Structural basis for carbohydrate binding properties of a plant chitinase-like agglutinin with conserved catalytic machinery.

A new chitinase-like agglutinin, RobpsCRA, related to family GH18 chitinases, has previously been identified in black locust (Robinia pseudoacacia) bark. The crystal structure of RobpsCRA at 1.85Å resolution reveals unusual molecular determinants responsible for the lack of its ancestral chitinase activity. Unlike other chitinase-like proteins, which lack chitinase catalytic residues, RobpsCRA has conserved its catalytic machinery. However, concerted rearrangements of loop regions coupled to non-conservative substitutions of aromatic residues central to the chitin-binding groove explain the lack of hydrolytic activity against chitin and the switch toward recognition of high-mannose type N-glycans. Identification of close homologs in flowering plants with conservation of sequence motifs associated to the structural adaptations seen in RobpsCRA defines an emerging class of agglutinins, as emphasized by a phylogenetic analysis, that are likely to share a similar carbohydrate binding specificity for high-mannose type N-glycans. This study illustrates the recent evolution and molecular adaptation of a versatile TIM-barrel scaffold within the ancestral GH18 family.

Rat liver carbohydrate alterations in streptozotocin-induced diabetic rats

Diabetes mellitus (DM) currently belongs to the most widespread human pathologies, affecting about 4% of the world adult population. Despite the pivotal role of the liver in the development of metabolic disorders, the influence of DM on hepatic glycoconjugates remains obscure. The aim of the present investigation was to use a set of lectins with different carbohydrate affinities to investigate impairment in rat liver glycoconjugates influenced by streptozotocin-induced diabetes mellitus. The lectin panel included 7 conventional lectins - Con A, SNA, RCA, WGA, PNA, SBA, and HPA, supplemented with the original fucose-specific lectin preparation from Laburnum anagyroides bark (LABA). Tissue samples were fixed in 4% neutral formalin, embedded in paraffin, and subjected to lectin-peroxidase-diaminobenzidine staining. In control rats a strong reactivity against Con A, LABA, SBA and SNA with cytoplasmic granularities of hepatocytes was detected, while RCA, WGA and HPA showed a strong reactivity with vascular endothelium, and WGA and HPA with bile capillaries. Experimental diabetes was associated with a redistribution of Con A and LABA receptor sites from centrolobular hepatocytes to hepatocytes with peripheral localization. Among the most remarkable observations was DMinduced exposure of lectin reactivity with hepatocyte and endothelial cell nuclei. The endothelial lining of sinusoidal hemocapillaries, of central veins, and portal tract vessels also displayed a significant and differential rearrangement of carbohydrate determinants when influenced by DM. Diabetes-induced activation of Kupffer cells was accompanied by the expression of SNA, PNA and SBA receptor sites within the cytoplasm of these cells, which was lectin-negative in control specimens. The results reported provide a new insight into the pathogenesis of DM-induced impairment of hepatic carbohydrates, and demonstrate the applicability of the original fucose-specific lectin preparation to experimental histopathology (AU)

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Characterization of vascular-specific RSs1 and rolC promoters for their utilization in engineering plants to develop resistance against hemipteran insect pests.

Rice sucrose synthase1, RSs1 (isolated from rice) and rolC (isolated from Agrobacterium rhizogenes) promoters were evaluated by binding analyses of their respective cis-elements with host nuclear transcription factors. The expression profile of an insecticidal protein driven by these promoters in transgenic plants was monitored. Motif-search analysis with available phloem-specific promoter sequences revealed the presence of two BoxII elements in RSs1. An octopine synthase element, a stem-specific, a root-specific and a light-responsive element were found in the rolC promoter, whereas the ASL box, GATA and 13 bp motifs were detected in both promoters. Binding analysis of these cis-elements (both in native and mutant forms) with the trans-factors present in the nuclear extracts from rice, tobacco and chickpea, followed by electrophoretic mobility shift assay, documented a highly specific cis-trans interaction. Both promoters were utilized to express Allium sativum leaf agglutinin (ASAL) gene in the three aforementioned plant systems. By immunohistochemistry and immunohistofluorescence, specific patterns of ASAL accumulation were detected in vascular tissues of single copy transgenic plants. Transgenic plants expressing ASAL in a phloem-specific manner demonstrated about 60-65% more insecticidal activity than control plants. The two promoters, which evolved independently from two distinctly unrelated origins, were found to maintain their functionality in a conserved manner. They were able to express the insecticidal protein coding ASAL as transgene both in monocot and dicot hosts. Thus, the two promoters are valuable as prospective phloem-specific promoters for use in plant biotechnological programmes.

