Neutralizing monoclonal antibodies that distinguish three antigenic sites on human cytomegalovirus glycoprotein H have conformationally distinct binding sites.

Seven neutralizing murine monoclonal antibodies specific for the glycoprotein H of human cytomegalovirus were produced and used to construct a topological map of two nonoverlapping antigenic sites that are bridged by a third antigenic site. Neutralization assays with 15 laboratory or clinical human cytomegalovirus strains indicated that the monoclonal antibodies recognize three antigenically variable and three conserved epitopes within the three antigenic sites. The variable-domain genes encoding monoclonal antibodies representing each of the three antigenic sites were cloned and sequenced, and molecular models of their binding sites were generated. Conformational differences in the antibody-binding sites suggested a structural basis for experimentally observed differences in gH epitope recognition.     

Characterization of the mucilage sheaths ofLemonniera aquatica by lectin-gold labelling

A pre-embedding lectin-gold labelling method was used to characterize the carbohydrate components in the mucilage ofLemonniera aquatica. A specific tissue processing protocol was developed, namely: a) primary fixation in 2% paraformaldehyde and 0.2% glutaraldehyde in PIPES buffer (pH 7.2) for 30 min; b) secondary fixation in 2% glutaraldehyde in the same buffer system for 1 h; c) post-fixation in 1% aqueous OsO₄ for 1h; d) embedment in Möllenhaur's resin. The three gold conjugated lectins used were: concanavalin A, wheat germ agglutinin andLimax flavus agglutinin, allowing detection of their complementary saccharides, namely α-d-mannose/α-d-glucose,N-acetyl-d-glucosamine (GluNAc), andN-acetylneuraminic acid (NANA), respectively.N-Acetyl-d-glucosamine and NANA residues were the major components of germ tube mucilage with only a small amount of α-d-manose/α-d-glucose. However, NANA was restricted to the mucilage in the region of germ tube emergence from the conidial arm. The abundance of GluNAc and NANA residues on hyphae and appressoria was less than that on the germ tube. Conversely, α-d-mannose/α-d-glucose was more abundant in the appressorial mucilage. Variability of mucilage composition was found to exist between different structures of the germinated conidium and also between different regions of the same structure. Further, the conidial cell wall ofL. aquatica is not chitinous, and lacks NANA and α-d-mannose/α-d-gluocse.     

Ultrastructure of asci and ascospores of the mangrove ascomyceteDactylospora haliotrepha

Ultrastructure of the marine LoculoascomyceteDactylospora heliotrepha is presented and compared withMarinosphaera mangrovei, Swampomyces armeniacus and other marine species. Ascospores are bi-celled and ridged. The ridges are outgrowths of the outer mesosporial layer and formed later in ascosporogenesis. The exosporial layer fragments to release mucilaginous material present between the spore wall ridges. Asci and pseudoparaphyses are held together by a fibrillar mucilaginous network. The endoascus is thicker than the ectoascus. Comparisons are made of the diameter of ascomata, size of asci and ascospores ofD. heliotrepha collected from mangroves in Hong Kong, Malaysia, the Philippines and Taiwan.     

Contents of Cd,Fe, Mn and Zn in the tissue ofKatelysia hiantina collected from Tolo Harbour,an almost land-locked sea

Summary Samples ofK.hiantina and sediments were collected from iron ore tailings and other nearby sites within Tolo Harbour for heavy metal analysis. It was found that sediments as well as the clams at the tailings contained higher concentrations of Cd, Fe, Mn and Zn. Positive correlations between the metals in the sediments and clam tissues were found. The clams collected from the tailings showed a bustantial loss of Fe, Mn and Zn when reared in clean filtered seawater for a period of 8 days.     

Changes in metabolism and hepatic ultrastructure induced by estradiol and testosterone in immature female Epinephelus akaara (Teleostei,Serranidae)

Summary Estradiol injections increase serum level of calcium, amino acid, glucose, protein, ammonia and creatinine in immature Epinephelus akaara, and also increase levels of total lipid, cholesterol, phospholipid and esterified fatty acids. Hepatic protein, glycogen and lipid concentrations also rise after estradiol treatment, and some hepatic enzymes participating in the metabolism of nitrogen, lipid and carbohydrate, show increased activity. Serum vitellogenin levels are increased. Testosterone treatment increases serum protein, total lipid, cholesterol, amino acid and ammonia levels, and also hepatic glycogen content, but in contrast to estradiol treatment, testosterone does not change serum vitellogenin, glucose, calcium, phospholipid, esterified fatty acid and creatinine levels, nor the hepatic lipid and protein content. A small number of hepatic enzymes shows an increased activity. Vitellogenic fish show biochemical changes similar to that of estradiol-treated fish, but are different from those of immature fish. Estradiol treatment induces ultrastructural changes in the hepatocytes of immature fish that are similar to those found in vitellogenic fish. These include a proliferation of rough endoplasmic reticulum and Golgi apparatus, and an increase in glycogen and lipid, all indicative of enhanced metabolic activity.     

Receptor activity of rotavirus nonstructural glycoprotein NS28.

