Separation of fructooligosaccharides using zeolite fixed bed columns

Recent studies have shown that the chromatographic separation of mixtures of monosaccharides and disaccharides may be improved by employing Y zeolites, a procedure which holds promise in the separation of oligosaccharides. In the present study, a column packed with zeolite was employed to study the separation of fructooligosaccharides (FOS). FOS were produced by an enzyme isolated from Rhodotorula sp., which produces GF₂ (kestose), GF₃ (nystose) and GF₄ (frutofuranosyl nystose). The identification and quantification of the sugars were carried out by ion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The separation of fructooligosaccharides was carried out using a fixed bed column packed with Ba²⁺-exchange Y zeolites. The effects of temperature (40–50 °C), injected volume per bed volume (2.55–7.64%), superficial velocity (0.1–0.15 cm min⁻¹) and eluent composition (40–60% ethanol) were investigated using a fractionary factorial design with separation efficiency as the response. The results showed that the most favorable conditions for the separation of the oligosaccharide–glucose mixture were 60% ethanol as eluent, temperature of 50 °C, superficial velocity of 0.1 cm min⁻¹ and 2.55% injection volume per bed volume of injection mixture, using two columns in series. The values for separation efficiency were 0.60 for oligosaccharide–glucose, 1.00 for oligosaccharide–fructose, 0.22 for oligosaccharide–sucrose, 0.43 for glucose–fructose, 0.82 for glucose–sucrose and 1.23 for fructose–sucrose.     

Finite element analysis of stress distribution in intact and porcelain veneer restored teeth

The aim of this study was to investigate the stress distribution generated in a veneer restoration of an upper central incisor compared to intact teeth using the finite element analysis after applying a lingual buccal loading at the incisal edge. Methods: Two models were developed: one model contained enamel, dentine, cementum, periodontal ligament, cortical and trabecullar bones, and the other model was a veneer restoration; both models were developed using MSC/Nastran software (MacNeal-Schwendler Corporation, Los Angeles, CA, USA) as the pre- and post-processor. A 10-N load was applied at the incisal edge from the lingual to the buccal side to simulate oral conditions in this area (protrusion). Results: Von Mises stresses were then analysed for three different regions: A-B (enamel elements under the veneer or second enamel layer), A'-B' (buccal enamel and/or veneer element layer) and C-D (lingual enamel elements layer). A higher stress mode was observed for both models at the lingual cervical region. Conclusions: The presence of a veneer restoration on the incisors is a good alternative to mimic the behaviour of enamel under protrusion loading conditions. The use of veneers to replace enamel during rehabilitations is recommended.     

Sperm quality of Colossoma macropomum after room‐temperature and cold storage

The objective of this study was to evaluate the effects of cold and room‐temperature storage on the quality of Colossoma macropomum sperm. The experiment was carried out in December (end of Spring), in Nova Mutum‐MT, Brazil, involving nine C. macropomum males (4 years old; 6.4 ± 1.5 kg average weight). The fish were selected and transferred to masonry tanks (4 m³) in a laboratory (water renewal rate: 10 L/s; average water temperature: 28°C). Subsequently, reproduction was induced using 2.5 mg of crude carp pituitary extract/kg and the semen was harvested 240 degree hours after hormonal induction. The following sperm characteristics were analyzed every 5 hr using Image J/casa software: total motility (MOT), curvilinear velocity (VCL), average path velocity (VAP), straight‐line velocity (VSL), straightness of sperm path (STR), wobble (WOB), progressive motility (PROG), beat cross frequency (BCF) and total number of spermatozoa (NSPZ). A fresh sample of semen from each animal was kept at room temperature (25.3 ± 1.2°C). For analysis of cooled semen, syringes were kept in cooling boxes at an average temperature of 16.9 ± 2.1°C. The reduction (p < 0.05) of MOT in semen kept at room temperature occurred at 10 hr (13.95%); in cooled semen, however, MOT declined at 15 hr (76.87%). At 15 hr, there was practically no MOT in the semen kept at room temperature (0.20%), whereas in the cooled semen this situation was observed only at 35 hr (2.91%) The MOT of cooled sperm was higher (p < 0.05) at all times (except zero time), compared with the semen maintained at room temperature. At 15 hr, the cooled spermatozoa showed higher (p < 0.05) VCL (142.18 μm/s) and BCF (29.72 Hz) than those maintained at room temperature (VCL: 51.18 μm/s; BCF: 19.57 Hz). After 15 hr, only the cooled sperm showed quality. In conclusion, semen cooling allows for extending the viability of C. macropomum spermatozoa from 5 to 10 hr without compromising their quality in most characteristics. At 15 and 25 hr of cooling, sperm viability is still observed, though with decreased quality.     

