Preparation of polysaccharide-enzyme conjugates for competitive binding assays

A variety of bacterial O-polysaccharides were covalently linked to enzymes and it was demonstrated with three discrete monoclonal antibodies that enzyme-glycoconjugates function as convenient labelled antigens in direct enzyme immunoassays, particularly competitive assays that quantify bacterial O-antigens. Two strategies, each involving reductive amination, were used to couple O-polysaccharides to enzymes, while retaining high enzymic activity. Reduction of the Schiff base formed between, 1,3-diaminopropane and the terminal reducing ketodeoxyoctanoic acid (KDO) residue present in the majority of the lipopolysaccharide (LPS) core domains, following mild acid removal of Lipid A, offered the most direct route to mono-aminated polysaccharide. Alternatively, mild periodate oxidation of KDO and heptose residues generated multiple aldehyde targets for Schiff base formation, without affecting the O-antigenic determinant. Hetero- and homobifunctional coupling reagents, sulphosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and discuccinimidyl suberate, activated polysaccharide for coupling to enzymes at amino and sulphydryl sites and produced conjugates that retained at least 95% of the original enzymic activity. The most suitable enzyme conjugates, especially for competitive inhibition EIA were those bearing one polysaccharide chain, and these were easily prepared from horse-radish peroxidase. Although the extent of conjugation of activated polysaccharide to -galactosidase and alkaline phosphatase could be controlled by reaction stoichiometry, the use of these enzymes was a less effective utilization of valuable antigen and enzyme.Issued as NRCC No. 31634     

Cytogenetic Definition and Morphogenetic Analysis of Delta, a Gene Affecting Neurogenesis in Drosophila Melanogaster

We have conducted a genetic analysis of a small interval of the third chromosome known to include Delta (Dl), a locus that affects the segregation of the ectoderm into neural and epidermal lineages during embryogenesis and the morphogenesis of some ectodermally derived structures, in Drosophila melanogaster. This analysis has led to the definition of seven independent complementation groups, one of which is Delta, within the interval extending from 91F6-13 to 92A2. Among the extant mutations in these seven loci, only mutations in Dl lead to the so-called neurogenic phenotype: hypertrophy of the nervous system and reduction of the epidermis. Combined cytogenetic and genetic analyses allow us to define absolute proximal (91F5-92A1) and distal (92A2) cytogenetic limits for the Dl locus. We have isolated hypomorphic and amorphic alleles of Dl and find that, for any given allele, there is an inverse correlation between neural hypertrophy and epidermal reduction in embryos and a direct correlation between the severity of embryonic phenotypes in mutant homozygotes and hemizygotes and the imaginal phenotype in heterozygous adults.     

A (1→3,1→4)-β-glucan-specific monoclonal antibody and its use in the quantitation and immunocytochemical location of (1→3,1→4)-β-glucans

Monoclonal antibodies were raised against a (1→3,1→4)-β-glucan-bovine serum albumin (BSA) conjugate. One antibody (BG1) selected for further characterization, was specific for (1→3,1→4)-β-glucan, displaying no binding activity against a (1→3)-β-glucan-BSA conjugate and minimal binding against a cellopentaose-BSA conjugate. A range of oligosaccharides was prepared by enzymatic digestion of (1→3,1→4)-β-glucan, purified by size exclusion chromatography and characterized by ¹H-NMR and anion exchange chromatography. These (1→3,1→4)-β-oligoglucosides, together with (1→3)-β- and (1→4)-β-oligoglucosides were used to characterize the binding site of the monoclonal antibody (BG1) by competitive inhibition. The monoclonal antibody showed maximal binding to a heptasaccharide with the structure Glc(1→3) Glc(1→4) Glc(1→4) Glc(1→3) Glc(1→4) Glc(1→4) Glc and was determined to have an affinity constant of 3.8 × 10⁴ M⁻¹ for this oligoglucoside. The monoclonal antibody (BG1) has been used to develop a sensitive sandwich ELISA for the specific quantitation of (1→3,1→4)-β-glucans. The assay operates in the range 1–10 ng ml⁻¹ and shows no significant cross-reaction with tamarind xyloglucan, wheat endosperm arabinoxylan or carboxymethyl-pachyman ((1→3)-β-glucan). When used with a second-stage, rabbit anti-mouse gold conjugate and viewed under the electron microscope, the monoclonal antibody probe was found to bind strongly to the walls of the aleurone in thin sections of immature wheat (Triticum aestivum) cv. Millewa grains but not to the middle lamella region. A previously described specific anti-(1→3)-β-glucan antibody (Meikle et al., 1991) bound to discrete patches on the aleurone walls, believed to be plasmodesmata.     

