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PubMed1997

Tissue prothrombin. Universal distribution in smooth muscle.

Immunohistochemical analysis of surgically obtained porcine tissue samples reveals ubiquitous staining for prothrombin in organs rich in smooth muscle content and universal staining of smooth muscle in tissue vasculature. The native character of tissue prothrombin is verified first by chromogenic substrate hydrolysis and hirudin inhibition after incubation of tissue extracts with taipan snake venom and phospholipid. Western analysis of tissue extracts confirms the native zymogen molecular weight. In addition, prothrombin purified in good yield from porcine uterus is activated by Echis carinatus venom which, like taipan venom, is 4-carboxyglutamic acid-sensitive. After correction for blood (gross heme) and interstitial fluid (albumin), excess functional prothrombin is observed in extracts of tissues having abundant smooth muscle. In contrast with factor X, the yield of prothrombin purified from porcine uterus greatly exceeds that attributable to contamination by whole blood. Northern blot analysis from selected bovine tissues extracted for polyadenylated messenger RNA is equivocal for prothrombin mRNA with the exception of liver, which is positive. It is concluded that functionally intact prothrombin is widely distributed among tissues owing to smooth muscle content, although the mechanism of emplacement and physiologic significance of prothrombin in these tissues remains unclear.

Animals↗
PubMed1978

Antibodies to histones in drug-induced and idiopathic lupus erythematosus.

When tissue sections are extracted with 0.1 N HCl, cellular nuclear proteins, including histones, are removed but nuclear DNA is retained. Histones can be reconstituted back to nuclear DNA in acid-extracted tissue sections so that the resulting nuclear substrate is composed only of DNA and histones and does not contain acidic nuclear protein antigens. The resulting DNA-histone tissue substrate can be used in the immunofluorescent method for specific detention of antibodies to histones. Sera from 23 patients with drug-induced lupus erythematosus (procainamide 19, isoniazid 2, nitrofurantoin 2) and 20 patients with idiopathic (not drug-induced) systemic lupus erythematosus (SLE) were studied. All 23 patients with drug-induced lupus erythematosus (LE) lost nuclear staining on acid-extracted sections. In contrast, only 12 of 20 with idiopathic SLE lost nuclear staining on acid-extracted tissues, and in the remaining 8, there was no significant fall in titer. In the drug-induced LE group, loss of nuclear staining was due to the absence of histones on the substrate because with histone-reconstituted sections, 22 of 23 again became positive for nuclear staining at titers equal to or at one doubling dilution below titers on unextracted tissues. In contrast, of the 12 idiopathic SLE sera which lost nuclear staining, only 5 regained nuclear staining on histone-reconstituted tissue sections. In idiopathic SLE, antinuclear antibodies are heterogeneous in specificities and may consist of antibodies to native DNA, histones, or nonhistone proteins. In contrast, antinuclear antibodies in drug-induced LE are less heterogeneous and mainly consist of antibodies to histones.

Antibodies, Antinuclear↗
PubMed2002

Ischemic cerebral tissue and MCP-1 enhance rat bone marrow stromal cell migration in interface culture.

OBJECTIVE: Intravascular administration of bone marrow stromal cells (MSCs) restores function in animal models of neural injury and neurodegeneration. Adult MSCs administered intravenously to rat migrate and express neural phenotypes in ischemic brain. The aim of the present study was to investigate the mechanisms targeting MSC migration into the ischemic brain. METHODS: Monocyte chemoattractant protein-1 (MCP-1), a chemoattractant factor, was measured in rat ischemic brain at various time points after middle cerebral artery occlusion (MCAo) using a specific enzyme-linked immunosorbent assay system (ELISA). In addition, using a microchemotaxis chamber, we measured whether ischemic brain tissue extracts induce migration of MSCs and whether brain tissue extracts incubated with antibodies against MCP-1 reduce MSC migration. RESULT: Our data indicate that ischemic brain MCP-1 levels significantly increase from 6 hours, peak at 48 hours after MCAo (p < 0.05), and thereafter gradually decrease. Brain tissue extract at 6 hours, 24 hours, and 48 hours after MCAo significantly increase MSC migration across the membrane of the microchemotaxis chamber compared to normal tissue (p < 0.05). However, when the ischemic brain tissue extracts are incubated with antibody against MCP-1, MSC migration is significantly reduced at 24 hours and 48 hours after MCAo compared to extracts without this antibody (p < 0.05). CONCLUSION: Our data suggest that MCP-1 contributes to MSC migration into the ischemic brain tissue environment.