Urtica dioica agglutinin: separation, identification, and quantitation of individual isolectins by capillary electrophoresis and capillary electrophoresis-mass spectrometry.

With benign prostatic hyperplasia (BPH) being a major health problem in ageing men, alternative therapeutic approaches (e.g., with phytopharmaceuticals) are of great interest. Based on pharmacological evidences, one of the most promising options in that respect are the lectins found in Urtica dioica (stinging nettle) roots. In this study the qualitative and quantitative analysis of individual isolectins in U. dioica extracts is described, which is the first report on using capillary electrophoresis (CE) for the analysis of lectins in plant material at all. By utilizing a 200 mM sodium acetate buffer (pH 3.75) a baseline separation and determination of four closely related isolectins was feasible within 20 min in the aqueous plant extracts. The individual compounds were identified based on reference compounds as well as data obtained from CE-mass spectrometry (MS) experiments. After modifying the optimized CE conditions to 100 mM ammonium formate buffer with pH 3.75 and a voltage of 15 kV, the isolectins were clearly assignable in positive electrospray ionization (ESI) mode. The quantitative results obtained by CE (the total lectin content varied from 0 to 0.42% in the samples) were accurate (recovery rates of spiked samples between 92.5 and 96.2%), precise (relative standard deviation < 5%) and in good agreement to those obtained by High-performance liquid chromatography (HPLC). As for peak resolution, assignable compounds and required separation time the newly developed CE method was clearly advantageous over the determination achieved by LC.

[Survey of Pinellia ternata and its adulterants].

The tubers of Pinellia ternata (Thunb.) Breit. distributed widely in China, have been used in traditional medicine. Recently, P. ternata is deficient in herb market and its adulterants are used. This article mainly summarizes some researches on P. ternata and its adulterants from four aspects, including pharmacognostics, chemical component, pharmacology and toxicity. Also some suggestions are presented about the study prospect of P. ternata.

Characteristics of polyarthritis in rabbits by hyperimmunization with attenuated Enterococcus faecalis.

To produce polyarthritis and rheumatoid factor like substance (RFLS), rabbits were hyperimmunized intravenously with 0.02% thimelosal (TMS)-treated Enterococcus faecalis (E. faecalis) as a persistent bacterial flora. Swelling of knee joints occurred at a rate of 41% (27/66), and of shoulder joints at a rate of 25% (17/66) while that of elbow joints occurred at a rate of 4.5% (3/66). On culturing of knee joint fluids, no colonies appeared while 2/4 fluid specimens from the shoulder joints gave positive colonies for 78 days after the first immunization; thereafter, no colonies appeared. On histological examination, in early stages, acute inflammatory reactions with degenerative changes of synovial tissue was observed. In later stages, chronic inflammatory changes, proliferation of synovial cells with pannus formation, destruction of articular cartilage and subchondral bone were observed. RFLS titer showed bi-phasic peaks at 11 days and 41 days after the first immunization. A high incidence of polyarthritis, particularly knee joints, occurred. Thus, hyperimmunization with attenuated E. faecalis as a normal intestinal flora may provide an animal model of chronic polyarthritis.

