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PubMed1995

Enzymatic conversion of IGF-I to des(1-3)IGF-I in rat serum and tissues: a further potential site of growth hormone regulation of IGF-I action.

We recently identified and characterized a protease present in rat serum which is capable of generating des(1-3)IGF-I. In this study, we have investigated the effects of GH deficiency and replacement on the activity of this protease in rat serum and tissue extracts. Protease activity was significantly higher in sera from hypophysectomized (hypox) rats than sham-operated rats (P < 0.001) and GH treatment of hypox rats (human GH, 100 micrograms/100 g body weight i.p. for 10 days) significantly reduced the levels towards normal. The addition of IGF-I to hypox rat serum to achieve IGF-I concentrations comparable with or greater than that seen in normal rat serum had no effect on the measured protease activity. Protease activity was also detected in tissue extracts. The level of protease activity in the various tissues from sham-operated rats demonstrated the following order: liver > testes > heart > skeletal muscle > lung > thymus > kidney > brain > spleen. In all tissue extracts examined, except that from the lung, the levels of protease activity were higher in extracts from hypox rats compared with sham-operated rats. The largest differences between tissue extracts from hypox and sham-operated rats were seen in spleen (4-fold higher), kidney (2.27-fold), testes (1.55-fold) and heart (1.31-fold). In the liver, kidney and testes, GH treatment significantly reduced protease activity. Since the pattern of serum IGF-binding proteins (IGFBPs) differ in hypox rats compared with normal rats, we determined whether these changes could result in enhanced serum binding of des(1-3)IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

Measurement of adenosine 3':5'-cyclic monophosphate by competitive binding to salt-dissociated protein kinase.

An assay for cyclic AMP is described which takes advantage of the high affinity of the dissociated receptor moiety of cyclic AMP-dependent protein kinase I for the nucleotide. The kinase is kept dissociated by salt (800 mM-NaCl/30mM-EDTA). In the presence of a simply prepared heat-stable protein fraction the binding reagent is stable for the time needed to reach equilibrium of binding. A simple procedure [precipitation with poly-(ethylene glycol) followed by DEAE-cellulose chromatography] is described for the separation of protein kinase I from other binding proteins for cyclic AMP in rabbit skeletal muscle. The sensitivity, precision, reproducibility and specificity of the assay compared favourably with those of other cyclic AMP assays. The main advantage of the present assay is its resistance towards non-specific interference from a number of salts, tissue-culture media and substances found in crude tissue extracts. The reliability of cyclic AMP measurement directly in crude tissue extracts was ensured by removal of the assayable cyclic AMP with cyclic nucleotide phosphodiesterase digestion or adsorption with antibody against cyclic AMP, by comparison with measurement in tissue extracts purified by chromatography on QAE-Sephadex or sequentially on Dowex 50, and aluminium oxide as well as by dilution and recovery experiments.

Binding, Competitive↗
PubMed1993

Radiopharmacological studies of 99mTc-CPI: experience with isolated rat atrial tissue.

Extraction, washout and chemical integrity of technetium(I) hexakis (2-carbomethoxy-2-isocyano propane), (99mTc-CPI) were evaluated in isolated contractile rat atrial tissue. 99mTc-CPI accumulated linearly over time at 32 degrees C with uptakes that were both concentration dependent and temperature sensitive. Uptake rates also increased with contractile strength, indicating a dependence of retention on metabolic status of the tissue. Retention indices showed that percent washout of 99mTc-CPI was much slower than control studies with 99mTcO4. HPLC analysis of retained radioactive products in atrial tissue showed most of the activity present as the 99mTc-CPI complex (75%). These observations in isolated rat atrial tissue help explain the interspecies differences in biodistribution of 99mTc-CPI and support the membrane potential dependent model for uptake and retention of technetium isonitrile complexes.

Animals↗
PubMed1975

Observations on the heat stability and electrophoretic pattern of alkaline phosphatases extracted from various tissues.

