Ultrastructural characterization of bovine umbilical cord blood cells

The umbilical cord blood (UCB) is an important source of pluripotent stem cells, which motivated researches on ontogeny and transplantation. The morphological characterization of umbilical cord cells is the first step to establish subsequent experiments on these areas. Although some information on humans can be found, no data on UCB is available for bovines. Therefore, this work is the first attempt to conduct an ultrastructural characterization of bovine umbilical cord blood. Blood was collected from the umbilical cord of twenty fetuses by punction of the umbilical vein. Samples were processed for whole leucocytes observation by centrifugation and the buffy coat was collected. Cells were washed and pelleted and prepared according to the standard protocol of the transmission electron microscopy. The presence of cells with morphologic characteristics compatible with the precursors from the erythrocytic, neutrophilic, eosinophilic, basophilic, and lymphocytic lineages was observed. Atypical cells with peculiar morphological features, strongly similar to apoptotic cells, were seen. Bovine neutrophils with three types of cytoplasmic granules were also found in the blood. The ultrastructural characteristics of observed bovine UCB cells where similar to those found in other species, suggesting that bovines could possibly constitute an experimental model for approaches on UCB cells research.

Method to obtain platelet-rich plasma from rabbits (Oryctolagus cuniculus )

Abstract: Platelet-rich plasma (PRP) is a product easy and inxpesnsive, and stands out to for its growth factors in tissue repair. To obtain PRP, centrifugation of whole blood is made with specific time and gravitational forces. Thus, the present work aimed to study a method of double centrifugation to obtain PRP in order to evaluate the effective increase of platelet concentration in the final product, the preparation of PRP gel, and to optimize preparation time of the final sample. Fifteen female White New Zealand rabbits underwent blood sampling for the preparation of PRP. Samples were separated in two sterile tubes containing sodium citrate. Tubes were submitted to the double centrifugation protocol, with lid closed and 1600 revolutions per minute (rpm) for 10 minutes, resulting in the separation of red blood cells, plasma with platelets and leucocytes. After were opened and plasma was pipetted and transferred into another sterile tube. Plasma was centrifuged again at 2000rpm for 10 minutes; as a result it was split into two parts: on the top, consisting of platelet-poor plasma (PPP) and at the bottom of the platelet button. Part of the PPP was discarded so that only 1ml remained in the tube along with the platelet button. This material was gently agitated to promote platelets resuspension and activated when added 0.3ml of calcium gluconate, resulting in PRP gel. Double centrifugation protocol was able to make platelet concentration 3 times higher in relation to the initial blood sample. The volume of calcium gluconate used for platelet activation was 0.3ml, and was sufficient to coagulate the sample. Coagulation time ranged from 8 to 20 minutes, with an average of 17.6 minutes. Therefore, time of blood centrifugation until to obtain PRP gel took only 40 minutes. It was concluded that PRP was successfully obtained by double centrifugation protocol, which is able to increase the platelet concentration in the sample compared with whole blood, allowing its use in surgical procedures. Furthermore, the preparation time is appropriate to obtain PRP in just 40 minutes, and calcium gluconate is able to promote the activation of platelets.

Sorption and desorption of picloram in soils under pastures in Brazil

The objective of this work was to determine the coefficients of sorption and desorption of picloram in Ultisol (PVA) and Oxisol (LVA), displaying different physical and chemical characteristics. Samples of soil were collected at the 0 20 cm depth in degraded pasture areas in Viçosa-MG. Firstly, the equilibrium time between the herbicide in solution and the herbicide which was sorbed in the soil was determined by the Batch Equilibrium method. The time required was 24 hours. Sorption and desorption studies were carried out under controlled laboratory conditions; the sorption evaluation consisted in adding 10.0 mL of herbicide solutions at different concentrations to tubes containing 2.00 g of soil, with vertical rotary agitation being maintained during the pre-determined equilibrium time. After centrifugation, supernatant extract cleaning and filtration, herbicide concentration was determined by high performance liquid chromatography (HPLC) with UV detection at 254 nm. Desorption was evaluated using the samples in the tubes after the sorption tests. The Freundlich model was used for interpretation of the sorption process. Ultisol showed higher adsorption coefficient (Kf a) compared with Oxisol, which may be attributed to the lower pH of the soil and its higher organic matter content. Desorption process occurred in both soils; the LVA allowed greater release of the previously sorbed molecules.

