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PubMed2005

A flow cytometric assay to monitor antimicrobial activity of defensins and cationic tissue extracts.

To determine the antibacterial activity of defensins and other antimicrobial peptides in biopsy extracts, we evaluated a flow cytometric method with the membrane potential sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. This assay enables us to discriminate intact non-fluorescent and depolarized fluorescent bacteria after exposure to antimicrobial peptides by measurement at the direct target, the cytoplasmic membrane and the membrane potential. The feasibility of the flow cytometric assay was evaluated with recombinant human beta-defensin 3 (HBD-3) against 25 bacterial strains representing 12 species. HBD-3 showed a broad-spectrum dose dependent activity and the minimal dose to cause depolarization ranged from 1.25 to >15 microg/ml HBD-3, depending on the species tested. The antibacterial effect was diminished with sodium chloride or dithiothreitol and could be abrogated with a HBD-3 antibody. Additionally, isolated cationic extracts from human intestinal biopsies showed a strong bactericidal effect against Escherichia coli K12, E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923, which was diminished towards E. coli at 150 mM NaCl, whereas the activity towards S. aureus ATCC 25923 remained unaffected at physiological salt concentrations. DTT blocked the bactericidal effect of biopsy extracts completely.

Anti-Bacterial Agents↗
PubMed1992

Two methods to avoid the effect of endogenous protein inhibitors during the assay of protein kinase C activity in tissue extracts.

Using H1 as substrate the protein kinase C activity of rat liver cell sap was increased about fourfold by treatment with DEAE-cellulose at pH 7.5 at an intermediate ionic strength due to removal of protein inhibitors. The activity of cell sap from rat spleen, brain or muscle was about doubled by the same treatment. In contrast, when a specific synthetic peptide substrate was used the corresponding increase of enzyme activity was not obtained when the inhibitors were removed. This shows that this type of substrates should be preferred for reliable assays of protein kinase C in crude extracts. The possible role of the protein inhibitors for the substrate specificity of protein kinase C is briefly discussed.

Animals↗
PubMed1983

The biological and clinical significance of tumor markers in tissue extracts.

A quantitative study of carcinoembryonic antigen (CEA) activity was made in plasma and in 25 colorectal tumors, 30 lung tumors, 20 breast tumors, and 20 fibrocystic dysplasias of the breast. The CEA content was detected by enzyme assays (EIA) in solid phase. In colorectal cancer the amount of CEA ranged from 40 to 800 ng/mg protein. In lung cancer, values ranged from 5 to 130 ng/mg protein. Differences in mean content of CEA between normal and cancer yielded the highest significant value of P less than 0.001. In 54 of 95 of cases, high concentrations of CEA in tissues were associated with high values in plasma. In fibrocystic dysplasia of the breast, the CEA concentration levels were lower than in cases of breast carcinoma. The CEA concentration values in fibrocystic dysplasia were associated with the epithelial proliferation and the degree of dysplasia. Therefore, the presence of elevated CEA tissues values may be indicative of patients with higher risk of malignant transformation.

Breast Neoplasms↗
PubMed1999

Application of polydivinylbenzene liquid chromatography columns to remove lipid material from fish tissue extracts for the analysis of semivolatile organics.

Liquid chromatography columns of 100% polydivinylbenzene (DVB) (packing) were used to remove lipid material from fish extracts before analysis of several semivolatile organic pollutants by gas chromatography-mass spectrometry (GC-MS). This packing material was found to be durable as the columns could be operated to about 900 p.s.i. resulting in high efficiency separation. Recoveries and relative standard deviations for 18 polynuclear aromatic hydrocarbons fortified into a fish extract and cleaned up by multiple DVB columns in series were in the range of 86 to 123% and 4 to 11%, respectively.

Animals↗
PubMedDate not supplied

[The effects of aqueous extracts of Raphanus niger on an experimental influenza infection in mice and on the enzyme polymorphism in lung tissue extracts].

Prophylactic effects of Raphanus niger water extract against A/PR 8/34 influenza virus experimental infection was tested in mice, by means of the following parameters: mortality mean survival time and hemagglutinating (HA) titre of lung extract. Variations of the isoenzymatic structures of peroxidase and acid and alkaline phosphatases were studied too. The results revealed the significant reduction of mortality and of hemagglutinating titre and the augmentation of mean survival time. Modifications of the isoenzymatic structures, especially of the phosphatases, are characteristic for the illness state as well as for the one induced by R. niger extract treatment.

