by binding the ligand, 3-[(4'-mercapto)butylthio]-1,1,1-trifluoropropan-2-one (MBTFP), to epoxy-activated Sepharose. The purification was greater than 700-fold with a 72% recovery, and the purified enzyme appeared as a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoelectrophoresis, reverse phase high performance liquid chromatography, and amino acid sequence analysis. The molecular weight was 66,000. The plasma JH esterase in wild-type, black, and white strains of M. sexta was similar when analyzed by immunotitration, wide range (pH 3.5-9.0) isoelectric focusing, and inhibition with MBTFP and 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP). Inhibition studies revealed a sensitive and insensitive form (I50 = 10(-9) and 10(-6) M, respectively) in these three biotypes. Narrow range isoelectric focusing (pH 4.0-7.0) indicated the presence of two major isoelectric forms with pI values of 6.0 and 5.5, but their inhibition kinetics with OTFP and O,O-diisopropyl phosphorofluoridate were identical.     

A comparison study of the interaction between β-lactoglobulin and retinol at two different conditions: spectroscopic and molecular modeling approaches

The interaction between bovin β-Lactoglobulin (β-LG) and retinol at two different pH values was investigated by multispectroscopic, zeta potential, molecular modeling, and conductometry measurements. The steady state and polarization fluorescence spectroscopy revealed that complex formation at two different pH values could occur through a remarkable static quenching. According to fluorescence quenching, one set of binding site at pH 2 and two sets of binding sites at pH 7 were introduced for binding of retinol to β-LG that show the enhancement of saturation score of β-LG to retinol in dimmer condition. The polarization fluorescence analysis represented that there is more affinity between β-LG and retinol at pH 7 rather than at pH 2. The effect of retinol on β-LG was studied by UV-visible, circular dichroism (CD), and synchronous fluorescence, which indicated that retinol induced more structural changes on β-LG at pH 7. β-LG–retinol complex formation at two different pH values was recorded via applying resonance light scattering (RLS) and zeta potential. Conductometry and RLS showed two different behaviors of interaction between β-LG and retinol at two different pH values; therefore, dimmer formation played important roles in different behaviors of interaction between β-LG and retinol. The zeta potential was the implied combination of electrostatic and hydrophobic forces which are involved in β-LG–retinol complex at two different pH values, and the hydrophobic interactions play a dominant role in complex formation. Molecular modeling was approved by all experimental results. The acquired results suggested that monomer and dimmer states of β-LG can be induced by retinol with different behaviors.     

Assessment of platelet function in patients with stroke using multiple electrode platelet aggregometry: a prospective observational study

The Qatari law, as in many other countries, uses brain death as the main criteria for organ donation and cessation of medical support. By contrast, most of the public in Qatar do not agree with the limitation or withdrawal of medical care until the time of cardiac death. The current study aims to examine the duration of somatic survival after brain death, organ donation rate in brain-dead patients as well as review the underlying etiologies and level of support provided in the state of Qatar. This is a retrospective study of all patients diagnosed with brain death over a 10-year period conducted at the largest tertiary center in Qatar (Hamad General Hospital). Among the 53 patients who were diagnosed with brain death during the study period, the median and mean somatic survivals of brain-dead patients in the current study were 3 and 4.5 days respectively. The most common etiology was intracranial hemorrhage (45.3%) followed by ischemic stroke (17%). Ischemic stroke patients had a median survival of 11 days. Organ donation was accepted by only two families (6.6%) of the 30 brain dead patients deemed suitable for organ donation. The average somatic survival of brain-dead patients is less than one week irrespective of supportive measures provided. Organ donation rate was extremely low among brain-dead patients in Qatar. Improved public education may lead to significant improvement in resource utilization as well as organ transplant donors and should be a major target area of future health care policies.     

Synthesis and immunological evaluation of self-adjuvanting glycolipopeptide vaccine candidates

Synthesis of four glycolipids with different number of lauroyl groups on glucose or cellobiose as scaffolds is described. Their immunological evaluations either admixed with or covalently linked to J8, a peptide antigen derived from the C-terminus of the antiphagocytic M-protein of group A streptococcus, are also investigated. Administration of mixtures of J8 and glycolipids to B10BR (H-2(k)) mice induced low-levels of J8-specific IgG antibodies. While glycolipopeptides, in which J8 was covalently linked to the synthetic glycolipids, demonstrated high-levels of antibody titers comparable with the co-administration of these glycolipopeptides with complete Freund's adjuvant, suggesting clearly the strong potency of the synthesized glycolipids as self-adjuvanting moieties.     

