Effects of 48/80 or reserpine on tissue antihistamine extracts.
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Animals↗
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Pentoxifylline improves the tissue oxygen extraction capabilities during endotoxic shock.
Pentoxifylline (PTX), a xanthine derivative used in the treatment of circulatory insufficiency, has been found to have protective effects in different models of sepsis. We hypothesized that this drug might improve the cellular oxygen availability following endotoxin challenge by increasing oxygen delivery (DO2) and/or tissue oxygen extraction. The oxygen extraction capabilities were studied during a reduction in blood flow induced by cardiac tamponade. Fourteen anesthetized, ventilated, and paralyzed dogs, received intravenous 2 mg/kg of Escherichia coli endotoxin followed by a continuous infusion of 20 ml/kg.h of saline. 30 min later tamponade was induced by repeated bolus injections of warm saline into the pericardial space. Seven dogs were pretreated with PTX as an intravenous bolus of 20 mg/kg, followed by a continuous infusion at 20 mg/kg.h, and the other seven dogs served as a control group. PTX largely attenuated the systemic and pulmonary vasoconstriction observed in the control group and resulted in significant increases in cardiac index, DO2 and oxygen consumption (VO2). PTX also improved ventilation/perfusion matching in the lungs as indicated by a higher PaO2 and PvO2 and a lower venous admixture than in the untreated group during cardiac tamponade (both p < .05). In addition, the critical DO2 (DO2 crit) was lower and the critical oxygen extraction ratio was higher in the PTX treated than in the control group (9.1 +/- 1.8 vs. 11.6 +/- 2.4 ml/kg.min, and 70.6 +/- 14.0 vs. 49.3 +/- 14.6%, both p < .05). The VO2/DO2 dependency slope was also steeper in the PTX-treated than in the control group (.80 +/- .28 vs. .43 +/- .19, p < .05).(ABSTRACT TRUNCATED AT 250 WORDS)
Analysis of Variance↗
Identification of high molecular weight neurophysins in extracts of human neurohypophyseal tissue.
Acid extracts of human neurohypophyseal tissue were found to contain neurophysin-immunoreactive proteins in the 17,000-20,000 dalton molecular weight range by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 17,000-20,000 dalton neurophysins persisted under reducing conditions. Limited chymotrypsin proteolysis of the 17,000-20,000 dalton neurophysins generated both increased 10,000 dalton neurophysin and also 4000-8000 dalton vasopressin immunoreactivity. These 'big' neurophysins appear to represent precursor forms of the neurophysins and the neurohypophyseal hormones, which are present within the neurosecretory granules of the human posterior pituitary.
Electrophoresis, Polyacrylamide Gel↗
Up-regulation of matrix metalloprotease-9 in middle ear cholesteatoma--correlations with growth factor expression in vivo?
The role of matrix metalloproteases and their regulation in the pathology of middle ear cholesteatoma is still unclear. Recently we have demonstrated that incubation of keratinocytes with cholesteatoma debris and granulation tissue extracts causes induction of gelatinase B (matrix metalloproteinase-9, MMP-9) secretion in vitro. Antibodies against a variety of growth factors revealed some inhibitory effect on MMP-9 induction, caused by debris or granulation tissue extracts. In order to investigate the coherence of growth factor expression and matrix metalloproteinase activity in vivo in middle ear cholesteatoma, we performed quantitative gelatin zymographic analysis with tissue homogenates of 37 cholesteatoma and nine external ear canal skin (EACS) samples. Furthermore we quantified levels of the cytokines IL-1alpha, IL-1beta, TNF-alpha, TGF-beta and EGF present in tissue extracts, using enzyme-linked immunosorbent assays (ELISA), and correlated cytokine concentrations with gelatinolytic activities. Zymographic analysis revealed a highly heterogeneous expression of gelatinase A and B in cholesteatoma specimens. As shown previously, MMP-9, but not MMP-2, was increased in cholesteatoma when compared to EACS samples. ELISA studies revealed a significantly elevated IL-1alpha level in cholesteatoma. Regression analysis involving gelatinolytic activity and cytokine concentrations in tissue homogenates showed no statistically significant correlation between expression of gelatinases and the cytokines IL1-alpha, IL1-beta, TNF-alpha, TGF-beta or EGF. The discrepancy between in vitro observations and the situation in vivo is discussed critically.
Cholesteatoma, Middle Ear↗
Identification of a plasminogen activator derived from nasopharyngeal carcinoma.
The activity of plasminogen activator (PA) in tissue extracts from nasopharyngeal carcinoma (NPC) was determined by means of the fibrin plate method. Development of PA activity was observed in 16 out of 25 cases investigated. Furthermore, using tissue extract of NPC with PA activity, characterization and identification of the activator were carried out by means of electrophoretic analysis, fibrin zymography and immunological analysis. The molecular weight of this PA was found to be 38,000 daltons. Additionally, a urokinase type of plasminogen activator was contained in the tissue extracts.