Brain bank of the Brazilian aging brain study group—a milestone reached and more than 1,600 collected brains

INTRODUCTION: Brain banking remains a necessity for the study of aging brain processes and related neurodegenerative diseases. In the present paper, we report the methods applied at and the first results of the Brain Bank of the Brazilian Aging Brain Study Group (BBBABSG) which has two main aims: (1) To collect a large number of brains of elderly comprising non-demented subjects and a large spectrum of pathologies related to aging brain processes, (2) To provide quality material to a multidisciplinar research network unraveling multiple aspects of aging brain processes and related neurodegenerative diseases. METHODS: The subjects are selected from the Sao Paulo Autopsy Service. Brain parts are frozen and fixated. CSF, carotids, kidney, heart and blood are also collected and DNA is extracted. The neuropathological examinations are carried out based on accepted criteria, using immunohistochemistry. Functional status are assessed through a collateral source based on a clinical protocol. Protocols are approved by the local ethics committee and a written informed consent form is obtained. RESULTS: During the first 21 months, 1,602 samples were collected and were classified by Clinical Dementia Rating as CDR0: 65.7%; CDR0.5:12.6%, CDR1:8.2%, CDR2:5.4%, and CDR3:8.1%. On average, the cost for the processing each case stood at 400 US dollars. To date, 14 laboratories have been benefited by the BBBABSG. CONCLUSION: The high percentage of non- demented subjects and the ethnic diversity of this series may be significantly contributive toward aging brain processes and related neurodegenerative diseases understanding since BBBABSG outcomes may provide investigators the answers to some additional questions.     

Microbiological quality of water and dialysate in a haemodialysis unit in Ponta Grossa-PR, Brazil

AIMS: The objective of the study was to determine the microbiological quality of samples of water and dialysate in a haemodialysis unit. METHODS AND RESULTS: Seventy-two samples each of water and dialysate were collected during November 2003 to April 2004. The following microbiological analyses were performed: test for total and faecal coliforms, which produced negative results for all the samples; counts of total heterotrophic bacteria, where three samples of water and two of dialysate showed levels higher than those permitted by national standards; and endotoxin assay, which revealed high quantities only in samples of water that preceded reverse osmosis. Nonfermenting Gram-negative bacteria were identified in 54 samples of dialysate and in 26 samples of water. The test for adhesion to an inert surface showed that various bacteria were capable of forming biofilms. Twenty-seven per cent of the bacteria were resistant to sodium hypochlorite at 500 ppm for 10-min contact time. Sixty per cent of the isolates were resistant to three or more antibiotics. CONCLUSIONS: Water and dialysate can be a source of infection for patients who need haemodialysis. SIGNIFICANCE AND IMPACT OF THE STUDY: An adequate system for water treatment, disinfection of the haemodialysis system and microbiological monitoring of the water and dialysate are necessary to reduce bacteraemia and pyrogenia outbreaks.     

DNA integrity and transgene expression after passage through the NOGA needle catheter used for therapeutic myocardial angiogenesis

