Morphological, Biochemical, and Functional Characterization of Bulk Isolated Glial Progenitor Cells

We describe a simple, rapid, and efficient method, based on separation on a Percoll centrifugation gradient, to purify glial progenitor cells from newborn rat brains. Cytofluorimetry analysis of the isolated cell population showed that 75 +/- 8 and 86 +/- 7% of the cells were A2B5- and R24-positive, respectively. Transmission electron microscopy examination of the purified cell population confirmed their homogeneity and illustrated their typical morphology, as previously described in situ. Assay of UDP-galactose-ceramide galactosyltransferase, 3'-phosphoadenosine 5'-phosphosulfate galactosylceramide sulfotransferase, and 2',3'-cyclic nucleotide 3'-phosphohydrolase activities showed that the levels of these enzymes were 446, 76, and 11 times lower, respectively, than the levels measured in mature oligodendrocytes. Low levels of mRNA coding for 2',3'-cyclic nucleotide 3'-phosphohydrolase and myelin proteolipid protein, but not for myelin basic protein, were present in the glial progenitor cells. At the time of isolation, 40% of the cells in the population were dividing, and the cells could easily be expanded in culture. After 3 weeks of culture in the presence of 1% fetal calf serum, 75% of the cells had differentiated into galactosylceramide-positive oligodendrocytes. When the culture took place in the presence of 10% fetal calf serum, only 2% of the cells expressed galactosylceramide, and 60% were glial fibrillary acidic protein-positive astrocytes; half of them were also A2B5 positive.     

Effect of Two Probiotic Strains of Lactobacillus on In Vitro Adherence of Listeria monocytogenes,Streptococcus agalactiae,and Staphylococcus aureus to Vaginal Epithelial Cells

The lactobacilli probiotics maintain a normal vaginal biota and prevent disease recurrence. This microorganisms form a pellicle on the vaginal epithelium that acts as a biologic barrier against colonization by pathogenic bacteria. In this paper were realized assays of exclusion, competition, and displacement. For these test, vaginal epithelial cells, two strains of lactobacilli and pathogenic bacteria (Staphylococcus aureus, Streptococcus agalactiae and Listeria monocytogenes) were used. The lactobacilli strains showed a great capacity of adherence, with a mean of 83.5 ± 26.67 Lactobacillus fermentum cells and 56.2 ± 20.87 Lactobacillus rhamnosus cells per vaginal epithelial cells. L. fermentum and L. rhamnosus were able to reduce the adherence of S. aureus, S. agalactiae and L. monocytogenes in a significant level in this assay (P < 0.01). The lactobacilli used in this study protect the vaginal epithelium through a series of barriers and interference mechanisms. The aim of present study was to assess the ability of vaginal Lactobacillus strains, selected for their probiotic properties, to block the adherence of pathogenic microorganisms in vitro by displacement, competition, and exclusion mechanisms.     

Lactobacillus plantarum 24, Isolated From the Marula Fruit (Sclerocarya birrea), has Probiotic Properties and Harbors Genes Encoding the Production of Three Bacteriocins

Lactobacillus plantarum 24, isolated from marula fruit grows at pH 4.0 and tolerates acid levels and bile concentrations normally present in the human gastro-intestinal tract. Wistar rats that have been administered L. plantarum 24 showed no signs of discomfort or abnormal behavior. Tissue samples from the liver, spleen and intestine appeared normal. Furthermore, strain 24 harbors the genes encoding plantaricins A, F, and NC8α, a gene encoding immunity to plantaricin, and an ABC transporter similar in sequence to that reported for plantaricin G. At least one antimicrobial peptide within the size range of plantaricins A, F, and NC8α has been detected on a tricine-SDS–PAGE gel. Little is known about the microbial population in marula. This is the first report of a L. plantarum strain from marula fruit with bacteriocin genes and probiotic properties.     

Isolation and Characterization of Putative Probiotic Bacterial Strain, Bacillus amyloliquefaciens, from North East Himalayan Soil Based on In Vitro and In Vivo Functional Properties

This study is aimed to isolate some novel probiotics from the soils of North East Himalayas. Eleven Gram positive isolates were obtained in MRS with Oxgall media from soil samples. Four of the isolates withstood the in vitro gastric juice pH 3.0 and 0.45% bile salt tolerance screening. Among these, PBT 3 showed high cell surface hydrophobicity and adhered to the Caco-2 cells. 16s rDNA gene sequences of this probiotic strains were identified as Bacillus amyloliquefaciens (accession no: JF836079). In in vivo bioefficacy evaluation by DSS-induced colitis animals, B. amyloliquefaciens significantly ameliorated the loss in body weight. Further, the treatment altered the levels of myeloperoxidase, lipoperoxides, and mucous content in the colon tissues compared with normal colon. It reduced the protein and mRNA levels of pro-inflammatory cytokines such as TNF-α and IL-1β. These biochemical findings were supported by histopathological evidences. Our study reports the use of B. amyloliquefaciens isolated from Himalayan soil as probiotic and its beneficial effect on IBD for the first time and suggests that this could be used as potential probiotics in functional foods or as a curative agent.     

Structural and Functional Properties of Cr 5, a New Lys49 Phospholipase A2 Homologue Isolated from the Venom of the Snake Calloselasma rhodostoma

Cr 5 PLA₂ homologous (K49) was isolated from Calloselasma rhodostoma venom in only one chromatographic step in reverse phase HPLC (RP-HPLC) (on μ-Bondapack C-18). A molecular mass of 13.965 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that Cr 5 had a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical residues of a basic PLA₂. The complete amino acid sequence of Cr 5 PLA₂ contains 120 residues, resulting in a calculated pI value of 5.55. This sequence shows high identity values when compared to other K49 PLA₂s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA₂s. The sequence found was SLVELGKMIL QETGKNPAKS YGAYGCNCGV LGRHKPKDAT DRCCFVHKCC YKKLTGCDPK KDRYSYSWKD KTIVCGENNP CLKEMCECDK AVAICLRENL DTYNKKYRYL KPFCKKADDC. In mice, Cr 5 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD₅₀ of Cr 5 was 0.070 mg/kg of the animal weight, by intracerebroventricular (i.c.v.) route. In vitro, the toxin caused rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. The isolation of this PLA₂ and the combined structural and functional information obtained classify Cr 5 as a new member of the K49 PLA₂ family, since it presents typical features from such proteins.     

A Low-Km 5'-Nucleotidase from Rat Brain Cytosolic Fraction: Purification, Kinetic Properties, and Description of Regulation by a Novel Factor that Increases Sensitivity to Inhibition by ATP and ADP

Abstract: A readily soluble 5'-nucleotidase was purified 1,800-fold from rat brain 105,000- g supernatant. The enzyme showed similarity to the 5'-nucleotidase ectoenzyme of plasma membranes. It exhibited a low K _m for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by α,β-methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol-specific phospholipase C increased its hydrophilicity. A single subunit of M_r = 54,300 ± 800 was observed, which is appreciably smaller than published values for the 5'-nucleotidase ectoenzyme or for other low- K _m"soluble" 5'-nucleotidases. The soluble 5'-nucleotidase showed an elution profile on AMP-Sepharose affinity chromatography or on Mono Q ion-exchange chromatography different from that of the brain ectoenzyme. Forty-two percent of the soluble 5'-nucleotidase in brain 105,000- g supernatant did not bind to a Mono Q ion-exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A-Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5'-nucleotidase toward the inhibitor ATP by 20-fold. This factor was also able to increase the inhibition of brain 5'-nucleotidase ectoenzyme by ATP.