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PubMed1983

Assay of interferon-mediated protein kinase activity from plasma and tissue extracts.

Interferon-treated mouse and human cells show enhanced levels of a protein kinase activity which is manifested by the phosphorylation of endogenous 67,000 and 72,000 Mr proteins, respectively. Enhanced levels of such kinase activity are also detectable in the plasma of patients treated with interferon and in the plasma and tissues of interferon-treated mice. A rapid and efficient method of assay for these protein kinase activities is described. The samples are first incubated with heparin (100 units/ml), which results in the inhibition of different protein kinase activities, but not the one mediated by interferon. The latter one is then assayed after partial purification on poly(rI):(rC)-Sepharose or poly(rG)-Sepharose. The protein kinase from human and mouse cells in culture and from the different tissues of mice binds specifically to poly(rI):(rC)-Sepharose. On the other hand, the protein kinase activity from both mouse and human plasma shows a higher affinity toward poly(rG)-Sepharose. These methods are successfully applied for the determination of the interferon-mediated protein kinase activity from tissue extracts and plasma.

Animals↗
PubMed1984

Neural crest-derived spinal and cranial sensory neurones are equally sensitive to NGF but differ in their response to tissue extracts.

The response of two distinct populations of neural crest-derived sensory neurones to nerve growth factor (NGF) and other neurotrophic activities present in extracts of chick tissues has been studied in vitro. Dorsal root ganglia (DRG) and the dorso-medial part of the trigeminal ganglion (DM-TG) from embryonic chicks of 6-11 days of incubation (E6-E11) were grown as either explant or dissociated neurone-enriched cultures. Over the age range studied NGF promoted survival and pronounced neurite outgrowth from both DRG and DM-TG neurones. Whilst extracts of chick eye, liver and spinal cord also elicited a marked response from E8 and older DRG neurones, DM-TG neurones were almost entirely unresponsive to the neurotrophic activity of these extracts.

Animals↗
PubMed1989

Calmodulin activity in whole body and fat body tissue extracts of Heliothis virescens larvae.

Calmodulin is an activator of many enzymatic activities. Total calmodulin activity in tissue extracts of Heliothis virescens larvae (5th instar), assayed by cyclic phosphodiesterase activation, was 0.48 unit/gm for whole body and 22.2 units/gm for fat body. Specific calmodulin activity was 0.1 unit/mg protein for whole body and 3.0 units/mg protein for fat body. The larval fat body is therefore the main site of calmodulin activity in this lepidopterous larva.

Adipose Tissue↗
PubMed2002

Human bone marrow stromal cell cultures conditioned by traumatic brain tissue extracts: growth factor production.

Treatment of traumatic brain injury (TBI) with bone marrow stromal cells (MSCs) improves functional outcome in the rat. However, the specific mechanisms by which introduced MSCs provide benefit remain to be elucidated. Currently, the ability of therapeutically transplanted MSCs to replace injured parenchymal CNS tissue appears limited at best. Tissue replacement, however, is not the only possible compensatory avenue in cell transplantation therapy. Various growth factors have been shown to mediate the repair and replacement of damaged tissue, so trophic support provided by transplanted MSCs may play a role in the treatment of damaged tissue. We therefore investigated the temporal profile of various growth factors, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), within cultures of human MSCs (hMSCs) conditioned with cerebral tissue extract from TBI. hMSCs were cultured with TBI extracts of rat brain in vitro and quantitative sandwich enzyme-linked immunosorbent assays (ELISAs) were performed. TBI-conditioned hMSCs cultures demonstrated a time-dependent increase of BDNF, NGF, VEGF, and HGF, indicating a responsive production of these growth factors by the hMSCs. The ELISA data suggest that transplanted hMSCs may provide therapeutic benefit via a responsive secretion of an array of growth factors that can foster neuroprotection and angiogenesis.

Analysis of Variance↗
PubMed1980

Chemotactic response of polymorphonuclear leukocytes to corneal tissue extracts.

The chemotactic response of polymorphonuclear leukocytes of normal subject, to corneal extracts of 0.2 and 0.4 mg/dl protein concentration were studied in blind-well type chemotactic chambers. E. coli endotoxin served for the positive, Rindex-5 fluid for the negative controls. The chemotactic response to corneal tissue extracts, as compared to random migration in the Rindex-5 medium, proved significant. It is suggested that chemotactic factors may be released from corneal grafts in vivo too, in consequence of diverse noxae. These factors are supposed to elicit a granulocytic invasion and to lead to an opacification of the graft as a result of a cellular reaction.

