Characterization of a mouse serotonin 5-HT3 receptor purified from mammalian cells

A serotonin 5-HT3 receptor was functionally expressed to high levels and on a large scale in mammalian cells with the Semliki Forest virus system. Conditions were optimized to maximize detergent solubilization of the receptor, while preserving ligand binding activity. An efficient one-step purification yielding approximately 50% of the histidine-tagged 5-HT3 receptor was achieved with immobilized metal ion chromatography. The expressed receptor, in both membranes and purified preparations, exhibited wild-type ligand binding properties, characterized by one class of binding sites. The purity of the receptor was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding a single band at 65 kDa, and was confirmed by the specific ligand binding activity of approximately 5 nmol/mg of protein. Deglycosylation of the receptor reduced the estimated relative molecular mass to 49 kDa. The apparent molecular mass of the functional receptor complex was determined by size exclusion chromatography to be 280 kDa, suggesting that the 5-HT3 receptor is a pentameric homooligomer. The secondary structure of the 5-HT3 receptor as determined by circular dichroism appeared to consist of mainly alpha-helices (50%) and beta-strands (24%), with minor contributions from nonregular structure (9%). The binding of either agonist or antagonist did not alter the secondary structure of the receptor.     

Preparation and properties of partially purified pulmonary cytochrome P-450 from rabbits

Cytochrome P-450 from rabbit pulmonary microsomes was purified approximately 32-fold. The purification method involved solubilization of microsomes using sodium cholate, and recovery of cytochrome P-450 in the precipitate formed between 25 to 42% saturation of the digested microsomes with ammonium sulfate in the absence of glycerol. Further purification was achieved by chromatography on DEAE-cellulose and hydroxylapatite using Emulgen 913 as an eluent. Partially purified preparations containing up to 7.4 nmol of cytochrome P-450 per mg of protein were essentially free of NADPH-cytochrome c reductase activity and cytochromes b5 and P-420. However, epoxide hydrase was found to co-purify with cytochrome P-450. The CO-difference spectrum of dithionite-reduced purified cytochrome showed the expected peak at 450 nm. However, the magnitude of the peak was dependent on added microsomal lipid fraction in the assay medium. Purified pulmonary cytochrome P-450 formed typical types I and II substrate difference spectra with benzphetamine and pyridine, respectively. Sodium dodecyl sulfate-gel electrophoresis of partially purified cytochrome P-450 gave two major bands when stained with Coomassie blue. The faster moving band which contained peroxidase activity had an estimated molecular weight of 49,000 +/- 1,200. The cytochrome P-450 fraction, when combined with solubilized pulmonary microsomal NADPH-cytochrome c reductase and lipid fractions, was active in the O-deethylation of 7-ethoxycoumarin and the N-demethylation of benzphetamine.     

Preparation of a purified antigenic cholera toxoid

Purified cholera enterotoxin was prepared by methods described by Finkelstein and Lo Spalluto (1970). This toxin was detoxified by treatment with heat and formaldehyde. Heating cholera toxin at 60 C for 25 min resulted in the formation of a polymer named procholeragenoid by Finkelstein et al. (1971). The weak toxic activity of this product was removed by treatment with formalin. No residual toxicity could be demonstrated in formalinized procholeragenoid by the rabbit ileal loop assay and the highly sensitive rabbit skin tests. This toxoid was nevertheless at least as antigenic in the rabbit as was the toxin. No reversion to toxicity was observed in vivo and in vitro at 4 C. The toxicity of formalinized procholeragenoid never exceeded 1/5,000 to 1/10,000 that of the toxin.     

Human neoplastic and normal cells in tissue culture. I. Cell lines derived from malignant melanomas and normal melanocytes

