Streptococcus Colonial Growth of Dental Plaque Inhibition Using Flavonoid Extract of Ants Nest (Myrmecodia pendans): An in Vitro Study

Objective: To know the activity of resistance of flavonoid content in ant nest plant in decreasing the number of colonies S. mutans oral cavity of children as a medic herbal material. Material and Methods: The subjects were plaque sample of 20 children aged 7-12 years. Research begins by making toothpaste from ant nest extract. Samples of children's dental plaque were inserted into BHIB media, after which incubated for 24 hours, 1/10 dilution with BHIB media three times, followed by TYC media planting and incubation of anaerob with temperature 370C for 48 hours. After that then count the number of colonies of S. mutans. Results: On ethyl acetate extract of ant nest incubated at room temperature with concentration 20%, 40%, 60%, 80%, 100% obtained a decrease from each treatment amount of Streptococcus mutans colony on TYC media with median value of each treatment was 89, 67, 64, 61, 59 and 51 for the ethyl acetate fraction, and 62, 61, 60, 59, 49 at the ethanol fraction. There was no significant difference between the six concentration groups (p>0.05). Conclusion: Flavonoids extract of ant nest plants have growth barrier on Streptococcus mutans bacteria, the greater the concentration given the greater the number of S. mutans colony.

Comparisons of IgA response in saliva and colostrum against oral streptococci species

Abstract The present study compared IgA specificity against oral streptococci in colostrum and saliva samples. Sixty-two mother-and-child pairs were included; samples of colostrum (C) and saliva (MS) were collected from the mothers and saliva samples were collected from babies (BS). The specificity of IgA against Streptococcus mutans and S. mitis were analyzed by western blot. Only 30% of babies’ samples presented IgA reactivity to S. mutans, while 74 and 80% of MS and C, respectively, presented this response. IgA reactivity to S. mutans virulence antigens (Ag I/II, Gtf and GbpB) in positive samples showed differences between samples for Gtf and especially for GbpB (p < 0.05), but responses to Ag I/II were similar (p > 0.05). The positive response of Gtf-reactive IgA was different between C (90%) and MS (58%) samples (p < 0.05), but did not differ from BS (p > 0.05). GbpB was the least detected, with 48 and 26% of C and MS, and only 5% of BS samples presenting reactivity (p > 0.05). Eight percent of MS and C samples presented identical bands to SM in the same time-point. In conclusion, the differences of IgA response found between C and MS can be due to the different ways of stimulation, proliferation and transportation of IgA in those secretions. The colostrum has high levels of IgA against S. mutans virulence antigens, which could affect the installation and accumulation process of S. mutans, mainly by supplying anti-GbpB IgA to the neonate.

A new alkalophilic isolate of Bacillus as a producer of cyclodextrin glycosyltransferase using cassava flour

Abstract Cyclodextrin glycosyltransferase (CGTase) catalyzes the conversion of starch into non-reducing cyclic sugars, cyclodextrins, which have several industrial applications. This study aimed to establish optimal culture conditions for β-CGTase production by Bacillus sp. SM-02, isolated from soil of cassava industries waste water lake. The optimization was performed by Central Composite Design (CCD) 2, using cassava flour and corn steep liquor as substrates. The maximum production of 1087.9 U mL−1 was obtained with 25.0 g L−1 of cassava flour and 3.5 g L−1 of corn steep after 72 h by submerged fermentation. The enzyme showed optimum activity at pH 5.0 and temperature 55 °C, and maintained thermal stability at 55 °C for 3 h. The enzymatic activity was stimulated in the presence of Mg+2, Ca+2, EDTA, K+, Ba+2 and Na+ and inhibited in the presence of Hg+2, Cu+2, Fe+2 and Zn+2. The results showed that Bacillus sp. SM-02 have good potential for β-CGTase production.

