A convenient microfiltration procedure for purification of bovine and ovine subcutaneous tissue lipid extracts.
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Animals↗
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Studies on proactivator from the paranasal mucous membrane in chronic sinusitis.
We succeeded in differentiating the proactivator (PA) in tissue extract of paranasal mucous membrane with chronic sinusitis by the gel filtration technique. Furthermore, the results demonstrated that PA in the tissue extracts of the paranasal mucous membrane with chronic sinusitis and antrochoanal polyp was unrelated to the antigenicity of plasminogen. In particular, it was clarified that the tissue extract of antrochoanal polyp as a source of PA was not related to the antigenicity of plasminogen.
Antigen-Antibody Complex↗
Elevated levels of the IGF-binding protein protease MMP-1 in asthmatic airway smooth muscle.
We have previously demonstrated that the asthma-associated proinflammatory eicosanoid leukotriene D4 (LTD4) is co-mitogenic with insulin-like growth factors (IGFs) in airway smooth-muscle (ASM) cells in vitro. This synergistic effect of LTD4 and IGF on ASM cell growth involves proteolysis of ASM-produced IGF binding proteins (IGFBPs), which are cell growth-inhibitory proteins. We also identified this IGFBP protease to be the matrix metalloproteinase-1 (MMP-1), and showed that this enzyme had a significant role in modulating IGF action in ASM cells. In the present study, we tested the hypothesis that ASM hyperplasia in vivo involves induction of MMP-1 leading to IGFBP proteolysis. We detected the presence of MMP-1 and measured its levels in human airway tissue sections prepared from nonasthmatic and asthmatic subjects. Six nonasthmatic and six asthmatic airway tissue samples were analyzed for immunoreactive MMP-1 through an immunohistochemical detection method. Both the bronchial and tracheal smooth-muscle cells from different regions of the same sample were examined and documented. The immunostaining for MMP-1 was significantly elevated in both the bronchial and tracheal smooth-muscle cells of the airway sections from asthmatic samples relative to that of the nonasthmatic samples. The differences in levels of MMP-1, IGFBP-2, IGFBP-3, and IGFBP proteolytic activity were quantified using densitometric analyses of the ASM tissue extracts that were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The MMP-1 levels in the asthmatic airway tissue extracts were 12-fold higher than those found in control samples. In addition, IGFBP-2 and IGFBP-3, which we have previously demonstrated to be proteolytic substrates of MMP-1, were found to be cleaved in asthmatic airway tissue extracts. Furthermore, the asthmatic airway extracts contained IGFBP proteolytic activity that was shown by immunodepletion studies to be due to MMP-1. These observations demonstrate that MMP-1 may play a significant role in inducing ASM hyperplasia and airway obstruction in asthma by modulating the IGF axis.
Adolescent↗
The use of reversed-phase high-performance liquid chromatography to detect proteolytic activity from human pancreas in a radioimmunoassay for corticotrophin-releasing factor.
Degradation of tracer during a radioimmunoassay (RIA) can result in false-positive concentrations of immunoreactivity being reported in a biological sample. A technique has been developed using reversed-phase high-performance liquid chromatography (HPLC) to detect proteolytic degradation of corticotrophin-releasing factor-41 (CRF-41) during incubation with tissue extracts under RIA conditions. Human pancreatic tissue was extracted in HCl or urea and incubated with 125I-labelled CRF-41 at neutral pH for 18 h. When samples were analysed by HPLC and fractions counted for radioactivity, tracer was extensively degraded. Heating extracts at 85 degrees C or adding lima bean trypsin inhibitor to the medium prevented degradation. Pancreatic tissue extracted in HCl was analysed by gel filtration and HPLC, and fractions were subjected to RIA for CRF-41. A peak of immunoreactivity was detected by both chromatographic methods. However, when this material was incubated with tracer and analysed by HPLC, the tracer was degraded, indicating that proteolytic activity remained after acid extraction and two forms of chromatography.
Chromatography, High Pressure Liquid↗
Localized survival of ciliary ganglionic neurons identifies neuronotrophic factor bands on nitrocellulose blots.
