Expression and characterization of β-N-acetylglucosaminidases from Bacillus coagulans DSM1 for N-acetyl-β-D glucosamine production / 生物工程学报

β-N-acetylglucosaminidases (NAGases) can convert natural substrates such as chitin or chitosan to N-acetyl-β-D glucosamine (GlcNAc) monomer that is wildly used in medicine and agriculture. In this study, the BcNagZ gene from Bacillus coagulans DMS1 was cloned and expressed in Escherichia coli. The recombinant protein was secreted into the fermentation supernatant and the expression amount reached 0.76 mg/mL. The molecular mass of purified enzyme was 61.3 kDa, and the specific activity was 5.918 U/mg. The optimal temperature and pH of the BcNagZ were 75 °C and 5.5, respectively, and remained more than 85% residual activity after 30 min at 65 °C. The Mie constant Km was 0.23 mmol/L and the Vmax was 0.043 1 mmol/(L·min). The recombinant BcNagZ could hydrolyze colloidal chitin to obtain trace amounts of GlcNAc, and hydrolyze disaccharides to monosaccharide. Combining with the reported exochitinase AMcase, BcNagZ could produce GlcNAc from hydrolysis of colloidal chitin with a yield over 86.93%.

Generation of novel hyaluronic acid biomaterials for study of pain in third molar intervention: a review

Hyaluronic acid (HA) has long been studied in diverse applications. It is a naturally occurring linear polysaccharide in a family of unbranched glycosaminoglycans, which consists of repeating di-saccharide units of N-acetyl-D-glucosamine and D-glucuronic acid. It is almost ubiquitous in humans and other vertebrates, where it participates in many key processes, including cell signaling, tissue regeneration, wound healing, morphogenesis, matrix organization, and pathobiology. HA is biocompatible, biodegradable, muco-adhesive, hygroscopic, and viscoelastic. These unique physico-chemical properties have been exploited for several medicinal purposes, including recent uses in the adjuvant treatment for chronic inflammatory disease and to reduce pain and accelerate healing after third molar intervention. This review focuses on the post-operative effect of HA after third molar intervention along with its various physio-chemical, biochemical, and pharmaco-therapeutic uses.

Non-pharmacological measures in preterm newborns submitted to arterial puncture / Medidas no farmacológicas en recién nacidos prematuros sometidos a la punción arterial / Medidas não farmacológicas em recém-nascidos pré-termo submetidos à punção arterial

ABSTRACT Objective: to assess pain in preterm newborns and to compare the neonatal and therapeutic variables with the total scores of the Neonatal Facial Coding System of preterm newborns submitted to arterial puncture exposed to music and 25% oral glucose. Method: a comparative study with 48 recordings of preterm newborns - Group 1, music (26); Group 2, glucose 25% (22) - individually analyzed by three trained nurses, after Kappa of at least 80%. Results: the variables and the pain scores of the groups did not present statistical significance (p < 0.05) according to the Neonatal Facial Coding System. 80.8% of the preterm infants in Group 1 had a higher quantitative score ≥ 3 in the neonatal variables (gender, type of delivery), and therapeutic variables (type of oxygen therapy, place of hospitalization, type of puncture). Conclusion: There was no difference when comparing the music and glucose 25% groups and the variables studied.

RESUMEN Objetivo: evaluar el dolor en recién nacidos prematuros y comparar las variables neonatales y terapéuticas con las puntuaciones totales del Neonatal Facial Coding System de los recién nacidos prematuros sometidos a una punción arterial expuestos a la música y glucosa al 25% por vía oral. Método: estudio comparativo con 48 fi lmaciones de los recién nacidos prematuros divididos en el Grupo 1 - música (26) y el Grupo 2 - glucosa al 25% (22). Las fi lmaciones fueron analizadas individualmente por tres enfermeras capacitadas después de coefi ciente Kappa de al menos 80%. Resultados: las variables y puntuaciones de dolor de los grupos no fueron estadísticamente signifi cativas (p<0,05) de acuerdo con el Neonatal Facial Coding System. En el Grupo 1, 80,8% de los recién nacidos prematuros mostraron mayores cantidades de puntuaciones ≥ 3 en las variables neonatales (sexo, tipo de parto) y las variables terapéuticas (tipo de la terapia de oxígeno, lugar de internación, tipo de punción). Conclusión: No hubo diferencias cuando se comparan los grupos de música y de glucosa al 25% y las variables estudiadas.