BACKGROUND: The NOGA (Biosense Webster, Markham, ON, Canada) injection catheter is an innovative navigational device that provides an ideal platform for intra-myocardial injection material. However, injection through a long (1.91 m), narrow (27G) nitinol needle could result in deterioration in the integrity and functionality of DNA. METHODS: To test this possibility, DNA in plasmid form (pcDNA3.1) containing the Lac Z transgene (250 micro l) was passed through the NOGA needle using a hand-held 1 cc syringe at a gentle hand injection pressure (43 +/- 3 PSI, 3.0 +/- 0.2 kg/cm(2)) or at maximal manual pressure (90 +/- 6 PSI, 6.3 +/- 0.4 kg/cm(2)), either once or 20 times. This DNA, compared to DNA not passed through the NOGA needle (control), was then used to transfect primary cultures of rat skin fibroblasts (FB) from Fisher 344 rats and the cells were subsequently stained for beta galactosidase (betagal). RESULTS: Transfection efficiency was significantly reduced by passing the DNA through the needle at both 43 +/- 3 PSI (78 +/- 4% of control, n = 10, P < 0.05 versus control) and 90 +/- 6 PSI (66 +/- 4 % of control, n = 10, P < 0.01 versus control, P < 0.02 versus 43 +/- 3 PSI). Passage of the DNA through the NOGA needle 20 times resulted in a transfection efficiency of only 5 +/- 1% of control (n = 20, P < 0.1 x 10(-11) versus control). Capillary Electrophoresis revealed that the reduction in transfection efficiency was due to a conformational change in the DNA from predominantly supercoiled to nicked and linearized DNA. Transfection efficiency as compared with control decreased as the concentration of the DNA solution which was passed through the needle was increased from 0.3 micro g/ micro l to 2.4 micro g/ micro l. Recovery experiments confirmed that the reduction in transfection efficiency was not due to loss of DNA by binding to the NOGA needle. CONCLUSION: These results suggest that DNA is susceptible to shear forces when injected through the NOGA needle even at nominal clinical injection pressures, suggesting that careful and controlled injections will be required to achieve optimal gene integrity and expression.     

Simulation of integrated first and second generation bioethanol production from sugarcane: comparison between different biomass pretreatment methods

Sugarcane bagasse is used as a fuel in conventional bioethanol production, providing heat and power for the plant; therefore, the amount of surplus bagasse available for use as raw material for second generation bioethanol production is related to the energy consumption of the bioethanol production process. Pentoses and lignin, byproducts of the second generation bioethanol production process, may be used as fuels, increasing the amount of surplus bagasse. In this work, simulations of the integrated bioethanol production process from sugarcane, surplus bagasse and trash were carried out. Selected pre-treatment methods followed, or not, by a delignification step were evaluated. The amount of lignocellulosic materials available for hydrolysis in each configuration was calculated assuming that 50% of sugarcane trash is recovered from the field. An economic risk analysis was carried out; the best results for the integrated first and second generation ethanol production process were obtained for steam explosion pretreatment, high solids loading for hydrolysis and 24–48 h hydrolysis. The second generation ethanol production process must be improved (e.g., decreasing required investment, improving yields and developing pentose fermentation to ethanol) in order for the integrated process to be more economically competitive.     

Biochemical,physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves

Background

Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H₂O₂ and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx).

Methods

Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography.

Results

Vu-2-Cys-Prx reduces H₂O₂ using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56–4.72; its NH₂-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% β-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys⁵² residue and the amino acids Pro⁴⁵, Thr⁴⁹ and Arg¹²⁸ are conserved as in other 2-Cys-Prx.

General significance

The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.     

Patterns of population genetic variation in sympatric chiltoniid amphipods within a calcrete aquifer reveal a dynamic subterranean environment

Calcrete aquifers from the Yilgarn region of arid central Western Australia contain an assemblage of obligate groundwater invertebrate species that are each endemic to single aquifers. Fine-scale phylogeographic and population genetic analyses of three sympatric and independently derived species of amphipod (Chiltoniidae) were carried out to determine whether there were common patterns of population genetic structure or evidence for past geographic isolation of populations within a single calcrete aquifer. Genetic diversity in amphipod mitochondrial DNA (cytochrome c oxidase subunit I gene) and allozymes were examined across a 3.5 km² region of the Sturt Meadows calcrete, which contains a grid of 115 bore holes (=wells). Stygobiont amphipods were found to have high levels of mitochondrial haplotype diversity coupled with low nucleotide diversity. Mitochondrial phylogeographic structuring was found between haplogroups for one of the chiltoniid species, which also showed population structuring for nuclear markers. Signatures of population expansion in two of the three species, match previous findings for diving beetles at the same site, indicating that the system is dynamic. We propose isolation of populations in refugia within the calcrete, followed by expansion events, as the most likely source of intraspecific genetic diversity, due to changes in water level influencing gene flow across the calcrete.