β-galactosidase immobilization on controlled pore silica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of disinfectant agents on dimensional stability of elastomeric impression materials.

STATEMENT OF PROBLEM: Difficulties in sterilizing impressions by traditional methods have led to chemical disinfection as an alternative, and some studies have shown that disinfectants may adversely affect impressions. PURPOSE: This study investigated the effect of disinfection methods on the dimensional stability of 6 elastomeric materials. MATERIAL AND METHODS: Impression materials were submitted to the following treatments: immersion in 5.25% sodium hypochlorite solution for 10 minutes, immersion in 2% glutaraldehyde solution for 30 minutes, and no immersion (control). After treatments, impressions were poured, and respective stone casts were measured with a Nikon Profile projector and compared with the master model. RESULTS: The elastomeric materials had different reproduction capacities, and the disinfecting treatments did not differ from the control.

Apoptosis in the early developing periodontium of rat molars

Development of the periodontium involves a series of complex steps that result in the formation of root dentine, cementum, bone and fibres of the ligament. These precisely controlled and timed events require the participation of the enamel organ derived epithelial cells of Hertwig's (HRS) and ectomesenchymal cells of the dental follicle. These events involve rapid turnover of the tissues and cells, including disappearance of epithelial cells of HRS. Thus, it seemed likely to us that programmed cell death (apoptosis) may play a role in the development of the periodontium. Fragments of first molars, obtained from 14- and 29-day-old rats, were fixed in glutaraldehyde-formaldehyde and processed for light and electron microscopy. For the TUNEL method for detection of apoptosis, specimens were fixed in 4% formaldehyde and embedded in paraffin. Results confirmed that epithelial cells of HRS maintain a close relationship with the forming dentine root, and that they may become trapped in the dentino-cemental junction. Some of the epithelial cells exhibited ultrastructural features which are consistent with the interpretation that they were undergoing programmed cell death, i.e. apoptosis. Periodontal fibroblast-like cells showed typical images of apoptosis and engulfed apoptotic bodies. TUNEL positive structures were present in all corresponding regions. It seems therefore that apoptosis of epithelial cells of HRS and fibroblast-like cells of the periodontal ligament constitutes an integral part of the developmental process of the tissues of the periodontium. (C) 2000 Wiley-Liss, Inc.

Scanning electron microscopy study of the choroid plexus in the monkey (Cebus apella apella)

The cells of the choroid plexus of the lateral ventricles of the monkey Cebus apella apella were examined through scanning electron microscopy at contributing to the description of such structures in primates. The animals were anesthetized previously with 3% hypnol intraperitoneally and after perfusion with 2.5% glutaraldehyde, samples of the choroid plexus were collected after exhibition of the central portion and inferior horn of the lateral ventricles. The ventricular surface of those cells presents globose form as well as fine interlaced protrusions named microvilli. Among those, it is observed the presence of some cilia. Resting on the choroid epithelial cells there is a variable number of free cells, with fine prolongations which extend from them. They are probably macrophages and have been compared to Kolmer cells or epiplexus cells, located on choroid epithelium. The choroid plexus of the encephalic lateral ventricles of the monkey Cebus apella apella at scanning electron microscopy is similar to that of other primates, as well as to that of other species of mammals mainly cats and rats, and also humans.

A new application of humic substances: Activation of supports for invertase immobilization

Invertase was immobilized on aminopropyl silica (APTS-SiO2) activated with humic substances (APTS-SiO2-HS) and on aminopropyl silica activated with glutaraldehyde (APTS-SiO2-GA). The resulting activity of both systems was compared. Humic substances (HS) used for the activation of the silica were extracted from soil of Cananéia, São Paulo State, Brazil, according to the procedure recommended by the International Humic Substances Society. Activity was determined by measuring the rate of formation of reduced sugars using the reaction with dinitrosalicylic acid (DNS). The amount of HS bound on the APTS-SiO2 was equal to 50 mg. The maximum amount of invertase immobilized on APTS-SiO2-HS was 15200 U/g while in the system APTS-SiO2-GA it was 13400 U/g. The experimental enzymatic activity was 3700 and 3300 U/g, for the systems APTS-SiO2-HS and APTS-SiO2-GA, respectively. Considering the increased amount and activity of immobilized enzyme compared with the glutaraldehyde method, it was concluded that this technique opens a new perspective in the preparation of supports for enzyme immobilization employing humic substances. © Springer-Verlag 2000.

