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PubMed1990

Degradation of individual intracellular proteins analyzed by two-dimensional gel electrophoresis and computerized video densitometry.

A technique is described for the analysis of degradation rates of individual intracellular proteins, based on pulse-chase-labeling of cells using radioactive amino acids [35S]methionine, two-dimensional polyacrylamide gel electrophoresis, fluorography and scanning of the fluorograms by a computerized video densitomter. As compared to scintillation counting of individual protein spots resolved by two-dimensional gel electrophoresis, this method allows a rapid and precise determination of the degradation rates of individual intracellular proteins. In the present study, degradation rates of individual intracellular proteins of normal human skin fibroblasts and skin fibroblasts from patients with Duchenne muscular dystrophy were compared. Rates of degradation for proteins PIIa, PIIb and PIIc recently described as cell-type-specific proteins were significantly enhanced (p less than 0.01) in fibroblast cultures of Duchenne muscular dystrophy origin.

Cells, Cultured↗
PubMedDate not supplied

A qualitative and quantitative protein database approach identifies individual and groups of functionally related proteins that are differentially regulated in simian virus 40 (SV40) transformed human keratinocytes: an overview of the functional changes associated with the transformed phenotype.

A qualitative and quantitative two-dimensional (2-D) gel database approach has been used to identify individual and groups of proteins that are differentially regulated in simian virus 40 (SV40) transformed human keratinocytes (K14). Five hundred and sixty [35S]methionine-labeled proteins (462 isoelectric focusing, IEF; 98 nonequilibrium pH gradient electrophoresis, NEPHGE), out of the 3038 recorded in the master keratinocyte database, were excised from dry, silver-stained gels of normal proliferating primary keratinocytes and K14 cells and the radioactivity was determined by liquid scintillation counting. Two hundred and thirty five proteins were found to be either up- (177) or down-regulated (58) in the transformed cells by 50% or more, and of these, 115 corresponded to known proteins in the keratinocyte database (J.E. Celis et al., Electrophoresis 1993, 14, 1091-1198). The lowest abundance acidic protein quantitated was present in about 60,000 molecules per cell, assuming a value of 10(8) molecules per cell for total actin. The results identified individual, and groups of functionally related proteins that are differentially regulated in K14 keratinocytes and that play a role in a variety of cellular activities that include general metabolism, the cytoskeleton, DNA replication and cell proliferation, transcription and translation, protein folding, assembly, repair and turnover, membrane traffic, signal transduction, and differentiation. In addition, the results revealed several transformation sensitive proteins of unknown identity in the database as well as known proteins of yet undefined functions. Within the latter group, members of the S100 protein family--whose genes are clustered on human chromosome 1q21--were among the highest down-regulated proteins in K14 keratinocytes. Visual inspection of films exposed for different periods of time revealed only one new protein in the transformed K14 keratinocytes and this corresponded to keratin 18, a cytokeratin expressed mainly by simple epithelia. Besides providing with the first global overview of the functional changes associated with the transformed phenotype of human keratinocytes, the data strengthened previous evidence indicating that transformation results in the abnormal expression of normal genes rather than in the expression of new ones.

Autoradiography↗
PubMedDate not supplied

Several small GTP-binding proteins are strongly down-regulated in simian virus 40 (SV40) transformed human keratinocytes and may be required for the maintenance of the normal phenotype.

High resolution two-dimensional (2-D) gel electrophoresis in combination with the blot overlay nucleotide binding assay was used to reveal low molecular weight GTP-binding proteins expressed by primary cultured, normal human keratinocytes. Forty one small GTP-binding proteins (30 isoelectric focusing, IEF; and 11 nonequilibrium pH gradient electrophoresis, NEPHGE) ranging in molecular weights from 18,000 to 30,000 and isoelectric points from 4.4 to 8.0 were detected and mapped in the master human keratinocyte database. Four GTP-binding proteins were identified by 2-D gel immunoblotting and these correspond to rap1 and 2 and two forms of rab6. ras-Proteins are most likely present in the [alpha 32P]GTP 2-D gel blots but their levels may be too low to be detected by immunoblotting. Quantitative changes in the relative expression levels of [alpha 32P]GTP-binding proteins in normal proliferating and simian virus 40 (SV40) transformed human keratinocytes (K 14) were determined by scintillation counting of the radioactive spots excised from the nitrocellulose blots. The results showed that thirteen of these proteins were not expressed in transformed K14 keratinocytes, implying that they may play a role in the maintenance of the normal cell phenotype.