The interplay of JNK and SCG10 during cortical development and in excitotoxic responses in brain

Initially identified as stress activated protein kinases (SAPKs), the c-Jun Nterminal kinases (JNKs) are currently accepted as potent regulators of various physiologically important cellular events. Named after their competence to phosphorylate transcription factor c-Jun in response to UVtreatment, JNKs play a key role in cell proliferation, cell death or cell migration. Interestingly, these functions are crucial for proper brain formation. The family consists of three JNK isoforms, JNK1, JNK2 and JNK3. Unlike brain specific JNK3 isoform, JNK1 and JNK2 are ubiquitously expressed. It is estimated that ten splice variants exist. However, the detailed cellular functions of these remain undetermined. In addition, physiological conditions keep the activities of JNK2 and JNK3 low in comparison with JNK1, whereas cellular stress raises the activity of these isoforms dramatically. Importantly, JNK1 activity is constitutively high in neurons, yet it does not stimulate cell death. This suggests a valuable role for JNK1 in brain development, but also as an important mediator of cell wellbeing. The aim of this thesis was to characterize the functional relationship between JNK1 and SCG10. We found that SCG10 is a bona fide target for JNK. By employing differential centrifugation we showed that SCG10 co-localized with active JNK, MKK7 and JIP1 in a fraction containing endosomes and Golgi vesicles. Investigation of JNK knockout tissues using phosphospecific antibodies recognizing JNK-specific phosphorylation sites on SCG10 (Ser 62/Ser 73) showed that phosphorylation of endogenous SCG10 was dramatically decreased in Jnk1-/- brains. Moreover, we found that JNK and SCG10 co-express during early embryonic days in brain regions that undergo extensive neuronal migration. Our study revealed that selective inhibition of JNK in the cytoplasm significantly increased both the frequency of exit from the multipolar stage and radial migration rate. However, as a consequence, it led to ill-defined cellular organization. Furthermore, we found that multipolar exit and radial migration in Jnk1 deficient mice can be connected to changes in phosphorylation state of SCG10. Also, the expression of a pseudo-phosphorylated mutant form of SCG10, mimicking the JNK1- phopshorylated form, brings migration rate back to normal in Jnk1 knockout mouse embryos. Furthermore, we investigated the role of SCG10 and JNK in regulation of Golgi apparatus (GA) biogenesis and whether pathological JNK action could be discernible by its deregulation. We found that SCG10 maintains GA integrity as with the absence of SCG10 neurons present more compact fragmented GA structure, as shown by the knockdown approach. Interestingly, neurons isolated from Jnk1-/- mice show similar characteristics. Block of ER to GA is believed to be involved in development of Parkinson's disease. Hence, by using a pharmacological approach (Brefeldin A treatment), we showed that GA recovery is delayed upon removal of the drug in Jnk1-/- neurons to an extent similar to the shRNA SCG10-treated cells. Finally, we investigated the role of the JNK1-SCG10 duo in the maintenance of GA biogenesis following excitotoxic insult. Although the GA underwent fragmentation in response to NMDA treatment, we observed a substantial delay in GA disintegration in neurons lacking either JNK1 or SCG10.

In vitro radioautographic studies of the biodistribution of radiopharmaceuticals on blood elements

In the present study we evaluated the binding of the radiopharmaceuticals sodium pertechnetate (Na 99mTcO4), methylenediphosphonic acid (99mTc-MDP) and glucoheptonate acid (99mTc-GHA) to blood elements using centrifugation and radioautographic techniques. Heparinized blood was incubated with the labelled compounds for 0, 1, 2, 3, 4, 6 and 24 h. Plasma (P) and blood cells (BC) were isolated and precipitated with 5% trichloroacetic acid (TCA), and soluble (SF) and insoluble fractions (IF) were separated. Blood samples were prepared (0 and 24 h) and coated with LM-1 radioautographic emulsions and percent radioactivity (%rad) in P and BC was determined. The binding of Na 99mTcO4 (%rad) to P was 61.2% (0 h) and 46.0% (24 h), and radioautography showed 63.7% (0 h) and 43.3% (24 h). The binding to BC was 38.8% (0 h) and 54.0% (24 h), and radioautography showed 36.3% (0 h) and 56.7% (24 h). 99mTc-MDP study presented 91.1% (0 h) to P and 87.2% (24 h), and radioautography showed 67.9% (0 h) and 67.4% (24 h). The binding to BC was 8.9% (0 h) and 12.8% (24 h), and radioautography showed 32.1% (0 h) and 32.6% (24 h). 99mTc-GHA study was 90.1% (0 h) to P and 79.9% (24 h), and radioautography showed 67.2% (0 h) and 60.1% (24 h). The binding to BC was 9.9% (0 h) and 20.1% (24 h), and radioautography showed 32.8% (0 h) and 39.9% (24 h). The comparison of the obtained results suggests that the binding to plasma and blood cells in the two techniques used (radioautography and centrifugation) is qualitatively in accordance