A novel and sensitive method has been developed to identify ciliary neuronotrophic factors (CNTFs) from tissue extracts after blotting to nitrocellulose paper. The CNTF proteins are required for the in vitro survival of embryonic chick ciliary ganglionic neurons. Tissue extracts containing such CNTFs are electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Purified ciliary ganglionic neurons are seeded on the surface of the nitrocellulose blot, and the culture is incubated for 24 hr in medium lacking CNTF. CNTF can be localized on the blot because it retains its ability to support the survival of the neurons cultured on the nitrocellulose. A band of viable neurons, easily visualized by staining with a vital dye, is supported by the blotted CNTF polypeptide. The number of neurons surviving on the blotted CNTF is related to the amount of CNTF originally loaded on the electrophoretic gel. As little as 2 ng (16 trophic units) of CNTF protein contained in crude tissue extracts can be loaded on the sodium dodecyl sulfate gel and still be recognized by the cultured neurons. This method was used to identify CNTF polypeptides from extracts of adult rat nerve (24,000 and 19,000 daltons) and from tissue found near experimentally induced adult rat brain lesions (24,000 daltons). The electrophoretic mobilities of these peptides are distinct from the previously purified chick eye CNTF polypeptide (20,400 daltons).
Animals↗
Acute excitotoxic injury induces expression of monocyte chemoattractant protein-1 and its receptor, CCR2, in neonatal rat brain.
Chemokines are a family of structurally related cytokines that activate and recruit leukocytes into areas of inflammation. The "CC" chemokine, monocyte chemoattractant protein (MCP)-1 may regulate the microglia/monocyte response to acute brain injury. Recent studies have documented increased expression of MCP-1 in diverse acute and chronic experimental brain injury models; in contrast, there is little information regarding expression of the MCP-1 receptor, CCR2, in the brain. In the neonatal rat brain, acute excitotoxic injury elicits a rapid and intense microglial response. To determine if MCP-1 could be a regulator of this response, we evaluated the impact of excitotoxic injury on MCP-1 and CCR2 expression in the neonatal rat brain. We used a reproducible model of focal excitotoxic brain injury elicited by intrahippocampal injection of NMDA (10 nmol) in 7-day-old rats, to examine injury-induced alterations in MCP-1 and CCR2 expression. RT-PCR assays demonstrated rapid stimulation of both MCP-1 and CCR2 mRNA expression. MCP-1 protein content, measured by ELISA in tissue extracts, increased >30-fold in lesioned tissue 8-12 h after lesioning. CCR2 protein was also detectable in tissue extracts. Double-immunofluorescent labeling enabled localization of CCR2 both to activated microglia/monocytes in the corpus callosum adjacent to the lesioned hippocampus and subsequently in microglia/monocytes infiltrating the pyramidal cell layer of the lesioned hippocampus. These results demonstrate that in the neonatal brain, acute excitotoxic injury stimulates expression of both MCP-1 and its receptor, CCR2, and suggests that MCP-1 regulates the microglial/monocyte response to acute brain injury.
Animals↗
Modified proenzymes as artificial substrates for proteolytic enzymes: colorimetric assay of bacterial collagenase and matrix metalloproteinase activity using modified pro-urokinase.
We describe a new principle for assessment of the activity of proteolytic enzymes of all classes and show the application of this principle for the quantitative assay of bacterial collagenase and human matrix metalloproteinases (MMPs). Central to this new principle is the presence of a proenzyme that can be activated into an active enzyme by a single proteolytic event. The regular activation sequence in the proenzyme is replaced using protein engineering by an artificial sequence recognized by the proteinase to be determined. The latter can act as an activator for the newly engineered proenzyme. In the present paper a simple colorimetric assay for the determination for MMPs is described based on this principle. With the aid of protein engineering, a modified pro-urokinase has been prepared in which the activation sequence normally recognized by plasmin (Pro-Arg-Phe-Lys upward arrowIle-Ile-Gly-Gly) has been replaced by a sequence expected to be recognized and hydrolysed by many MMPs (Arg-Pro-Leu-Gly upward arrowIle-Ile-Gly-Gly). The active urokinase resulting from activation of the modified pro-urokinase by a MMP could be measured either directly, using a specific chromogenic peptide substrate for urokinase, or indirectly via urokinase-catalysed plasminogen activation. The response of the assay to equal molar quantities of active MMPs decreases in the order MMP-2>MMP-9>MMP-1>MMP-3>MMP-7. The detection limit for MMP-9 was below 15 pM, corresponding to 3. 75x10(-15) mol per assay. Using the assay, increased MMP activity was detected in synovial tissue extracts from rheumatoid arthritis patients compared with those from osteoarthritis patients, and in stomach tumour extracts as compared with normal stomach tissue extracts.
Arthritis, Rheumatoid↗
Binding of polyreactive antibodies to self versus foreign antigens.
