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PubMedDate not supplied

[Isolation of fibrin assembly inhibitor from plasma and tissue extracts].

Procedures for isolation of an inhibitor of the fibrin self association are described. The procedures involved dialysis across cuprophane or cellophane T-100, ion exchange chromatography (the first method) or gel filtration (the second method). The product containing amino acid impurities was obtained after dialysis across cuprophane and ion exchange chromatography; a homogeneous inhibitor was isolated by means of dialysis across cellophane T-100 and gel filtration through Sephadex G-50.

Amino Acids↗
PubMed1988

A nonimmunoglobulin precipitin to tissue extracts in pathological human sera.

An alpha-globulin component was noted in pathological human sera, which produced gel precipitation reactions with extracts of human and animal liver. The highest incidence of the precipitin was found in malaria (95%), renal graft rejection (81%), and rheumatoid arthritis (57%). The precipitinogen was thermostable and ethanol soluble; of two precipitation lines formed by this component, one merged into identity reaction with a line produced by commercial lecithin of bovine origin. The possible diagnostic application of the reactions noted was considered.

Alpha-Globulins↗
PubMed1995

The hybrid xenograft/autograft bioprosthetic heart valve: in vivo evaluation of tissue extraction.

The major functional problem with bioprostheses is poor long-term durability. Bioprosthetic valves fail because of calcification and mechanical fatigue, both of which result from the glutaraldehyde fixation process. In an effort to develop a biologically active, non-cross-linked bioprosthetic valve, we devised a cellular extraction process. We tested the mechanical integrity of the processed valves and cultured both human and porcine cells on this material. To test the potential for calcification, we implanted strips of fresh, extracted, and glutaraldehyde-treated porcine heart valve tissue subcutaneously into 3-week-old Sprague Dawley rats for 21 days. We used atomic absorption spectroscopy to measure the extent of calcium accumulation and histopathologic assessment to evaluate the antigenic response. We found that the cell extraction process significantly reduced the propensity of the material to calcify in vivo (mean +/- standard deviation, 4.12 +/- 1.02 mg/g calcium extracted versus 10.75 +/- 3.9 mg/g calcium fresh versus 79.6 +/- 18.3 mg/g calcium glutaraldehyde fixed) but increased the antigenicity, as evidenced by increased cellular activity and resorption. Although they may reduce calcification, conventional detergent-based cell extraction techniques do not completely remove porcine aortic valve antigens and may in fact increase the antigenicity of the valve cusp material.

Animals↗
PubMedDate not supplied

[Identification of the catecholamines in tissue extracts of the trematode, Fasciola hepatica].

Dopamine was identified in extracts from Fasciola hepatica by the fluorometric method. It was found that the dopamine concentration in F. hepatica was 0.668 +/- 0.027 micrograms/g of wet weight. The head region (including the oral and ventral suckers) contained 1.658 +/- 0.275 micrograms/g of dopamine (0.016 +/- 0.002 micrograms per parasite), while the posterior region (located behind the ventral sucker) contained 0.278 +/- 0.070 micrograms/g of dopamine (0.014 +/- 0.004 micrograms per parasite). When the worms were treated with DL-DOPA, the concentration of dopamine increased. In helminths treated with reserpine a marked decline of dopamine level was observed. Adrenaline and noradrenaline were not detectable in F. hepatica.

Animals↗
PubMedDate not supplied

[In vitro determination of the biological activity of the electrophoretic fractions of embryonic tissue extracts].

The possibility of direct combining the protein fractions after electrophoresis in polyacrylamide gel (PAAG) with the ectoderm of avian embryos in vitro was shown. The extract of eye vesicle of 30-33 hrs chick embryos were subjected to electrophoresis in 20% PAAG in capillaries (diameter 300 mu). The columns of PAAG were cut in equal parts and these pieces with protein fractions were combined with the trunk ectoderm of 2 days old chick embryos. The preliminary analysis has shown that some fractions of the eye vesicle exert a selective neutralizing effect on the trunk ectoderm (formation of neural tubes). The PAAG itself had no toxic or inducing effect on the trunk ectoderm cells. The lens-inducing effect of the eye vesicle fractions was not observed.