Immobilization of lipases and assay in continuous fixed bed reactor

Lipases are versatile enzymes regarding the range of reactions they catalyse and substrates on which they act. They are as well important as catalyst in organic synthesis. Their immobilization on appropriate supports confer them greater stability besides the possibility of operating in continuous reactors. In order to explore these abilities, the reactions involving hydrolysis of p-nitrophenyl acetate (PNPA) and transesterification of PNPA with n-butanol were chosen. Lipases from two different sources were assayed, namely: microbial (Candida rugosa, CRL, Sigma Type VII) and pancreatic (PPL, Sigma, Type 11). Two immobilization methods were also used, namely: 1) adsorption, using as support the following silica derivatives (150-300μm e 450μ): phenyl, epoxy, amino and without derivation, and 2) covalent binding, using glutaraldehyde as binding agent and silica amino as support. This later method led to better results. Hydrolytic activity was 6.1 U/gsupport for CRL and 0.97U/gsupport for PPL, and of transesterification, 2,8U/gsupport for CRL and 1,9U/gsupport for PPL. Stability of the immobilized enzyme as a function of temperature was evaluated for CRL at 40°C and 50°C and for PPL at 32°C and 40°C. The assays were initially carried out batchwise, both for soluble and immobilized enzymes, aiming to the obtention of parameters for the continues reactor. Lipases immobilized by covalent binding were used in the assays of operacional stability in continuos reactors. For PPL in aqueous medium, at 32°C, and CRL in organic medium at 40°C, both operating continuously, no significant loss of activity was detected along the analysis period of 17 days. In the case of CRL in aqueous medium at 40°C there was a loss of activity around 40% after 18 days. For PPL in organic medium at 40°C the loss was 33% after 20 days. Compairing both sources with each other, very different results were obtained. Higher activitiy was found for CRL, both for hydrolysis and for transesterification reactions, with higher stability in organic medium. PPL showed lower activity as well as higher stability in aqueous medium. The immobilization method by covalent binding showed to be the most appropriate. Immobilized lipases are therefore relatively stable both in aqueous and organic medium.

Effect of disinfection treatments on the hardness of soft denture liner materials

Purpose: To evaluate the effects of disinfection treatments with chemical solutions (2% glutaraldehyde, 5% sodium hypochlorite, and 5% chlorhexidine) and microwave energy on the hardness of four long-term soft denture liners. Materials and Methods: Forty rectangular specimens of four soft lining materials (Molloplast-B, Ufi Gel P, Eversoft, and Mucopren soft) were made for each material. Ten samples of each material were immersed in different disinfectant solutions for 10 minutes or placed in a microwave oven for 3 minutes at 500 W. The hardness values were obtained with a Shore A durometer, before the first disinfection cycle (control), and also after two cycles of disinfection. Data were submitted to analysis of variance and Tukey's test (p < 0.01). Results: The highest value of hardness was obtained for Molloplast-B, independent of the disinfection technique. Mucopren soft demonstrated intermediate values and Ufi Gel P and Eversoft the lowest values of Shore A hardness. For Molloplast-B, the disinfection using glutaraldehyde demonstrated the highest value of hardness. The number of disinfections had no effect on the hardness values for all the materials studied and disinfection techniques. Conclusions: The application of two disinfection cycles did not change the Shore A hardness values for all the materials. The glutaraldehyde solution demonstrated the highest values of Shore A hardness for the Molloplast-B, Mucopren soft, and Ufi Gel P materials, while Eversoft did not present any differences in hardness when submitted to different disinfection treatments. Copyright © 2007 by The American College of Prosthodontists.

The materno-fetal interface in llama (Lama guanicoe glama)

Samples from 9 llamas (28 through 36 weeks of gestation) were collected and fixed in 4% buffered paraformaldehyde (light microscopy) and in 2.5% buffered glutaraldehyde (transmission and scanning electron microscopy). The material was processed in paraplast and slides (5μm) were stained with HE, PAS, Masson-Trichrome, acid phosphatase and Perl's. The uteroferrin was immunolocalized. The results show that llama placenta is chorioallantoic, diffuse, folded and epitheliochorial, and the fetus is covered with an epidermal membrane. The trophoblast cells have variable morphology: cubic, rounded and triangular cells, with cytoplasm containing PAS-positive granules. Binucleated cells with large cytoplasm and rounded nuclei, as well as giant trophoblastic cells with multiple nuclei were also observed. Numerous blood vessels were observed beneath the cells of the uterine epithelium and around the chorionic subdivided branches. Glandular activity was shown by PAS, Perl's, and acid phosphatase positive reactions in the cytoplasm and glandular lumen, and by immunolocalization of the uteroferrin in the glandular epithelium. The uterine glands open in spaces formed by the areoles, which are filled by PAS-positive material. The llama fetus was covered by the epidermal membrane, composed of stratified epithelium, with up to seven layers of mono-, bi- or trinucleated cells. The high level of maternal and fetal vascularization surfaces indicates an intense exchange of substances across both surfaces. The metabolic activity shown in the uterine glands suggests an adaptation of the gestation to the high altitudes of the natural habitat of this species.

