Isolation of a ß-galactoside-binding lectin from cat liver

A lectin from cat liver has been identified and purified by affinity chromatography on asialofetuin-Sepharose. One hundred micrograms of lectin was obtained from one cat liver with a purification factor of 1561. The lectin agglutinates trypsin-treated rabbit and cow erythrocytes. Hemagglutination was inhibited only by saccharides containing ß-galactosyl residues, of which the 1-amine-1-deoxy-ß-D-galactose was the most potent one by inhibiting hemagglutination at a concentration of 12.5 mM, followed by melibiose, trehalose and galactose. The lectin has a subunit molecular mass of 14.4 kDa determined by SDS-PAGE under reducing conditions and a pI of 4.85. Compared with the composition of lectins from calf heart and porcine heart, cat liver lectin contains approximately the same amount of cysteine, half the amount of glycine, twice as much arginine and threonine, and three times the amounts of tyrosine and methionine. Cat liver lectin contains four cysteine residues per subunit, all of them in the reduced form. Their lack of reactivity towards thiol-reactive supports suggests they are not exposed on the lectin surface. The protein apparently has a blocked N-terminus. The purified lectin was stable for up to 20 months stored at +4ºC in buffer supplemented with 4 mM ß-mercaptoethanol. Results indicated that this lectin belongs to the family of soluble ß-galactoside-binding lectins, also known as galectins, which are expressed in a wide range of vertebrate tissues.

Isolation and culture of umbilical vein mesenchymal stem cells

Bone marrow contains a population of stem cells that can support hematopoiesis and can differentiate into different cell lines including adipocytes, osteocytes, chondrocytes, myocytes, astrocytes, and tenocytes. These cells have been denoted mesenchymal stem cells. In the present study we isolated a cell population derived from the endothelium and subendothelium of the umbilical cord vein which possesses morphological, immunophenotypical and cell differentiation characteristics similar to those of mesenchymal stem cells isolated from bone marrow. The cells were isolated from three umbilical cords after treatment of the umbilical vein lumen with collagenase. The cell population isolated consisted of adherent cells with fibroblastoid morphology which, when properly stimulated, gave origin to adipocytes and osteocytes in culture. Immunophenotypically, this cell population was found to be positive for the CD29, CD13, CD44, CD49e, CD54, CD90 and HLA-class 1 markers and negative for CD45, CD14, glycophorin A, HLA-DR, CD51/61, CD106, and CD49d. The characteristics described are the same as those presented by bone marrow mesenchymal stem cells. Taken together, these findings indicate that the umbilical cord obtained from term deliveries is an important source of mesenchymal stem cells that could be used in cell therapy protocols.

Routine post-weaning handling of rats prevents isolation rearing-induced deficit in prepulse inhibition

Rats reared under isolation conditions from weaning present a number of behavioral changes compared to animals reared under social conditions (group housing). These changes include deficits in prepulse inhibition (PPI) of the startle reflex to a loud sound. PPI refers to the reduction of the magnitude of the startle reflex when a relatively weak stimulus (the prepulse) precedes by an appropriate time interval the intense startle-elicing stimulus (the pulse). PPI is useful for studying sensorimotor integration. The present study evaluated the effect of handling on the impairment of PPI induced by isolation-rearing. Male Wistar rats (N = 11-15/group) were housed in groups (5 per cage and handled three times a week) or isolated (housed individually) since weaning (21 days) for 10 weeks when they reach approximately 150 g. The isolated rats were divided into "minimally handled" animals (handled once a week for cleaning purposes only) or "handled" animals (handled three times a week). This handling consisted of grasping the rat by the tail and moving it to a clean cage (approximately 5 s). A statistically significant reduction (52%) in the PPI test was found only in the isolated group with minimal handling while no difference was seen between grouped animals and isolated handled animals. These results indicate that isolation rearing causes disruption in the PPI at adult age, which serves as an index of attention deficit. This change in the sensory processing of information induced by post-weaning isolation can be prevented by handling during the development of the animal.