Adult↗
Studies on in vitro synthesis and secretion of human chorionic gonadtropin and its subunits.
Human chorionic tissues were cultivated in vitro in they presence of 3H-proline in order to study the synthesis of the hCG and its subunits by the placenta. After terminating the culture, tissue extracts and media were individually gel-filtrated on Sephadex G-100. The eluted fractions were radioimmunoassayed for hCG, hCGalpha and hCGbeta and were measured for 3H-radioactivity. Label incorporation was determined by immunoprecipitation. Elution profiles of tissue extracts showed the existence of large immunologic forms of hHCG, hCGalpha and hCGbeta emerging near the void volume. The amounts of these large immunologic species in chorionic tissue gradually decreased during the course of cultivation. 3H-proline was almost exclusively incorporated into the large immunologic forms of hCG, hCGalpha and hCGbeta within the chrionic tissue during the 5-hour exposure. A great quantity of hCGalpha was found in the media after the 3-day culture, while the amount of hCGbeta found in the media was minute. After a 15-minute pulse, the 3H-radioactivity peak within the chorionic tissue appeared in the void volume, conicidental with hCG immunoreactivity. During the chase period, there was shift of the 3H-radioactivity peak associated with hCG immunoreactivity from the void volume to the more retarded area. In the media until after the 60-minute chase, no labeled hCG and its subunits appeared. Within the media after 3-hour chase, no labeled hCG peak associated with 3H-radioactivity was more retarded on Sephadex G-100 than that within the tissue extract after the 15-MINUTE pulse. These results suggest that the large immunologic forms of hCG, HCGalpha and hCGbeta are synthesized as the earliest detectable biosynthetic forms and that the may then be converted to small molecule species. In the culture of molar trophoblastic tissues after a 15-minute pulse, considerable amounts of hCG and its subunits accompanied by high 3H-radioactively had already been secreted into the media. These observations suggest that protein synthesis by molar trophoblastic tissue is markedly enhanced as compared with that by normal chorionic tissue and that immunoreactive materials synthesized in molar trophoblastic tissue may be secreted more radily than those synthesized a in normal chorionic tssue.
Chorionic Gonadotropin↗
Purification and identification of two structural variants of porcine tissue plasminogen activator by affinity adsorption on fibrin.
Porcine tissue plasminogen activator has been purified from delipidized heart tissue by affinity adsorption to fibrin. A crude fraction is prepared from an acid tissue extract by precipitation with ammonium sulphate. The tissue activator of this fraction is isolated by adsorption on fibrin and elution with KSCN. The procedure also includes chromatography on arginine-Sepharose and two gel-filtration steps. The final product has a specific activity of 250 000 IU/mg (+/- 16 000) as compared to an international urokinase reference preparation. The yield calculated from the active ammonium sulphate precipitate is about 28%. An approx. 7 000-fold increase of specific activity is obtained, most of which is achieved in the fibrin step. The native tissue plasminogen activator consists of a single chain molecule with a molecular weight of 64 000 as measured by SDS-polyacrylamide gel electrophoresis. In a previous report, it was claimed that the activator is composed of two disulphide-connected polypeptide chains. These results were due to a preparation artefact, caused by proteolytic activity present in the tissue extracts. The introduction of the protease inhibitor aprotinin and 6-amino-hexanoic acid in the purification procedure has abolished the effect of the protease contaminant, leading to the production of a one-chain activator. Treatment with plasmin transforms the native, one-chain tissue activator into a variant composed of two chains of about equal size (Mr 32 000) connected by disulphide bonding. This modified activator is indistinguishable from the one obtained at insufficient protection against proteolytic enzymes. The cleavage by plasmin causes about an 8-fold increase of amidolytic activity as measured on H-D-Val-Gly-Arg-p-nitroanilide. The fibrinolytic activity as measured by clot lysis in only slightly increased. The physiological significance of the cleavage is discussed.
Amino Acids↗
Benzoquinone, the substance essential for antibacterial activity in aqueous extracts from succulent young shoots of the pear Pyrus spp.