Forty-six cell lines derived from 31 human melanomas obtained from 28 patients were cultured. Fourteen of 16 lines have produced malignant tumors when injected into nude (thymus-deficient) mice. Tumors in 5 of the nude mice metastasized to distant lymph nodes and/or to the lungs of the mouse host. Extreme variability from line to line was observed for doubling time (34 to 106 hr), plating efficiency (0-86%), and melanin production. All tested lines had type B glucose-6-phosphate dehydrogenase, thereby excluding HeLa cell contamination. HeLa cells have been grown for some time in our laboratory. Our results clearly demonstrated that HeLa cell contamination does not occur invariably in heteroploid lines growing in a laboratory simultaneously with Hela cells, provided that proper care is taken to avoid such occurrence. Multiple cell lines derived from the same tumor had identical phosphoglucomutase enzyme phenotype, which suggested a lack of significant cross-contamination between the lines. Four long-term cultures of normal human uveal embryo melanocytes have also been established and characterized. Although all produced melanin after reaching saturation density, they differed from the melanoma cells morphologically; they were flat, not refringent, and lacked piling up and plating ability. When melanoma cells were exposed to bromodeoxyuridine (BUDR) for long periods, a phenotypic change toward non-neoplastic characteristics was observed. Cells became flat and not refringent and, when injected into nude mice, tumors appeared after a long latent period. These changes were completely reversible in vitro and in vivo. The BUDR-treated cultures were undistinguishable from the untreated mother cultures after 2 to 3 passages. Lines derived from tumors in nude mice (obtained by injection of BUDR-treated cells) were again indistinguishable from the untreated mother line. Normal melanocytes were mostly euploid; all the melanoma cells were aneuploid. All 29 cell lines derived from 14 patients had an average chromosome number higher than 46. Detailed group-by-group chromosome analysis always showed an excess of C chromosomes, which suggested that hyperreduplication of one or more C chromosomes is a specific characteristic of human melanomas.     

Functional regulation of tyrosinase and LAMP gene family of melanogenesis and cell death in immortal murine melanocytes after repeated exposure to ultraviolet B

The aim of the present study was to evaluate the effect of human follicle stimulating hormone (hFSH) on cellular proliferation in the chick embryo ovary. Chick embryos (Babcock B300) were injected on chorioallantoic membrane with a single dose of hFSH (2.0 IU/ embryo) at Days 7, 9, or 13 of incubation or with hCG (2.0 IU/embryo) at Day 13 of incubation. At 17 days of incubation and within 24 h after hatching, left ovaries were dissected and completely dissociated. Cells from the whole ovary were classified into germ cells (primary oocytes), typical steroidogenic cells, and poorly differentiated somatic cells and counted with the aid of a hemocytometer. Aliquots of the cell suspension from the whole left ovary were analyzed by flow cytometry, in order to determine the percentage of cells at each phase of the cell cycle. In addition, samples of the suspension (1.0 x 10(6 )cells) were incubated for 2 h in basal and stimulated conditions measuring 17beta-estradiol secretion in the medium. The ovarian cell number at 17 days of incubation showed that hFSH treatment at Day 7 did not modify the cell number in any of the subpopulations evaluated; treatment at Day 9 resulted in an increase in poorly differentiated somatic cell number, without changes in steroidogenic and germ cells, whereas hFSH treatment at Day 13 augmented the number of poorly differentiated, steroidogenic, and germ cells. The percentage of cells in S-phase was increased 12 and 15 h after hFSH treatment (Day 13). Secretion of 17beta-estradiol was increased in the hFSH-treated group (Day 13) measured at 17 days of incubation. The increase in cell number of the three subpopulations was still observed in the left ovary of the newly hatched chicken. Treatment with hCG at Day 13 of incubation did not change the number of poorly differentiated, steroidogenic, and germ cells in the left ovary, neither in the 17-day-old chick embryo nor in the newly hatched chicken. The 17beta-estradiol secretion in hCG-treated embryos was similar to controls. The present study is the first evidence of an effect of FSH on somatic and germ cell number, together with an increase in 17beta-estradiol production during chick embryo ovary development.     

Hepatitis C virus E2 protein purified from mammalian cells is frequently recognized by E2-specific antibodies in patient sera

The envelope protein of hepatitis C virus (HCV) is composed of two membrane-associated glycoproteins, E1 and E2. To obtain HCV E2 protein as a secretory form at a high level, we constructed a recombinant chinese hamster ovary (CHO) cell line expressing a C-terminal truncated E2 (E2t) fused to human growth hormone (hGH), CHO/hGHE2t. The hGHE2t fusion protein was purified from the culture supernatant using anti-hGH mAb affinity chromatography at approximately 80% purity. The purified hGHE2t protein appeared to be assembled into oligomers linked by intermolecular disulfide bond(s) when density gradient centrifugation and SDS-polyacrylamide gel electrophoresis were employed. When the purified fusion protein was used for testing its ability to bind to antibodies specific for HCV by enzyme-linked immunosorbent assay, the protein was recognized by antibodies in sera from 90% of HCV-positive patients. Treatment of hGHE2t protein by beta-mercaptoethanol, but not by heat and SDS, significantly reduced its reactivity to the antibodies of patient sera, suggesting that intermolecular and/or intramolecular disulfide bonds are important for its ability to recognize its specific antibody and that the E2 protein contains discontinuous antigenic epitope(s).     