Chemotaxis, Leukocyte↗
PubMed1986

A semi-automated fluorimetric method for measuring acetylcholinesterase activity in small volumes of cerebrospinal fluid and tissue extracts using acetylcholine as substrate.

A fluorimetric method for the assay of acetylcholinesterase (AChE; EC 3.1.1.7) in tissue extracts and cerebrospinal fluid, using acetylcholine as the substrate, is described. The method is based on the measurement of hydrogen peroxide, formed by the oxidation of choline resulting from the action of AChE on acetylcholine, by means of horseradish peroxidase and 4-hydroxy-3-methoxyphenylacetic acid (HVA) to yield a fluorescent derivative. Choline, if present in any sample to be analysed, is first removed by a modification of the peroxidase reaction during preincubation.

Acetylcholine↗
PubMed1993

On-line recognition and quantitation of coeluting hypoxanthine and guanine in reversed-phase high-performance liquid chromatography of placental tissue extracts: photodiode-array detection and spectral analysis of coeluting peaks.

In a reversed-phase HPLC method, optimized for resolution and quantitation of nucleosides and nucleobases in human term placental extracts, guanine coeluted with hypoxanthine. Three numerical techniques that treat spectral data generated by photodiode-array detection to yield indices of peak purity were compared for sensitivity of on-line recognition of overlapped guanine and hypoxanthine peaks in chromatograms of 7.0 microM hypoxanthine containing 0.075-0.54 microM guanine. These techniques were purity parameter, the average wavelength of a peak spectrum (lambda w), peak area ratio, and peak absorbance ratio. Purity parameter of a 7.0 microM hypoxanthine peak containing 1% guanine was significantly different from that of pure hypoxanthine (P < 0.01); peak area and peak absorption ratios were four and seven times, respectively, less sensitive than purity parameter. In chromatograms of human term placental extracts, purity parameter values generated on-line signalled the presence of guanine in hypoxanthine peaks. Quantitation of coeluting hypoxanthine and guanine was performed on-line from dual-wavelength detection at 249 and 278 nm via calibration curves for both analytes at these wavelengths. Hypoxanthine and guanine concentrations in seven samples from five human term placentas were 41-161 and 3-76 nmol/g, respectively. These findings demonstrate that on-line signalling of hypoxanthine and guanine coelution is achieved by purity parameter, area ratio, and absorbance ratio, that purity parameter is the most sensitive index of coelution, and that quantitation of the coeluting analytes can be performed on-line. These approaches should be applicable to recognition and quantitation of coeluting analytes in chromatograms of mixtures of nucleosides and nucleobases such as occur in extracts of other tissues.

Chromatography, High Pressure Liquid↗
PubMed1991

A phosphatase resistant substrate for the assay of protein kinase C in crude tissue extracts.

Protein kinase C (PKC) is routinely assayed, after it is partially purified over DEAE-cellulose chromatography to eliminate any interfering protein kinases and phosphatases, by measuring the transfer of gamma-phosphate of [gamma-32P]ATP to H1 histone. Recently, it has been shown that a synthetic peptide, comprising residues 4-14 of myelin basic protein (MBP4-14), is a very selective PKC substrate which is not phosphorylated effectively by cyclic AMP-dependent protein kinase, casein kinase I and II, Ca2+/calmodulin dependent protein kinase II or phosphorylase kinase [Yasuda, I., Kishimoto, A., Tanaka, S-I., Tominaga, M., Sakurai, A. and Nishizuka, Y. (1990) BBRC 166, 1220-1227]. We report here that once MBP4-14 is phosphorylated, it is not dephosphorylated by okadaic acid-sensitive phosphatases (protein phosphatases 1, 2A and 3) or other protein phosphatases such as calcineurin and/or PP 2C present in hippocampal homogenates. Therefore, MBP4-14 can be used for PKC assay in crude extracts of neural tissue.