Aqueous extracts of the tissue of succulent young shoots of the pear Pyrus spp. exhibited strong antibacterial activity against the bacterium Erwinia amylovora bv. 4. This activity was investigated quantitatively by a newly developed bioassay method. It was found that the activity changed with the age of the tissue. Extracts of the youngest leaves and stems from the shoot tops showed the strongest activity, and the activity decreased with age of the leaves and stems. The activity also changed with increase in time after preparation of the extract, increasing rapidly in the first hour after preparation, reaching a maximum at about 4 h, and then decreasing slowly. The substance essential for the antibacterial activity was isolated from the extract by steam distillation in vacuo and through charcoal powder column chromatography. It was identified as benzoquinone (2,5-cyclohexadiene-1,4-dione) by NMR-spectra, mass spectra and HPLC analysis. The phenolic metabolism from arbutin to hydroquinone and then to benzoquinone in the aqueous extracts was analyzed quantitatively by HPLC. The changes in the contents of benzoquinone in the extracts of leaves and stems with tissue aging and with increase in time after preparation of the extracts paralleled the changes in antibacterial activity as determined by the quantitative bioassay.
Aging↗
Micellar high-performance liquid chromatographic determination of folylpolyglutamate hydrolase activity.
A rapid and sensitive method for the quantitative determination of folylpolyglutamate hydrolase activity in crude tissue extracts was developed. The procedure is based on high-performance liquid chromatographic separation of folate analogue mono- and polyglutamates on a reversed-phase column using sodium dodecyl sulfate in water as the mobile phase. Interfering substances in tissue extracts were removed by gel filtration on centrifugally-eluted mini-columns of Sephadex G-25 prior to incubation of polyglutamate substrate with tissue extract hydrolase. Reactions were terminated by denaturation of the enzyme in sodium dodecyl sulfate, which subsequently served as the micellar solvent system for chromatographic separation of substrate from reaction products.
Animals↗
Presence of calcitonin-like immunoreactivity (iCT) in human prostate gland: evidence for iCT secretion by cultured prostate cells.
Immunoreactive calcitonin (iCT) has been detected in human prostate tissue extracts as well as seminal plasma. The present studies were undertaken to examine whether iSCT (immunoreactive salmon CT-like human peptide) co-exists with iHCT (thyroid CT-like substance) in human prostate tissue extracts, and whether these substances are secreted by primary prostate cells in culture. Since the local secretion of these substances seems to increase in some neoplasms, a second objective of the study was to examine whether basal secretion of iCTs from primary prostate cells is increased in carcinoma. The present results have shown that both iHCT and iSCT were present in prostate tissue extracts. The mean iHCT levels in extracts of benign hyperplastic prostates (BPH) were 0.59 ng/g prostate, and these were significantly lower than iHCT concentrations in prostatic carcinoma (PC) (2.53 ng/g). No significant differences in their iSCT contents were observed. However, the results from culture of over 90 individual prostate tissue specimens from BPH or PC indicate that primary prostate cells secreted detectable quantities of iSCT and the basal release of this material from PC prostate cultures was almost four-fold higher than that from BPH prostate cultures. These results suggest that a CT-like immunoreactive material is secreted by primary prostate cells in culture, and the basal secretion of this material is significantly higher in PC cells as compared to BPH cells. Endogenous secretion of prostatic CT, and the elevation of its expression in PC suggest that it may serve as a regulatory factor in the pathophysiology of the prostate gland.
Aged↗
[Tests of lipid extracts of neoplastic tissue in medical therapy of cancer].
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Lipids↗
[Effect of various extracts of normal tissues on migration of leukocytes in vivo].
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Humans↗
Estrogen producing activity in epithelial ovarian tumors and dermoid cysts.
In 28 cases of postmenopausal women with non-functioning ovarian tumors, the estradiol (E2) level was assayed in peripheral plasma and tumor tissue extract. E2 precursors were also assayed in the tissue extract, and localization of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in the tissue was investigated. The plasma E2 levels were 153.8 +/- 205.9 pg/ml in 15 benign tumor cases, 244.3 +/- 336.5 pg/ml in 13 malignant tumor cases, and both of which were significantly higher than 12.3 +/- 3.6 pg/ml in normal postmenopausal women (p less than 0.01). The postoperative plasma E2 level (33.3 +/- 32.7 pg/ml) was significantly lower than the preoperative one (228.3 +/- 319.0 pg/ml). Twelve of 19 cases (68.2%) showed a postoperative decline in the E2 level to less than a half of the preoperative level. E2 and its precursors were found in tumor tissue. These findings demonstrate that E2 is produced by the tumor tissue itself. In addition, 3beta-HSD activity was localized in the stroma of ovarian tumors, indicating that E2 synthesis is mainly carried out in stromal cells rather than in epithelial cells.
3-Hydroxysteroid Dehydrogenases↗
Subfractionation of heart tissue: the "in sequence" myofilament protein extraction of myocardial tissue.
Proteomic analysis of heart tissue is complicated by the large dynamic range of its proteins. The most abundant proteins are the myofilament proteins, which comprise the contractile apparatus. This chapter describes a protocol for fractionation of heart tissue that extracts the myofilament proteins into a separate sample fraction, allowing analysis of lower-abundance proteins. Importantly, this is performed in a manner that is compatible with two-dimensional electrophoresis and high-performance liquid chromatography, two of main technologies of proteomics. The method produces three fractions based on solubility at different pHs: (1) cytoplasmic-enriched extract (neutral pH), (2) myofilament-enriched extract (acidic pH), and (3) membrane protein-enriched pellet. Fractionation of heart tissue in this manner provides the basis for in-depth proteomic analysis.