Autoradiography↗
PubMed2006

Measurement of specific radioactivity in proteins separated by two-dimensional gel electrophoresis.

We report a method to quantify the specific radioactivity of proteins that have been separated by 2-DE. Gels are stained with SyproRuby, and protein spots are excised. The SyproRuby dye is extracted from each spot using DMSO, and the fluorescence is quantified automatically using a plate reader. The extracted gel piece is then dissolved in hydrogen peroxide and radioactivity is quantified by liquid scintillation counting. Gentle agitation with DMSO for 24 h was found to extract all the SyproRuby dye from gel fragments. The fluorescence of the extract was linearly related to the amount of BSA loaded onto a series of 1-D gels. When rat muscle samples were run on 2-DE gels, the fluorescence extracted from 54 protein spots showed a good correlation (r = 0.79, p < 0.001) with the corresponding spot intensity measured by conventional scanning and image analysis. DMSO extraction was found not to affect the amount of radioactive protein left in the gel. When a series of BSA solutions of known specific radioactivity were run on 2-DE gels, the specific radioactivity measured by the new method showed a good correlation (r = 0.98, p < 0.01, n = 5) with the specific radioactivity measured directly before loading. Reproducibility of the method was measured in a series of 2-DE gels containing proteins from the livers of rats and mice that had been injected with [35S]methionine. Variability tended to increase when the amount of radioactivity in the protein spot was low, but for samples containing at least 10 dpm above background the CV was around 30%, which is comparable to that obtained when measuring protein expression by conventional image analysis of SyproRuby-stained 2-DE gels. Similar results were obtained whether spots were excised manually or using a spot excision robot. This method offers a high-throughput, cost-effective and reliable method of quantifying the specific radioactivity of proteins from metabolic labelling experiments carried out in vivo, so long as sufficient quantities of radioactive tracer are used.

Animals↗
PubMed1983

Unscheduled DNA synthesis and DNA repair studies of peroxyacetic and monoperoxydecanoic acids.

Peroxyacetic (PAA) and monoperoxydecanoic (MPDA) acids were assayed for induction of unscheduled DNA synthesis (UDS) by liquid scintillation counting of hot-acid-extractable DNA and by light microscope autoradiography. Both compounds were also assayed for induction of DNA repair synthesis by differential density labeling ultracentrifugation. Uniformly negative results were obtained for MPDA. Conflicting results were obtained for PAA using the UDS techniques. Negative results were consistently obtained, however, in three separate assays using two different PAA samples with the more definitive differential density DNA repair synthesis technique. Hydrogen peroxide, which is present as a contaminant in the PAA samples, was also assayed for induction of UDS by autoradiography and for induction of DNA repair synthesis by differential density labeling. Our results with this compound are in agreement with published data and were consistently positive using both techniques. We conclude that neither MPDA nor PAA induce DNA repair synthesis and suggest that the conflicting PAA results may be due to the presence of hydrogen peroxide in commercial samples of PAA.

Acetates↗
PubMed1985

Diphtheria toxin resistance in human lymphocytes and lymphoblasts in the in vivo somatic cell mutation test.