Using molecular markers to assess Streptococcus mutans variability and the biological risk for caries

AIM: To characterize the genetic variability of Streptococcus mutans isolates and to correlate this variability with different colonization profiles observed during dental caries in a sample of children. METHODS: S. mutans samples were isolated from the saliva of 30 children with varying histories of dental caries, and they were characterized according to morphological and biochemical markers and the sequences of their 16S-23S intergenic spacer region. The genetic variability of the isolates was first assessed using Random Amplified Polymorphic DNA (RAPD) markers. Next, the isolates were differentiated by sequencing a specific region of the gene encoding the enzyme glucosyltransferase B (gtfB). RESULTS: Characterization using RAPD markers uncovered significant genetic variability among the samples and indicated the existence of clusters, which allowed us to reconstruct both the origin and clinical history of the disease. By sequencing the 16S-23S intergenic region, it was found that all of the isolates belonged to the species S. mutans. Based on the genetic similarity of the isolates and pattern of amino acid variations identified by partial sequencing of the gtfB gene, base-pair changes were identified and correlated with different virulence patterns among the isolates. CONCLUSIONS: The partial sequencing of the gtfB gene can be a useful tool for elucidating the colonization patterns of S. mutans. As amino acid variations are likely to be correlated with differences in biological risk, molecular characterization, such as that described in this paper, could be the key for assessing the development of dental caries in children...

Cyclodextrin glucanotransferase immobilization onto functionalized magnetic double mesoporous core-shell silica nanospheres

Background Cyclodextrin glucanotransferase (CGTase) from Amphibacillus sp. NPST-10 was covalently immobilized onto amino-functionalized magnetic double mesoporous core-shell silica nanospheres (mag@d-SiO2@m-SiO2-NH2), and the properties of the immobilized enzyme were investigated. The synthesis process of the nanospheres included preparing core magnetic magnetite (Fe3O4) nanoparticles, coating the Fe3O4 with a dense silica layer, followed by further coating with functionalized or non-functionalized mesoporous silica shell. The structure of the synthesized nanospheres was characterized using TEM, XRD, and FT-IR analyses. CGTase was immobilized onto the functionalized and non-functionalized nanospheres by covalent attachment and physical adsorption. Results The results indicated that the enzyme immobilization by covalent attachment onto the activated mag@d-SiO2@m-SiO2-NH2, prepared using anionic surfactant, showed highest immobilization yield (98.1%), loading efficiency (96.2%), and loading capacity 58 µg protein [CGTase]/mg [nanoparticles]) which were among the highest yields reported so far for CGTase. Compared with the free enzyme, the immobilized CGTase demonstrated a shift in the optimal temperature from 50°C to 50-55°C, and showed a significant enhancement in the enzyme thermal stability. The optimum pH values for the activity of the free and immobilized CGTase were pH 8 and pH 8.5, respectively, and there was a significant improvement in pH stability of the immobilized enzyme. Moreover, the immobilized CGTase exhibited good operational stability, retaining 56% of the initial activity after reutilizations of ten successive cycles. Conclusion The enhancement of CGTase properties upon immobilization suggested that the applied nano-structured carriers and immobilization protocol are promising approach for industrial bioprocess for production of cyclodextrins using immobilized CGTase.

A new low molecular mass alkaline cyclodextrin glucanotransferase from Amphibacillus sp. NRC-WN isolated from an Egyptian soda lake