Automated grading of left ventricular segmental wall motion by an artificial neural network using color kinesis images

The present study describes an auxiliary tool in the diagnosis of left ventricular (LV) segmental wall motion (WM) abnormalities based on color-coded echocardiographic WM images. An artificial neural network (ANN) was developed and validated for grading LV segmental WM using data from color kinesis (CK) images, a technique developed to display the timing and magnitude of global and regional WM in real time. We evaluated 21 normal subjects and 20 patients with LVWM abnormalities revealed by two-dimensional echocardiography. CK images were obtained in two sets of viewing planes. A method was developed to analyze CK images, providing quantitation of fractional area change in each of the 16 LV segments. Two experienced observers analyzed LVWM from two-dimensional images and scored them as: 1) normal, 2) mild hypokinesia, 3) moderate hypokinesia, 4) severe hypokinesia, 5) akinesia, and 6) dyskinesia. Based on expert analysis of 10 normal subjects and 10 patients, we trained a multilayer perceptron ANN using a back-propagation algorithm to provide automated grading of LVWM, and this ANN was then tested in the remaining subjects. Excellent concordance between expert and ANN analysis was shown by ROC curve analysis, with measured area under the curve of 0.975. An excellent correlation was also obtained for global LV segmental WM index by expert and ANN analysis (R² = 0.99). In conclusion, ANN showed high accuracy for automated semi-quantitative grading of WM based on CK images. This technique can be an important aid, improving diagnostic accuracy and reducing inter-observer variability in scoring segmental LVWM.

Isolation and intracellular localization of insulin-like proteins from leaves of Bauhinia variegata

Evidence based on immunological cross-reactivity and anti-diabetic properties has suggested the presence of insulin-like peptides in plants. The objective of the present study was to investigate the presence of insulin-like proteins in the leaves of Bauhinia variegata ("pata-de-vaca", "mororó"), a plant widely utilized in popular medicine as an anti-diabetic agent. We show that an insulin-like protein was present in the leaves of this plant. A chloroplast protein with a molecular mass similar to that of bovine insulin was extracted from 2-mm thick 15% SDS-PAGE gels and fractionated with a 2 x 24 cm Sephadex G-50 column. The activity of this insulin-like protein (0.48 mg/mL) on serum glucose levels of four-week-old Swiss albino (CF1) diabetic mice was similar to that of commercial swine insulin used as control. Further characterization of this molecule by reverse-phase hydrophobic HPLC chromatographic analysis as well as its antidiabetic activity on alloxan-induced mice showed that it has insulin-like properties. Immunolocalization of the insulin-like protein in the leaves of B. variegata was performed by transmission electron microscopy using a polyclonal anti-insulin human antibody. Localization in the leaf blades revealed that the insulin-like protein is present mainly in chloroplasts where it is also found associated with crystals which may be calcium oxalate. The presence of an insulin-like protein in chloroplasts may indicate its involvement in carbohydrate metabolism. This finding has strengthened our previous results and suggests that insulin-signaling pathways have been conserved through evolution.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

Nutritional status and body composition after 6 months of patients switching from continuous ambulatorial peritoneal dialysis to automated peritoneal dialysis

Our objective was to determine if automated peritoneal dialysis (APD) leads to changes in nutritional parameters of patients treated by continuous ambulatory peritoneal dialysis (CAPD). Twenty-six patients (15 males; 50.5 ± 14.3 years) were evaluated during CAPD while training for APD and after 3 and 6 months of APD. Body fat was assessed by the sum of skinfold thickness and the other body compartments were assessed by bioelectrical impedance. During the 6-month follow-up, 12 patients gained more than 1 kg (GW group), 8 patients lost more than 1 kg (LW group), and 6 patients maintained body weight (MW group). Except for length on dialysis that was longer for the LW group compared with the GW group, no other differences were found between the groups at baseline. After 6 months on APD, the LW group had a reduction in body fat (24.5 ± 7.7 vs 22.1 ± 7.3 kg; P = 0.01), body cell mass (22.6 ± 6.2 vs 21.6 ± 5.8 kg, P = 0.02) and phase angle (5.4 ± 0.9 vs 5.1 ± 0.8 degrees, P = 0.004). In the GW group, body fat (25 ± 7.6 vs 27.2 ± 7.6 kg, P = 0.001) and body cell mass (20.1 ± 3.9 vs 20.8 ± 4.0 kg, P = 0.05) were increased. In the present study, different patterns of change in body composition were found. The length of previous dialysis treatment seems to be the most important factor in determining these nutritional modifications.