Actin Cytoskeleton↗
A fully automatable enzymatic method for DNA extraction from plant tissues.
BACKGROUND: DNA extraction from plant tissues, unlike DNA isolation from mammalian tissues, remains difficult due to the presence of a rigid cell wall around the plant cells. Currently used methods inevitably require a laborious mechanical grinding step, necessary to disrupt the cell wall for the release of DNA. RESULTS: Using a cocktail of different carbohydrases, a method was developed that enables a complete digestion of the plant cell walls and subsequent DNA release. Optimized conditions for the digestion reaction minimize DNA shearing and digestion, and maximize DNA release from the plant cell. The method gave good results in 125 of the 156 tested species. CONCLUSION: In combination with conventional DNA isolation techniques, the new enzymatic method allows to obtain high-yield, high-molecular weight DNA, which can be used for many applications, including genome characterization by AFLP, RAPD and SSR. Automation of the protocol (from leaf disks to DNA) is possible with existing workstations.
Botany↗
31P NMR of tissue phospholipids: a comparison of three tissue pre-treatment procedures.
Extracted tissue phospholipid 31P NMR profiles, obtained from individual porcine lenses subjected to two preservation procedures (acetone desiccation and freeze-drying) and a perchloric acid-extraction procedure, were compared to those from freshly excised lens specimens. Each profile yielded quantitative data on 12 lens phospholipids: PC, LPC, PC plas, PE, LPE, PE plas, PS, SPH, PI, LPI, PG, and CL. A specimen group size of at least 9 lenses was required for secure statistical inter-group comparisons by the Scheffé procedure, due to specimen 31P NMR profile variability, interpreted as arising from specimen biological variability. The phospholipid profiles of lenses preserved by acetone desiccation were essentially identical to those from the freshly excised control lenses. Freeze-dried lens profiles differed significantly in four components, while profiles from perchloric acid-extracted lenses differed in six. It is concluded that specimen preservation by acetone disiccation is a useful method for preserving tissue phospholipids for subsequent 31P NMR profile analysis, while freeze-drying is not. Lipid extraction following a tissue acid extraction is also of little or no value in the determination of tissue phospholipid profiles.
Animals↗
2',5'-Oligo(A)-activated endoribonuclease. Tissue distribution and characterization with a binding assay.
We have measured the binding of highly radioactive (2'-5')pppA4-5[32P]cytidine 3',5'-diphosphate to human, mouse, and rabbit cell and tissue extracts. A binding activity for this oligonucleotide is present in all extracts examined and high levels of this activity are found in some cells of lymphoid origin. In particular, the mouse thymoma cell line W7 has about 10 times higher binding activity than other cell lines. The oligonucleotide is bound with a single high affinity constant, as shown by Scatchard plot analyses. Cell extracts fractionated by centrifugation on glycerol gradients show a single peak of binding activity, which co-sediments with (2'-5')oligo(A)-dependent endoribonuclease (RNase L). This enzyme apparently binds the oligonucleotide, as shown by experiments with an analog of (2'-5')oligo(A) which competes in the binding and inhibits the RNase L. This endonuclease cannot be directly assayed in unfractionated cell or tissue extracts with high levels of other nuclease activities. The binding assay for the oligonucleotide may provide an estimate of RNase L levels in such cell and tissue extracts.
Adenine Nucleotides↗
The changes in aldolase isoenzyme pattern during development of the human kidney and small intestine--demonstrated in organ extracts and tissue sections.
Aldolases A, B and C were determined by immunotitration analysis in extracts of human kidney and small intestine and demonstrated immunohistochemically in tissue sections of the same organs at various stages of development. By both techniques a change of isoenzyme pattern during development of the kidney and the small intestine was observed, leading from the predominance of A-type towards the predominance of B-type aldolase. In the extracts of kidney and small intestine the specific activity of aldolase B--but not that of aldolase A--rises with age by about one order of magnitude. The histochemical investigation showed that the developmental change in aldolase pattern in the organ extracts is caused by the differentiation of proximal tubulus cells in the kidney and the differentiation of epithelial cells in the small intestine. Within these cells an increase in the concentration of aldolase B and a decrease in that of aldolase A takes place during development. The possible physiological role of this cellular change in aldolase isoenzyme pattern is discussed. Aldolase C was found only in low concentrations in fetal organs. Only in the kidney, a specific localization within the proximal tubules could be demonstrated.
Fetus↗