Animals↗
PubMed1983

Activating proteins for ganglioside GM2 degradation by beta-hexosaminidase isoenzymes in tissue extracts from different species.

The existence of activator proteins that stimulate hydrolysis of ganglioside GM2 by beta-hexosaminidase was demonstrated in kidney extracts from four species (rat, mouse, cattle and pig). The extent to which these preparations, as well as their human counterpart, promote ganglioside GM2 catabolism by autologous and heterologous hexosaminidase isoenzymes was compared. It was found that these activators can replace each other functionally, although the animal activator proteins do not cross-react immunochemically with an antiserum against the human protein. All preparations examined catalysed the transfer of ganglioside GM2 between liposomal membranes, indicating that the animal activator proteins act by a mechanism similar to the human GM2 activator.

Animals↗
PubMed2003

Contributions of lung tissue extracts to invasion and migration of human hepatocellular carcinoma cells with various metastatic potentials.

PURPOSE: The MHCC97 cell line contains two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potentials, for which the pulmonary metastatic rate was 100% vs 40% between the two human hepatocellular carcinoma (HCC) clones. In an effort to elucidate the mechanism of organ-specific metastasis, we studied the effect of lung extracts from C57BL/6 mice on migration and invasion using the MHCC97 cell line. METHODS: Determination of migration and invasion induced by lung extracts to MHCC97 cell lines was examined by chemoinvasion assay. The organization of cytoskeleton was tested using filamentous actin (F-actin) polymerization assay and flow cytometry. The activity of matrix metalloproteinase (MMPs) was analyzed by zymogram. Fluorescence double staining was employed for MMPs and F-actin colocalization in MHCC97-H cells induced by lung extracts. RESULTS: The number of cells in response to extracts of lung, liver, kidney, and spleen in MHCC97-H cells was 64+/-10, 6+/-2, 22+/-4, and 3+/-1, respectively. Of the extracts, lung extracts showed significant differences to promote the migratory and invasive ability for MHCC97-H cells( p<0.001). The number of cells in response to lung extracts was threefold higher in MHCC97-H than that of cells in MHCC97-L ( p<0.001). Confocal laser scan microscope of MHCC97-H cells and MHCC97-L cells stimulated with lung extracts revealed pseudopodia formation around the cell front at the indicated time point. With the time increasing, the pseudopodia formation became increasingly more obvious and distinct. Compared with unstimulated cells, analysis of FACS showed a transient 1.9-fold and 1.7-fold increase in F-actin within 30 s in MHCC97-H cells and MHCC97-L cells, respectively. Confocal laser scan microscopy of MHCC97-H cells stimulated in suspension with lung extracts revealed intense F-actin staining in the periphery of the cells and redistribution of F-actin towards a leading edge. After the cells were incubated with lung extracts, not only expressions of active and latent form of MMP-9 were upregulated, but that of latent form of MMP-2 was increased in the MHCC97-H cells and MHCC97-L cells. The levels of latent and active form of MMP-9(18.8+/-1.2, 100.1+/-1.1), and latent form of MMP-2(22.4+/-1.3) were much higher in MHCC97-H cells than those of MHCC97-L cells, which were 7.8+/-0.3, 40.8+/-2.2, and 8.2+/-0.4, respectively. MMP-9 was mainly localized perinuclear pool when the MHCC97-H cells were incubated with serum-free medium. After the cells were stimulated with lung extracts, MMP-9 were expressed and colocalized with F-actin at the front of extending pseudopodia. CONCLUSIONS: Our results indicate that the expression of matrix metalloproteinase and the pseudopodia formation in MHCC97-H cells may correlate to the metastatic potential; thus, the host environment may contribute to the preferential metastasis of HCC cells to the lung depending on the high level of MMP-9 and migratory ability.