RESUMO Objetivo: avaliar a dor em recém-nascidos pré-termo e comparar as variáveis neonatais e terapêuticas com os escores totais da Neonatal Facial Coding System de recém-nascidos pré-termo submetidos à punção arterial exposto à música e glicose 25% oral. Método: estudo comparativo com 48 fi lmagens de recém-nascidos pré-termo - Grupo 1, música (26); Grupo 2, glicose 25% (22) - analisadas individualmente por três enfermeiras treinadas, após Kappa de no mínimo 80%. Resultados: as variáveis e os escores de dor dos grupos não apresentaram signifi cância estatística (p < 0,05) de acordo com o Neonatal Facial Coding System. 80,8% dos prematuros do Grupo 1 apresentaram um maior quantitativo de escores ≥ 3 nas variáveis neonatais (sexo, tipo de parto) e, variáveis terapêuticas (tipo de oxigenoterapia, local de internação, tipo de punção). Conclusão: Não houve diferença ao se comparar os grupos da música e da glicose 25% e as variáveis estudadas.

Potential coordination role between O-GlcNAcylation and epigenetics

Dynamic changes of the post-translational O-GlcNAc modification (O-GlcNAcylation) are controlled by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) and the glycoside hydrolase O-GlcNAcase (OGA) in cells. O-GlcNAcylation often occurs on serine (Ser) and threonine (Thr) residues of the specific substrate proteins via the addition of O-GlcNAc group by OGT. It has been known that O-GlcNAcylation is not only involved in many fundamental cellular processes, but also plays an important role in cancer development through various mechanisms. Recently, accumulating data reveal that O-GlcNAcylation at histones or non-histone proteins can lead to the start of the subsequent biological processes, suggesting that O-GlcNAcylation as 'protein code' or 'histone code' may provide recognition platforms or executive instructions for subsequent recruitment of proteins to carry out the specific functions. In this review, we summarize the interaction of O-GlcNAcylation and epigenetic changes, introduce recent research findings that link crosstalk between O-GlcNAcylation and epigenetic changes, and speculate on the potential coordination role of O-GlcNAcylation with epigenetic changes in intracellular biological processes.

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

Garlic supplemented diet attenuates gentamicin nephrotoxicity ın rats

Purpose To demonstrate the effect of a 4% pulverized garlic supplemented diet on the nephrotoxicity induced by gentamicin in rats. Materials and Methods Twenty four healthy male Wistar rats, weighing between 220 - 260grams, were divided into three groups. The rats were randomly assigned to either the gentamicin injection without garlic supplementation group (Group I, n = 8), gentamicin injection with garlic supplementation group (Group II, n = 8), and control group (Group III, n = 8). Urine from the rats was collected and the volume (mL), microalbumin (mg/L), creatinine (mg/dL), Na (mmol/L), K (mmol/L), Cl (mmol/L), P (mg/dL), N-acetyl glucosamine (NAG) (U/L) and pH values were measured. Then urea (mg/dL), creatinine (mg/dL), total protein (g/dL) and cystatin (mg/L) values were measured for the blood samples obtained from tail veins. Results The median NAG value for the control group (52.050 U/L) was similar to value for Group II (56.400 U/L), which received gentamicin and the garlic diet. However, the median NAG value for Group I (77.030 U/L), which received gentamicin without garlic supplementation, was determined to be statistically significantly higher (p = 0.010) than the value for the control group. In addition, the mean cystatin value for Group II (1.360 U/L) was found to be statistically significantly lower than the value for the Group I (2.240 U/L) (p = 0.015). Conclusions In this study we showed the effect of 4% pulverized garlic supplemented diet for preventing nephrotoxicity induced by gentamicin in rats by using as parameters NAG in urine samples and cystatin C in serum samples. .