Acid Phosphatase↗
PubMed1984

Scanning electron microscopic study of polyvinyliden fluoride degradation by ocular tissue extracts.

A scanning electron microscopic study showed that the surface of the polyvinyliden fluoride thread, used to make loops for intraocular lenses in Japan, became significantly rough after treatment with bovine iris-ciliary body extracts. The polyvinyliden fluoride degrading factor in the extract was nondialyzable, heat-labile and active at an acidic pH, suggesting that a lysosomal system might be involved in this phenomenon. Bovine extracts from the retinal pigment epithelium and choroid degraded the surface of the polyvinyliden fluoride substantially, while extracts from the cornea, iris, ciliary body and neuroretina only moderately digested it. Lenticular extract did not affect the polyvinyliden fluoride surface. It is possible that the polyvinyliden fluoride loop undergoes degradation in the eye after a long time.

Animals↗
PubMed1990

A simple quantitative dot-immunobinding assay for glial and neuronal marker proteins in SDS-solubilized brain tissue extracts.

Immunoassays for quantitative determinations of the S-100 protein, the glial fibrillary acidic protein, the neuron specific enolase and the neurofilament proteins with molecular weight of 68 and 200 kDa in hot SDS sonicated rat brain extracts have been developed and characterized. The assays utilize a dot immunobinding technique, poly- or monoclonal antibodies and 125I-protein A. The SDS-sonication procedure was not found to affect the radioactivity recovery in the assay of the soluble S-100 protein or the neuron specific enolase. All 5 antigens can be measured with a within-assay variance below 10%. Even at a coefficient of variation less than or equal to 5%, the working ranges are approximately 30-100-fold with regard to the different antigens. It was found that gelatin-coated nitrocellulose membranes considerably increase the recovered radioactivity in the assay of the purified bovine S-100 protein, possibly by protein-protein interaction. This effect was not observed when SDS-sonicated rat brain extracts were assayed. The assay appears to be reproducible, convenient and rapid, and provides a high degree of precision in the determination of large number of samples.

Animals↗
PubMed1984

Parenterally administered salivary tissue extracts reduce outputs of Na+ and K+ associated with bile salt-induced damage of rat oxyntic mucosa.

Extirpation of the major salivary glands in rats has been shown to exacerbate the ionic fluxes associated with pharmacologically-induced damage to the gastric mucosal barrier. In the present study we have investigated the effect of the administration of a crude extract of submandibular-sublingual salivary glands on gastric secretion and gastric mucosal barrier integrity in the rat. The extract was administered in a dose (4 mg protein) which would reduce established acid secretion by 30%. Chronic parenteral (intraperitoneal) administration of salivary gland extract but not phosphate buffer resulted in a reduction in the luminal appearance of Na+ and K+ associated with bile salt-induced damage (5 mM sodium taurocholate in 150 mM HCl) to the gastric mucosa of sialoadenectomized animals. Oral administration of the extract depressed its protective action. Boiling the extract prior to parenteral administration also abolished its ability to protect the mucosa. Acute administration of the extract by intraperitoneal injection only 30 min prior to bile salt treatment was also ineffective. These results suggest that chronic parenteral but not enteral treatment with a crude salivary gland extract aids in the maintenance of the gastric mucosal barrier in sialoadenectomized rats. The inability of acute administration of the extract to reduce the net fluxes of Na+ and K+ suggests that its mode of action is not entirely dependent upon an inhibition of gastric acid secretion.

Animals↗
PubMed1983

Scanning electron microscopic study of Dacron degradation by ocular tissue extracts.

A scanning electron microscopic study showed that the surface of Dacron thread became significantly roughened after treatment with bovine iris-ciliary body extracts. The Dacron-degrading factor in the extract was nondialysable, heat-labile, and active at an acidic pH, suggesting that lysosomal enzymes may be a factor in this phenomenon. Bovine extracts from the iris, ciliary body, and sensory retina degraded the surface of Dacron most substantially, while the Dacron surface was moderately digested by extracts from the cornea and retinal pigment epithelium. Lenticular and choroidal extracts did not affect the Dacron surface. Possibly the factor that degrades Dacron may be different from that which affects nylon, and the Dacron suture may not be preferable for corneal surgery.