Aside from their ability to bind to multiple antigens, the classic hallmark of polyreactive antibodies is their autoreactivity. Because of their ability to bind a number of common autoantigens, it has long been speculated that polyreactive antibodies are involved in the clearance of self-antigens. However, it has been demonstrated more recently that polyreactive antibodies are also capable of binding to some foreign and synthetic antigens. Although data regarding the relative reactivity of polyreactive antibodies with self versus foreign antigens is lacking, it is generally thought that both activities may play an important biological role. In this study, the relative reactivity of polyclonal human polyreactive IgM with human proteins and tissue extracts versus foreign (xenogeneic) proteins and tissue extracts was probed. The binding of affinity purified anti-ssDNA IgM from adult human serum and the binding of polyreactive IgM in human cord serum and in human adult serum were evaluated. Using competitive and direct binding assays, human polyreactive IgM were found to be generally more reactive with foreign (xenogeneic) proteins than with self or allogeneic proteins. These data shed light on the fundamental nature of polyreactive antibodies, and may provide additional insight into their putative biological roles.
Animals↗
Production and characterization of a monoclonal antibody specific for the polyamine spermidine and its application in ELISA.
To enable the immunoassay of spermidine in tissue extracts, a monoclonal antibody, JAC-1, specific for free spermidine was raised, using a spermidine-ovalbumin conjugate as immunogen. This antibody was characterized, and found to possess a high degree of specificity for spermidine, with only 4% molar cross-reactivity with spermine. A competitive ELISA using this antibody was developed. This assay is able to detect as little as 10 pmol spermidine extracted from small, circa 10 mg, tissue samples. The assay is unaffected by the presence of up to a 4-fold molar ratio of spermine, whereas the corresponding spermine competitive ELISA is adversely affected by a 0.5-fold molar ratio of spermidine. The spermidine competitive ELISA using JAC-1 was used to estimate the spermidine content of several plant and animal tissue extracts and the results compared with HPLC data. This novel assay is a useful development in the assay of polyamines since, compared to routinely employed HPLC methods, it offers increased convenience, rapidity, and capacity for large numbers of samples.
Animals↗
Adaptation of an enzymatic kit for the assay of cholesterol in tissue lipid extracts.
In the assay for total cholesterol in lipid extracts by enzymatic methods, it was found necessary to redissolve the lipid prior to the reagent addition. Isopropyl alcohol was found to be best at promoting the colorimetric reaction. As little as 1.45 micrograms free cholesterol and 1.8 micrograms esterified cholesterol could be determined after 15 min incubation with the isopropyl alcohol-reagent. Using a formula for the correction of pigment absorbance, it was also possible to apply the enzymatic kit method to red blood cell lipid extracts.
Cholesterol↗
Localisation and temporal changes in prostaglandin G/H synthase-1 and -2 content in ovine intrauterine tissues in relation to glucocorticoid-induced and spontaneous labour.
Parturition in the ewe is preceded by an increase in the synthesis of prostaglandins (PGs) by gestational tissues. To establish the uterine source of these PGs, placental cotyledons, fetal membranes and maternal uterine tissues were collected from ewes (n=6) at spontaneous parturition. Solubilised tissue extracts were prepared and analysed by Western blots using polyclonal antibodies to PG G/H synthase-1 and -2 (PGHS-1 and PGHS-2). PGHS-1 was expressed by all intrauterine tissues at term labour. Densitometric analysis of Western blot autoradiographs showed that the fetal membranes and maternal cervix contained the largest amounts of PGHS-1. PGHS-1 enzyme content of ovine amnion was significantly greater than that of either chorion or allantois (P<0.05). PGHS-1 protein content of myometrial, endometrial and cotyledonary tissue extracts was minimal. Formation of the PGHS-2 isozyme was confined to placental tissue at term labour. PGHS-2 protein levels in sheep placenta were significantly higher than those of PGHS-1 in all intrauterine tissues examined. This result supports the hypothesis that PGHS-2 is a major contributor to PG formation at term labour. To elucidate the developmental changes in PGHS-1 and PGHS-2 relative to labour onset, an experimental paradigm of glucocorticoid-induced delivery was used. Previous characterisation and validation of this labour model demonstrated that direct, transabdominal, intrafetal injection of the synthetic glucocorticoid betamethasone (5.7 mg in 1 ml aqueous vehicle) on day 131 of gestation induced labour onset in 56.6+/-0.8 h (mean+/-s.e.m.). As the latent period to induced-labour was known, the time course of enzyme formation could be ascertained. Sheep (n=20) were killed by barbiturate injection at various time intervals post-injection (0, 14, 28, 42 and 56 h). Tissue extracts collected at post-mortem examination were prepared and analysed by Western blots. PGHS-2 was induced in ovine cotyledon in a time-dependent fashion following glucocorticoid injection (P<0.05). There was a 12-fold increase in abundance between the time of betamethasone administration (0 h) and established labour (56 h). The PGHS-2 isozyme was not detected in any of the other tissues examined. In contrast, formation of the PGHS-1 isozyme did not change in relation to induced-labour in any of the intrauterine tissues. This finding is consistent with constitutive formation of PGHS-1. Previous studies have demonstrated a rise in PG production in association with glucocorticoid-induced labour and spontaneous delivery. The results of the present study indicate that this rise in PG production is due to increased formation of the PGHS-2 isozyme in ovine cotyledon. PGHS-2 appears to be induced by exogenous glucocorticoid administration and/or the mechanisms controlling ovine parturition. The role of PG formation by the fetal membranes is yet to be elucidated.