Microwave-induced fast decalcification of rat bone for electron microscopic analysis: An ultrastructural and cytochemical study

Bone decalcification is a time-consuming process. It takes weeks and preservation of the tissue structure depends on the quality and velocity of the demineralization process. In the present study, a decalcification methodology was adapted using microwaving to accelerate the decalcification of rat bone for electron microscopic analysis. The ultrastructure of the bone decalcified by microwave energy was observed. Wistar rats were perfused with paraformaldehyde and maxillary segments were removed and fixed in glutaraldehyde. Half of specimens were decalcified by conventional treatment with immersion in Warshawsky solution at 4oC during 45 days, and the other half of specimens were placed into the beaker with 20 mL of the Warshawsky solution in ice bath and thereafter submitted to irradiation in a domestic microwave oven (700 maximum power) during 20 s/350 W/±37°C. In the first day, the specimens were irradiated 9 times and stored at 40°C overnight. In the second day, the specimens were irradiated 20 times changing the solution and the ice after each bath. After decalcification, some specimens were postfixed in osmium tetroxide and others in osmium tetroxide and potassium pyroantimonate. The specimens were observed under transmission electron microscopy. The results showed an increase in the decalcification rate in the specimens activated by microwaving and a reduction of total experiment time from 45 days in the conventional method to 48 hours in the microwave-aided method.

Analysis of the nucleolar cycle in the seminiferous epithelium of rodents

The nucleolus is a subcompartment of the nucleus and the site of ribosome biogenesis. During the mitotic and meiotic cell cycles, a disorganization and later reorganization of the nucleolar material occur, an event called nucleologenesis. In the spermatogenesis of mammals and other vertebrates, there is evidence of the disorganization of the nucleolus at the end of meiosis I, which supplies material for the cytoplasmic formation of an organelle called the chromatoid body (CB). The CB is a structure characteristic of spermatogenic cells and seems to be responsible for RNA metabolism in these cells and for some events of spermiogenesis, such as the formation of the acrosome, cellular communication between spermatids, and the formation of the spermatozoon middle piece and tail. The aim of this paper was to obtain information about the cytochemical and ultrastructural nature of the nucleolar cycle and the distribution of cytoplasmic RNAs in the seminiferous tubule cells of Rattus novergiucus, Mus musculus and Meriones unguiculatus. The testis was fixed in Bouin and Karnovsky solutions for conventional histological analysis and for cytochemical study that included: periodic acid-Schiff, hematoxylin-eosin, Feulgen reaction, silver-ion impregnation, Gomori's reticulin stain, toluidine blue, modified method of critical electrolyte concentration, and basic and acid fast green. The blocks of testis fixed in glutaraldehyde were used for ultrastructural analysis by transmission electron microscopy. Ultrathin sections were double-stained with uranyl acetate and lead citrate. All the techniques used provided information on the origin and function of the CB in the spermatogenic cells. Therefore, considering the persistence of the RNA and nucleolar ribonucleoproteins during spermatogenesis of Rattus novergicus, Mus musculus and Meriones unguiculatus, our findings corroborate the statement that these molecular complexes are very important in the spermiogenesis phases. It can be suggested that these ribonucleoprotein corpuscles (chromatoid bodies) are of nuclear origin and have a role in the successive series of events that occur in the formation of the spermatozoon. Furthermore, these results reinforce the conservation of the mechanisms involved in preserving necessary levels of protein stocks in different stages of cell differentiation, from spermatid to spermatozoon, in these rodent species. ©FUNPEC-RP.

A scanning electronic microscopy study of the action of different desensitizing agents on bovine dentin

This study sought to use scanning electronic microscopy (SEM) to evaluate the dentinal tubule occlusion potential of different desensitizing agents. Ten slices of bovine dentin were divided into six fragments, cleaned (using ultrasound), and etched for 15 seconds with a 35% phosphoric acid solution. All but one of the groups received a different desensitizing agent; the sixth group served as a control and received no additional treatment. After the agents were applied, the dentin specimens were analyzed by SEM and scores were assigned based on the extent of tubular obliteration. Only three agents demonstrated tubular sealing that was significantly different from that of the control group.