Acetylcholinesterase mediated susceptibility of soldiers and workers of formosan subterranean termite (Isoptera: Rhinotermitidae) to chlorpyrifos

Target site studies were undertaken to examine the difference in susceptibility of Formosan subterranean termite, Coptotermes formosanus Shiraki, workers and soldiers to chlorpyrifos. Workers exhibited significantly greater acetylcholinesterase activity per insect than soldiers (118.63 +/- 48.51 versus 47.98 +/- 22.59 mOD/min/insect equivalent). Likewise, enzyme activity (mean +/- SD) per milligram of protein was greater in workers than soldiers (440.30 +/- 267.43 versus 311.53 +/- 149.83 mOD/min/mg protein). The enzyme of soldiers was more sensitive to the acetylcholinesterase (AChE) inhibitors eserine and chlorpyrifos-oxon than that of workers. The I50s of chlorpyrifos-oxon were 2.66 and 4.59 nM for soldiers and workers, respectively, whereas the I50s of eserine were 16.56 and 25.41 nM for soldiers and workers, respectively. The amount of protein was significantly higher in workers than in soldiers with mean values of 0.270 +/- 0.102 and 0.154 +/- 0.054 mg/insect equivalent, respectively. We suggest that the differential response of workers and soldiers to chlorpyrifos may be due to the difference in AChE sensitivity to inhibition and the amount of protein between them.     

Seoul Virus-Infected Rat Lung Endothelial Cells and Alveolar Macrophages Differ in Their Ability To Support Virus Replication and Induce Regulatory T Cell Phenotypes

Hantaviruses cause a persistent infection in reservoir hosts that is attributed to the upregulation of regulatory responses and downregulation of proinflammatory responses. To determine whether rat alveolar macrophages (AMs) and lung microvascular endothelial cells (LMVECs) support Seoul virus (SEOV) replication and contribute to the induction of an environment that polarizes CD4⁺ T cell differentiation toward a regulatory T (Treg) cell phenotype, cultured primary rat AMs and LMVECs were mock infected or infected with SEOV and analyzed for viral replication, cytokine and chemokine responses, and expression of cell surface markers that are related to T cell activation. Allogeneic CD4⁺ T cells were cocultured with SEOV-infected or mock-infected AMs or LMVECs and analyzed for helper T cell (i.e., Treg, Th17, Th1, and Th2) marker expression and Treg cell frequency. SEOV RNA and infectious particles in culture media were detected in both cell types, but at higher levels in LMVECs than in AMs postinfection. Expression of Ifnβ, Ccl5, and Cxcl10 and surface major histocompatibility complex class II (MHC-II) and MHC-I was not altered by SEOV infection in either cell type. SEOV infection significantly increased Tgfβ mRNA in AMs and the amount of programmed cell death 1 ligand 1 (PD-L1) in LMVECs. SEOV-infected LMVECs, but not AMs, induced a significant increase in Foxp3 expression and Treg cell frequency in allogeneic CD4⁺ T cells, which was virus replication and cell contact dependent. These data suggest that in addition to supporting viral replication, AMs and LMVECs play distinct roles in hantavirus persistence by creating a regulatory environment through increased Tgfβ, PD-L1, and Treg cell activity.     

The computer as a laboratory for the physical chemistry of membranes

A mini-review is given of some recent advances in the use of computer-simulation approaches to the study of physico-chemical properties of lipid bilayers and biological membranes. The simulations are based on microscopic molecular interaction models as well as random-surface models of fluid membranes. Particular emphasis is put on those properties that are controlled by the many-particle character of the lamellar membrane, i.e. correlations and fluctuations in density, composition and large-scale conformational structure. It is discussed how dynamic membrane heterogeneity arises and how it is affected by various molecular species interacting with membranes, such as cholesterol, drugs, insecticides, as well as polypeptides and integral membrane proteins. The influence of bending rigidity and osmotic-pressure gradients on large-scale membrane conformation and topology is described.     

Genome sequencing and mapping reveal loss of heterozygosity as a mechanism for rapid adaptation in the vegetable pathogen Phytophthora capsici

The oomycete vegetable pathogen Phytophthora capsici has shown remarkable adaptation to fungicides and new hosts. Like other members of this destructive genus, P. capsici has an explosive epidemiology, rapidly producing massive numbers of asexual spores on infected hosts. In addition, P. capsici can remain dormant for years as sexually recombined oospores, making it difficult to produce crops at infested sites, and allowing outcrossing populations to maintain significant genetic variation. Genome sequencing, development of a high-density genetic map, and integrative genomic or genetic characterization of P. capsici field isolates and intercross progeny revealed significant mitotic loss of heterozygosity (LOH) in diverse isolates. LOH was detected in clonally propagated field isolates and sexual progeny, cumulatively affecting >30% of the genome. LOH altered genotypes for more than 11,000 single-nucleotide variant sites and showed a strong association with changes in mating type and pathogenicity. Overall, it appears that LOH may provide a rapid mechanism for fixing alleles and may be an important component of adaptability for P. capsici.