Analysis of Variance↗
Abnormal behavior of polyamines on gel filtration: a cautionary note.
The polyamines, spermine and spermidine, have been shown to persist in various tissue extracts despite procedures such as dialysis and ultrafiltration which normally remove such low molecular weight compounds. We have found that polyamines in tissue extracts and the standard compounds alone can migrate as much higher molecular weight compounds on gel filtration under a variety of conditions. Thus, even relatively pure fractions obtained from tissue extracts may be contaminated with, or consist entirely of, polyamines, which are potent inhibitors of cell proliferation under certain conditions.
Chromatography, Gel↗
Statement on the detection of viruses by electron microscopy in human leukaemic tissues and extracts and in similar material from domestic animals.
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Animals↗
Detection of genotoxic substances in bivalve molluscs from the Saguenay Fjord (Canada), using the SOS chromotest.
Few studies have employed bioassays to investigate the accumulation of genotoxins in aquatic biota that inhabit areas contaminated with industrial and municipal wastes. This study employed the SOS Chromotest, a short-term bacterial genotoxicity assay, to investigate the presence of genotoxins in bivalve molluscs from the Saguenay Fjord (Canada). Genotoxicity analyses were performed on dichloromethane extracts of Mya arenaria and Mytilus edulis collected downstream from several aluminum refineries and forestry products industries known to produce and release genotoxic substances. The results confirmed that bivalve molluscs inhabiting downstream regions are contaminated with both direct-acting and pro-genotoxic substances. In several cases, SOS response induction factors exceeded 3.0. The results failed to reveal a clear downstream trend of decreasing genotoxicity with increasing distance from the presumed industrial sources(s). A significant relationship (r2 = 0.61, p < 0.007) between a demographic variable (population near shoreline) and lipid-corrected genotoxic potency suggest that the accumulated direct-acting genotoxins may be of municipal origin. Significant relationships between tissue extract genotoxicity (r2 = 0.75, p < 0.003) and tissue PAH contamination (r2 = 0.77, p < 0.0001) and drainage basin area suggests that the bivalves are accumulating airborne contaminants deposited on the surface of the relevant drainage basins. In spite of contamination with genotoxic PAHs, the addition of rat liver microsomal enzymes reduced the genotoxic potency of all samples investigated (31-94% decrease). The results also revealed a significant relationship between tissue extract genotoxicity and PAH concentration (r2 = 0.72, p < 0.0005). Further analyses confirmed that a variable portion (7-97%) of the S9-activated tissue extract genotoxicity can be attributed to the detected PAHs. Although the sources, identity and effects of genotoxins accumulated by bivalves of the Saguenay Fjord remain to be determined, the study has confirmed the utility of the SOS Chromotest in environmental monitoring of aquatic biota.
Animals↗
Two-dimensional electrophoresis of synovial tissue, synovial fluid and serum in patients with rheumatoid arthritis and related diseases.
Using the technique of two-dimensional (2D) electrophoresis with consecutive silver staining, we investigated samples of serum, synovial fluid and synovial tissue obtained from 19 patients suffering from rheumatoid arthritis (RA) or non-RA arthritis. From these experiments we have drawn the following conclusions. 2D electrophoresis of serum, synovial fluid and synovial tissue extracts taken from patients suffering from joint diseases is a reproducible method. Repeated runs of the same sample reveal an essentially constant protein spot pattern. The time period between surgery and tissue preparation did not influence the number of protein spots when less than 15 h was involved. The protein spot number is always lower in synovial fluid than in either synovial tissue or serum in RA and non-RA patients. The mean value for the number of spots is 68 for the inflamed tissue irrespective of the cause of arthritis (RA and non-RA group taken together) and 47 for the control group. This difference is significant. We were able to definitely identify 7 spots in the tissue extract. We did not find RA-specific protein spots in either serum, synovial fluid or tissue extracts from the synovial membrane. The only significant difference between RA patients and either non-RA or control group patients concerning the protein spot pattern is the increased size of the immunoglobulin spot (mainly IgG) in RA. In addition, we discuss possible reasons for failure of the 2D electrophoresis technique to detect disease-specific protein patterns.