Animals↗
PubMed1991

[Changes in tissue oxygen extraction in patients with acute respiratory insufficiency].

We analyzed the tissue oxygen extraction in 25 patients with acute respiratory failure. Fourteen met the clinical criteria for the adult respiratory distress syndrome (ARDS). The 11 remaining patients had acute respiratory failure with causes different from ARDS. In all cases the changes in the oxygen extraction ratio (O2ER) and in the oxygen consumption (VO2) were evaluated after changing oxygen availability (O2A) with positive end-expiratory pressure (PEEP) and dobutamine infusion. The patients with ARDS showed a change in VO2 parallel to O2A changes, with a significant correlation (r = 0.85); however, no changes were found in O2ER (r = 18). In the patients without ARDS, the changes in O2A did not modify the VO2 (r = 0.02) but there was a significant inverse relationship between DO2 and O2ER (r = -0.70). These findings suggest an abnormal regulation of tissue oxygen extraction and an abnormal dependence of VO2 on O2A in cases with ARDS. Dobutamine therapy, in addition to inotropic effects, could improve a situation of hidden hypoxia, as it is a vasodilator that might act on microvasculature.

Acute Disease↗
PubMed1976

Detection of tumor-specific antigens in human mucinous cystadenocarcinoma of the ovary by immunodiffusion.

Rabbit antiserum to a tissue extract of human mucinous cystadenocarcinoma of the ovary reacted with tissue extracts of normal ovary and various ovarian malignancies, and ascitic or cystic fluids of ovarian origin by Ouchterlony double gel diffusion and precipitin inhibition techniques. The tumor-associated antigen(s) of mucinous cystadenocarcinoma, which were demonstrated by Ouchterlony double diffusion, were not present in tissue extract of pooled normal ovaries and cystic fluid of benigh tubo-ovarian cyst. An organ-associated tumor antigen as well as the type-specific tumor antigen may exist in mucinous cystadenocarcinoma of the ovary. The mucinous cystadenocarcinoma was not very immunologically different but was distinguishable from serous cystadenocarcinoma and other types of ovarian cancer by double gel diffusion. Precipitin-inhibition reactions demonstated that the adsorbed antiserum to human ovarian mucinous cystadenocarcinoma mixed with tissue extracts of dysgerminoma and serous cystadenocarcinoma, and ascitic fluid of papillary embryonal adenocarcinoma of the ovary could not eliminate the specific precipin line developed with tissue extract of mucinous cystadenocarcinoma.

Antigens, Neoplasm↗
PubMed1982

Search for retrovirus expression in men--failure to demonstrate retrovirus-specific antigens in normal and malignant tissue.

Extracts for different normal and malignant human tissues were checked for retrovirus antigens by Sepharose bead immuno-fluorescence assay. No convincing evidence could be obtained for the presence of type D and mammalian type C virus antigens in the tissues tested by three different immunoassays. It is concluded from these results that such viruses have no ubiquitous distribution in human beings.

Animals↗
PubMed1988

Analysis of growth factors in renal cell carcinoma.

Tissue extracts prepared from human renal tissue, renal cell carcinoma and serum-free conditioned media of ACHN cells and A498 cells, cell line originated from human renal cell carcinoma, stimulated DNA synthesis of BALB/c 3T3 cells. The activity (growth factor activity) was significantly higher in renal cell carcinoma than in normal tissues. Radioreceptor assay revealed that the contents of epidermal growth factor and type alpha transforming growth factor in the tissue extracts from renal cell carcinoma and conditioned media from renal cell carcinoma cell lines were below detectable level. Most of growth factor activity of the tissue extracts and conditioned media showed high affinity for heparin-Ultrogel, indicating that the major growth factor activity was due to heparin-binding growth factor(s). In addition, renal cell carcinoma contained growth factor activity for ACHN cells, which did not show specific affinity for heparin-Ultrogel.