Animals↗
PubMed1981

Nuclear androgen receptors in human prostatic tissue. Extraction with heparin and estimation of the number of binding sites with different methods.

A procedure for the estimation of nuclear androgen receptors in benign prostatic hyperplastic tissue is described, which employs extraction of receptors from nuclei with buffers containing heparin. Extraction of a nuclear pellet with a heparin-containing (1 g/l) buffer appeared to have definite advantages over 0.4 mol/l KCl extraction. Heparin appeared to be twice as efficient in extracting androgen receptors. In addition aggregated receptor proteins, formed after storage at -80 degrees C, were partly deaggregated by heparin. Specific isolation of the androgen receptor was performed using either agar gel electrophoresis, protamine sulphate precipitation or LH-20 gel filtration. A comparison was made between the amounts of estimated receptors with these different techniques. Protamine sulphate precipitation resulted in the highest estimates of receptor-bound 5 alpha-[3H]dihydrotestosterone (3H-DHT). Treatment of the labelled nuclear extracts with a charcoal suspension prior to the receptor assay resulted in lower amounts of estimated androgen receptors. A method for routine evaluation of nuclear androgen receptors in prostatic tissue has been evaluated, which involves extraction of nuclear pellets with a heparin-containing (1 g/l) buffer, exchange labelling of the nuclear extracts for 20 h at 10 degrees C and quantification of the receptors with protamine sulphate precipitation.

Cell Nucleus↗
PubMed1983

Creatine kinase and lactate dehydrogenase: stability of isoenzymes and their activity in stored human plasma and prostatic tissue extracts and effect of sample dilution.

We examined the effect of storing human plasma or extracts of prostate at -90 degrees C on the activity of creatine kinase and lactate dehydrogenase and isoenzyme distribution. Enzyme activities were unaltered during storage for as long as six weeks. If these preparations were thawed only once at 2 to 4 degrees C, they could be stored for as long as 165 days at -90 degrees C with no change in isoenzyme distribution. Inexplicably, apparent isoenzyme distribution of prostatic lactate dehydrogenase was sensitive to sample dilution, whereas the isoenzyme distribution of lactate dehydrogenase in plasma was not. Our observations emphasize the importance of validating details of analytical protocols that are to be used for quantification of new types of specimens.

Creatine Kinase↗
PubMed1994

Influence of a corpus luteum tissue extract on rabbit ovarian mesothelial cells.

This study investigates rabbit ovarian mesothelial (OM) cells exposed in vitro to a crude corpus luteum extract (CLE; 60 micrograms/ml). The growth of OM cells was evaluated by measuring the change in cell number (mean % +/- standard error of mean, SEM), the number of cell population doublings (CPD +/- SEM), and the cell population doubling time in hours (CPDT +/- SEM) after 7.5 days of culture in a serum-poor medium. Quantitative estimates of surface morphology changes were obtained by analyzing the total number (mean no. +/- SEM), density (mean no./100 microns 2 +/- SEM), and length-to-diameter ratio (mean L/D +/- SEM) of microvilli. OM cells in control medium formed loosely cohesive monolayers, and grew 152.53 +/- 11.01% with a CPD of 0.59 +/- 0.08 and a CPDT of 117.29 +/- 6.43 hours. The exposed surface area of these cells was over 8,000 microns 2 and was covered in its epinuclear region by long and slender microvilli with a L/D of 6.01 +/- 0.29. The total number of microvilli in each control cell was 1977.52 +/- 120.49 with a density of 0.58 +/- 0.03/100 microns 2 in the epinuclear region and of 0.05 +/- 0.003/150 microns 2 in the remaining surface area (5,161.62 +/- 354.43 microns 2). In contrast, CLE-rich cells cultures grew 329.57 +/- 16.65%, with a CPD of 1.71 +/- 0.07 and a CPDT of 53.43 +/- 2.93 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Simple and rapid preparation of infected plant tissue extracts for PCR amplification of virus, viroid, and MLO nucleic acids.