Adenosine Triphosphate↗
PubMed1998

High-performance liquid chromatographic determination of beta-alanine, beta-aminoisobutyric acid and gamma-aminobutyric acid in tissue extracts and urine of normal and (aminooxy)acetate-treated rats.

A method is described for the simultaneous determination of beta-alanine, beta-aminoisobutyric acid and gamma-aminobutyric acid in biological materials. Amino acids including these beta- and gamma-amino acids were derivatized with 4-dimethylaminoazobenzene-4'-sulfonyl (dabsyl) chloride and dabsyl amino acids formed were separated by reversed-phase high-performance liquid chromatography. Dabsyl derivatives of these beta- and gamma-amino acids were well separated from other dabsyl-amino acids. The method was applied to the determination of these beta- and gamma-amino acids in trichloroacetic acid extracts of various tissues and to the urine of normal rats and those injected with (aminooxy)acetate (AOA). AOA injection (15 mg per kg of body mass) produced remarkable increase in beta-alanine contents in liver, kidney and urine (10.2, 4.6 and 25.7 times, respectively).

Aminoisobutyric Acids↗
PubMed1991

The effect of horse placental tissue extracts and equine chorionic gonadotrophin on the proliferation of horse lymphocytes stimulated in vitro.

Commercial preparations of equine chorionic gonadotrophin (eCG) and extracts of horse placenta taken at 80 days gestation were incorporated into mixed lymphocyte culture and mitogen stimulation assays of horse peripheral blood mononuclear cells. A dose-related inhibition of lymphocyte proliferation, indicative of immunosuppressive activity, was observed in both systems, both with commercial eCG preparations and tissue extracts. Negligible inhibitory activity was observed with an extract of term placenta. The inhibitory activity of the placental samples was not related to their eCG content as measured by immunoassay. The fact that one pregnant horn allantochorion extract containing 50 IU/ml of eCG showed an identical dose-dependent lymphoproliferation-inhibitory activity to an extract of a second such allantochorion containing only 1.7 IU/ml of eCG suggests that this hormone does not inhibit lymphocyte proliferation and that commercially available eCG contains inhibitory contaminants.

Animals↗
PubMed1977

Specific restoration of delayed hypersensitivity by lymphoid tissue extracts.

Mice lose demonstrable delayed hypersensitivity (DH) to DNFB, picryl chloride, or sheep red blood cells. Reconstitution of immune responsiveness can be accomplished by administration of cell-free lysates of spleens from mice with active DH to structurally related, but not to unrelated antigens. Peritoneal exudate cell lysates from mice with active DNFB-DH also restore DH to this antigen. Sera from sensitized mice, and sera and lymphoid tissue extracts from unsensitized mice are without activity. The restorative property of splenic lysates from DNFB-sensitized mice is unstable at 56 degrees C, not sedimented at 90,000 X G and inactivated by trypsin or magnesium ions. The presence of unexpressed, restorable DH may provide a biologic basis for the so called "transfer factor" phenomenon.

Animals↗
PubMed2000

Radioimmunoassay for rat galanin: immunochemical and chromatographic characterization of immunoreactivity in tissue extracts.

Galanin is a regulatory peptide with wide distribution in the central and peripheral nervous system and with numerous biological effects. Several radioimmunoassays based on antisera raised against porcine galanin have been used to measure immunoreactivity in rat tissues. However, considerable lack of parallelism has been observed between the porcine standard and rat tissue extracts, which may decrease the reliability of the quantitative data. The purpose of the present study was therefore to raise antibodies against rat galanin and establish a competitive radioimmunoassay for rat galanin. Two antisera, RatGal4 and RatGal5, were characterized in detail. The homogeneity of the immunoreactive material from several tissues was also investigated with column chromatography. At reverse-phase high-pressure liquid chromatography more than 95% of the immunoreactive material from rat CNS eluted as a single peak in the position of synthetic rat galanin, whereas almost half of the immunoreactive material from the intestine eluted in positions different from the synthetic peptide. Extracts of rat brains as well as jejunum diluted in parallel with the standard curve for both antisera. We conclude that measurements of rat galanin based on these antisera are therefore more reliable than those based on antisera raised against porcine galanin.

Animals↗
PubMed2000

A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts.

Because G-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1-EGFP receptor in a dose-dependent fashion (EC(50) = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1-EGFP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs.

Animals↗