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.

An improved HPLC method for the quantitation of 6-mercaptopurine and its metabolites in red blood cells

A procedure is described for the rapid determination of the intra-erythrocyte concentration of 6-mercaptopurine (6-MP) and its metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). Erythrocytes (8 x 10(8) cells) in 350 µl Hanks solution containing 7.5 mg dithiothreitol were treated with 50 µl 70% perchloric acid. The precipitate was removed by centrifugation (13,000 g) and the supernatant hydrolyzed at 100°C for 45 min. After cooling, 100 µl was analyzed directly by HPLC using a Radialpack Resolve C18 column eluted with methanol-water (7.5:92.5, v/v) containing 100 mM triethylamine. 6-TG, 6-MP and the hydrolysis product of 6-MMP, 4-amino-5-(methylthio)carbonyl imidazole, were monitored at 342, 322 and 303 nm using a Shimadzu SPD-M10A diode array UV detector. The analytes eluted at 5.3, 6.0 and 10.2 min, respectively. The calibration curves were linear (r² > 0.998), and the analytical recoveries were 73.2% for 6-TG, 119.1% for 6-MP and 97.4% for 6-MMP. The intra- and inter-assay variations were highest for 6-MP (9.6 and 14.3%, respectively). The lowest detectable concentrations were 3, 3 and 25 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The quantification limits (coefficients of variation <15%) were 8, 10 and 70 pmol/8 x 10(8) erythrocytes for 6-TG, 6-MP and 6-MMP, respectively. The method was applied to the analysis of 183 samples from 36 children under chemotherapy for acute lymphoblastic leukemia. The concentrations of the metabolites in the red cells of the patients ranged from 0 to 1934 pmol/8 x 10(8) erythrocytes for 6-TGN, and from 0 to 105.8 and 0 to 45.9 nmol/8 x 10(8) erythrocytes for 6-MP and 6-MMP, respectively. The procedure gave results that were in agreement with those obtained with other methods designed to detect cases of non-compliance with treatment, including patient interviews and medical evaluation, among others, demonstrating its applicability to monitoring the treatment of leukemic children.

Enucleated L929 mouse fibroblasts support invasion and multiplication of Shigella flexneri 5a

Invasive bacteria can induce their own uptake and specify their intracellular localization; hence it is commonly assumed that proximate modulation of host cell transcription is not required for infection. However, bacteria can also modulate, directly or indirectly, the transcription of many host cell genes, whose role in the infection may be difficult to determine by global gene expression. Is the host cell nucleus proximately required for intracellular infection and, if so, for which pathogens and at what stages of infection? Enucleated cells were previously infected with Toxoplasma gondii, Chlamydia psittaci, C. trachomatis, or Rickettsia prowazekii. We enucleated L929 mouse fibroblasts by centrifugation in the presence of cytochalasin B, and compared the infection with Shigella flexneri M90T 5a of nucleated and enucleated cells. Percent infection and bacterial loads were estimated with a gentamicin suppression assay in cultures fixed and stained at different times after infection. Enucleation reduced by about half the percent of infected cells, a finding that may reflect the reduced endocytic ability of L929 cytoplasts. However, average numbers of bacteria and frequency distributions of bacterial numbers per cell at different times were similar in enucleated and nucleated cells. Bacteria with actin-rich tails were detected in both cytoplasts and nucleated cells. Lastly, cytoplasts were similarly infected 2 and 24 h after enucleation, suggesting that short-lived mRNAs were not involved in the infection. Productive S. flexneri infection could thus take place in cells unable to modulate gene transcription, RNA processing, or nucleus-dependent signaling cascades.

Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

Osteogenesis induced in goat bone marrow progenitor cells by recombinant adenovirus coexpressing bone morphogenetic protein 2 and basic fibroblast growth factor

Bone morphogenetic protein 2 (BMP2) and basic fibroblast growth factor (bFGF) have been shown to exhibit a synergistic effect to promote bone repair and healing. In this study, we constructed a novel adenovirus with high coexpression of BMP2 and bFGF and evaluated its effect on osteogenic differentiation of goat bone marrow progenitor cells (BMPCs). Recombinant adenovirus Ad-BMP2-bFGF was constructed by using the T2A sequence. BMPCs were isolated from goats by density gradient centrifugation and adherent cell culture, and were then infected with Ad-BMP2-bFGF or Ad-BMP2. Expression of BMP2 and bFGF was detected by ELISA, and alkaline phosphatase (ALP) activity was detected by an ALP assay kit. In addition, von Kossa staining and immunocytochemical staining of collagen II were performed on BMPCs 21 days after infection. There was a high coexpression of BMP2 and bFGF in BMPCs infected with Ad-BMP2-bFGF. Twenty-one days after infection, ALP activity was significantly higher in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. Larger and more mineralized calcium nodules, as well as stronger collagen II staining, were observed in BMPCs infected with Ad-BMP2-bFGF than in those infected with Ad-BMP2. In summary, we developed a novel adenovirus vector Ad-BMP2-bFGF for simultaneous high coexpression of BMP2 and bFGF, which could induce BMPCs to differentiate efficiently into osteoblasts.

Processing of spent nickelcatalyst for fat recovery

Spent nickel catalyst (SNC) has the potential of insulting the quality of the environment in a number of ways. Its disposal has a pollution effect. Optimum recovery of fat from SNC, could save the environment and reduce the oil loss. Hexane has been the solvent of choice for oil extraction. Alternative solvents considered to have been safer have been evaluated. Hexane, isopropanol, ethanol and heptane were examined using soxhlet extraction. While hexane is more efficient in oil recovery from SNC, isopropanol proved to be very good in clear separation of oil from waste material and also provides high solvent recovery compared to other solvents. Isopropanol extraction with chill separation of miscella into lower oil-rich phase, and an upper, solvent-rich recyclable phase save mush energy of vaporization for distilling. An aqueous extraction process with immiscible solvent assisted was tested. Solvent like hexane added to SNC, and water added later with continuous stirring. The mixture was stirred for about 30 minutes, prior to centrifugation. Aqueous process extracted less amount of oil compared to solvent extraction.

Shelf life of minimally processed carrot and green pepper

The postharvest losses of horticultural products justify the use of preservation techniques. The processing not only adds value to the products, but also makes the products more convenient to the consumers. The objective of this research was to define the methodologies for the minimal processing of carrot and green pepper as to the type and intensity of the adoption of conservation techniques, and to monitor the products after processing through microbiological, physicochemical and nutritional analysis. The vegetables were washed and they were immersed in cold (7ºC) water with 100 mg L-1 free chlorine for sanitation, followed by centrifugation for 5 min. The product was put into BOPP/LDPE (biaxially orientated polypropylene/low-density polyethylene) plastic bags, which were sealed under atmospheric air, vacuum and modified atmosphere (2% O2, 10% CO2, 88% N2) and stored at 1ºC±1ºC. The approximate composition of the vegetables stayed stable during the storage period, in the three tested treatments. The contents of vitamin C for the samples of minimally processed carrot and green pepper did not present differences among treatments. The contents of beta-carotene decreased slightly during the storage period for the minimally processed carrot and green pepper. After processing, carrot and green pepper had psychrotrophic counts of 10²-10(5) and 10³-10(6) CFU g-1, respectively. Anaerobic mesophiles and total coliforms were found in green peppers, representing 1.6x10³ - 7.4x10(5) and <10.g-1 - 7.4x10(5), respectively. Total and fecal coliforms, anaerobic mesophiles and Salmonella were not found in carrots. Salmonella was not found in green pepper.

