Examination of the kinetics of herpes simplex virus glycoprotein D binding to the herpesvirus entry mediator, using surface plasmon resonance

Previously, we showed that truncated soluble forms of herpes simplex virus (HSV) glycoprotein D (gDt) bound directly to a truncated soluble form of the herpesvirus entry mediator (HveAt, formerly HVEMt), a cellular receptor for HSV. The purpose of the present study was to determine the affinity of gDt for HveAt by surface plasmon resonance and to compare and contrast the kinetics of an expanded panel of gDt variants in binding to HveAt in an effort to better understand the mechanism of receptor binding and virus entry. Both HveAt and gDt are dimers in solution and interact with a 2:1 stoichiometry. With HveAt, gD1(306t) (from the KOS strain of HSV-1) had a dissociation constant (KD) of 3.2 x 10(-6) M and gD2(306t) had a KD of 1.5 x 10(-6) M. The interaction between gDt and HveAt fits a 1:1 Langmuir binding model, i.e., two dimers of HveAt may act as one binding unit to interact with one dimer of gDt as the second binding unit. A gD variant lacking all signals for N-linked oligosaccharides had an affinity for HveAt similar to that of gD1(306t). A variant lacking the bond from cysteine 1 to cysteine 5 had an affinity for HveAt that did not differ from that of the wild type. However, variants with double cysteine mutations that eliminated either of the other two disulfide bonds showed decreased affinity for HveAt. This result suggests that two of the three disulfide bonds of gD are important for receptor binding. Four nonfunctional gDt variants, each representing one functional domain of gD, were also studied. Mutations in functional regions I and II drastically decreased the affinity of gDt for HveAt. Surprisingly, a variant with an insertion in functional region III had a wild-type level of affinity for HveAt, suggesting that this domain may function in virus entry at a step other than receptor binding. A variant with a deletion in functional region IV [gD1(Delta290-299t)] exhibited a 100-fold enhancement in affinity for HveAt (KD = 3.3 x 10(-8) M) due mainly to a 40-fold increase in its kinetic on rate. This agrees with the results of other studies showing the enhanced ability of gD1(Delta290-299t) to block infection. Interestingly, all the variants with decreased affinities for HveAt exhibited decreased kinetic on rates but only minor changes in their kinetic off rates. The results suggest that once the complex between gDt and HveAt forms, its stability is unaffected by a variety of changes in gD.     

Assessing the condition of hydrogenerator stator winding insulationusing the ramped high direct-voltage test method

Ramped high direct-voltage tests have proven to be an effective and nondestructive means for reducing in-service stator winding failures. The measured current versus applied voltage response obtained during these off-line tests can be used to detect cracks and fissures, moisture absorption, tape separations, conductive surface contamination, lack of epoxy cure, and internal delamination. Ramped voltage test results have been used to determine when corrective actions are needed and what the appropriate actions are. When done properly, the test can identify stator windings that are approaching failure without accelerating the deterioration process     

Disulfide bond structure determination and biochemical analysis of glycoprotein C from herpes simplex virus

A biochemical analysis of glycoprotein C (gC of herpes simplex virus was undertaken to further characterize the structure of the glycoprotein and to determine its disulfide bond arrangement. We used three recombinant forms of gC, gC1(457t), gC1(delta33-123t), and gC2(426t), each truncated prior to the transmembrane region. The proteins were expressed and secreted by using a baculovirus expression system and have been shown to bind to monoclonal antibodies which recognize discontinuous epitopes and to complement component C3b in a dose-dependent manner. We confirmed the N-terminal residues of each mature protein by Edman degradation and confirmed the internal deletion in gC1(delta33-123t). The molecular weight and extent of glycosylation of gC1 (457t), gC1(delta33-123t), and gC2(426t) were determined by treating each protein with endoglycosidases and then subjecting it to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. The data indicate that eight to nine of the predicted N-linked oligosaccharide sites on gC1(457t) are occupied by glycans of approximately 1,000 Da. In addition, O-linked oligosaccharides are present on gC1(457t), primarily localized to the N-terminal region (amino acids [aa] 33 to 123) of the protein. gC2(426t) contains N-linked oligosaccharides, but no O-linked oligosaccharides were detected. To determine the disulfide bond arrangement of the eight cysteines of gC1(457t),the protein was cleaved with cyanogen bromide. SDS-PAGE analysis followed by Edman degradation identified three cysteine-containing fragments which are not connected by disulfide linkages. Chemical modification of cysteines combined with matrix-assisted laser desorption ionization mass spectrometry identified disulfide bonds between cysteine 1 (aa 127) and cysteine 2 (aa 144) and between cysteine 3 (aa 286) and cysteine 4 (aa 347). Further proteolysis of the cyanogen bromide-generated fragment containing cysteine 5 through cysteine 8, combined with mass spectrometry and Edman degradation, showed that disulfide bonds link cysteine 5 (aa 386) to cysteine 8 (aa 442) and cysteine 6 (aa 390) to cysteine 7 (aa 419). A similar disulfide bond arrangement is postulated to exist in gC homologs from other herpesviruses.     