Synapsin-1 and tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes

O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, beta-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, 603QASQAGPGPR612, and the serine residue at 692 of the tau peptide, 688SPVVSGDTSPR698, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.

Influence of nagE and manX knockout with red homologous recombination on the microbial production of glucosamine by Escherichia coli / 生物工程学报

Glucosamine (GlcN), also called amino sugar, is a compound derived from the substitution of a hydroxyl group of glucose molecule with an amino group. GlcN finds a wide-range of applications in health food and pharmaceutical industries. In our previous research, a recombinant Escherichia coli-glms-gnal was constructed for the efficient production of GlcN and N-acetylglucosamine (GlcNAc), the latter can be readily deacetylated to GlcN under mild acidic conditions. However, the results indicated that the titer of GlcN and GlcNAc decreased significantly due to the transportation of GlcN and GlcNAc from the culture broth to the inside of cells. To alleviate or block the transportation process, nagE gene (encoding for the GlcNAc-specific transporter) and manX gene (encoding for the mannose transporter) were knocked out with the Red homologous recombination method, and two engineered strains, E. coli-glms-gna1-delta nagE (with nagE gene deletion) and E. coli-glms-gna1-delta nagE-delta manX (with nagE and manX genes deletion), were successfully constructed. The two strains were cultured in a 7-L fermentor for the production of GlcN and GlcNAc. The maximal GlcN concentration of control strain E. coli-glms-gnal reached 4.06 g/L, and the maximal GlcNAc concentration reached 41.46 g/L. The maximal GlcN and GlcNAc concentration of E. coli-glms-gna1-delta nagE reached 4.38 g/L and 71.80 g/L, respectively, which were 1.08-fold and 1.70-fold of those of E. coli-glms-gnal, respectively. The maximal GlcN and GlcNAc concentration of E. coli-glms-gnal-delta nagE-delta manX reached 4.82 g/L and 118.78 g/L, respectively, which were 1.20-fold and 2.86-fold of those of E. coli-glms-gnal, respectively. These results suggested that the deletion of nagE and manX could significantly increase the extracellular accumulation of GlcN and GlcNAc. The results obtained here maybe useful for the microbial GlcN production in an industrial scale.

Wheat germ agglutinin anchored chitosan nanoparticles and its conjugation with N-acetylglucosamine / 药学学报

This study is undertaken to modify the chitosan nanoparticles (CS-NPs) with wheat germ agglutinin (WGA), and investigate the conjugation between WGA-CS-NPs and N-acetylglucosamine (NAG). CS-NPs were prepared by ionotropic gelation process and then conjugated with WGA under the activation of glutaricdialdehyde. The mean diameter of the CS-NPs was approximately 113.5 nm and the poly-dispersity index (PDI) was 0.18. The binding yield of WGA to CS-NPs was comprised between 27.8% and 87.9% depending mostly on the addition of 0.3% (w/v) glutaraldehyde solution. A competitive inhibition experiment of WGA-CS-NPs to bovine submaxillary gland mucin (BSM) was taken to illuminate the binding activity of WGA-CS-NPs to the sugar of N-acetylglucosamine. After the addition of NAG, the binding rates between CS-NPs and BSM almost didn't change, while the binding rates between WGA-CS-NPs and BSM dropped down significantly, which confirmed the specific binding characteristics of WGA to NAG.

Participation of N-acetyl-D-glucosamine carbohydrate moieties in the recognition of Schistosoma mansoni sporocysts by haemocytes of Biomphalaria tenagophila