Effects of dietary sucrose concentration on aphid honeydew carbohydrate levels and rates of excretion

Honeydew droplets freshly excreted by two aphid species, Myzus persicae (Sulz.) and Acyrthosiphon pisum (Harr.), feeding on nutritionally adequate, artificial diets containing 10–40% w/v sucrose, were collected in oil. The volume, frequency of excretion, and carbohydrate content of the droplets were determined by measuring the diameter of the droplets in the oil, by recording the number of droplets excreted per aphid per hour, and by using a quantitative colorimetric method for estimating carbohydrates. Rates for the intake or excretion of fluid, the intake and absorption of sucrose, and the excretion of carbohydrate were consequently calculated for the two species on each dietary sucrose concentration. The percentage of dietary sucrose absorbed by the aphids ranged from 36% on diet with 40% sucrose to 97% on diet with 10% sucrose. Such honeydew/diet relationships may allow one to use honeydew carbohydrate concentrations to estimate the sucrose concentrations of the phloem sap ingested by aphids feeding on host plants.

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Résumé Des gouttelettes de miellat fraîchement excrétées par deux espèces de pucerons, Myzus persicae (Sulz.) et Acyrthosiphon pisum (Harr.), pucerons nourris sur des régimes nutritifs synthétiques contenant 10–40% de saccharose, ont été récupérées dans de l'huile. Le volume des gouttelettes a été déterminé en mesurant leur diamètre individuel dans l'huile, la fréquence d'excrétion établie par le nombre de gouttelettes excrétées par puceron par heure, et la teneur en glucides par une méthode d'analyse colorimétrique quantitative. Les taux d'ingestion et d'excrétion de liquide, d'ingestion et d'absorption de saccharose ainsi que les taux d'excrétion de glucides ont été par la suite calculés pour les deux espèces d'aphides nourris sur les différentes concentrations de saccharose. Le pourcentage de saccharose alimentaire absorbé par les aphides a été de 36% sur le régime contenant 40% de ce glucide, mais s'est élevé à 97% sur le régime ne contenant que 10% de saccharose. D'après ces relations miellat/régime nutritif, et en connaissant la teneur du miellat en glucides, on peut faire une approximation de la concentration du saccharose dans la sève élaborée du phloème ingérée par les aphides.

     

Comparative trial of volunteer and professional treatments of dysphasia after stroke.

A study of the treatment of dysphasia after stroke compared the progress of two groups of disabled patients. One received conventional treatment from qualified speech therapists and the other from non-professional volunteers. Methods of assessing communication difficulties were also compared and the impact of aphasic illness on families examined. No important differences in the results of treatment were seen between the two groups. The volunteers, however, often had to assume some of the responsibilities of social workers, and transport to hospital created practical and economic problems. It is concluded that the two forms of treatment provide essentially the same benefit, although doubt must still remain because relatively few patients were studied.     

Luminometric measurement of population activity of genetically modified Pseudomonas fluorescens in the soil

Genetically modified cells of Pseudomonas fluorescens, chromosomally marked with genes for bioluminescence, were inoculated into sterile soil microcosms. During incubation for 90 days, viable cell concentration did not change significantly but light output, measured by luminometry, decreased, indicating reduced metabolic activity due to lack of substrates. Amendment with nutrients resulted in parallel increases in both luminescence and dehydrogenase activity. Luminometry therefore enables rapid monitoring of the activity of populations of luminescence-marked microbial inocula in the soil, with greater sensitivity and selectivity than traditional techniques.     