It has been shown that circulating peripheral blood lymphocytes can be used for the enumeration of 6-thioguanine-resistant cells that presumably arise by mutation in vivo. This somatic cell mutation test has been studied in lymphocytes from human populations exposed to known mutagens and/or carcinogens. The sensitivity of the test could be further enhanced by including other gene markers, since there is evidence for locus-specific differences in response to mutagens. Resistance to diphtheria toxin (Dipr) seemed like a potential marker to incorporate into the test because the mutation acts codominantly, can readily be selected in human diploid fibroblasts and Chinese hamster cells with no evidence for cell density or cross-feeding effects, and can be assayed for in nondividing cells by measuring protein synthesis inhibition. Blood samples were collected from seven individuals, and fresh, cryopreserved, or Epstein-Barr virus (EBV)-transformed lymphocytes were tested for continued DNA synthesis (3H-thymidine, autoradiography) or protein synthesis (35S-methionine, scintillation counting). Both fresh and cryopreserved lymphocytes, stimulated to divide with phytohemagglutinin (PHA), continued to synthesize DNA in the presence of high doses of diphtheria toxin (DT). Similarly, both dividing (PHA-stimulated) and nondividing fresh lymphocytes carried on significant levels of protein synthesis even 68 hr after exposure to 100 flocculating units (LF)/ml DT. The findings were confirmed in cord blood lymphocytes, ruling out the possibility that diphtheria immunization could have led to a selection of Dipr lymphocytes. One lymphoblast line (EBV-transformed lymphocytes) showed a reduction in protein synthesis to 0.2% of controls only at 192 hr after exposure to 100 LF/ml. The results suggest that human T and B lymphocytes may not be as sensitive to DT protein synthesis inhibition as human fibroblast and Chinese hamster cells. For this reason, Dipr may not be a suitable marker for the somatic cell mutation test.

Cells, Cultured↗
PubMed1994

Serum activity induces Schwann cell proliferation in vitro.

The present series of experiments demonstrate that a polypeptide activity present in rat serum induces a proliferative response in cultured rat Schwann cells. Schwann cells in multi-well tissue culture plates were incubated in medium containing 10% heat-inactivated fetal bovine serum and serial dilutions of normal rat serum, and control preparations were incubated in the same culture medium without rat serum. Rates of cell proliferation were assayed by measuring DNA incorporation of tritiated thymidine using liquid scintillation counting. A prominent dose-dependent proliferative response was observed among Schwann cells incubated with rat serum and rat plasma dilutions as compared to controls; this activity is abolished by heat inactivation and by proteolytic digestion, and was not affected by dialysis against a cellulose ester membrane that excludes molecules larger than 10,000 daltons. In contrast, no increase in DNA uptake of tritiated thymidine was observed when astrocyte and oligodendrocyte cultures were incubated with serial dilutions of rat serum. No proliferative effect was observed when rat Schwann cells were incubated with a dilution of standard adult bovine serum. These results suggest there is an intravascular plasma polypeptide with a molecular weight greater than 10,000 daltons that specifically stimulates Schwann cell proliferation, and it is proposed that this factor may be the mitogen responsible for the Schwann cell proliferative response known to occur after nerve injury.

Animals↗
PubMed2002

Adsorbed poly(ethyleneoxide)-poly(propyleneoxide) copolymers on synthetic surfaces: spectroscopy and microscopy of polymer structures and effects on adhesion of skin-borne bacteria.

Poly(ethyleneoxide)-copoly(propyleneoxide) (PEO-PPO) polymer coatings were evaluated for their resistance to the attachment of the marker organism Serratia marcescens and the skin-borne bacteria Staphylococcus epidermidis. The copolymers were adsorbed onto poly(styrene) films-chosen as simplified physicochemical models of skin surfaces-and their surface characteristics probed by contact angle goniometry, attenuated total reflectance-Fourier transform infrared (ATR-FTIR), atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). These functional surfaces were then presented to microbial cultures, bacterial attachment was assessed by fluorescence microscopy and AFM, and the structures of the polymer films examined again spectroscopically. Surface characterization data suggest that the adsorbed copolymer was partially retained at the surface and resisted bacterial attachment for 24 h. Quantitative evaluation of cell attachment was carried out by scintillation counting of (14)C-labeled microorganisms in conjunction with plate counts. The results show that a densely packed layer of PEO-PPO copolymer can reduce attachment of skin commensals by an order of magnitude, even when the coating is applied by a simple adsorptive process. The work supports the hypothesis that adhesion of microorganisms to biological substrates can be reduced if a pretreatment with an appropriate copolymer can be effected in vivo.

Adsorption↗
PubMed1993

Toxicokinetics and biotransformation of pentachlorophenol in the topsmelt (Atherinops affinis).