Background: Cyclodextrin glucanotransferase (CGTase) is one of the most industrially important enzymes used in the commercial production of cyclodextrins (CDs). Alkaliphilic bacteria have attracted much interest in the last few decades because of their ability to produce extracellular enzymes that are active and stable at high pH values. Here, we report the isolation of a new CGTase from alkaliphilic bacteria collected from Egyptian soda lakes and describe the purification and biochemical characterization of this CGTase. Results: Screening for CGTase-producing alkaliphilic bacteria from sediment and water samples collected from Egyptian soda lakes located in the Wadi Natrun valley resulted in the isolation of a potent CGTase-producing alkaliphilic bacterial strain, designated NRC-WN. Strain NRC-WN was belonging to genus Amplibacullus by 16S rDNA sequence analysis (similarity: ca. 98%). Among the tested nitrogen and carbon sources, peptone (0.15%, w/v) and soluble starch (0.4%, w/v) allowed maximal CGTase production by Amphibacillus sp. NRC-WN. CGTase was successfully purified from Amphibacillus sp. NRC-WN up to 159.7-fold through a combination of starch adsorption and anion exchange chromatography, resulting in a yield of 84.7%. SDS-PAGE analysis indicated that the enzyme was purified to homogeneity and revealed an estimated molecular mass of 36 kDa, which makes it one of the smallest CGTases reported in the literature. The purified enzyme exhibited maximum activity at 50ºC and was stable up to 70ºC, retaining 93% of its initial activity after treatment for 1 hr. Furthermore, Ca2+ ions (10 mM) significantly enhanced the thermal stability of the CGTase. The purified enzyme was active and stable over a wide pH range, showing maximal activity at pH 9.5. The enzyme was significantly stimulated by Zn2+, Ca2+ and Co2+ but was completely inhibited in the presence of Fe3+ and mercaptoethanol. The Km and Vmax values of the purified CGTase were estimated to be 0.0434 mg/ml and 3,333.3 mg β-CD/ml/min, respectively. β-CD was the predominant product of starch degradation by the Amphibacillus sp. NRC-WN CGTase, followed by α-and γ-CDs. Conclusions: A new low molecular mass alkaline CGTase was purified from a newly identified alkaliphilic Amphibacillus sp. NRC-WN isolate from the Egyptian soda lakes. The enzyme showed promising thermal and pH stability and a high affinity toward starch as a natural substrate.

Immobilization of cyclodextrin glucanotransferase on aminopropyl-functionalized silica-coated superparamagnetic nanoparticles

Background: Cyclodextrin glycosyltransferase (CGTase) from Amphibacillus sp. NPST-10 was successfully covalently immobilized on aminopropyl-functionalized silica coated superparamagnetic nanoparticles; and the properties of immobilized enzyme were investigated. The synthesis process included preparing of core magnetic magnetite (Fe3O4) nanoparticles using solvothermal synthesis; followed by coating of Fe3O4 nanoparticles with dense amino-functionalized silica (NH2-SiO2) layer using in situ functionalization method. The structure of synthesized Fe3O4@NH2-SiO2 nanoparticles was characterized using TEM, XRD, and FT-IR analysis. Fe3O4@NH2-SiO2 nanoparticles were further activated by gluteraaldehyde as bifunctional cross linker, and the activated nanoparticles were used for CGTase immobilization by covalent attachment. Results: Magnetite nanoparticles was successfully synthesized and coated with and amino functionalized silica layer (Fe3O4/NH2-SiO2), with particle size of 50-70 nm. The silica coated magnetite nanoparticles showed with saturation magnetization of 65 emug-1, and can be quickly recovered from the bulk solution using an external magnet within 10 sec. The activated support was effective for CGTase immobilization, which was confirmed by comparison of FT-IR spectra of free and immobilized enzyme. The applied approach for support preparation, activation, and optimization of immobilization conditions, led to high yields of CGTase immobilization (92.3%), activity recovery (73%), and loading efficiency (95.2%); which is one of the highest so far reported for CGTase. Immobilized enzyme showed shift in the optimal temperature from 50 to 55ºC, and significant enhancement in the thermal stability compared with free enzyme. The optimum pH for enzyme activity was pH 8 and pH 7.5 for free and immobilized CGTase, respectively, with slight improvement of pH stability of immobilized enzyme. Furthermore, kinetic studies revealed that immobilized CGTase had higher affinity toward substrate; with k m values of 1.18 ± 0.05 mg/ml and 1.75 ± 0.07 mg/ml for immobilized and free CGTase, respectively. Immobilized CGTase retained 87% and 67 of its initial activity after 5 and 10 repeated batches reaction, indicating that immobilized CGTase on Fe3O4/NH2-SiO2 had good durability and magnetic recovery. Conclusion: The improvement in kinetic and stability parameters of immobilized CGTase makes the proposed method a suitable candidate for industrial applications of CGTase. To best of our knowledge, this is the first report about CGTase immobilization on silica coated magnetite nanoparticles.