The reconstitution of microtubules from purified calf brain tubulin

The in vitro reconstitution of calf brain tubulin, purified by the method of Weisenberg et al. [(1968), Biochemistry 7, 4466-4479; (1970), Biochemistry 9, 4110-4116] as modified by Lee et al. [(1973), J. Biol. Chem. 248, 7253-7262], was successful in a medium consisting of 10(-2) M sodium phosphate, 10(-4) M GTP, and concentrations of magnesium ions ranging from 0.5 to 16 X 10(-3) M at 37 degrees. Filaments resembling native microtubules were formed. The filaments are in equilibrium with the associating species of tubulin and the equilibrium can be shifted to depolymerization by lowering the temperature to 20 degrees. Filament formation is inhibited by calcium ions which also cause disassembly of the formed filaments. The effects of calcium ion can be reversed by the addition of [ethylenebis-oxyethylenenitrilo)]tetraacetic acid. The formation of filaments is favored by the presence of 3.4 M glycerol; only twisted abnormal filaments are observed in the presence of 1 M sucrose. The high molecular weight components observed in the sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of many tubulin preparations were shown not to be essential for the formation of the filaments.     

Distribution and localization of galectin purified from Rana catesbeiana oocytes

Galectins are a family of lectins that recognize beta-D-galactosides independently of calcium ions, and are widely distributed in animals. To characterize a galectin previously purified from oocytes of Rana catesbeiana (American bullfrog), we studied its distribution and localization in several tissues from this frog. Hemagglutination assay and western blotting showed that this lectin is present in many tissues including the liver, skin, kidney, skeletal muscle, and sciatic nerve, but is particularly concentrated in the ovary. Light microscopic immunohistochemistry showed that this lectin is localized in such places as cell-cell junctions, basement membranes, extracellular matrix, or secretory substances in several organs, indicating that this galectin is mainly distributed extracellularly. However, in the ovary, light microscopy showed that this lectin is present in or associated with the yolk platelet. Electron microscopy further revealed that it is localized in the periphery of the yolk platelet (the yolk plasm), but not in the cortical granule. These results indicate that Rana oocytes contain abundant galectin in their yolk platelets in contrast to Xenopus laevis oocytes, which have been found not to contain galectins but other classes of lectins in their yolk platelets and cortical granules.     

Preparation of highly purified microbules and evidence for a novel molecular arrangement at low temperatures

Microtubules were prepared by in vitro polymerization-depolymerization cycles, 1.0 M NaCl which totally depolymerizes was then added to the preparation. After removal of NaCl new arrangements of tubulin were observed at 4 degrees C: simple and double rings as well as fibrils. At 37 degrees these structures disappeared and tubulin polymerized into microtubules. The highly microtubules contain tubulin, tubulin associated proteins of 300,000 and 330,000 molecular weight, minor proteins of low molecular weight and proteins similar to the Tau factors. This raises a question of the role played by low molecular weight polypeptides. Are they products of proteolysis of rather factors of polymerisation?     

Antitumorigenic effect of a mammalian lignan precursor from flaxseed

Secoisolariciresinol diglycoside (SD), a mammalian lignan precursor found in high-fiber foods, was isolated from flaxseed and tested for effects on mammary tumorigenesis in rats fed a high-fat (20%) diet. Ingestion of purified SD at 1.5 mg/day for 20 weeks starting 1 week after treatment with the carcinogen dimethylbenzanthracene resulted in a 37% reduction (p < 0.05) in the number of tumors per tumor-bearing rat and a 46% reduction (p < 0.05) in the number of tumors per tumor-bearing rat and a 46% reduction (p < 0.05) in the number of tumors per number of rats in each group. Urinary mammalian lignan excretion significantly increased (p < 0.0001) with SD treatment, indicating the conversion of SD to mammalian lignans. No enlargement or gross abnormalities of the major organs were observed in the SD-treated rats. This study showed, for the first time, that SD has an antitumor effect when provided at the early promotion stage of tumorigenesis and may contribute to the health benefits of high-fiber foods.     