Use of Moringa oleífera Lamarck leaf extract as sugarcane juice clarifier: effects on clarifed juice and sugar

The objective of this study was to evaluate the effect of Moringa oleifera Lam. leaf extract on the sedimentation of impurities in the treatment of sugarcane juice and the effects on sugar quality and on the clarified juice. The experimental design used was a 4x2 factorial arrangement with four replications. The main treatments performed included the extracted original sugarcane juice, the synthetic polyelectrolyte (Flomex 9076), the leaf extract, and a control. The secondary treatments consisted of the sugarcane varieties RB92579 and RB867515. The clarification process used was simple defecation, in which the flocculating agents and the juice, limed and heated, were poured simultaneously into a decanter. The microbiological and chemico-technological characteristics of the extracted and clarified juices were evaluated. The clarified juice was concentrated up to 60° Brix (syrup) and subjected to boiling in a pilot pan using seeds to perform the graining: The sugar was recovered by centrifugation and analyzed for microbiological and chemico-technological characteristics. It was concluded that the use of the Moringa oleifera Lam. leaves extract resulted in a better quality of clarified juice and sugar.

Structural effects of neutral lipids on the divalent cation-induced interactions of phosphatidylserine - containing bilayers

As Ca2+ and phosphatidylserine (PS) are known to induce the adhesion of bilayer vesicles and form collapsed multibilayer structures in vitro, it was the aim of this study to examine how that interaction and the resultant structures might be modified by neutral lipid species. X-ray diffraction data from multilamellar systems suggest that phosphatidylcholine (PC) and diacylglycerol (DG) might be in the collapsed phase up to a concentration of -30 mole % and that above this concentration these neutral lipids may modify Ca2+-induced bilayer interactions. Using large unilamellar vesicles and long incubations in excess Ca2+ to ensure equilibration, similar preliminary results were again obtained with PC, and also with phosphatidylethanolamine (PE). A combination of X-ray diffraction, thin-layer chromatography, density gradient centrifugation and freeze-fracture electron microscopy, used in conjunction with an osmotic stress technique, showed that (i) -30 mole % PC can be accomodated in the Ca(DOPS)2 phase; and (ii) higher PC levels modify Ca2+-induced bilayer interactions resulting in single lamellar phases of larger dimension and reduced tendency for REV collapse. Importantly, the data suggest that PC is dehydrated during the rapid collapse process leading. to Ca(DOPS)2 formation and exists with this dehydrated phase. Similar results were obtained using PS isolated from bovine brain. Preliminary studies using two different phosphatidylethanolamine (PE) species indicated accomodation by Ca(DOPS)2 of -25-30 mole 0/0 PE and bulk phase separation, of species favouring a non-bilayer phase, at higher levels. Significantly, all PS/PE vesicles appear to undergo a complete Ca2+-induced collapse, even with contents of up to 90 mole % PE. These data suggest that PE may have an important role in fusion mechanisms in vivo. In sum the data lend both structural and stoichiometric evidence for th~ existence of laterally segregated neutral lipid molecules within the same bilayers as PS domains exposed to Ca2+.

The identification, distribution and persistence of oxamyl and its degradation products in planted corn seed, seedling root and soil from oxamyl-treated corn seeds

A simple method was developed for treating corn seeds with oxamyl. It involved soaking the seeds to ensure oxamyl uptake, centrifugation to draw off excess solution, and drying under a stream of air to prevent the formation of fungus. The seeds were found to have an even distribution of oxamyl. Seeds remained fungus-free even 12 months after treatment. The highest nonphytotoxic treatment level was obtained by using a 4.00 mg/mL oxamyl solution. Extraction methods for the determination of oxamyl (methyl-N'N'-dimethyl-N-[(methylcarbamoyl)oxy]-l-thiooxamimidate), its oxime (methyl-N',N'-dimethyl-N-hydroxy-1-thiooxamimidate), and DMCF (N,N-dimethyl-1-cyanoformanade) in seed" root, and soil were developed. Seeds were processed by homogenizing, then shaking in methanol. Significantly more oxamyl was extracted from hydrated seeds as opposed to dry seeds. Soils were extracted by tumbling in methanol; recoveries range~ from 86 - 87% for oxamyl. Root was extracted to 93% efficiency for oxamyl by homogenizing the tissue in methanol. NucharAttaclay column cleanup afforded suitable extracts for analysis by RP-HPLC on a C18 column and UV detection at 254 nm. In the degradation study, oxamyl was found to dissipate from the seed down into the soil. It was also detected in the root. Oxime was detected in both the seed and soil, but not in the root. DMCF was detected in small amounts only in the seed.