Complement dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling

Oligosaccharides are implicated in the development of the immune response notably in complement activation. Anti-tumoural immunotherapy by monoclonal antibodies (mAbs) offers some advantages to chemotherapy including cell targeting but some of them are inefficient to generate cytotoxicity dependent complement (CDC) known to be important in the antibody’s efficacy. The aim of this study is to give a CDC activity of mAb by linkage of a complement activating oligosaccharide to this antibody via a hetero-bifunctional linker allowing control of the conjugation reaction. We worked on non Hodgkin Burkitt’s lymphoma as cancer source, Fab fragments of rituximab devoid of complement activity as mAb and the trisaccharide Gal alpha(1→3)Gal beta(1→4)GlcNAc as immunogenic glycan. The bioconjugate Fab-Gal was characterized by biochemical methods and we demonstrated that the α-Gal epitope was recognized by seric immunoglobulins. After checking the recognition capacity of the Fab-Gal conjugate for the CD20 epitope, in vitro assays were performed to evaluate the activation of the complement cascade by the Fab-Gal conjugate. The effect of this bioconjugate was confirmed by the evaluation of the proliferation response of Burkitt’s cell line. The relative facility realization of this strategy represents new approaches to increase activities of mAbs.

Effects of substrates and reaction conditions on production of cyclodextrins using cyclodextrin glucanotransferase from newly isolated Bacillus agaradhaerens KSU-A11

The effects of reaction conditions on cyclodextrins (CDs) production by CGTase from newly isolated Bacillus agaradhaerens KSU-A11 is reported. Among six types of starch tested, potato starch gave highest starch conversion into CDs. In addition, CDs yield was about three fold higher when using gelatinized potato starch in comparison to raw starch. The total CDs production was increased with increasing pH, showing maximum starch conversion at pH 10. Furthermore, the proportion of gamma-CD was relatively higher under slightly acidic-neutral conditions than at alkaline pH with a maximum proportion of 35.6 percent at pH 7 compared to 7.6 percent at pH 10. Maximum starch conversion into CDs was seen at reaction temperature of 55ºC. Lower reaction temperature led to higher proportion of gamma-CD with maximum percentage at 35ºC. Cyclization reaction was significantly promoted in the presence CaCl2 (10 mM), while in the presence of ethyl alcohol there was significant decrease in CD production particularly at high concentration. beta-CD was the major product up to 1 hr reaction period with traces of alpha-CD and no detectable gamma-CD. However, as the reaction proceed, gamma-CD started to be synthesised and alpha-CD concentration increased up to 4 hrs, where the CDs ratios were 0.27:0.65:0.07 for alpha-CD:beta-CD:gamma-CD, respectively. In addition, optimum CGTase/starch ratio was obtained at 80 U/g starch, showing highest starch conversion into CDs. All the parameters involved have been shown to affect the products yield and/or specificity of B. agaradhaerens KSU-A11 CGTase.

