Isolation and characterization of the ecto-5′-nucleotidase from a rat glioblastoma cell line

Summary 5-Nucleotidase has been purified from rat glioblastoma cells (Rugli cells). The enzyme has been solubilized from plasma membranes by using Triton X-100 and CHAPS. Two affinity chromatographies on concanavalin A and 5-AMP-Sepharose render the purified enzyme with a high specific activity (76.36 mol AMP-min^–1-mg^–1). The purified enzyme gives a single polypeptide band on SDS-PAGE with an apparent molecular mass of 74 kDa. Active forms with an apparent molecular mass of 135 kDa and 268 kDa are observed when the purified enzyme is analyzed by gel filtration in the presence of either 0.6% sodium deoxycholate or 0.1% Triton X-100, respectively. The purified 5-nucleotidase presents optimum activity at pH 7.8–8.1 either in the presence or in the absence of Me²⁺. A linear Arrhenius plot is observed in the 25–46° C temperature range and an activation energy of 33.7 KJ/mol is calculated. The enzyme is inhibited by EDTA; the activity is partially restored by different divalent cations as Zn²⁺, Mn²⁺, and Co²⁺. The hydrolysis of nucleosides 5-monophosphate shows Michaelis kinetic. The enzyme is inhibited by nucleosides di- and triphosphate. 5-Nucleotidase is a glycoprotein, being its activity inhibited at different extent by various lectins.     

Clonal derivatives of a human B-lymphoblastoid cell line producing prolactin—A cytogenetic characterization

The IM-9-P cell line is a variant of the human B-lymphoblastoid cell line IM-9 which ectopically secretes prolactin (hPRL). The heterogeneous line IM-9-P and three sublines of clonal origin, two of them positive and one negative for PRL gene expression, were subjected to cytogenetic analysis and compared with the reference line IM-9 which showed a normal female diploid karyotype. G-banding revealed several rearrangements in the chromosomes. Nine altered chromosomes including one stable marker chromosome were common to all analysed karyotypes of IM-9-P cells and their clones. A second marker chromosome mar2 occurred only in the karyotypes of the hPRL producing clones, but not in the non-producing clone. None of the visible alterations involve chromosome 6 which carries the PRL gene in humans.     

Isolation and characterization of 17β-hydroxy steroid dehydrogenase from human erythrocytes

1. The 17beta-hydroxy steroid dehydrogenase was solubilized during haemolysis of erythrocytes and was isolated from the membrane-free haemolysate. Membrane preparations isolated in different ways did not contain 17beta-hydroxy steroid dehydrogenase activity. The 17beta-hydroxy steroid dehydrogenase activity in the haemolysate was concentrated by repeated ammonium sulphate precipitation and gel filtration on Sephadex G-150. The 17beta-hydroxy steroid dehydrogenase activity of the purified preparation per unit weight of protein was 350-3000 times higher than the activity of the crude erythrocyte haemolysate. The 20alpha-hydroxy steroid dehydrogenase activity was lost during this purification procedure. 2. The 17beta-hydroxy steroid dehydrogenase was NADP-dependent and had a pH optimum for conversion of testosterone between 8.5 and 10. For the molecular weight of the enzyme a value of 64000 was calculated from Sephadex chromatography results. 3. p-Chloromercuribenzoate inhibited the enzymic activity. The oxidative activity of the enzyme for the 17beta-hydroxyl group was only partly inhibited when a large excess of 17-oxo steroids was added. The catalysing activity of the enzyme was influenced by the NADP(+)/NADPH ratio. The oxidation of the 17beta-hydroxyl group in the presence of NADP(+) proceeded faster than the reduction of the 17-oxo group with NADPH. When both reduced and oxidized cofactors were present the oxidation of the 17beta-hydroxyl group was inhibited to a considerable extent. 4. The enzyme had a broad substrate specificity and not only catalysed the conversion of androstanes with a 17beta-hydroxyl group, or 17-oxo group, but also the conversion oestradiolleft arrow over right arrowoestrone. In addition the steroid conjugates dehydroepiandrosterone sulphate and oestrone sulphate were also converted. There were no indications that more than one 17beta-hydroxy steroid dehydrogenase was present in the partially purified preparation.     

Characterization of a new human diploid cell line—IMR-91

Summary A human diploid fibroblast cell line has been established from the lung tissue of a male fetus. This has been characterized and frozen away in large quantity. A smaller quantity of fibroblastlike cells from skin has also been established, partially characterized, and placed in frozen storage from the same fetus. This project is in support of the National Institute on Aging research in general cell biology. The present lines designated IMR-91 lung and IMR-91 skin complement the previous human diploid fibroblast culture (IMR-90) established from a female fetus. The lack of random inactivation of one of the two X chromosomes in the present male line reduces the genetic heterogeneity inherent in the female line.     

Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture — primary culture cells markedly differ from fourth-passage cells

To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase⁺/74% Thy-1/CD90⁺ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1β. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.     

Starfish saponins—XI. Isolation and partial characterization of the saponins from the starfish Marthasterias glacialis

• 1.1. The saponin mixture isolated from Marthasterias glacialis was resolved through a series of chromatographic steps into four principal individual components, named marthasteroside A₁, A₂, B and C.
• 2.2. The isolated sulphate steroidal glycosides were characterized by ¹H-NMR, ¹³C-NMR, Fast Atom Bombardment mass spectrometry and GLC analysis of the sugars after acid hydrolysis.
• 3.3. Marthasteroside A₁ and A₂ contained the aglycone thornastrol A and six sugar units. The second group of compounds, marthasteroside B and C, contained five sugar units; the aglycone of marthasteroside B was identified as marthasterone, while that of marthasteroside C was identified as dihydromarthasterone. In all compounds the sulphate group is attached at C-3 of the steroid.

     

Isolation and characterization of an α-satellite repeated sequence from human chromosome 22

We constructed a library in IL47.1 with DNA isolated from flow-sorted human chromosome 22. Over 50% of the recombinants contained the same highly repetitive sequence. When this sequence was used to probe Southern blots of EcoRI-digested genomic DNA, a ladder of bands with increments of about 170 bp was observed. This sequence comigrates with satellite III in Ag⁺/Cs₂SO₄ gradients and may account for at least part of the 170 bp Hae III ladder seen in isolated satellite III DNA. Partial sequence analysis revealed homology to the 171 bp monomeric repeat unit of -R1-DNA and the X specific -satellite consensus sequence. After low stringency in situ hybridization, silver grains were found over the centromeres of a number of chromosomes. Under high stringency conditions, however, the labeling was concentrated over the centromeric region of chromosome 22. This localization was confirmed using DNA from a panel of human/hamster cell lines which showed that the homologous 2.1 and 2.8 kb EcoR1 restriction fragments were chromosome 22 specific. These clones therefore contain chromosome 22 derived -satellite sequences analogous to other chromosome-specific satellite sequences described previously.     

Isolation,characterization and opiate activity of β-endorphin from human pituitary glands

The isolation of a 31-amino acid peptide from human pituitary glands has been described. Its amino acid sequence has been proposed to be identical to the sequence of the carboxyl-terminal 31 amino acids of human β-lipotropin. The peptide, designated as β_h-endorphin, possesses significant opiate activity.     

Isolation and characterization of an inter-α-trypsin inhibitor-IgG complex from human serum

Inter-α-trypsin inhibitor is a human serum protease inhibitor of M_r 180 000 which may release physiological derivatives. A complex between IgG and an inter-α-trypsin inhibitor derivative of M_r 30000 has been recently detected in human serum and was found to be inactive against trypsin, in contrast with the known inhibitory activity of the free 30-kDa derivative. The present study deals with detailed characterization of an inter-α-trypsin inhibitor-IgG complex following its purification by affinity chromatography techniques (anti-inter-α-trypsin inhibitor immunoadsorbent and Protein A-Sepharose) in mild conditions. The resulting product reacted simultaneously with anti-IgG and anti-inter-α-trypsin inhibitor antibodies. This complex contained M_r 180 000 inhibitor at least to some extent. It migrated in the β-γ zone in agarose; its molecular weight was estimated to be 1500 000 or more; part of it displayed covalent bonding between inter-α-trypsin inhibitor and IgG; it had a trypsin inhibitor activity. Immunoelectrophoresis allowed one to demonstrate the native complex in serum owing to the use of anti-inter-α-trypsin inhibitor and anti-γ radioactively labelled antibodies. The double immunoreactivity thus evidenced proved to be heterogeneous with respect to its level and location in the native as well as in the purified complex.     

Isolation and characterization of a gene encoding α-tubulin from Candida albicans

A gene encoding the α-tubulin of Candida albicans has been cloned and characterized. Nucleotide sequence analysis reveals the presence of an intron within the structural gene and predicts the synthesis of a polypeptide of 448 amino acid residues. Comparison of nucleotide and amino acid sequences with the Saccharomyces cerevisiae α-tubulin encoding genes shows a 75% homology and about 92% similarity respectively. In contrast to S. cerevisiae, C. albicans appears to possess only one gene for α-tubulin which is able to functionally complement a S. cerevisiae cold-sensitive tub1 mutant.     