[Transformation of glycyrrhizic acid. Synthesis of glycopeptides of glycyrrhizic acid monomethyl ether, having anti-inflammatory and immunostimulating action]. / Transformatsii glitsirrizinovoi kisloty. Sintez glikopeptidov monometilovogo éfira glitsirrizinovoi kisloty, proiavlliaiushchikh protivosvospatitel'noe i immunostimuliruiushchee deistvie.

Three triterpene glycopeptides, glycyrrhizic acid monomethly ester derivatives containing peptide fragment of N-acetylmuramoyldipeptide and its analogues, are synthesized. They possess high prednizolone-like antiinflammatory activity and display pronounced stimulative effect on humoral factors of immunity.

Lectin affinity high-performance liquid chromatography: interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin A, Datura stramonium agglutinin, and Vicia villosa agglutinin.

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.

Protection of the nursing pig against experimentally induced enteric colibacillosis by vaccination of dam with fimbrial antigens of E coli (K88, K99 and 987P).

Pregnant gilts were vaccinated with two doses of alhydrogel adsorbed fimbrial antigens of Escherichia coli (K88ab, K88ac, K99 and 987P) supplemented with beta toxoid of Clostridium perfringens type C. Their piglets, and piglets of nonvaccinated gilts, were subsequently orogastrically challenged with one or other of the four fimbrial types of enteropathogenic E coli. Some of the vaccinated animals were reinjected with a single dose of the vaccine during second gestation and their piglets, and piglets of non-vaccinated sows, were challenged the same way as were litters of gilts. Blood serum and colostra were examined for antibodies to the four fimbrial antigens of E coli and for antitoxin to beta toxin of C perfringens type C. It was found that: (1) a highly significant reduction in mortality and morbidity was achieved in vaccinated litters against all four challenge strains of E coli; (2) excretion of K88ab and K88ac but not of K99 and 987P challenge strains was significantly reduced; (3) revaccination of sows by a single dose of the vaccine during second gestation conferred complete protection against mortality and highly significant protection against morbidity; (4) no correlation was noted between colostral or seroagglutinins to fimbrial antigens of E coli and mortality rates in litters challenged with homologous fimbrial types of E coli, but good correlation was found between colostral precipitins to K88 antigens and mortality rates in litters; (5) antitoxin value in 97 per cent of colostrum of vaccinated sows was 10 iu equivalent of C perfringens type C toxin or more per ml of colostrum.

[Effect of levamisole on white mice and on the immunological reactivity of guinea pigs]. / Vliianie na levamizola vurkhu beli mishki i imunologichnata reaktivnost na morski svincheta.

Investigations were carried out on the protective action of therapeutic doses of levamisole (Pharmachim) on mice with a LD100 Salmonella gallinarum infection. Studied was also the effect of levamisole on the immunologic response of guinea pigs infected with Salmonella cholerae suis. It was found that therapeutic amounts of 3 mg/kg levamisole applied to mice following respective patterns protected them against a lethal dose of S. gallinarum. The animals showed enhanced resistance to the infection--20 to 50 per cent of them survived as against 100 per cent mortality with the controls. The guinea pigs showed upon treatment with levamisole certain changes with some of the factors of unspecific response--enhanced phagocytic and lysozyme activity as judged by the development of infection with a S. cholerae suis culture. At the same time the agglutinin titer that substantiated the immunologic response was negligibly changed as compared to the controls. On the base of the results obtained an attempt was made to elucidate the mechanism of the protective action of levamisole.

Evidence for amelioration of steroid-mediated immunosuppression by ascorbic acid.

Four experiments were conducted to determine if supplemental ascorbic acid (AA) would ameliorate the immunosuppression induced by exogenous cortisol (COL). Supplemental AA was provided in the diets at levels of 0 and 1000 ppm. Administration of COL significantly lowered plasma AA at both 0 and 10 days postimmunization, and AA supplementation elevated plasma AA at both times. Cortisol-treated chicks exhibited significantly reduced agglutinins to sheep red blood cells but not to Brucella abortus. Supplemental AA significantly ameliorated the immunosuppression associated with exogenous COL and may serve as an antiimmunosuppressive agent in the chicken.