The toxicokinetics and biotransformation of pentachlorophenol (PCP) were determined in the topsmelt (Atherinops affinis). In a static system, topsmelt (n = 9) were exposed to 50 micrograms/L of [U-14C]PCP for 24 hours to determine the absorption rate constant (Ka), the whole-body bioconcentration (at steady-state conditions), the elimination rate constant (Ke), and the elimination half-life (t1/2). Kinetics were determined by direct quantitation of radioactivity in the exposure water. Following exposure, fish were placed in a flow-through metabolism chamber for 24 hours to allow depuration of retained residues, which were collected on XAD-4 resin. Excreted residues were identified and quantified by high-pressure liquid co-chromatography, fraction collection, and liquid scintillation counting. The Ka and Ke, calculated using a simplified model, were 0.012 +/- 0.005/h and 0.014 +/- 0.003/h, respectively, while the 24 hour total concentration factor was 278.0 +/- 182.0 and the t1/2 was 52.7 +/- 11.2. During 24 hours of exposure to clean seawater, topsmelt depurated 32.9% of retained residues, and while PCP was primarily excreted unchanged (64.9%), significant amounts of both pentachlorophenylsulfate (18.9%) and pentachloro-beta-D-glucuronide (16.2%) were also formed.

Absorption↗
PubMed1986

In vivo biosynthesis of clathrin and other coated vesicle proteins from rat liver.

A biosynthetic study of rat liver coated vesicle (CV) proteins was undertaken by using in vivo labeling with L-[35S]methionine. CVs were isolated and purified by using standard procedures and characterized by electron microscopy, sedimentation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography, or by gel slicing and liquid scintillation counting. After 5 1/2 min of labeling (the earliest time examined), incorporation of radioactive clathrin heavy-chain (180-kD (kilodalton] subunits as well as a 90-kD CV-associated protein into purified CVs was demonstrated. The level of labeled 180-kD clathrin in coated vesicles increased rapidly during the first 2 hr of labeling and then continued to rise at a slower rate between 4 and 16 hr. This slow accumulation of labeled clathrin heavy chains in the CV pool may reflect early compartmental sequestration of a fraction of newly synthesized clathrin with delayed assembly into free CVs. By 16 hr of labeling, clathrin 180-kD chains and the 90-kD CV-associated protein accounted for approximately 48 and 26%, respectively, of the radioactivity in all CV proteins. Two proteins of MWa 68 kD and 53 kD showed marked declines in cpm/unit protein between 30 min and 4 hr, raising the possibility that these species may be transferred out of CVs during or after transport without loss of the other CV proteins. The possibility is also raised that clathrin heavy chains may be recycled during CV formation. Possible heterogeneity within individual CV preparations with respect to protein composition and derivation from both plasma membrane and Golgi regions are proposed.

Animals↗
PubMed1994

Quantitation of hepatitis B virus DNA in serum by ammonium sulfate precipitation and molecular hybridization.

We developed a method to quantitate hepatitis B virus (HBV) DNA in serum by ammonium sulfate fractionation and DNA hybridization. Serum samples were precipitated with 45% saturated ammonium sulfate, resuspended in buffer, and spotted on a nylon membrane. Following denaturation in alkali, HBV DNA sequences on the membrane were detected by hybridization with a 32P-labeled DNA probe of the entire HBV genome. Bound radioactivity was measured with liquid scintillation counting. Ammonium sulfate fractionation of positive samples increased assay sensitivity by 10-30-fold compared to no treatment. Sensitivity for detection of cloned HBV DNA added to negative serum was 0.2 pg. Recovery of cloned HBV DNA added to negative serum before fractionation was equivalent to direct spotting of DNA onto the membrane in the absence of serum. This method enhanced HBV DNA recovery from serum into small volumes, thereby expanding the potential analytic range of spot hybridization assays.

Adult↗
PubMed1977

Production of 5-hydroxyindoleacetic acid from serotonin by cultured endothelial cells.