Rotavirus morphogenesis involves the budding of subviral particles through the rough endoplasmic reticulum (RER) membrane of infected cells. During this process, particles acquire the outer capsid proteins and a transient envelope. Previous immunocytochemical and biochemical studies have suggested that a rotavirus nonstructural glycoprotein, NS28, encoded by genome segment 10, is a transmembrane RER protein and that about 10,000 Mr of its carboxy terminus is exposed on the cytoplasmic side of the RER. We have used in vitro binding experiments to examine whether NS28 serves as a receptor that binds subviral particles and mediates the budding process. Specific binding was observed between purified simian rotavirus SA11 single-shelled particles and RER membranes from SA11-infected monkey kidney cells and from SA11 gene 10 baculovirus recombinant-infected insect cells. Membranes from insect cells synthesizing VP1, VP4, NS53, VP6, VP7, or NS26 did not possess binding activity. Comparison of the binding of single-shelled particles to microsomes from infected monkey kidney cells and from insect cells indicated that a membrane-associated component(s) from SA11-infected monkey kidney cells interfered with binding. Direct evidence showing the interaction of NS28 and its nonglycosylated 20,000-Mr precursor expressed in rabbit reticulocyte lysates and single-shelled particles was obtained by cosedimentation of preformed receptor-ligand complexes through sucrose gradients. The domain on NS28 responsible for binding also was characterized. Reduced binding of single-shelled particles to membranes was seen with membranes treated with (i) a monoclonal antibody previously shown to interact with the C terminus of NS28, (ii) proteases known to cleave the C terminus of NS28, and (iii) the Enzymobead reagent. VP6 on single-shelled particles was suggested to interact with NS28 because (i) a monoclonal antibody to the subgroup I epitope on VP6 reduced particle binding, (ii) a purified polyclonal antiserum raised against recombinant baculovirus-produced VP6 reduced ligand binding, and (iii) a monoclonal antibody to a conserved epitope on VP6 augmented ligand binding. These experimental data provide support for the hypothesized receptor role of NS28 before the budding stage of rotavirus morphogenesis.     

The effect of 3-aminobenzamide on X-ray induction of chromosome aberrations in Down syndrome lymphocytes

Human lymphocytes from normal and Down syndrome (DS) subjects were examined to determine the effect of 3-aminobenzamide (3AB) on X-ray-induced chromosome aberrations. Lymphocytes were treated with 150 or 300 rad of X-rays in the presence of 3 mM 3AB for various times after irradiation, and then the cells were analyzed for the presence of chromosome aberrations in mitotic cells. 3-Aminobenzamide had no effect on the frequency of chromosome aberrations produced by X-rays in G0 lymphocytes from normal subjects. In contrast, lymphocytes from DS patients displayed an increase in the frequency of chromosome aberrations as a result of treatment with X-rays in the presence of 3AB. These observations indicate that DS lymphocytes are more sensitive to the inhibition of poly(ADP)ribose synthetase than normal lymphocytes.     

Ca2+-mediated activation of human erythrocyte membrane Ca2+-ATPase

Ca2+-ATPase of human erythrocyte membranes, after being washed to remove Ca2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca2+. Stimulation of the ATPase by a membrane-associated Ca2+-dependent proteinase was thus suggested. Much less 138 kDa Ca2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca2+ than without Ca2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca2+-mediated stimulation of membrane Ca2+-ATPase. While Km(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca2+ concentration for half-maximal activation of the enzyme was observed.     

Cylinder wall thickness difference discrimination by an echolocating Atlantic bottlenose dolphin

Summary The capability of an Atlantic bottlenose dolphin Tursiops truncatus to discriminate wall thickness differences of hollow cylinders by echolocation was studied. A standard cylinder of 6.35 mm wall thickness was compared with cylinders having wall thicknesses that differed from the standard by ± 0.2, ± 0.3, ± 0.4, and ± 0.8 mm. All cylinders had an O.D. of 37.85 mm, and a length of 12.7 cm. The dolphin was required to station in a hoop while the standard and comparison targets, separated by an angle of ± 11° from a center line, were simultaneously presented at a range of 8 m. The dolphin was required to echolocate and indicate the side of the standard target. Target location on each trial was randomized. Interpolation of the dolphin performance data indicated a wall thickness discrimination threshold (at the 75% correct response level) of –0.23 mm and +0.27 mm. Backscatter measurements suggest that if the dolphin used time domain echo cues, it may be able to detect time differences between two echo highlights to within approximately ± 500 ns. If frequency domain cues were used, the dolphin may be able to detect frequency shifts as small as 3 kHz in a broadband echo having a center frequency of approximately 110 kHz. Finally, if the dolphin used time-separation pitch (TSP) cues, it may be able to detect TSP differences of approximately 450 Hz.Discrimination tests with the thinner comparison targets were also conducted in the presence of broadband masking noise. For an echo energy-to-noise ratio of 19 dB the dolphin's performance was comparable to its noise-free performance. At an energy-to-noise ratio of 14 dB the dolphin was unable to achieve the 75% correct threshold with any of the comparison targets.Abbreviations c sound velocity - DI _R receive directivity index - difference between highlight intervals of two targets - th wall thickness difference between standard and comparison targets; - E energy flux density - E _e echo energy flux density - E _e /N _L echo energy to noise ratio - E(f) frequency spectrum of artificial echo - e(t) artificial echo - N _J ambient noise spectral density - N _L received noise spectral density - O.D. outer diameter - p instantaneous acoustic pressure - R target range - SE source energy flux density in dB - s(t) dolphin sonar signal - time between first and second echo highlights - TS _E target strength based on energy - TSP time-separation pitch