Animals↗
PubMed1979

Radioimmunoassay of glycosphingolipids: application for the detection of forssman glycolipid in tissue extracts and cell membranes.

Development of a radioimmunoassay for detecting glycosphingolipids has been difficult, primarily because of transfer of radiolabeled glycolipid antigen to the unlabeled antigen pool. This difficulty has been overcome by the use of a radiolabeled glycolipid-polymer. Thus, an assay system has been developed for measuring picomolar quantities of Forssman hapten glycolipid. This assay is based on competition for rabbit anti-Forssman antibodies between Forssman glycolipid and a radiolabeled Forssman polyacrylic hydrazide polymer. Antigen-antibody complexes are removed quickly and efficiently by binding to formalin-fixed Staphylococcus aureus and subsequent centrifugation. One nanogram of Forssman glycolipid can be readily detected both in plasma membrane preparations and in purified glycolipid fractions. The isoantigenic expression of Forssman glycolipid in human gastrointestinal tissues has been reported previously (Hakomori et al., 1977). Using the radioimmunoassay, the Forssman status of several additional cases has been determined quantitatively.

Animals↗
PubMed1976

A method for the study of the inhibition of DNA synthesis by rabbit tissue extracts.

This paper describes a method to measure DNA synthesis in the jejunum. Everted rings of rat jejunum were incubated in the presence of (3H) thymidine. The specific activity of jejunal DNA was assayed at the end of incubation. This method was found to be valid when (1) incubation time was 30 min, (2) (3H) thymidine concentration was 90 ng/ml, and (3) paired flasks containing four, respectively, contiguous rings were compared. This method was used to investigate the existence of an intestinal chalone. An aqueous extract (S-105)of rabbit small intestine mucosa was prepared and tested for DNA synthesis inhibition in jejunum. A significant inhibition was noted, which was an hyperbolic function of the quantity of extract present in the media. The study of S-105 inhibition on other organs (kidney, liver, colon) failed to demonstrate organ specificity. However, aqueous extracts from intestinal organs were found to exert a greater inhibition than aqueous extracts form other organs. It is suggested that S-105 contains other nonspecific inhibitory fractions; our data prove the necessity of an extensive purification to demonstrate the existence of a specific chalone that regulates intestinal cell proliferation.

Animals↗
PubMed2000

Attachment-inducing capacities of fish tissue extracts on oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae).

When oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae) were incubated in wells with lyophilized extracts of fish skin epithelia on the bottom, some attached to the well bottom with the haptor unfolded and shed the ciliated epidermal cells. Based on these morphological changes in oncomiracidia, we developed a new assay method to examine the attachment-inducing capacities of various fish extracts for oncomiracidia. Attachment-inducing capacities were found only in extracts of fish skin epithelium and not in other fish extracts. No significant difference in capacities was observed among extracts of skin epithelia of 4 fish species. Wheat germ lectin and concanavalin A suppressed capacities in extracts of skin epithelia of Japanese flounder and yellowtail, respectively. Suppressed capacities were recovered by adding sugars that bound specifically to these lectins. These results indicate that some sugar-related chemical substances that exist specifically in fish epithelium induce the attachment of N. girellae oncomiracidia.

Adhesiveness↗
PubMed1984

[Possible mechanism of action of a water-soluble tissue extract on arterial pressure in the rat].

A double hypo-hypertension effect is seen when a hydrosoluble splenic extract at pH 5 is injected to rats intravenously. The hypotensive effect may be caused by local vasodilating agents, such as histamine. The hypertensive one does not appear in adrenoprive animals or in rats treated with inhibitors of prostaglandin synthesis which suggests that the mechanism for this effect is complex.

Adrenalectomy↗
PubMed1978

Mesenchymal tumor hypoglycemia: the effect of a tumor tissue extract on the insulin and glucagon secretion from the isolated perfused porcine pancreas.

Severe fasting hypoglycemia and hypoinsulinemia occurred in a sixty-six year old woman with an intrahepatic mesenchymal tumor. An extract from the tumor was potent in inhibiting the arginine-induced glucagon secretion from the isolated perfused porcine pancreas. This observation supports the theory recently advanced that mesenchymal tumor hypoglycemia in some cases is due to a still unknown factor secreted from the tumor, which directly inhibits pancreatic glucagon secretion.

Aged↗