Endothelial cells cells from bovine aorta and human umbilical vein and fibroblasts from human foreskin were cultured and subsequently evaluated for ability to metabolize serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA). Cells were incubated for three hours with 4 X 10(-6) M [14C] 5-HT creatinine sulfate. [14C] 5-HIAA was separated from labeled 5-HT by column chromatography and measured for scintillation counting. Production of 5-HIAA by bovine aorta cells was 39.0+/-7.5 (S.E.M., n=6) nmoles per 10(9) cells per hour. Production of 5-HIAA was markedly inhibited by the presence of 10(-4) M iproniazid (an inhibitor of monoamine oxidase) or 10(-4) M imipramine (an inhibitor of amine transport). 5-HIAA was the only product of 5-HT metabolism detected by thin layer chromatography. Production of 5-HIAA by human umbilical vein endothelial cells was 5.4+/-2.0 nmoles per 10(9) cells per hour (n=5) and by human foreskin fibroblasts was 3.9+/-1.4 nmoles per 10(9) cells per hour (n=5). The results obtained during incubation in the presence and absence of inhibitors indicate that bovine aorta endothelial cells maintained in tissue culture are able to transport serotonin with subsequent production of 5-HIAA. By contrast, human umbilical vein endothelial cells and fibroblasts exhibited relatively low rates of 5-HT uptake and metabolism.

Animals↗
PubMed1982

Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and growth factors in mouse 3T3 cells.

Addition of the cAMP derivatives butcAMP or 8BrcAMP to quiescent cultures of Swiss 3T3 causes synergistic stimulation of DNAk synthesis with insulin, phorbol esters, vasopressin, epidermal growth factor, or fetal bovine serum (2-5%). In the presence of insulin, 8BrcAMP, and butcAMP stimulate [3H]-thymidine incorporation into acid-precipitable material in a dose-dependent manner. The effect of these agents is specific since 8Br5'AMP, 5'AMP, butyrate, or 8BrcGMP fail to stimulate DNA synthesis under identical experimental conditions. Furthermore, the mitogenic effects of the cAMP derivatives were markedly potentiated by 1-methyl-3-isobutyl xanthine and 4-(3-butoxy-4-methoxy benzyl)-2-imidazolidine, both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. The growth-promoting effects of the cAMP derivatives were demonstrated by [3H]-thymidine incorporation (either by scintillation counting or by autoradiography), by flow cytofluorometric analysis, and by increase in cell number. When quiescent Swiss 3T3 cells were exposed to butcAMP and insulin, DNA synthesis began after a lag of 17h. The result of sequential additions of cAMP derivatives and insulin to quiescent 3T3 cells suggest that these agents must act simultaneously in G0/G1 to stimulate entry into DNA synthesis in these cells. The findings support the proposition that an increase in cellular levels of cAMP (but not cGMP) act sas a mitogenic stimulus for confluent and quiescent Swiss 3T3 cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1976

Octopamine: presence in firefly lantern suggests a transmitter role.

The lantern of the adult firefly is innervated by a nerve supply with endings similar to other known monoaminergic nerves. However, catecholamines could not be detected in the nerve endings and noradrenaline and adrenaline were relatively weak agonists in eliciting a flash of light when applied to the lantern in vitro. Firefly lanterns were assayed for octopamine using a sensitive, specific enzymatic assay in which octopamine is enzymatically converted to 3H-synephrine and quantitated by liquid scintillation counting. The presence of substantial amounts (125 ng/g tissue) of octopamine was demonstrated and confirmed by thin layer chromatography. On the basis of this and earlier work, it is suggested that the nerves to the firefly lantern are octopaminergic.

Animals↗
PubMed1978

Effects of puromycin and actinomycin D on fertilized rabbit eggs cultured in vitro.