Lectin-carbohydrate binding may be involved in the recognition of Schistosoma mansoni sporocysts by haemocytes of Biomphalaria; therefore, we tested if this interaction is associated with snail resistance against Schistosoma infection. In vitro data showed that most of the S. mansoni sporocysts cultured with haemocytes from Biomphalaria glabrata BH, a highly susceptible snail strain, had a low number of cells that adhered to their tegument and a low mortality rate. Moreover, the addition of N-acetyl-D-glucosamine (GlcNAc) did not alter this pattern of adherence and mortality. Using haemocytes and haemolymph of Biomphalaria tenagophila Cabo Frio, we observed a high percentage of sporocysts with adherent cells, but complete encapsulation was not detected. Low concentrations of GlcNAc increased haemocyte binding to the sporocysts and mortality, which returned to basal levels with high concentrations of the carbohydrate. In contrast, haemocytes plus haemolymph from B. tenagophila Taim encapsulated cellular adhesion index of level 3 and destroyed over 30 percent of the S. mansoni sporocysts in culture. Interestingly, the addition of GlcNAc, but not mannose, to the culture medium resulted in the significant inhibition of cellular adhesion to the parasite tegument and the reduction of parasite mortality, suggesting that GlcNAc carbohydrate moieties are important to the recognition of S. mansoni by B. tenagophila Taim.

Glucosamine induces cell death via proteasome inhibition in human ALVA41 prostate cancer cell

Glucosamine, a naturally occurring amino monosaccharide, has been reported to play a role in the regulation of apoptosis more than half century. However the effect of glucosamine on tumor cells and the involved molecular mechanisms have not been thoroughly investigated. Glucosamine enters the hexosamine biosynthetic pathway (HBP) downstream of the rate-limiting step catalyzed by the GFAT (glutamine:fluctose-6-phosphate amidotransferase), providing UDP-GlcNAc substrates for O-linked beta-N-acetylglucosamine (O-GlcNAc) protein modification. Considering that O-GlcNAc modification of proteasome subunits inhibits its activity, we examined whether glucosamine induces growth inhibition via affecting proteasomal activity. In the present study, we found glucosamine inhibited proteasomal activity and the proliferation of ALVA41 prostate cancer cells. The inhibition of proteasomal activity results in the accumulation of ubiquitinated proteins, followed by induction of apoptosis. In addition, we demonstrated that glucosamine downregulated proteasome activator PA28gamma and overexpression of PA28gamma rescued the proteasomal activity and growth inhibition mediated by glucosamine. We further demonstrated that inhibition of O-GlcNAc abrogated PA28gamma suppression induced by glucosamine. These findings suggest that glucosamine may inhibit growth of ALVA41 cancer cells through downregulation of PA28gamma and inhibition of proteasomal activity via O-GlcNAc modification.

Evaluation of efficacy and complications of N-Acetylglucosamine for hemostasis of femoral arterial catheterization wounds

Reports to the FDA of local vascular complications associated with the use of hemostatis devices following cardiac catheterization and resulting in serious injuries have raised concerns about the safety of these devices. A review of the medical literature also has posed cause for concern. We performed this study to assess the efficacy and risks of local adverse events in hemostatis following cardiac catheterization with N-Acetyglucosamine-facilitated manual compression versus manual compression alone. N-Acetylglucosamine was used for hemostasis in 205 patients who underwent diagnostic cardiac catheterization performed via femoral access at a single site. Another 205 patients underwent diagnostic cardiac catheterization and received routine manual compression and served as the control group. Ambulation was permitted 12 hours after manual compression and 6 hours after using N-Acetylglucosamine. Poisson regression analysis using four different outcomes was used to assess the risk associated with the type of hemostasis, while controlling for confounding variables. Overall, the unadjusted incidence of any vascular complication was 4.8% for the manual compression and 5.4% for the N-Acetylglucosamine group. No serious adverse events were seen in this study. N-Acetylglucosamine does not appear to pose a greater risk for local vascular complications flowing cardiac catheterization than the manual compression method. This method permitted early ambulation and was as safe as manual compression

á-N-acetylglucosamine-linked O-glycans of sialoglycoproteins (Tc-mucins) from Trypanosoma cruzi Colombiana strain

Trypanosoma cruzi sialoglycoproteins (Tc-mucins) are mucin-like molecules linked to a parasite membrane via a glycosylphosphatidylinositol anchor. We previously determined the structures of Tc-mucin O-glycan domains from several T. cruzi strains and observed significant differences among them. We now report the amino acid content and structure of Tc-mucin O-glycan chains from T. cruzi Colombiana, a strain resistant to common trypanocidal drugs. Amino acid analysis demonstrated the predominance of threonine residues (42%) and helped to identify the O-glycans as belonging to a Tc-mucin family that contain a ²-galactofuranose (²-Galf) residue attached to an á-N-acetylglucosamine (á-GlcNAc) O-4, with the most complex glycan, a pentasaccharide-GlcNAc-ol with a branched trigalactopyranose chain, on the GlcNAc O-6. The presence of ²-Galf on O-glycans from T. cruzi Colombiana mucins supports the use of glycosylation as a phylogenetic marker for the classification of Colombiana in the T. cruzi I group.