Study of bacteria Gluconobacter sp.: isolation, purification, phenotypic and molecular identification

The importance of the study of acetic bacteria, on species of the Gluconobacter genus is based on its industrial application, as these possess the capacity of bioconversion of sorbitol to sorbose, enabling the process of vitamin C production. The study involved samples collected in industries of soft drinks, flowers, fruits and honey, followed by purification, phenotypic identification, molecular identification with the use of primer defined from Nucleotide Sequence Database consultation. Strains preserved were identified as members of the Acetobacteraceae family, Gluconobacter genus. 110 strains had been isolated of substrate: Pyrostegia venusta (ker-gawler), honey, Vitis vinifera (grape), Pyrus communis (pear), Malus sp. (apple) and in two samples of soft drinks. Of this total 57 strains had been recovered in manitol medium (manitol, yeast extract, peptone), 12 in YMG medium (glucose, manitol, yeast extract, ethanol, acetic acid), 41 in enrichment medium (De Ley and Swings) and later in the GYC medium (glucose, yeast extract and calcium carbonate). 68 strains were identified as Gram negative bacilli rods. Of these, 31 were characterized biochemically as belonging to the Acetobacteriaceae family as they were catalase positive, oxidase negative and producers of acid from glucose. The characterization of these strains was complemented with the biochemistry tests: gelatin liquefaction, nitrate reduction, indole and H2S production, oxidation of ethanol to acetic acid and molecular tests for genus identification. Only eight strains were characterized as pertaining to the Gluconobacter genus. The strains are maintained in collection cultures at the Microbiology Laboratory of the Biology Department at the São Paulo State University (UNESP) in Assis, stored in malt extract at -196 ºC.

Anthocyanins standards (cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside) isolation from freeze-dried açaí (Euterpe oleraceae Mart.) by HPLC

Availability of analytical standards is a critical aspect in developing methods for quantitative analysis of anthocyanins. The anthocyanins cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside were isolated from samples of freeze-dried açaí (Euterpe oleraceae Mart.), which is a round and purple well-known palm fruit in Brazil, and then used as standards. The isolation of the anthocyanins was performed by High Performance Liquid Chromatography (HPLC), using an adapted six-channel selection valve. The identification of anthocyanin pigments in açaí was based on mass spectrometric data for molecular ions and MS-MS product ions and on previous published data. After the collection procedure, standards with a high purity grade were obtained and an external standard curve of each anthocyanin was plotted.

Isolation and recovery of glycomacropeptide from milk whey by means of thermal treatment

During enzymatic process of cheese manufacturing, rennin cleaves κ-casein releasing two fractions: para-κ-casein and glycomacropeptide (GMP), which remains soluble in milk whey. GMP is a peptide with structural particularities such as chain carbohydrates linked to specific threonine residues, to which a great variety of biological activities is attributed. Worldwide cheese production has increased generating high volumes of milk whey that could be efficiently used as an alternative source of high quality peptide or protein in foodstuff formulations. In order to evaluate isolation and recovery on whey GMP by means of thermal treatment (90 °C), 18 samples (2 L each) of sweet whey, resuspended commercial whey (positive control) and acid whey (negative control) were processed. Indirect presence of GMP was verified using chemical tests and PAGE-SDS 15%. At 90 °C treated sweet whey, 14, 20 and 41 kDa bands were observed. These bands may correspond to olygomers of GMP. Peptide recovery showed an average of 1.5 g/L (34.08%). The results indicate that industrial scale GMP production is feasible; however, further research must be carried out for the biological and nutritional evaluation of GMP's incorporation to foodstuff as a supplement.