Scanning Electron Microscopic study of Piper betle L. leaves extract effect against Streptococcus mutans ATCC 25175

INTRODUCTION: Previous studies have shown that Piper betle L. leaves extract inhibits the adherence of Streptococcus mutans to glass surface, suggesting its potential role in controlling dental plaque development. OBJECTIVES: In this study, the effect of the Piper betle L. extract towards S. mutans (with/without sucrose) using scanning electron microscopy (SEM) and on partially purifed cell-associated glucosyltransferase activity were determined. MATERIAL AND METHODS: S. mutans were allowed to adhere to glass beads suspended in 6 different Brain Heart Infusion broths [without sucrose; with sucrose; without sucrose containing the extract (2 mg mL-1 and 4 mg mL-1); with sucrose containing the extract (2 mg mL-1 and 4 mg mL-1)]. Positive control was 0.12 percent chlorhexidine. The glass beads were later processed for SEM viewing. Cell surface area and appearance and, cell population of S. mutans adhering to the glass beads were determined upon viewing using the SEM. The glucosyltransferase activity (with/without extract) was also determined. One- and two-way ANOVA were used accordingly. RESULTS: It was found that sucrose increased adherence and cell surface area of S. mutans (p<0.001). S. mutans adhering to 100 µm² glass surfaces (with/without sucrose) exhibited reduced cell surface area, fuffy extracellular appearance and cell population in the presence of the Piper betle L. leaves extract. It was also found that the extract inhibited glucosyltransferase activity and its inhibition at 2.5 mg mL-1 corresponded to that of 0.12 percent chlorhexidine. At 4 mg mL-1 of the extract, the glucosyltransferase activity was undetectable and despite that, bacterial cells still demonstrated adherence capacity. CONCLUSION: The SEM analysis confrmed the inhibitory effects of the Piper betle L. leaves extract towards cell adherence, cell growth and extracellular polysaccharide formation of S. mutans visually. In bacterial cell adherence, other factors besides glucosyltransferase are involved.

Production of glucosyltransferase B and glucans by streptococcus mutans strains isolated from caries-free individuals

La glucosiltranferasa B es una enzima producida por Streptococcus mutans, que a partir de la sacarosa, cataliza la síntesis de glucanos insolubles los cuales dan soporte a la biopelícula, siendo uno de los principales factores de virulencia para la generación de la caries dental. Sin embargo, no se ha esclarecido su papel en los individuos libre de caries, portadores delmicroorganismo. El objetivo de este estudio fue determinar la producción de glucosiltransferasa B y la producción de glucanos por cepas de Streptococcus mutans aisladas de biopelícula de 30individuos libres de caries. Las cepas fueron cultivadas en caldo Todd Hewitt y las proteínas extracelulares fueron obtenidas por precipitación con sulfato de amonio las proteínas asociadas amembrana por extracción con urea. La presencia de GtfB fue determinada por peso molecular por SDS–PAGE, confirmada por Western Blot utilizando un anticuerpo específico y la producciónde polisacáridos por separación electroforética, incubación con sacarosa y coloración de Schiff. Los resultados muestran que el 96.7 por ciento de las cepas de Streptococcus mutans producen una banda a la altura del peso molecular correspondiente a las Gtf,de las cuales son reactivas por western blot el 63.4 por ciento El 93.3 por cientode las cepas producen polisacáridos. Conclusiones: la cepas de Streptococcus mutans aisladas de biopelícula de individuos sanos producen factores de virulencia asociados a la caries dental como glucosiltransferasa B y glucanos lo que indica que hay condiciones en la cavidad oral diferentes a estos factores que mantienen al individuo libre de caries dental, los cuales deben ser investigados en la búsqueda de estrategias para controlar la enfermedad.

Kinetic characteristics of beta-cyclodextrin production by cyclodextrin glycosyltransferase from newly isolated Bacillus sp. C26