A rapid, simple method for preparing plant tissues infected with viruses, viroids, or MLOs using a commercial product known as Gene Releaser is described. The Gene Releaser polymeric matrix method produced plant extracts suitable for PCR amplification without the use of organic solvents, ethanol precipitation, or additional nucleic acid purification techniques. Modification of maceration methods and/or extraction buffers resulted in the PCR amplification of potato spindle tuber, apple scar skin, and dapple apple viroids, as well as, genomic segments of plum pox potyvirus, grapevine virus B, grapevine leafroll-associated virus III, and elm yellows MLO. These pathogens were amplified from tissue of woody and herbaceous hosts such as peach, apricot, apple, grapevine, elm, periwinkle and potato. The application of this product for use with intractable tissue avoids lengthy and laborious extraction procedures. In our hands, about 20 samples could be prepared for PCR or RT-PCR in 1-2 h versus 1-3 days.

Base Sequence↗
PubMed1982

A scanning electron microscopic study of nylon degradation by ocular tissue extracts.

A scanning electron microscopic study showed that the surface of the nylon used for corneoscleral wound closure was smooth, while the nylon suture thread used to fix the intraocular lens against the iris showed cracks and roughness on its surface. The surface of the nylon thread was also roughened after treatment with bovine iris-ciliary body extracts. The nylon-degrading factor in the extract was nondialyzable, heat-labile, active at acidic pH, and inhibited in part by leupeptin, suggesting that lysosomal enzymes may be a factor in this phenomenon. Of the bovine ocular tissues, ciliary body extract degraded the surface of the nylon thread most significantly. The nylon surface was moderately digested by the retinal pigment epithelial extract, and slightly eroded by the extracts from the iris, sensory retina, and choroid. Corneal and lenticular extracts did not affect the nylon surface. Nylon suture thread may be hydrolyzed by lysosomal enzymes and should be considered a late-degrading suture.

Animals↗
PubMed1982

[Effect of heterogeneous inducers on the ectoderm of the early gastrula in Rana temporaria in vitro. IV. The inductive effect of embryonic tissue extracts after ribonuclease and proteinase treatment].

An attempt was undertaken to determine the chemical nature of the neuralizing and lens-inducing effect of the retina and brain extracts from 7-8 day old chick embryos. These extracts were treated with immobilized enzymes (RNAse, proteinase K) and the effect of treatment was then estimated in the organ cultures (reacting tissue - early gastrula ectoderm). The effect of the enzymes was studied in different experimental variants which allowed to exclude the effect of the own proteases and temperature on the inducing activity of the extracts under study. The neuralizing activity of the retinal extract was shown to preserve after its treatment with RNAse but to be almost fully lost (or decrease) after proteolytic hydrolysis. Proteinase suppressed as well completely the lens-inducing effect of the extract from the chick embryo brain. A conclusion is drawn that the inducing activity of the extracts under study is due to proteins, rather than to RNA.

Animals↗
PubMed1966

The interconversion of glycine and serine by plant tissue extracts.

1. Extracts prepared from a variety of higher-plant tissues by ammonium sulphate fractionation were shown to catalyse the interconversion of glycine and serine. This interconversion had an absolute requirement for tetrahydrofolate and appeared to favour serine formation. 2. The biosynthesis of serine from glycine was studied in more detail with protein fractionated from 15-day-old wheat leaves. Synthesis of [(14)C]serine from [(14)C]glycine was not accompanied by labelling of glyoxylate, glycollate or formate. 3. The synthesis of serine from glycine was stimulated by additions of formaldehyde, and [(14)C]formaldehyde was readily incorporated into C-3 of serine in the presence of tetrahydrofolate. 4. The results are interpreted as indicating that serine biosynthesis involves a direct cleavage of glycine whereby the alpha-carbon is transferred via N(5)N(10)-methylenetetrahydrofolate to become the beta-carbon of serine.

Carbon Isotopes↗
PubMed1976

A method for tissue extraction and determination of prostate concentrations of endogenous androgens by radioimmunoassay.