Binding, Competitive↗
PubMed1991

Detection of (2'-5')oligoadenylate binding proteins by nondenaturing polyacrylamide gel electrophoresis and affinity blotting onto nitrocellulose.

A latent endoribonuclease, RNase L, binds to and is activated by (2'-5')oligoadenylates ((2'-5')(A)n, n = 2-15). Binding to a labeled derivative of (2'-5')(A)n, [32P](2'-5')(A)3pCp, is detected as a protein-ligand complex observed following nondenaturing polyacrylamide gel electrophoresis. One major binding complex and two minor binding complexes are readily seen in cytoplasmic extracts from Ehrlich ascites tumor cells, murine tissue extracts and rabbit liver tissue extracts. At least one of the more rapidly migrating complexes appears to be a proteolytic degradation product of the larger [32P](2'-5')(A)3pCp binding protein. Cell and tissue extracts containing [32P](2'-5')(A)3pCp binding activity can be immobilized onto nitrocellulose filters and [32P](2'-5')(A)3pCp binding activity detected using a simple, rapid, economical affinity blot assay. Detection of [32P](2'-5')(A)3pCp binding proteins following electrophoresis on nondenaturing polyacrylamide gels and the affinity blot assay significantly improve and simplify the analysis of (2'-5')(A)n binding proteins.

Adenine Nucleotides↗
PubMed1990

Tissue oxygen extraction in patients with adult respiratory distress syndrome and multiple organ failure.

Multiple organ failure (MOF) associated with adult respiratory distress syndrome (ARDS) secondary to sepsis, trauma, etc., continues to be a syndrome with poor prognosis. It is generally accepted that tissue hypoxia is associated with the subsequent development of MOF. The purpose of this study was to find the relationship between the reduced tissue oxygen extraction and the severity of the syndrome. By correlating either the tissue oxygen extraction per unit volume of blood, CaO2-CvO2, or the oxygen extraction ratio, (CaO2-CvO2)/CaO2, with the intrapulmonary shunt, Qs/Qt, we found that there were significant inverse correlations between these two parameters of tissue oxygen extraction and the severity of the illness in patients with ARDS and MOF. The observed reduction in tissue extraction of oxygen may be related to the development of deranged metabolism in ARDS and MOF.

Adult↗
PubMed1989

The generation of valosin-like peptides from a precursor protein in vitro as an extraction artifact.

Valosin is a 25 amino acid peptide recently isolated from the porcine gastrointestinal tract. The molecular forms of valosin-like immunoreactivity (VLIR) were examined following different tissue extraction procedures. Fractionation of tissue extracted with cold 0.1 M sodium hydroxide by Sephadex G50 gel permeation chromatography revealed a large form of VLIR (Kav = 0). Smaller forms of VLIR, Kav = 0.36 and 0.57 were obtained in tissue extracted by boiling in 0.5 M acetic acid. Acidification and boiling of the 0.1 M sodium hydroxide tissue extracts also generated smaller forms of VLIR of Kav = 0.36 and 0.57. Partially purified preparations of the large forms of VLIR extracted with sodium hydroxide could be disrupted into a smaller form of Kav = 0.57 by acidification and boiling. This smaller molecular form co-eluted with the synthetic 25 amino acid valosin standard. We conclude that valosin does not occur naturally but is an artifact generated by cleavage of a larger protein precursor upon acid extraction of tissues. Workers should be aware of the need to verify their extraction procedures when characterising novel peptides to avoid potential pitfalls such as acid/thermal cleavage of proteins.

Chromatography, Gel↗
PubMed2004

Parameters affecting substance P measurement in heart, lung, and skin.