Escherichia coli-associated porcine neonatal diarrhea: antibacterial activities of colostrum from genetically susceptible and resistant sows.

Antibacterial properties of colostrum from genetically resistant and susceptible sows in a herd in which only the susceptible sows had acquired natural immunity to K88-positive Escherichia coli have been investigated. Significant differences in antiadhesive and opsonic activities occurred. Colostrum from susceptible sows inhibited the binding of 125I-labeled K88 antigen to brush borders significantly better than did the colostrum from resistant dams. Colostrum from susceptible dams effected more efficient in vitro opsonic phagocytosis and killing of K88 E. coli than did colostrum from resistant dams. Differences in bactericidal properties of colostrum between the two groups of pigs were not significant. Fractionation of colostrum from susceptible dams by gel filtration and ion-exchange chromatography revealed that the fractions rich in immunoglobulin M had the highest opsonic activity, whereas those containing predominantly immunoglobulins G and A were of lower activity.

Differences in inhibition of the growth of commensal and enteropathogenic strains of Escherichia coli by lactotransferrin and secretory immunoglobulin A isolated from human milk.

Immunoglobulins from bovine and human colostrum and milk and lactotransferrin (LTF) from human milk were investigated for bacteriostatic activity against Escherichia coli growing in a tissue culture medium. When tested separately, LTF or secretory immunoglobulin A (sIgA) from pooled human milk showed only slight bacteriostatic activity against human commensal or enteropathogenic strains of E. coli. Together, they had a considerable bacteriostatic effect, but only against strains of enteropathogenic serotype. This activity of the sIgA from pooled human milk was consistent for all enteropathogenic serotypes tested, but sIgA isolated from individual milk samples was inactive against some serotypes, and this specificity was associated with antibody to the O antigens. The activity of the sIgA was stable to heat at 56 degrees for 2 h but was lost progressively on heating at 65 degrees for 10 min or longer. Bovine colostral IgGl was without bacteriostatic effect alone. Together with LTF, it was active against a strain pathogenic to calves but not against human enteropathogenic strains. Tests on rabbit antisera raised against commensal enteropathogenic strains of E. coli showed that for the enteropathogens the bacteriostatic activity (in association with LTF) was high and was specific for the serotype of the eliciting strain, but bacteriostatic activity was low or absent in the antisera to commensal strains in spite of the presence of high titres of agglutinating antibodies to these strains.

Antibacterial antibodies in swine colostrum.

Antibacterial antibody activities were examined in 138 samples of swine colostrum by O-agglutinin test with serotypes of Escherichia coli (O-6, 0-45, 0-81, 0-83, 0-115, 0-138, O-139, and O-126), Salmonella typhimurium, and Salmonella cholerae suis. The mean agglutinin titer of E coli O-83 was the highest; E coli O-115 was next highest. The mean agglutinin titer of S cholerae suis was lowest; S typhimurium was next to lowest. Correlation coefficients among these O-agglutinin titers were between 0.109 and 0.693. Correlations of S typhimurium were the poorest; those of E coli O83, E coli O-138, and S cholerae suis were also poor. By principal component analysis, it was possible to classify ten kinds of O-agglutinins into four groups. The cumulative proportion of the first four principal components accounted for 77.97% of the total variance. Cumulative coefficients of factor loadings were from 65.25% to 99.50% of these O-agglutinins.

Spectrum of Escherichia coli agglutinins in dam's sera, colostrum and calf's sera in Fresian cows and buffaloes in Egypt.