A method for simultaneously determining concentrations of major androgens in prostate has been developed. Extraction techniques used to isolate the androgens from minced tissue include homogenization with high-speed blades in Delsal's solvent mixture, adsorption to silica gel, followed by column and one thin-layer chromatography (TLC). Radioimmunoassays (RIA) of small aliquots of TLC eluates are used to quantitate picogram amounts of 5alpha-dihydrotestosterone (DHT) and 5alpha-androstanediols (Diol) and to estimate testosterone (T) and androstenedione (Ad). Contamination of blanks was reduced to RIA sensitivity limits primarily by treatment of glassware in a self-cleaning oven. The specificity of the method for each androgen was established by TLC separations of known prostate metabolites, antisera specificities, and parallelism of sample aliquots to androgen RIA standards. The overall precision, in terms of coefficients of variation, was 21% for DHT and 24% for Diol. T and Ad could not be measured with acceptable precision because their very low concentrations in prostate (less than or equal 0.5 ng/g tissue) were less than RIA sensitivity limits. Accuracy studies indicated recoveries ranging from 96% for Diol to 121% for DHT. In human benign hypertrophic prostate tissue, DHT averaged 153 ng/g soluble protein (5.8 ng/g tissue) which was 17 times higher than values obtained in human spleen and kidney; Diol in prostate showed no consistent differences from values noted in kidney or spleen.

Androgens↗
PubMed2001

A cystatin-based affinity procedure for the isolation and analysis of papain-like cysteine proteinases from tissue extracts.

Cysteine-proteinases (CP) of the papain family can be affinity-adsorbed by egg white cystatin C coupled to Sepharose 4B, thus allowing their selective isolation from either tissue or cultured cell extracts as well as biolological fluids and culture media. CP complexed by immobilized cystatin are further analyzed by means of SDS-PAGE and Western blot followed by serial or parallel immunological detection. The single-step affinity adsorption of papain-like enzymes has the advantage, over immunoprecipitation techniques, of yielding the simultaneous and comprehensive picture of most CP, as both precursor and mature forms, in a given sample. Moreover, cell extraction in the presence of immobilized cystatin ensures a fast complexation of CP, avoiding artifacts, due to conversion, degradation, and, eventually, subtraction of constitutive enzymes from the sample because of their interactions with endogenous inhibitors. This will provide a pattern that might reflect more closely the real CP levels in intact cells. The method may be useful in the field of biochemistry, cell biology, and, possibly, clinical chemistry to perform rapid analyses of papain-like enzymes and to monitor changes in both cellular and extracellular CP profiles along with different physiopathological conditions.

Adsorption↗
PubMed1965

Diphosphoinositide and triphosphoinositide in animal tissues. Extraction, estimation and changes post mortem.

1. A method is presented for the determination of the di- and tri-phosphoinositide in animal tissues. 2. The polyphosphoinositides are quantitatively extracted into chloroform-methanol-hydrochloric acid solvent after a preliminary chloroform-methanol (1:1, v/v) extraction to remove the bulk of the other phospholipids. On washing this extract with n-hydrochloric acid the polyphosphoinositides pass completely into the lower chloroform-rich phase. Their concentrations in the lower phase are determined by chromatography on formaldehyde-treated paper or chromatography and ionophoresis of the acid hydrolysis products. 3. When guinea-pig brain is extracted by the method of Folch (1942), considerable hydrolysis of the triphosphoinositide and accumulation of diphosphoinositide occurs during the initial acetone extraction. 4. The tri- and di-phosphoinositide contents of rat and guinea-pig brain decline substantially within a few minutes after death. 5. The concentrations of tri- and di-phosphoinositide in rat brain are not changed by insulin-hypoglycaemia or electrical stimulation. 6. Examination of frozen rat tissues showed that the brain contained the highest concentration of polyphosphoinositides. Much smaller amounts are present in kidney, and only trace quantities in liver and lung. None could be detected in spleen, heart and skeletal muscle.

Animals↗