Isolation and characterization of A new β-melanotropin from horse pituitary glands

A new β-melanotropin was isolated from horse pituitaries and its primary structure has been determined. The amino acid sequence of the new peptide differed from that of β-melanotropin in the deletion of the NH₂-terminal aspartic acid. Thus, it is designated as [Des-Asp¹]-β-melanotropin. It possessed higher lipolytic activity when compared with β-melanotropin.     

Isolation and characterization of a novel GH67 α-glucuronidase from a mixed culture

Hemicelluloses represent a large reservoir of carbohydrates that can be utilized for renewable products. Hydrolysis of hemicellulose into simple sugars is inhibited by its various chemical substituents. The glucuronic acid substituent is removed by the enzyme α-glucuronidase. A gene (deg75-AG) encoding a putative α-glucuronidase enzyme was isolated from a culture of mixed compost microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The DEG75-AG enzyme had optimum activity at 45?°C. Unlike other α-glucuronidases, the DEG75-AG had a more basic pH optimum of 7-8. When birchwood xylan was used as substrate, the addition of DEG75-AG increased hydrolysis twofold relative to xylanase alone.     

Initiation of human parturition—VII partial characterization of progesterone metabolizing enzymes of human amnion and chorion laeve

The kinetics of the metabolism of progesterone by the reducing enzymes present in the human fetal membranes was evaluated utilizing tissue samples obtained from early and late pregnancies. The specific activities of 5α-reductase, 3β- and 20α-hydroxysteroid oxidoreductase were greater in amnion and chorion laeve tissue obtained from a 16 weeks pregnancy than in those obtained from a 36 1/2 week and from a term pregnancy. Amnion and chorion laeve tissue 20α-hydroxysteroid oxidoreductase enzymes utilized either NADH or NADPH as the source of reducing equivalents (K_m = 0.2–0.3 mM) while amnion tissue 5α-reductase activity required NADPH as the obligatory cofactor (K_m = 0.1mM). The apparent pH optima for the 20α-hydroxysteroid oxidoreductase present in amnion and chorion laeve tissues were similar, while the apparent pH optima for the 5α-reductase enzyme was slightly higher in chorion laeve than in amnion tissue. In 15 min incubations, the apparent maximal activity of amnion 5α-reductase was found at a temperature of 49°C. whereas chorion laeve tissue 3β- and 20α-hydroxysteroid oxidoreductase had maximal activity at 37°C. From progesterone saturation kinetics an apparent K_m of 0.20–0.25 mM for amnion and chorion laeve 20α-hydroxysteroid oxidoreductase, and an apparent K_m of 0.4–1.2 μM for amnion and chorion laeve tissue 5α-reductase activity was computed. Amnion 5α-reductase activity appeared to be particulate in nature.     

Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line

Periodontal ligament (PDL) is a specialized connective tissue that influences the lifespan of the tooth. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, but little is known about the effects of TGF-β1 on PDL cells. Our aim has been to demonstrate the expression of TGF-β1 in rat PDL tissues and to evaluate its effects on the proliferation and gene expression in human PDL cells (HPLCs) and a human PDL stem/progenitor cell line, line 1-11, that we have recently developed. The expression of TGF-β1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-β1, TGF-β type I receptor, and TGF-β type II receptor genes. Although exogenous TGF-β1 stimulated the proliferation of HPLCs, it did not upregulate the expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col I), or fibrillin-1 (FBN1) mRNA or of α-SMA protein in HPLCs, whereas expression for these genes was attenuated by an anti-TGF-β1 neutralizing antibody. In contrast, exogenous TGF-β1 reduced the proliferation of cell line 1-11, although it upregulated the expression of α-SMA, Col I, and FBN1 mRNA and of α-SMA protein in this cell line. In addition, interleukin-1 beta stimulation significantly reduced the expression of TGF-β1 mRNA and protein in HPLCs. Thus, TGF-β1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions.     

Isolation and characterization of dioscin-α-l-rhamnosidase from bovine liver

A novel dioscin-α-l-rhamnosidase was isolated and purified from fresh bovine liver. The activity of the enzyme was tested using diosgenyl-2,4-di-O-α-l-rhamnopyranosyl-β-d-glucopyranoside as a substrate. It was cleaved by the enzyme to two compounds, rhamnoses and diosgenyl-O-β-d-glucopyranoside. The optimal conditions for enzyme activity were that temperature was at 42 °C, pH was at 7, reaction time was at 4 h, and the substrate concentration was at 2%. Furthermore, metal ions such as Fe³⁺, Cu²⁺, Zn²⁺, Ca²⁺ and Mg²⁺ showed different effects on the enzyme activity. Mg²⁺ acted as an activator whereas Cu²⁺, Fe³⁺, and Zn²⁺ acted as strong inhibitors in a wide range of concentrations from 0 to 200 mM. It was interesting that Ca²⁺ played a role as an inhibitor when its concentration was at 10 mM and acted as an activator at the other concentrations for the enzyme. Moreover, the molecular weight of enzyme was determined as 75 kDa.     