Adult↗
Effect of alpha-1-acid glycoprotein, albumin and palmitic acid on the brain and salivary gland extraction of warfarin in rats.
The effect of plasma protein binding of warfarin on its transfer into the brain and salivary gland was investigated using alpha-1-acid glycoprotein and human serum albumin (HSA) in combination or not with palmitic acid. The tissue extraction of [14C] warfarin relative to [3H]water was determined by intracarotid injection technique in male Wistar rats. The tissue extraction of warfarin varied inversely with the concentration of added serum protein, (HSA and alpha-1-acid glycoprotein), and addition of palmitic acid to HSA diminished the extraction. The fraction of drug uptaked by tissue (tissue available fraction) was always dramatically greater than the in vitro free drug fraction, and this was interpreted as an enhanced in vivo drug dissociation from the binding protein. The fraction of drug uptake by salivary gland was closer to the in vitro free fraction than the fraction of drug uptake by brain tissue. The addition of palmitic acid to HSA induced parallel changes in the in vitro free fraction of warfarin and in the brain tissue or salivary gland extraction of warfarin. These data indicate that a part of protein-bound warfarin (as determined in vitro) is available for tissue extraction via an enhanced in vivo dissociation of the drug-protein complex in the tissue microcirculation. The in vitro data were fitted to a saturable model of binding whereas the in vivo data could satisfactorily fit a model dealing with a nonsaturable model of binding, and this is probably the result of the several-fold increase in the in vivo dissociation constant.
Animals↗
Differentiation of proactivator from tissue plasminogen activator with low molecular weight in the paranasal mucous membrane.
It has been shown that large amounts of fibrinolytic enzyme are contained in tissue extracts of the paranasal mucous membrane in patients with chronic sinusitis. However, the fibrinolytic enzyme in the tissue extracts has not yet been characterized by biochemical techniques. In the present study, proactivator was differentiated from tissue plasminogen activator with low molecular weight in extracts of the paranasal mucous membrane and the existence of the proactivator was thus demonstrated in tissue extracts. By analogy with the proactivator in antrochoanal polyps, this proactivator may play an important role in the proliferation and enlargement of the paranasal mucous membrane.
Enzyme Precursors↗
Demonstration of intracellular and secreted forms of large human chorionic gonadotrophin alpha subunit in cultures of normal placental tissue.
Free human chorionic gonadotrophin (hCG) alpha-subunit in tissue extracts and incubation media of normal placentae before and after four days of culture was investigated by Sephadex G-100 chromatography. Prior to incubation, tissue extracts of first-trimester and term placentae contained a large form and a small form of alpha-subunit relative to a reference urinary hCG alpha-subunit preparation. The large alpha-subunit comprised 30.6 +/- 1.4 per cent s.e.m. and the small alpha-subunit, 66.9 +/- 4.1 per cent of total intracellular alpha-subunit. After 96 h of incubation, the large alpha-subunit comprised 77.6 +/- 9.8 per cent of the total intracellular alpha-subunit, and the small 21.0 +/- 8.1 per cent. The incubation medium contained a single form of alpha-subunit which eluted with a larger apparent molecular weight than the urinary hCG alpha-subunit. Following neuraminidase treatment, the large alpha-subunit from tissue extracts eluted as a smaller molecule in the position of the urinary hCG alpha reference. A change in Ve/Vo of the small alpha-subunit was not detected. Identical neuraminidase treatment of the large alpha-subunit from incubation medium resulted in an increase in the Ve/Vo, but the large alpha-subunit continued to elute from Sephadex G-100 before the urinary hCG alpha-subunit. However, large alpha-subunit from culture medium did elute in the position of urinary hCG alpha following mild acid hydrolysis. These studies demonstrate that a large form of alpha-subunit is present in first-trimester and term placental tissue both before and after incubation in vitro and that the large form of alpha-subunit is secreted by the normal placenta. The differential effects of neuraminidase treatment on the intra- and extracellular forms of large alpha-subunit coupled with our findings from acid hydrolysis of the extracellular form suggest that alpha-subunit secreted into the culture medium may contain additional carbohydrate residues not present on the intracellular form.
Choriocarcinoma↗