Transient downregulation of protein O-N-acetylglucosaminylation by treatment of high-dose nicotinamide in human cells

Nicotinamide at millimolar concentrations affects cell survival in various conditions, and is being utilized therapeutically in many human diseases. However, the effect of an acute treatment of nicotinamide at such high dose on gene expression and cellular metabolism has rarely been determined previously. In this study, we found that levels of O-N-acetylglucosamin(O- GlcNAc)ylated proteins including Sp1 acutely decreased upon treatment of 10 mM nicotinamide. Concomitantly, Sp1 protein level decreased rapidly through accelerated proteasome-mediated proteolysis. Cotreatment of glucosamine or 2-deoxyglucose, which inhibits protein deGlcNAcylation, effectively blocked the decrease induced by nicotinamide. Interestingly, the decline in the levels of Sp1 and protein O- GlcNAcylation was only transient lasting for two days post treatment, and this pattern matched closely the rapid fluctuation of the cellular [NAD(+)]. Our results suggest a possible link between cellular nicotinamide metabolism and protein O-GlcNAcylation, and an existence of cellular [NAD(+)] homeostasis.

Effects of Poly-N-acetyl Glucosamine(pGlcNAc) Patch on Wound Healing in db/db Mouse

PURPOSE: Poly-N-acetyl glucosamine(PGlcNAc) nanofiber-based materials, produced by a marine microalga, have been characterized as effective hemostatic and angiogenic agents. The similarity between PGlcNAc patch and the natural extracellular matrix allows it to support new healthy tissue growth in an injured area and to encourage fluid absorption. In this study, we hypothesized that a poly-N-acetyl glucosamine fiber patch(PGlcNAc patch) may enhance wound healing in the db/db mouse. METHODS: PGlcNAc patches were applied on one square centimeter, full-thickness, skin wounds in the db/db mouse model. Wounds(n=15 per group) were dressed with a PGlcNAc nanofiber patch for 1 hour(1h), 24 hours(24h) or left untreated(NT). After the application time, patches were removed and wounds were allowed to heal spontaneously. The rate of wound closure was evaluated by digital analysis of unclosed wound area in course of time. At day 10, wounds(n=7 per group) were harvested and quantified with immunohistochemical markers of proliferation(Ki-67) and vascularization (platelet endothelial cell adhesion molecule, PECAM-1). RESULTS: Wounds dressed with PGlcNAc patches for 1 hour closed faster than control wounds, reaching 90% closure in 16.6 days, nine days faster than untreated wounds. Granulation tissue showed higher levels of proliferation and vascularization following 1h treatment than the 24h and NT groups. In addition to its hemostatic properties, the PGlcNAc material also appears to accelerate wound closure in healing-impaired genetically diabetic mice. CONCLUSION: This material, with its combination of hemostatic and wound healing properties, has the potential to be effective agent for the treatment of complicated wounds.

O-GlcNAc modification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat primary adipocytes