Rabbit zygotes were cultured in media containing varying concentrations of puromycin and actinomycin D in order to determine what effect these agents have on events of fertilization and early development. Concentrations of puromycin greater than 10(-5) M inhibited mitosis, cytokinesis of fertilized eggs and leucine incorporation. In approximately 50% of the zygotes treated with puromycin (10(-5) M or greater) the female pronucleus remained at its site of formation, the cortex, and did not migrate centrad. In these instances the male pronucleus moved from its site of development to the female pronucleus. Concentrations of actinomycin D greater than 10(-7) M also inhibited mitosis and cytokinesis of fertilized eggs. Breakdown of the nuclear envelopes and association of the paternally- and maternally-derived chromatin did not occur in either puromycin- or actinomycin D-treated embryos. Actinomycin D at all of the concentrations employed (10(-9) to 10(-4) M) had little effect upon the structure of nucleolus-like bodies within male and female pronuclei. Male and female pronuclei of puromycin (10(-4) M) and actinomycin D (10(-5) M) treated zygotes, incubated with tritiated thymidine and prepared for scintillation counting and autoradiography, synthesized DNA at a reduced level when compared to control specimens. Although "side effects" of puromycin and actinomycin D treatment are not excluded, these results suggest that nascent proteins may be required for events of fertilization.

Animals↗
PubMed1980

Changes in RNA and protein synthesis in the neural retina during lens regeneration in newts.

Since neural retina stimulates regeneration of a lens from the dorsal iris in newts, RNA and protein synthesis in the neural retina was investigated during this process. Incorporation of 3H-uridine and 3H-leucine using liquid scintillation counting was employed to compare RNA and protein synthesis in the neural retina from sham-operated control eyes with that in eyes during lens regeneration. An initial increase in 3H-uridine uptake was seen one to three days after lentectomy. This was followed by greater incorporation of 3H-leucine, indicating increased protein synthesis between 5 to 15 days after lens removal. A decrease in 3H-uridine uptake was also seen at 5 to 12 days after lentectomy. After 20 days both the RNA and protein synthesis returned to the normal level. Since the increase in protein synthesis is preceded by an increase in RNA synthesis, the two processes might be related. The results indicate significant changes in the synthesis of macromolecules by the neural retina following lentectomy. These may be indirectly related to the production of the neural retinal factor with stimulates lens differentiation.

Animals↗
PubMed1987

Fucosylated glycoconjugates in mouse preimplantation embryos.

Preimplantation mouse embryos were metabolically labelled with 3H or 14C-fucose to investigate the synthesis of fucosylated macromolecules. Scintillation counting revealed that there was a progressive increase in both total fucose taken up by the embryo and incorporation of fucose into TCA-precipitable material as embryos developed from the 4-cell to the blastocyst stage. This was reflected in the increasing intensity of bands on autoradiographs of radioactive fucose labelled proteins separated on 10% SDS-PAGs between the 4-cell embryo (at which stage bands were first detectable) and the blastocyst. Minor qualitative changes in fucoproteins were detected at the time of compaction and additional bands appeared at the blastocyst stage. Preliminary analysis of fucolipids in 6- to 8-cell embryos indicated that an approximately equal amount of fucose was incorporated into lipid and protein. Autoradiographs of semi-thin sections of 3H-fucose-labelled embryos showed substantial amounts of radioactive material in the vicinity of the plasma membrane both adjacent to other cells and facing the zona pellucida. These data would support a predominant role for fucoconjugates in cell surface events in the preimplantation embryo from the 8-cell stage.

Animals↗
PubMed1994

Effects of 5-fluorouracil on macromolecular synthesis during secondary palate development in quail.

A study was undertaken to examine the growth of normal and 5-fluorouracil-treated quail secondary palate during embryogenesis. The rates of DNA, RNA, and protein synthesis were measured in the developing quail palate by liquid scintillation counting of radiolabelled thymidine, uridine, or leucine. In addition, shelf volume was determined morphometrically. The results showed that in control palates the shelf volume increased rapidly between days 5 and 7 of incubation. Drug treatment on day 4 did not alter the shelf volume until day 9 of incubation, at which time the treated shelves were smaller than controls. In control palates, the rate of DNA synthesis decreased steadily between days 5 and 9 of incubation. A burst in RNA synthesis on day 7 of incubation was followed by an increase in protein synthesis. Administration of FU seems to exert its effect via disturbing the synthesis of RNA and protein, instead of disruption of DNA synthesis, to ultimately affect the shelf area, and thus palate morphogenesis in quail. Comparison of avian and mammalian data indicated that differences in their palate morphogenesis are also reflected in the different temporal patterns of various macromolecular synthesis.

Animals↗