Substance P (SP), a neuropeptide that is widely distributed both peripherally and centrally, mediates several pathophysiological processes. Among current assays for SP, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) have been most widely used. Several previous studies, mostly performed with nerve extracts or organ perfusates, determined that acidity of the extraction buffer as well as the number extractions performed constitute factors influencing accurate measurements. We used an ELISA protocol in this study to analyze methodological aspects of SP measurement in extracts from heart, skin, and lung. The extraction procedure had two steps, an acid extraction followed by a column extraction. We could effectively measure SP with extract from as little as 10 mg of tissue. For each tissue examined, different variables influenced the SP measured. For all tissues, the weight of tissue extracted was critical; the more tissue extracted, the lower the sensitivity of the assay. This problem could be overcome in skin by omitting the column extraction. When mechanical loses were considered (e.g., loss during extraction and SP retained by the column after elution), column extraction improved SP measurements only with lung tissue. The amount of SP remaining in the sample after the first extraction also varied among tissues. The first acid extraction effectively isolated 80% of total SP from skin. In contrast, the first extraction with lung tissue recovered only 58%. Because both acid and heat effectively release SP from nerve endings, this could reflect the presence of non-neuronal SP, especially in lung. High-dose capsaicin treatment, which depletes SP in nerve endings, caused 42% loss of SP in skin independent of amount of tissue extracted Our results suggest that a second acid extraction of tissue should be performed and that column extraction is clearly detrimental with skin samples.

Animals↗
PubMed1994

Characterization of platelet activity in neuroblastoma.

A study was conducted to characterize the platelet aggregation induced by neuroblastoma tissue to investigate the mechanism of hypercoagulability in patients with neuroblastoma. The patients whose tumor tissues were examined had been shown clinically to have enhanced platelet activity. Platelet aggregation induced by neuroblastoma tissue extract was compared with that of other pediatric tumors. The effects of pretreatment with an antithrombin agent and prostacyclin (PGI2) on the platelet aggregation induced by tumor tissue extracts were also evaluated. Tissue extracts of 12 of 15 neuroblastomas, 3 of 3 Wilms' tumors, and 1 pheochromocytoma were demonstrated to have an activity that potentiated platelet aggregation in vitro. The platelet aggregation induced by tissue extracts of neuroblastomas and other tumor tissues was suppressed almost completely by pretreatment with a PGI2 analogue. The aggregation induced by neuroblastomas and the pheochromocytoma was also suppressed by pretreatment with an antithrombin agent, argatroban, whereas the aggregation induced by Wilms' tumors was not suppressed by this agent. These results suggest that (1) malignant tumors in children also have some chemical substances that sensitize platelet activity, such as those in adult cancers, and (2) thrombin is one of the mediators stimulating platelet aggregation in cases of neuroblastoma, although it is unlikely to be a contributing factor in other pediatric malignancies such as Wilms' tumor.

Antithrombins↗
PubMed1999

Quantitative reevaluation of telomerase activity in cancerous and noncancerous gastrointestinal tissues.

Telomerase activity has been examined extensively in a variety of human cancerous and noncancerous tissues. However, it was sometimes difficult to measure telomerase activity quantitatively with the methods used and in the tissues examined. We examined telomerase activity quantitatively in gastrointestinal tissues by using the hybridization protection assay combined with the telomeric repeat amplification protocol (TRAP) to assess the diagnostic utility of measuring telomerase activity and to determine the relationship between telomerase activity and human telomerase reverse transcriptase (hTERT) expression. We report here that (i) polymerase chain reaction (PCR) inhibitors in the tissue extracts used for the telomerase assay were practically nullified by using tissue extract at 0.1 microg of protein/assay; (ii) RNase activity in tissue extracts should be blocked with 0.5 U of RNase inhibitor/microg tissue protein for the quantitative telomerase assay; (iii) no inhibitors of telomerase were found in tissue extracts other than RNase and PCR inhibitors (iv) higher telomerase activity in cancerous tissue than in noncancerous tissue from the same patients was observed in both gastric and colorectal tissues, but the telomerase activity varied from low to high levels in cancerous tissues, and it was not practical to set a general cut-off level for cancer diagnosis; (v) hTERT was expressed in both cancerous and noncancerous tissues, and (vi) the telomerase activity levels were generally lower than expected from the hTERT expression levels, suggesting posttranscriptional regulation of expression of telomerase activity.