Isolation and characterization of autoagglutination factor of Yersinia pestis Hms− cells

An approach for isolation of an autoagglutination factor (AF) from Hms(-) cells of the plague agent has been developed. Purified AF has been obtained and characterized in physicochemical properties. The AF is found to be a complex of a 17.5-kD protein with a low molecular weight peptide component, which binds iron ions and shows siderophore activity. This low molecular weight component is responsible for hydrophobic properties and immunochemical activity of the AF, as well as for its ability to interact with the plague diagnosticum L-413c bacteriophage.     

Isolation and characterization of extracellular α-galactosidases from Penicillium canescens

Two alpha-galactosidases were purified to homogeneity from the enzymatic complex of the mycelial fungus Penicillium canescens using chromatography on different sorbents. Substrate specificity, pH- and temperature optima of activity, stability under different pH and temperature conditions, and the influence of effectors on the catalytic properties of both enzymes were investigated. Genes aglA and aglC encoding alpha-galactosidases from P. canescens were isolated, and amino acid sequences of the proteins were predicted. In vitro feed testing (with soybean meal and soybean byproducts enriched with galactooligosaccharides as substrates) demonstrated that both alpha-galactosidases from P. canescens could be successfully used as feed additives. alpha-Galactosidase A belonging to the 27th glycosyl hydrolase family hydrolyzed galactopolysaccharides (galactomannans) and alpha-galactosidase C belonging to the 36th glycosyl hydrolase family hydrolyzed galactooligosaccharides (stachyose, raffinose, etc.) of soybean with good efficiency, thus improving the digestibility of fodder.     

Purification and partial characterization of β-glucosidase from papaya fruit

β-Glucosidase (I) was isolated from Carica papaya fruit pulp and purified ca 1000-fold to electrophoretic homogeneity. The procedure used ammonium sulphate fractionation followed by chromatography on Phenyl-Sepharose CL-4B and Sephacryl S-200 to separate α-mannosidase (II) and, in part, β-galactosidase (III) from (I). Final separation of (III) from (I) was achieved by preparative isoelectric focusing (PIEF). The glycosidases had pI of 5.2 (I), 4.9 (II) and 6.9 (III). M,s of 54 000 (I), 260 000 (II) and 67 000 (III) were determined by gel filtration. The M, of (I) estimated by SDS-PAGE was 27 000 suggesting that (I) consisted of two subunits. The optimum pH and optimum temperature of (I) were 5.0 and 50°, respectively, and the enzyme followed typical Michaelis kinetics with K_m and V_max of 1.1 × 10⁻⁴ M and 1.8 × 10⁻⁶ mol/hr, respectively, for p-nitrophenyl-β-d-glucoside (40°).     

Isolation and characterization of marine biofilm forming bacteria from a ship’s hull

Background

Diverse aquatic microorganisms are capable of colonizing living and non-living surfaces leading to the formation of biofilms. Commonly visualized as a slimy layer, these biofilms are filled with hundreds of other microorganisms compared to free living planktonic cells. Microbial surface colonization and surface-associated metabolic activities also exert several macroscale deleterious effects, including biofouling, biocorrosion and the persistence and transmission of harmful or pathogenic microorganisms and virulence determinants. The present study deals with the isolation and screening of marine bacteria for biofilm formation. The screened isolates were characterized and identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila by 16S rRNA sequencing.

Methods

Biofilm forming bacteria were isolated by spread plate technique and subjected to screening by microtiter plate assay. The potent biofilm formers were identified by molecular characterization using 16S rRNA gene sequencing.

Results

Twelve bacterial isolates were obtained by pour plate technique and subjected to biofilm assay. Among the 12 isolates three isolates which showed maximum biofilm formation were subjected to molecular characterizationby 16S rRNA gene sequencing method. The isolates were identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila. The EPS produced by the three biofilm forming bacteria was extracted and the protein and carbohydrate content determined.

Conclusion

Among the isolates screened, isolate 8 (Kocuria rhizophila) produced maximum protein and carbohydrate which was also in accordance with the results of microtiter plate assay.