The kinetic characteristics of beta-cyclodextrin production by a cyclodextrin glycosyltransferase (CGTase) produced by Bacillus sp. C26, a new isolate from a soil sample was investigated. Considering highest yield and initial production rate of beta-cyclodextrin, among the starches examined, soluble starch, tapioca starch, sago starch, corn starch and rice starch, tapioca starch was the best substrate for this CGTase. The optimum temperature for tapioca starch gelatinization prior to its use as a substrate for beta-cyclodextrin production was 65ºC. The yield and initial production rate of beta-cyclodextrin increased with increasing starch concentration up to 6 percent and an enzyme concentration up to 48 U/g-starch. The kinetic parameters of Vmax and Km of beta-cyclodextrin production from tapioca starch by CGTase were 1.59 mg/mL/h and 22.3 mg/mL, respectively. Considering high initial production rate and high yield of beta-cyclodextrin, the optimum reaction temperature was at 50ºC. This study provided the necessary kinetic information that may be useful to define the most suitable condition for industrialized production of beta-cyclodextrin with the high yield and productivity.

Glucanos extracelulares bacterianos: estructura, biosíntesis y función / Extreacellular bacterial glucans: structure, biosynthesis and function

La caries dental es una de las enfermedades más frecuentes en el ser humano. En su etiología multifactorial, desempeñan un papel importante determinadas bacterias cariogénicas, que en interacción con la superficie del diente promueven su desmineralización. Dentro de los mecanismos mediadores de la adhesión bacteriana, se encuentra la producción de polisacáridos extracelulares bacterianos. En particular los glucanos sintetizados por las glucosiltransferasas, no solo permiten la adherencia, sino que también constituyen una fuente nutricional para las bacterias, por lo tanto, la actividad de dichas enzimas se considera un factor de virulencia bacteriana en la caries dental. Esta revisión bibliográfica tiene el objetivo de esclarecer los aspectos relacionados con la estructura, biosíntesis y función de los glucanos, y enfatizar en la aplicación de estos conocimientos en la prevención de la caries dental(AU)

Dental caries is one of the most common diseases in the human being. Certain cariogenic bacteria play an important role in its multifactorial etiology, since in their interaction with the dental surface they promote its demineralization. The production of extracellular bacterial polyssacharides is among the mechanisms mediating bacterial adhesion. The glucans synthesized by glycosyltransferases not only allow the adherence, but they also are a nutritional source for bacteria and that's why the activity of such enzymes is considered a factor of bacterial virulence in dental caries. This bibliographic review is aimed at making clear the aspects related to the structure, biosynthesis and function of glucans and at giving emphasis to the application of this knowledge in the prevention of dental caries(AU)

Comparative analyses of the structure of the 1, 3-beta-glucan synthase gene in Paracoccidioides brasiliensis isolates

The evolutionary origin and significance of spliceosomal introns have been the subject of many investigations. Two theories, [quot ]introns-early[quot ] theory and [quot ]introns-late[quot ] theory, have been proposed to explain the evolution of introns in eukaryotic genes. Intron position is generally conserved in paralogue and orthologue genes. Some introns occur at similar but not necessarily identical positions in homologous genes, which were separated by great evolutionary distances. This event can be explained by insertion, loss or movement of the intron over short distances. Intron loss and gain events are unique in evolution and can be useful as markers for phylogenetic analyses. The insertion of introns at an identical position suggests a common ancestor gene. Here we analyzed, using PCR and RT-PCR, the structure of the 1,3-beta-glucan synthase gene (FKS) in several clinical isolates of Paracoccidioides brasiliensis (Pb): isolates Pb 01, Pb 4940, Pb 8515, Pb 8311, Pb 8334, Pb 4268, Pb 1668, and Pb E. Our results showed that seven of the isolates examined showed identical structures concerning the position of introns in PbFKS1. PbFKS4940 showed the intron described at the 3' end and had lost that one at the 5' end. The presence of the PbFKS4940 transcript suggests that it could be a functional gene. These data suggest a divergent evolution for introns with regard to the 1,3-beta-glucan synthase gene in P. brasiliensis isolates.