Effectiveness of six different disinfectants on removing five microbial species and effects on the topographic characteristics of acrylic resin

Purpose: The aim of this study was to evaluate the effectiveness of disinfectant solutions (1% sodium hypochlorite, 2% chlorhexidine digluconate, 2% glutaraldehyde, 100% vinegar, tabs of sodium perborate-based denture cleanser, and 3.8% sodium perborate) in the disinfection of acrylic resin specimens (n = 10/group) contaminated in vitro by Candida albicans, Streptococcus mutans, S. aureus, Escherichia coli, or Bacillus subtilis as measured by residual colony-forming unit (CFU). In a separate experiment, acrylic resin was treated with disinfectants to monitor potential effects on surface roughness, Ra (μm), which might facilitate microbial adherence. Materials and Methods: Three hundred fifty acrylic resin specimens contaminated in vitro with 1×10 6 cells/ml suspensions of standard strains of the cited microorganisms were immersed in the disinfectants for 10 minutes; the control group was not submitted to any disinfection process. Final counts of microorganisms per ml were performed by plating method for the evaluation of microbial level reduction. Results were compared statistically by ANOVA and Tukey's test (p ≤ 0.05). In a parallel study aiming to evaluate the effect of the tested disinfectant on resin surface, 60 specimens were analyzed in a digital rugosimeter before and after ten cycles of 10-minute immersion in the disinfectants. Measurements of superficial roughness, Ra (μm), were compared statistically by paired t-test (p ≤ 0.05). Results: The results showed that 1% sodium hypochlorite, 2% glutaraldehyde, and 2% chlorhexidine digluconate were most effective against the analyzed microorganisms, followed by 100% vinegar, 3.8% sodium perborate, and tabs of sodium perborate-based denture cleanser. Superficial roughness of the specimens was higher after disinfection cycles with 3.8% sodium perborate (p = 0.03) and lower after the cycles with 2% chlorhexidine digluconate (p = 0.04). Conclusion: Within the limits of this experiment, it could be concluded that 1% sodium hypochlorite, 2% glutaraldehyde, 2% chlorexidine, 100% vinegar, and 3.8% sodium perborate are valid alternatives for the disinfection of acrylic resin. © 2008 by The American College of Prosthodontists.

Cell immobilization improves isomaltulose production

The Laboratory of Industrial Biotechnology at the Biological Sciences Department in Sao Paulo State University, Brazil is working to improve the technologies involved with isomaltulose production. The study evaluated enzymatic reaction parameters with the goal of improving isomaltulose production which is grown with a medium of 1% cane molasses and 0.5% yeast extract thereby using calcium alginate, glutaraldehyde and polyethyleneimine. The best results were obtained using P. rubrum immobilized pellets in calcium alginate with 705 and 60% sucrose solution. The developed technology apparently allows the reuse of the cell-containing enzymes more times compared to conventional technologies, which ultimately results in decreased costs. The researchers are also involved in alcohol and biopolymer production and seeking interested industrial collaborators.

Effect of disinfectant solutions on a denture base acrylic resin.

The aim of this study was to evaluate the hardness, roughness and mass loss of an acrylic denture base resin after in vitro exposure to four disinfectant solutions. Forty specimens (Clássico, Brazil) were prepared and randomly assigned to 4 groups n = 10) according to the disinfectant solution: G1: control, stored in distilled water at 37 degrees C; G2: 1% sodium hypochlorite; G3: 2% glutaraldehyde; G4: 4% chlorhexidine. G2 to G4 were immersed for 60 minutes in the disinfectant solution. Measurements were carried out both before and after immersion in the solution. The surface was analyzed with a surface roughness tester (Surfcorder SE 1700 KOZAKALAB), a microdurometer FM-700 (Future Tech) and a scanning electron microscope (DSM 962-ZEISS). Loss of mass was determined with a digital weighing scale. After disinfection procedures, values were analyzed statistically. The acrylic denture base resin may be vulnerable to surface changes after in vitro immersion in the disinfectant solutions studied.

Produção de glicose e frutose por invertase de Saccharomyces cerevisiae imobilizada em suporte MANAE-agarose

Invertase from Saccharomyces cerevisiae was immobilized on agarose beads, activated with various groups (glyoxyl, MANAE or glutaraldehyde), and on some commercial epoxy supports (Eupergit and Sepabeads). Very active and stable invertase derivatives were produced by the adsorption of the enzyme on MANAE-agarose, MANAE-agarose treated with glutaraldhyde and glutaraldehyde-agarose supports. At pH 5.0, these derivatives retained full activity after 24h at 40°C and 50 °C. When assayed at 40°C and 50°C, with the pH adjusted to 7.0, the invertase-MANAE-agarose derivative treated with glutaraldehyde retained 80% of the initial activity. Recovered activities of the derivatives produced with MANAE, MANAE treated with glutaraldehyde and glutaraldehyde alone were 73.5%, 44.4% and 36.8%, respectively. These three preparations were successfully employed to produce glucose and fructose in 3 cycles of sucrose hydrolysis.