Adenocarcinoma↗
PubMed1985

Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydromyricetin to Its Flavan-3,4-Diol (Leucodelphinidin) and to (+)-Gallocatechin by Reductases Extracted from Tissue Cultures of Ginkgo biloba and Pseudotsuga menziesii.

Extracts of callus or cell suspension cultures from petioles of Ginkgo biloba catalyzed the production of (+)-gallocatechin (2,3-trans-3,5,7,3',4',5'-hexahydroxy-flavan) from (+)-dihydromyricetin (5'-hydroxy-dihydroquercetin) along with the expected 3,4-cis-diol intermediate, leucodelphinidin, in a NADPH-dependent double-step reductase reaction at pH 7.4. The latter diol, isolated from the above incubation mixture, produced (+)-gallocatechin in a NADPH-dependent reaction. Extracts from tissue cultures derived from needles of Pseudotsuga menziesii (Douglas fir) also produced significant amounts of the 3,4-diol from dihydromyricetin. (+)-Dihydromyricetin, purified via paper chromatography from leaves of Leptarrhena pyrolifolia, was reduced by NaBH(4) to the presumed 3,4-trans-diol and acid epimerized to the 3,4-cis-diol.

Journal Article↗
PubMed1994

[Effects of pentoxifylline on leukocyte in the lungs of dog with fat tissue extract-induced respiratory distress syndrome].

We studied the effects of pentoxifylline (PTX) on the polymorphonuclears (PMN) and alveolar macrophages (AM) in the lung of dog with fat tissue extract induced respiratory distress syndrome (RDS). Results showed that PTX decreased PMN accumulation and infiltration in the lung; the total leukocytes in BALF dropped from 2.92 x 10(8) in the experimental group to 1.21 x 10(8) in the PTX-treated group (P < 0.001); the percentage of PMN also reduced from 35.4% to 14.3% (P < 0.01); the production of IL-1 and TNF by AM in the PTX-treated group was markedly inhibited as compared with the experimental group (P < 0.001, P < 0.05). We conclude that PTX has an inhibitory effect on PMN and AM in the lung of dog with RDS, and PTX may have potential therapeutic effect on ARDS.

Animals↗
PubMed1970

Thin-layer chromatography of tissue lipids without extraction.

Frozen tissue sections were applied directly to silica gel plates, and the lipids were separated by developing the plates in different solvent systems. Quantitation of the lipid classes was achieved by direct transmission densitometry after the plates were treated with a chromic acid-sulfuric acid spray.

Adipose Tissue↗
PubMedDate not supplied

[Preparative isolation of progesterone, testosterone and estrogens from marine invertebrate tissues].

Extraction and quantitative determination of progesterone, testosterone and oestrogens have been made in tissues of cephalopod molluscs and crustaceans. Extraction was made by acetone and ethyl ether. Purification was performed by thin layer chromatography on aluminium oxide and silicagel in different systems. The quantitative assay of progesterone and oestrogens was made by fluorimetric and radioimmunological techniques, that of testosterone--by spectrophotometric determination of hydrozones and radioimmunological procedure. Progesterone and testosterone were obtained from the gonads and liver of cephalopods and from the whole body of crustaceans. During sexual maturation of the animals concentration of these hormones tends to increase. Oestrone and oestradiol were found only in the gonads of cephalopods.

Animals↗
PubMed1983

Tumor-reactive IgE antibodies in plasma of patients with gastrointestinal carcinomas.

A total of 208 plasma samples from 115 patients with gastrointestinal carcinomas and nine patients with other intestinal disease were examined for the presence of IgE tumor antibodies by a solid-phase radioimmunoassay. Approximately one-third of the patients gave significant reactions with gastrointestinal carcinoma extracts compared with normal tissue extracts. Absorption with tumor and normal tissue extracts, with type AB human red cells, and with CEA indicated tumor specificity in some of the samples so examined. None of the 50 serum samples tested from normal blood donors contained tumor-specific IgE. IgE tumor antibodies decreased or completely disappeared in the majority of patients 8-13 days after surgical treatment.

Antibodies, Neoplasm↗