Sera collected from 18 pregnant buffaloes and Fresian cows during the last two weeks before parturition and from the newborn calves during the first two weeks after birth. All the samples together with the colostral whey of the first three days were examined for the presence of E. coli agglutinins using the tube agglutination and passive hemagglutination tests. The latter test was more sensitive and demonstrated higher titres in all cases, and consequently more O groups. Agglutinins to various E. coli O groups were detected in all samples. It is worthy to note that agglutinins to all E. coli O groups used as antigens were detected in 13 dam's sera, 17 colostrum and in one buffaloe calf. The titres were higher in colostrum than in dam's or calf's sera. The highest titre was 1:640. The O groups 101, 115, 117, and 26 were the most common among Fresian cows whereby the O groups 101 and 115 showed the highest titres. In buffaloes the O groups 101, 115, 8 and 26 were the most common with the O8 having the highest titre.

Role of environment in the development of "natural" hemagglutinins in Minnesota miniature swine.

"Natural" hemagglutinin titers against a panel of fixed erythrocyte antigens were determined for groups of Minnesota miniature swine reared conventionally, in a specific pathogen-free facility, and in germfree isolators. Sera were assayed for hemagglutination (HA) titers by the microtiter method against 12 species of erythrocytes stabilized by treatment with pyruvic aldehyde and formaldehyde. These erythrocytes were stable for up to 2 years and gave slightly enhanced HA titers as compared to fresh, unfixed erythrocytes. Of the sera from conventional swine tested, the highest "natural" HA titers were directed towards rabbit, cat, swine dog, and burro erythrocytes (greater than 1:1,000), intermediate titers were detected against human A, B, and O, and sheep, pig, and chicken erythrocytes (1:64 to 1:1,000), whereas the lowest titers were found against ox and goat erythrocytes (less than 1:8). Titers obtained with sera from specific pathogen-free swine were 2- to 16-fold lower than those of conventional swine, but were of a similar distribution with regard to the species of erythrocyte tested. Germfree swine sera uniformly exhibited HA titers less than 1:4 against all species of erythrocytes. The majority of these hemagglutinins were immunoglobulin M class but there were some agglutinins of immunoglobulin A class and a slight amount of immunoglobulin G class. Specificity of these agglutinins was examined by absorption tests. The results are consistent with the hypothesis that natural hemagglutinins develop due to dietary or microbial antigenic stimulation, or both.

[Reactogenicity and immunological effectiveness study of a complex paratyphoid B antigen]. / Izuchenie reaktogennosti i immunologicheskoi éffektivnosti kompleksnogo paratifoznogo B antigena.

Reactogenic property and immunological efficacy of the paratyphoid preparation containing a complex of O-, K- and H-antigens obtained by single-stage antigens extraction were studied in a limited group of volunteers (22 persons). The antigen gave no untoward reactions and proved to be safe when given orally in doses of 25 to 150 mg. Paratyphoid B antigen was characterized by a marked immunization activity and stimulated formation of specific paratyphoid O-, K- and H-agglutinins and antibodies of the IgA,- IgG,- and IgM-classes.

Effective immunity to dental caries: dose-dependent studies of secretory immunity by oral administration of Streptococcus mutans to rats.

Rats (COBS/CD) provided Formalin-killed Streptococcus mutans 6715, C211 in their drinking water (10(8) to 10(9) equivalent colony-forming units [CFU] per ml) had high levels of specific antibodies in saliva, colostrum, and milk. Rats provided a lower concentration of S. mutans antigen (10(7) CFU per ml) in water had agglutinin titers in secretions that were similar to those in controls. Gnotobiotic rats provided S. mutans antigen in food (10(7) to 10(8) equivalent CFU per g of diet) manifested a secretory immune response as evidenced by the presence of specific immunoglobulin A antibodies in saliva, colostrum, and milk. Gnotobiotic rats provided a higher concentration of antigen (10(9) CFU per g) in food had levels of specific antibodies in their secretions that were similar to those in controls. No significant antibody activity to S. mutans was observed in sera of any group of animals. Furthermore, the presence of specific salivary immunoglobulin A antibodies in gnotobiotic rats correlated with a reduction in the level of plaque, numbers of viable S. mutans in plaque, and levels of S. mutans-induced dental caries. This paper discusses the importance of antigen dosage for induction of a secretory immune response that is protective against S. mutans-induced dental caries.