Expresión de isoantígenos glucoconjugados ABH en neoplasias gastrointestinales / Expression of glycoconjugated ABH isoantigens in gastrointestinal neoplasia

El tejido epitelial gastrointestinal normal presenta estructuras glicoesfingolipídicas que son propias de los antígenos ABH, las cuales confieren propiedades biológicas esenciales, dirigen el recambio y el tráfico transcelular y tienen gran importancia para la interacción entre células durante el desarrollo, crecimiento y diferenciación. Está descripto que la glicosilación aberrante es un atributo común del crecimiento neoplásico y uno de los principales determinantes del fenómeno relacionado con el cáncer, como es el crecimiento invasivo de la metástasis. El objetivo de este trabajo fue estudiar la expresión de los antígenos ABH en células epiteliales de neoplasias gastrointestinales. Se trabajó con catorce muestras de tumores gastrointestinales en tacos de parafina aplicando la técnica SRCA (Specific Red Cell Adherence). Se demostró que en 13 de las 14 muestras hubo pérdida total o parcial, o cambio de la expresión antigénica

The quality control of glycoprotein folding in the endoplasmic reticulum, a trip from trypanosomes to mammals

The present review deals with the stages of synthesis and processing of asparagine-linked oligosaccharides occurring in the lumen of the endoplasmic reticulum and their relationship to the acquisition by glycoproteins of their proper tertiary structures. Special emphasis is placed on reactions taking place in trypanosomatid protozoa since their study has allowed the detection of the transient glucosylation of glycoproteins catalyzed by UDP-Glc:glycoprotein glycosytransferase and glucosidade II. The former enzyme has the unique property of covalently tagging improperly folded conformations by catalyzing the formation of protein-linked Glc(1)Man(7)GlcNAc(2), Glc(1)Man(8)GlcNac(2) and Glc(1)Man(9)GlcNAc(2) from the unglucosylated proteins. Glucosyltransferase is a soluble protein of the endoplasmic reticulum that recognizes protein domains exposed in denatured but not in native conformations (probably hydrophobic amino acids) and the innermost N-acetylglucosamine unit that is hidden from macromolecular probes in most native glycoproteins. In vivo, the glucose units are removed by glucosidase II. The influence of oligosaccharides in glycoprotein folding is reviewed as well as the participation of endoplasmic reticulum chaperones (calnexin and calreticulin) that recognize monoglucosylated species in the same process. A model for the quality control of glycoprotein folding in the endoplasmic reticulum, i.e., the mechanism by which cells recognize the tertiary structure of glycoproteins and only allow transit to the Golgi apparatus of properly folded species, is discussed. The main elements of this control are calnexin and calreticulin as retaining components, the UDP-Glc:glycoprotein glucosyltransferase as a sensor of tertiary structures and glucosidase II as the releasing agent.

Production of cyclomaltodextrin glucanotransferase from an alkalophilic Bacillus sp. in rice and corn flakes

Cyclomaltodextrin glucanotranferase (CGTase) catalyzes the degradation of starch to form alpha-, beta- and gamma-cyclodextrin. Based on cyclodextrin formation, an alkalophilic Bacillus sp. ATCC 21783 was used for its high CGTase production ability, using corn and rice flakes as substrates. Maximum enzyme production was achieved after 96 hours at pH 9.0, temperature 37 degrees C, and 1 percent(w/v) of either substrate together with the addition of trub. The specific enzyme activity was determined by High Pressure Liquid Chromatography (HPLC) and expressed as using International Units based on total cyclodextrin formation. Optimum conditions for this determination were studied, finding that the best results are obtained at pH 5.0, 7.0 and 9.0, temperature 55 degrees C and 3 hours of incubation in 1 percent (w/v) of rice flakes as starch source

Cell wall deficiency in slime strains of Neurospora crassa: osmotic inhibition of cell wall synthesis and Beta-D-glucan synthase activity