A factor synthesized by rabbit periosteal fibroblasts stimulates bone resorption and collagenase production by connective tissue cells in vitro

Unstimulated monolayer cultures of confluent rabbit periosteal fibroblasts synthesize a factor that stimulates bone resorption in vitro. Furthermore it stimulates rabbit chondrocytes and mouse osteoblasts to synthesize collagenase. The factor has no effect on dead bone in culture, and its activity on live bone is mediated principally by osteoclasts, since it is 75% inhibited by salmon calcitonin. Characterization of the factor by gel filtration and isoelectric focusing indicates an M_r in the range 15 000–25 000 and a pI corresponding to approx. pH 4.7. These biological and physicochemical properties are similar to those reported for a factor released by peripheral blood monocytes. However, whereas human monocyte factor in both the crude and partially-purified state exhibits interleukin-1 activity, crude and fractionated periosteal fibroblast-conditioned medium does not. This is the first report of a conditioned medium containing a molecule like the monocyte-factor which appears to have no interleukin 1 activity. The factor may be synthesized by a wide range of cell types, and could have an important role in mediating connective tissue degradation during both physiological and pathological resorption.     

Colony aging affects the reproductive performance of Swiss Webster females used as recipients for embryo transfer

The objective was to evaluate the influence of colony aging in a Swiss Webster (SW) outbred stock used as recipients for embryo transfer. In the first study, a retrospective analysis was performed throughout several generations during a 38-month period in 2,398 embryos transferred to 108 SW recipients. A decrease in the percentage of live pups from transferred embryos was found at the end of the period. Impairment occurred due to the incidence of maternal cannibalism that increased from 0% to 67-100% (P<0.05), while pregnancy rate (pregnant/transferred recipients) and number of pups per delivered female were not affected throughout the period (P=NS). A following study was carried out to compare the reproductive performance of SW stock vs. B6D2F1 hybrid females in a 5-year interval. The study was conducted on a total of 893 embryos transferred to 40 females (20 SW and 20 B6D2F1) in Year #1, and 514 embryos transferred to 30 females (15 SW and 15 B6D2F1) in Year #5. No cases of maternal cannibalism were found on Year #1 in any of the strains (0/10 and 0/10). However, an incidence of 44,4% (4/9) was seen on Year #5 for SW, while for B6D2F1 the incidence was 0% (0/12) (P<0.05). Further examination of the uterus showed endometrial cysts and abnormal implantation sites in SW on Year #5 but not in B6D2F1 females. In conclusion, this study reports an impairment of the reproductive performance of an early aged SW outbred stock colony mainly due to the occurrence of maternal cannibalism. This finding has important implications for embryo transfer programs conducted in mouse facilities.     

Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1^+/+ and Abca1^−/− mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.     

Immunohistochemical determination of estrogen receptor-α in canine vaginal biopsies throughout proestrus,estrus, and early diestrus

The aim of this study was to describe the presence of estrogen receptor-α (ERα) in several vaginal histological compartments in healthy adult bitches throughout three estrous cycle stages (proestrus, estrus, and early diestrus) and to relate ERα presence with serum progesterone and estradiol-17β concentrations. For this purpose, serial blood samples and vaginal biopsies were taken from five bitches every 48 hours, starting at the clinical onset of proestrus, marked by the beginning of serosanguineous vaginal secretion. Serum progesterone and estradiol-17β concentrations were determined by RIA, whereas detection of steroid receptors was carried out through immunohistochemistry. Subjective image analysis was conducted by two independent observers in the following histological compartments: superficial, intermediate, and deep epithelia and superficial (loose) and deep (dense) stroma (connective tissue). Nuclear ERα immunoreactivity was detected in every histological compartment and estrous cycle stage studied. ERα expression varied among histological compartments and during stages of the cycle. Receptor expression was associated with estradiol-17β and progesterone serum profiles. Most relevant cyclic changes were detected in the superficial and deep epithelia and in the dense connective tissue. The highest ERα expression was detected during diestrus, although each compartment had a different pattern throughout the other cycle stages. Thus, vaginal ERα expression in the bitch varied throughout proestrus, estrus, and early diestrus according to the histological compartment involved.