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.     

Influence of chronic alcohol abuse on body weight and energy metabolism: is excess ethanol consumption a risk factor for obesity or malnutrition?

OBJECTIVES: To evaluate the influence of chronic alcohol abuse on body composition and energy metabolism in patients affected by chronic alcoholism (group A) compared with a group of healthy social drinkers (group B). SETTING: A university hospital clinic in Italy. SUBJECTS: A total of 32 alcoholics without clinical or laboratory signs of liver cirrhosis and malabsorption. MEASUREMENTS: Body composition was assessed by anthropometric measurements. Resting energy expenditure (REE) and substrate oxidation rate was measured by indirect calorimetry. Daily caloric intake was computed on the basis of a food diary compiled over 7 days. RESULTS: Alcoholics showed a significantly lower body weight (P < 0.05) and a significant lower fat mass (P < 0.05) compared with controls. A higher waist-to-hip ratio was found in group A than in group B, both as a whole group (P < 0.01) or separated by gender (females, P < 0.01) and males, P < 0.001), indicating a prevalence of fat distribution in the abdominal region in alcoholics. REE was significantly higher in group A than in group B (P < 0.05). The non-protein respiratory quotient was significantly lower in group A than in group B (P < 0.001) with a consequent higher utilization of lipids (P < 0.01) and a lower carbohydrate oxidation (P < 0.05) in group A. The energy intake provided only by food ingestion was found to be significantly higher in group B (P < 0.01), whilst the total caloric intake, computed as food intake plus alcohol intake, was higher in group A (P < 0.01). CONCLUSIONS: Alcoholics, as compared with social drinkers, showed a lower body weight due essentially to a fat mass reduction, a higher REE value normalized by fat-free mass, and a preferential utilization of lipids as energy substrate. These findings might suggest that chronic ethanol abuse is able to determine an impairment of nutritional status due, at least in part, to an alteration of the substrate oxidation.     

Capillary electrophoretic study of the complex formation between DNA and cationic surfactants

Due to the growing interest in the use of cationic surfactants for the construction of liposomal genetic delivery systems, the study of complex formation between DNA and quaternary ammonium detergents is of fundamental importance. In this context, we undertook the study of this complex formation using capillary zone electrophoresis (CZE) with suppressed electroosmotic flow, a technique that allowed us to both monitor the change in mobility of DNA as a function of added surfactant in a precise and reproducible manner and evaluate the potential of CZE to reflect the change in hydrodynamic friction upon binding. Nevertheless, CZE must be applied with caution for binding studies where strong cooperativity occurs, because of the presence of peak splitting at concentrations close to the half-point of binding. Also, a comparison between this experiment and Manning's polyelectrolyte transport properties theory on one hand and Tirado and Garcia de la Torre expression for hydrodynamic friction of rod-like molecules on the other hand is given.     

Differential diagnosis of the transplant rejection and herpesvirus-related infections in kidney transplant recipients

An algorythm of differential diagnosis conduction of the transplant rejection reaction and the diseases caused by herpes viruses in recipients after the kidney transplantation was elaborated, basing on the analysis of results of the clinical, laboratory, immunological and serological examination methods.     

Positional regulators of bone tissue--a basis for the autoregulatory mechanism of the development and reproduction of osteoporosis

A hypothesis on autoregulatory mechanism of reproduction of bone matrix structural changes was proposed resulting from the theoretical study based on author's own data and data documented in literature. This mechanism seems to underly osteoporosis development. All etiological factors of osteoporosis reported were united into a common pathogenetic mechanism on the basis of this conception.     

Possible mechanism of hypothalamic regulation of endocrine pancreatic function in mice of the diabetic strain C57bl/KsJ

In mice hetero- and homozygotic in relation to the diabetic gene db, it was shown that the regulation of the pancreatic endocrine function by hypothalamus is mediated at least by two mechanisms: neurohumoral one connected with hormone secretion by paraventricular and supraoptical nuclei and by a neuroconductivity mechanism connected with the ventromedial nucleus and lateral hypothalamic area.