Humidity-Regulating Trays: Moisture Absorption Kinetics and Applications for Fresh Produce Packaging

For packaged fresh produce, inappropriate high relative humidity (RH) levels and condensation of water vapour cause premature spoilage. Humidity-regulating trays were developed to solve this issue. They were made from a thermoformed multilayer structure: polyethylene (outside)/foamed hygroscopic ionomer (active layer) with 0 or 12 wt% NaCl/hygroscopic ionomer (sealing layer, inside). Moisture absorption kinetics of the humidity-regulating trays with 0 and 12 wt% NaCl (T-0 and T-12, respectively) was investigated under different RH conditions (76, 86, 96 and 100 %) at 13 °C for 16 days. Additional trays containing 7 g of distilled water were closed with a high barrier lidding film, and the headspace RH was continuously monitored as a function of time. As control, a polypropylene (control-PP) tray was used. Strawberries and tomatoes were used to test capability of the trays to regulate in-package RH. The amount of water absorbed by the T-0 and T-12 trays was 7.6 and 13.2 g, respectively. Active hygroscopic ionomer layer was effective in water vapour absorption, and the integration of NaCl into this active layer increased the water vapour absorption capacity of the tray. The Weibull model adequately described the moisture sorption kinetics of the individual packaging trays as a function of time. The headspace RH of trays covered with a lidding film was found to be 89.8, 99.6 and 100 % in the T-12, T-0 and control-PP trays, respectively. The T-12 trays containing fresh produce best regulated the in-package RH below 97 % and maintained overall quality, but at the expense of slightly higher product weight loss (2–3 wt% for strawberry, 1 wt% for tomatoes) compared to the control-PP trays (0.3–0.6 wt%).     

Functional region IV of glycoprotein D from herpes simplex virus modulates glycoprotein binding to the herpesvirus entry mediator

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry and has four functional regions (I to IV) important for this process. We previously showed that a truncated form of a functional region IV variant, gD1(Delta290-299t), had an enhanced ability to block virus entry and to bind to the herpesvirus entry mediator (HveAt; formerly HVEMt), a cellular receptor for HSV. To explore this phenotype further, we examined other forms of gD, especially ones with mutations in region IV. Variant proteins with deletions of amino acids between 277 and 300 (region IV), as well as truncated forms lacking C-terminal residues up to amino acid 275 of gD, were able to block HSV entry into Vero cells 1 to 2 logs better than wild-type gD1(306t). In contrast, gD truncated at residue 234 did not block virus entry into Vero cells. Using optical biosensor technology, we recently showed that gD1(Delta290-299t) had a 100-fold-higher affinity for HveAt than gD1(306t) (3.3 x 10(-8) M versus 3.2 x 10(-6) M). Here we found that the affinities of other region IV variants for HveAt were similar to that of gD1(Delta290-299t). Thus, the affinity data follow the same hierarchy as the blocking data. In each case, the higher affinity was due primarily to a faster kon rather than to a slower koff. Therefore, once the gDt-HveAt complex formed, its stability was unaffected by mutations in or near region IV. gD truncated at residue 234 bound to HveAt with a lower affinity (2.0 x 10(-5) M) than did gD1(306t) due to a more rapid koff. These data suggest that residues between 234 and 275 are important for maintaining stability of the gDt-HveAt complex and that functional region IV is important for modulating the binding of gD to HveA. The binding properties of any gD1(234t)-receptor complex could account for the inability of this form of gDt to block HSV infection.     

An incremental economic dispatch method for cascaded hydroelectricpower plants

The Bureau of Reclamation has undertaken a program to improve water resource management at its hydroelectric power plants. One feature of the resource optimization program involves the development of economic dispatch methods which will enable plant operators to meet power and water demands while increasing overall operating efficiency. The economic dispatch method described has been designed and simulated using a computer model of the Lower Colorado River Basin's hydraulic and electric power systems. Simulation results demonstrate that the economic dispatch method yields significant efficiency improvements with sufficient speed for practical application     

Respiration and Storage Quality of Fresh-Cut Apple Slices Immersed in Sugar Syrup and Orange Juice

Storing fresh-cut apple slices in suitable fruit juice or sugar syrup is a general practice. However, application of this approach is mainly based on empirical knowledge, while systematic and comprehensive analyses of the relevant effects of this storage technique on keeping quality-related physiological properties of fresh-cut products is still missing. Hence, the aim of this study was to evaluate the impacts of complete immersion of fresh-cut apples in sugar syrup and fruit juice solution on respiratory behaviour and other relevant quality attributes (colour, tissue strength, and soluble solid and acidity). Sugar syrup and pure orange juice showed a high potential to store and protect fresh-cut apples. Results showed that only pure orange juice positively affected the produce quality by preventing browning effects. In addition, sugar syrup of 13.4–20% most effectively prevented browning of apple slices and guaranteed high product quality retention during storage. The application of different liquid media provides a practical means to prevent browning and maintain product quality.