Comparative action spectrum for ultraviolet light killing of mouse melanocytes from different genetic coat color backgrounds

PURPOSE: This study reports the authors' experience with long-term follow-up of 100 consecutive peripherally inserted, subcutaneous arm ports for central venous access. MATERIALS AND METHODS: One hundred patients with subcutaneous arm ports inserted by interventional radiologists were retrospectively studied. Data were collected from the patients' medical records and from telephone canvassing. Using each insertion period as an observation, the complication rates per 100 catheter days were determined with 95% confidence intervals (CIs). RESULTS: One hundred subcutaneously implanted ports were placed in 98 patients; three devices (three patients) were lost to follow-up, leaving 97 devices in 95 patients. Total exposure time was 23,842 days (mean, 246 days; range, 2-865 days). Seven infectious and two noninfectious complications occurred with seven (7.2%) devices in six patients (6.3%), yielding 0.038 complications per 100 catheter days at risk (95% CI; 0.011-0.069) and 0.029 infections per 100 catheter days at risk (95% CI; 0.008-0.058). A successful clinical outcome was defined as a functional port at removal, time of death, or at study closure (minimum of 6 months of follow-up), which was not removed because of a complication. This successful outcome was achieved in 91 ports (93.8%). Procedural-related complications, defined as those occurring up to 30 days after insertion, occurred in only one port (thrombophlebitis and catheter tip infection-day 9). All other patients received several months of service from their port. Fifteen devices were placed in 13 patients with HIV for 3,486 days, with a total complication rate of 0.11 per 100 catheter days (95% CI; 0.0-0.28), all of which were infections. Devices in HIV-positive patients were associated with higher total complication (20% vs 4.9%) and infection rates (20% vs 3.7%) than devices in patients without HIV infection. This gives a relative risk 8.17 x (P = .04) greater for infectious complications for devices placed in HIV-infected individuals. CONCLUSIONS: Subcutaneous arm ports placed by interventional radiologists are effective for central venous access with excellent functionality (93.8% achieved a successful long-term outcome) and a very low procedural complication rate. Although infections were more frequent in HIV-infected individuals, these devices are associated with a very low incidence of both immediate and long-term complications, including infection, for all patients.     

Isolation and identification of glycosaminoglycans associated with purified nuclei from rat liver

The effects of nitrogen bases on the regulation of phospholipid metabolism in neuroblastoma cell cultures were investigated. An increase in the total cellular phospholipids was observed up to 24 h following plating. Addition of monomethyl- and dimethylethanolamine bases resulted in a stimulation of the synthesis of their corresponding phospholipids. The average rates of synthesis of phosphatidylmonomethyl- and phosphatidyldimethylethanolamine were 0.09 and 0.12 nmol/microgram DNA per h, respectively. The labeling patterns of the various phospholipid species from ortho[32P]phosphate have been determined. They suggest that the synthesis of the analogs proceeded entirely via a phosphate mediated pathway rather than through a base exchange mechanism. A number of distinct patterns for the incorporation of bases into acyl-, alkyl- and alkenyl-containing phosphoglyceride species were indicated. The polar head group composition appeared to be intimately related to the type of bond of the hydrocarbon residue.     

Properties of alpha-mannosidase partially purified from the apple snail, Pomacea canaliculata

Pomacea canaliculata alpha-mannosidase (260 kDa), composed of at least two isoforms with different pI, was partially purified. The activity was maximum at pH 4 and unaltered after incubation at 60 degrees C for 60 min. ZnCl2, CaCl2, NaCl, and SH-reagents increased the activity, while MnCl2 and EDTA inhibited it. The enzyme catalyzed the hydrolysis of alpha 1-2, alpha 1-3, and alpha 1-6 mannosidic linkages.     

Generation of purified neural precursors from embryonic stem cells by lineage selection

Mouse embryonic stem (ES) cells are non-transformed cell lines derived directly from the pluripotent founder tissue in the mouse embryo, the epiblast [1-3]. Aggregation of ES cells triggers the generation of a diverse array of cell types, including neuronal cells [4-7]. This capacity for multilineage differentiation is retained during genetic manipulation and clonal expansion [8]. In principle, therefore, ES cells provide an attractive system for the molecular and genetic dissection of developmental pathways in vitro. They are also a potential source of cells for transplantation studies. These prospects have been frustrated, however, by the disorganised and heterogeneous nature of development in culture. We have therefore developed a strategy for genetic selection of lineage-restricted precursors from differentiating populations. Here, we report that application of such lineage selection enables efficient purification of neuroepithelial progenitor cells that subsequently differentiate efficiently into neuronal networks in the absence of other cell types.     