1. The RCP-3 S/H mutant of Neurospora crassa was obtained by vegetative selection in medium of high osmolarity of a mycelial form an fz, sg, os-1 ("smile"-like) segregant. The mutant exhibits spheroplast-hyphal dimorphism conditioned by the osmolarity of the culture medium (pietro et al. (1990). Journal of General Microbiology, 136: 121-129). The carbohydrate composition of the cell wall of the mutant was different from that of the wild type in the absence of an alkalisoluble galactosaminoglycan polymer. Furthermore the mutant cell wall had a somewhat lower content of ß-glucan relative to that of chitin. 2. Increasing concentrations of sorbitol in the culture medium of the mutant inhibited by 10-fold the formation of cell wall relative to toal biomass. The cell wall of the mutant cultured in the presence of sorbitol lacked mannose-and galactose-containing polymers, and also showed progressively lower amounts of ß-glucan relative to chitin. 3. The activity of membrane-bound (1-3)-ß-D-glucan synthase from the mutant grown in the absence of sorbitol shared several properties with the wil type enzyme (i.e., Km app, Vmax, stability at 30ºC, activation by GTPyS, and dissociability by treatment with NaCl and Tergitol NP-40 into a membrane-bound catalytic center and GTP-binding activating protein). On the other hand, the enzyme from the mutant but not that from the wild type was inactivated by about 15 per cent by treatment with NaCl and detergent. 4. At high concentrations of sorbitol (1.0M) the RCP-3 S/H mutant exclusively produced spheroplasts devoid of (1-3)-ß-D-glucan synthase activity. The defect was at the level of the membrane-bound catalytic center. The activity of the GTP-binding activating factor was apparently normal in these cells. 5. These results suggest that the definitive loss of cell wall in the N. crassa "slime" RCP-3 S/H mutant was due to a defect in (1-3)-ß-D-glucan synthase activity which wass exaggerated in the presence of high osmolyte concentrations

Composicion fosfolipidica de la membrana microsomal hepatica u su relacion con la actividad de la bilirrubina UDP-Glucuroniltransferasa en colestasis humana / Phospholipid composition of the hepatic microsomal membrane and its relationship to bilirubin UDP glucuronyltransferase in human cholestasis

Han sido descriptos diversos cambios estructurales y morfológicos sobre el hepatocito cuando se utilizaron modelos de coelstasis experimental asimismo se han documentado alteraciones en la actividad de las denominadas "enzimas unidas a membrana" frente a los cambios en el entorno lipídico. En el presente estudio se estableció la actividade de la bilirrubina UDP-Glucuraniltransferasa y el perfil fosfolipídico microsomal en hepatocitos de individuos control y en pacientes con colestasis extrahepática prolongada. No se hallaron diferencias significativas en la actividad total de Bilirrubina UDP-Glucuroniltransferasa entre ambos grupos de pacientes (normales: 1,11 ñ 0,66; colestáticos: 1,93 ñ 0,82 nmoles bilirrubina conjugada/mg proteína en 10 min). Sin embargo cuando el producto final de la reacción se analizó, los controles presentaron 80% de bilirrubina diglucuronido (20% bilirrubina monoglucuronido), resultando ausente este metabolito en los pacientes colestáticos (100% bilirrubina monoglucuronido). El análisis cualitativo de los fosfolípidos reveló una disminución en el contenido de fosfatidilcolina (-9,5%) y fosfatidiletanolamina (-62,9%) y, paralelamente, incrementos en fosfatidilserina (+143,4%) y esfingomielina (+475,6%) en los microsomas dle grupo colestático cuando se comparó con el grupo control. Por el contrario el contenido de fósforo total resultó similar en ambos grupos estudiados (normales: 979, 55 ñ 42,15; colestáticos: 958, 26 ñ 30,86 umol Pi/mg proteina). Los datos presentados en este estudio demuestran que la capacidad de bilirrubina y el perfil fosfolipídico microsomal se ven seriamente afectados durante la colestasis humana. En próximas experiencias se intentará esclarecer la relación entre ambos hechos como así también delucidar el mecanismo de los cambios inducidos por la acumulación de bilis