It has been known that O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of proteins plays an important role in transcription, translation, nuclear transport and signal transduction. The increased flux of glucose through the hexosamine biosynthetic pathway (HBP) and increased O-GlcNAc modification of protein have been suggested as one of the causes in the development of insulin resistance. However, it is not clear at the molecular level, how O-GlcNAc protein modification results in substantial impairment of insulin signaling. To clarify the association of O-GlcNAc protein modification and insulin resistance in rat primary adipocytes, we treated the adipocytes with O-(2-acetamido-2deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), a potent inhibitor of O-GlcNAcase that catalyzes removal of O-GlcNAc from proteins. Prolonged treatment of PUGNAc (100 micrometer for 12 h) increased O-GlcNAc modification on proteins in adipocytes. PUGNAc also drastically decreased insulin-stimulated 2-deoxyglucose (2DG) uptake and GLUT4 translocation in adipocytes, indicating that PUGNAc developed impaired glucose utilization and insulin resistance in adipocytes. Interestingly, the O-GlcNAc modification of IRS-1 and Akt2 was increased by PUGNAc, accompanied by a partial reduction of insulin-stimulated phosphorylations of IRS-1 and Akt2. The PUGNAc treatment has no effect on the expression level of GLUT4, whereas O-GlcNAc modification of GLUT4 was increased. These results suggest that the increase of O-GlcNAc modification on insulin signal pathway intermediates, such as IRS-1 and Akt2, reduces the insulin-stimulated phosphorylation of IRS-1 and Akt2, subsequently leading to insulin resistance in rat primary adipocytes.

Recognition of n-acetylglucosamine (GLyNAc) and poly-i-acetyllactosamine residues in vessels of the rat pineal gland

Las lectinas son proteínas que contienen áreas singulares para el reconocimiento de secuencias de azúcares en los glicoconjugados. La lecitina del tomate Lycopersicon esculentum (LEL) es capaz de reconocer específicamente los residuos de N-acetil-glucosamina (Gly-Nac) y poli-N-acetil-lactosamina. Utilizamos la técnica histoquímica para LEL conjugada a la biotina con el propósito de investigar en la glándula pineal de ratones adultos y durante el desarrollo, las estructuras morfológicas capaces de unirse a esta lecitina. Nuestros resultados experimentales mostraron un material de coloración por la LEL, solamente en la superficie de las células endoteliales de todos los vasos sanguíneos y en todas las regiones de la glándula. La excepción ocurrió en los ratones con un día pos-natal (PN1), donde solamente los vasos de la región más periférica de la glándula presentaban coloración marrón amarillenta por la LEL, pero ninguno presentaba esta coloración en la región más central de la glándula. La reacción apareció especialmente en el espacio interno de las pseudo-rosetas, demostrando así que este espacio está, seguramente, representado por un vaso.

Characterization and identification of chitinase producing Streptomyces venezuelae P10.

In an attempt to isolate chitinase producers from soil, a streptomycete strain was found potent using natural chitin as the substrate. Chitinolytic activity was tested directly on agar plates, also with crude enzyme. Chitinase assay showed that the isolate could produce 0.8 U/ml of the enzyme. The morphological, cultural, physiological and biochemical characters of the isolate P10 were studied, and identified as Streptomyces venezuelae P10.

Effect of sugars on the association between cowpea vicilin (7S storage proteins) and fungal cells

Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of vicilins associated to chitinous structures present in yeast cells. Our results showed that vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine.

Histochemical detection of sugar residues in lizard teeth (Liolaemus gravenhorsti): a lectin-binding study

The structural diversity of the many oligosaccharide chains of surface glycoconjugates renders them likely candidates for modulators of cell-interactions, cellular movements, differentiation, and cellular recognition. A selection of different lectins was used to investigate the appearance of cellular distribution and changes in sugar residues during tooth development in the polyphyodont lizard, Liolaemus gravenhorsti. Lectins from three groups were used: (1) N-acetylgalactosamine specificity: BS-1, PNA, RCA-120; (2) N-acetylglucosamine specificity: ECA; and (3) fucose specificity: UEA 1 and LTA.. Digital images were processed using Scion Image. Grayscale graphics in each image were obtained. The lectins used showed a strong, wide distribution of the L-fucose and N-acetylgalactosamine at the cell surface and in the cytoplasm of multinucleate odontoclast cell, while mononuclear odontoclast cells showed no binding, suggesting some roles that the residues sugar might play in the resorption of dentine or with multinucleation of odontoclast after the attachment to the dentine surface in this polyphyodont species. Further studies must be planned to determine the specific identities of these glycoconjugates,and to elucidate the roles played by these sugar residues in the complex processes related to odontogenesis in polyphyodont species.