Sulphogalactolipid sulphohydrolase activity of arylsulphatase purified from a marine gastropod Charonia lampas

Sulphatide, cerebroside 3-sulphate was hydrolyzed at a considerable rate by arylsulphatase (aryl-sulphate sulphohydrolase, EC 3.1.6.1) purified from a marine gastropod, Charonia lampas. However, it was scarcely hydrolyzed by glycosulphatase (sugar-sulphate sulphohydrolase, EC 3.1.6.3) from the same origin. The same was observed with seminolipid, a sulphoglycerogalactolipid. The enzymatic characteristics of both sulphogalactolipid and sulphohydrolase activities of the arylsulphatase were determined as follows. The enzyme activities are stimulated by the addition of sodium taurodeoxycholate and MnCl2. The pH optimum of sulphatide sulphohydrolase activity was pH 5.0, while seminolipid sulphohydrolase activity had maximum activity at pH 5.5. Both of these pH versus activity curves were broad. The Km value was 6.22-10-5 M for both substrates. However, the V values were sulphatide were lower by a factor of one-third than those with seminolipid. These enzyme activities were inhibited by substrates of the arysulphatase, i.e., p-nitrophenyl sulphate, p-nitrocatechol sulphate, ascorbate 2-sulphate and each other sulphogalactolipid, but not by glucose 6-sulphate. Sulphate and phosphate anions inhibited both of the enzyme activities.     

Preparation of highly purified cytochrome P4501A1 from leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular and immunochemical properties

Cytochrome P4501A1 was purified to electrophoretic homogeneity from the liver microsomes of feral fish leaping mullet (Liza saliens) collected in Izmir Bay, Aegean coast of Turkey. Purification of cytochrome P4501A1 involved anion exchange chromatography of Emulgen 913-cholate solubilized microsomes on first- and second-DEAE-cellulose columns, hydrophobic interaction chromatographies of the partially purified cytochrome P4501A1 on Porapak Q and phenyl-Sepharose CL-4B and further purification on adsorption chromatography on the hydroxylapatite column. Finally, it is further concentrated and purified on the third DEAE-cellulose column. The purified cytochrome P4501A1 was characterized with respect to spectral, electrophoretic, immunochemical and biocatalytic properties. Cytochrome P4501A1, purified 32-fold with a specific content of 15-17 nmoles P450 (mg protein)-1, produced a single band on SDS-polyacrylamide gel electrophoresis having monomer molecular weight of 58,000 +/- 500. Absolute absorption spectrum of the purified cytochrome P4501A1 fractions showed maximal absorption at 417.5 nm and CO-difference spectrum of dithionite-reduced cytochrome P4501A1 gave a peak at 448 nm. Purified P4501A1 was found to be active in the O-deethylation of 7-ethoxyresorufin in the reconstituted system containing purified fish liver cytochrome P450 reductase and synthetic lipid. However, it was unable to catalyze the oxidation of the other monooxygenase substrates such as benzphetamine and aniline known to be specific for the other isozymes. Purified L. saliens liver microsomal cytochrome P4501A1 showed strong cross-reactivity with the antibodies directed against the cytochrome P4501A1 homologues purified from other teleost species such as rainbow trout and scup. Spectral, electrophoretic, immunochemical and biocatalytic properties of the purified cytochrome P4501A1 strongly suggested that it is the CYP1A1 in the L. saliens liver.     

Demonstration and genotyping of pestivirus RNA from mammalian cell lines

Mineralization occurred both in fetal rat calvarial cells and UMR 106 osteoblastic cells when they were cultured in medium containing L-ascorbate and beta-glycerophosphate as evidenced by von Kóssa staining as well as deposition of calcium ions and inorganic phosphate in the cells. When compared with corresponding non-mineralized cell cultures, both the mineralized cultures of calvarial cells and UMR 106 cells did not exhibit any change in intracellular bone-specific alkaline phosphatase activities which were measured by wheatgerm lectin precipitation method. Our results support the hypothesis that mineralization may not exert any direct negative feedback on matrix protein synthesis in osteoblasts during bone formation.