Isolation and characterization of bacterial strains with a hydrolytic profile with potential use in bioconversion of agroindustial by-products and waste

There is a trend towards the use of novel technologies nowadays, mainly focused on biological processes, for recycling and the efficient utilization of organic residues that can be metabolized by different microorganisms as a source of energy. In the present study the isolation of bacterial strains from six different agro-industrial by-products and waste was performed with the objective of evaluating their hydrolytic capacities and suitability for use in bioconversion of specific substrates. The 34 isolated strains were screened in specific culture media for the production of various hydrolytic enzymes (lipase, protease, cellulase, and amylase). It was found that 28 strains exhibited proteolytic activity, 18 had lipolytic activity, 13 had caseinolytic activity, 15 had amylolytic activity, and 11 strains exhibited cellulolytic activity. The strains that showed the highest hydrolytic capacities with biotechnological potential were selected, characterized genotipically, and identified as Bacillus, Serratia, Enterococcus, Klebsiella, Stenotrophomonas, Lactococcus, and Escherichia genera. It was concluded that the strain isolates have a high potential for use in the bioconversion of agro-industrial waste, both as a pure culture and as a microbial consortium.

Isolation of a yeast strain able to produce a polygalacturonase with maceration activity of cassava roots

The objective of the present study was the isolation of a yeast strain, from citrus fruit peels, able to produce a polygalacturonase by submerged fermentation with maceration activity of raw cassava roots. Among 160 yeast strains isolated from citrus peels, one strain exhibited the strongest pectinolytic activity. This yeast was identified as Wickerhamomyces anomalus by 5.8S-ITS RFLP analysis and confirmed by amplification of the nucleotide sequence. The yeast produced a polygalacturonase (PG) in Erlenmeyer shake flasks containing YNB, glucose, and citrus pectin. PG synthesis occurred during exponential growth phase, reaching 51 UE.mL-1 after 8 hours of fermentation. A growth yield (Yx/s) of 0.43 gram of cell dry weight per gram of glucose consumed was obtained, and a maximal specific growth rate (µm) of 0.346 h-1 was calculated. The microorganism was unable to assimilate sucrose, galacturonic acid, polygalacturonic acid, or citrus pectin, but it required glucose as carbon and energy source and polygalacturonic acid or citrus pectin as inducers of enzyme synthesis. The crude enzymatic extract of Wickerhamomyces anomalus showed macerating activity of raw cassava. This property is very important in the production of dehydrated mashed cassava, a product of regional interest in the province of Misiones, Argentina.

Automated static image analysis as a novel tool in describing the physical properties of dietary fiber

Abstract The growing interest in the usage of dietary fiber in food has caused the need to provide precise tools for describing its physical properties. This research examined two dietary fibers from oats and beets, respectively, in variable particle sizes. The application of automated static image analysis for describing the hydration properties and particle size distribution of dietary fiber was analyzed. Conventional tests for water holding capacity (WHC) were conducted. The particles were measured at two points: dry and after water soaking. The most significant water holding capacity (7.00 g water/g solid) was achieved by the smaller sized oat fiber. Conversely, the water holding capacity was highest (4.20 g water/g solid) in larger sized beet fiber. There was evidence for water absorption increasing with a decrease in particle size in regards to the same fiber source. Very strong correlations were drawn between particle shape parameters, such as fiber length, straightness, width and hydration properties measured conventionally. The regression analysis provided the opportunity to estimate whether the automated static image analysis method could be an efficient tool in describing the hydration properties of dietary fiber. The application of the method was validated using mathematical model which was verified in comparison to conventional WHC measurement results.

Automated sugar analysis

Abstract Sugarcane monosaccharides are reducing sugars, and classical analytical methodologies (Lane-Eynon, Benedict, complexometric-EDTA, Luff-Schoorl, Musson-Walker, Somogyi-Nelson) are based on reducing copper ions in alkaline solutions. In Brazil, certain factories use Lane-Eynon, others use the equipment referred to as “REDUTEC”, and additional factories analyze reducing sugars based on a mathematic model. The objective of this paper is to understand the relationship between variations in millivolts, mass and tenors of reducing sugars during the analysis process. Another objective is to generate an automatic model for this process. The work herein uses the equipment referred to as “REDUTEC”, a digital balance, a peristaltic pump, a digital camcorder, math programs and graphics programs. We conclude that the millivolts, mass and tenors of reducing sugars exhibit a good mathematical correlation, and the mathematical model generated was benchmarked to low-concentration reducing sugars (<0.3%). Using the model created herein, reducing sugars analyses can be automated using the new equipment.

Salmonella peritonitis in a patient on automated peritoneal dialysis

We describe the clinical course of a case of peritonitis caused by Salmonella sp. after an episode of intestinal salmonellosis, and a brief review of the literature is also done.