Various tissues were extracted with either normal saline or heat inactivated serum (HIS) and the heat stability and electrophoretic migration of the alkaline phosphatase enzymes (AP) of the extracts were compared to the heat stability and electrophoretic properties of serum AP. The electrophoretic pattern of HIS extracts of liver and bone differed from that of saline extracts but the pattern was unaffected if HIS was added to the saline extracts. The heat stabilities of the tissue AP also differed depending on whether they were extracted with saline or HIS. However, serum AP heat stability can help differentiate between liver and bone disease. It is concluded that the comparison of serum and tissue AP heat stabilities or the comparison of serum and tissue AP electrophoretic patterns as criteria for identification of the tissue source of the serum enzyme may be misleading since both these parameters vary, depending on the medium used for extracting the tissue and the extract(s) may contain a mixture of enzymes different from that in serum. It is further concluded from the electrophoretic studies on tissue AP that the increased serum AP in patients with hepatobiliary disease was unlikely to be due to regurgitation of bile but due to increased synthesis and release of alpha 1 and alpha 2 AP isoenzymes from liver, bile ducts or gall bladder. In patients with bone disease the increased serum AP is derived from bone. The source of the serum AP of "normal" subjects may be either liver or vascular tissue or both.

Alkaline Phosphatase↗
PubMed1988

CA 125 in tissues and amniotic fluid during pregnancy.

CA 125 was assayed in amniotic fluid and tissue extracts by immunoradiometric assay, and immunohistochemical studies were performed on paraffin-embedded sections of endometrium, decidua, and fetal membranes with the monoclonal antibody OC 125 used as primary antibody. The concentration of CA 125 in amniotic fluid changes during pregnancy so that levels of 800 to 1000 U/ml are found before 12 weeks. Thereafter, levels of 4000 to 10,000 U/ml are detected routinely. As term approaches, amniotic fluid CA 125 concentrations fall to a range of 1000 to 2000 U/ml. Levels of CA 125 in tissue extracts of secretory endometrium and decidua were 65,000 and 29,500 U/gm of tissue, respectively. CA 125 was readily detected on the apical surfaces of glandular epithelium and in the secretions of endometrial glands obtained throughout the menstrual cycle. It was also detected in the lumina of decidualized glands throughout pregnancy. No antigen was detectable within glandular epithelial cells. We have previously reported high concentrations of CA 125 in chorionic tissue extracts (42,000 U/gm) and low concentrations in amniotic tissue extracts (275 U/gm). In contrast to those findings, immunohistochemical techniques detected CA 125 within the intercellular canaliculi that surround amniotic epithelial cells but not in chorion. We conclude that the likely source of amniotic fluid CA 125 is the decidua and that it gains access to the amniotic fluid via the intercellular canalicular system that traverses the amniotic epithelium.

Amniotic Fluid↗
PubMed1987

Detection of IgE antibody-forming cells by passive cutaneous anaphylaxis using cell extract from lymphoid organs.

IgE and IgG2a antibody-forming cells in the lymphoid organs of rats were detected by passive cutaneous anaphylaxis (PCA) reaction using the extract from the tissue. The amount of antigen-specific IgE and IgG2a antibodies in the tissue extract could be expressed as PCA titers in which extract from 10(8) cells/ml was used as starting dilution. The total amount of antigen-specific IgE antibody produced in mouse IgE-producing hybridoma cells was calculated by maximum dilution of the extract to the minimal amount of IgE antibody inducing PCA reaction, i.e., 4 ng/ml. In the case of the extract from mouse IgE antibody-forming hybridomas, the minimal number of hybridoma cells to be detected by this method proved to be 2-3 X 10(4) cells/ml.

Animals↗
PubMed1999

Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR.

Polymerase chain reaction (PCR) techniques are used increasingly for the diagnosis of Mycobacterium tuberculosis infection and can be used on the DNA obtained from both frozen and formalin fixed, paraffin wax embedded tissues. However, the extraction of DNA by means of the conventional phenol/chloroform method is time consuming and requires the use of potentially dangerous chemical reagents. This paper describes a method based upon the use of magnetic beads for the extraction of M tuberculosis DNA from both routinely formalin fixed, paraffin wax embedded tissues and frozen tissues. Magnetic bead extracted DNA from brain, lymph node, and lung tissues collected from patients with human immunodeficiency virus and tuberculosis was compared with that extracted using the phenol/chloroform method. The magnetic bead extraction procedure requires less than two hours, including the time necessary to dewax the tissue sections. In all cases, the DNA extracted with both methods was amplified successfully by PCR for the M tuberculosis IS6110 sequence. Magnetic bead DNA extraction can be used on both frozen and archival tissues: the method is reliable, simple, sensitive, and rapid; in addition, it does not use hazardous procedures or specialised laboratory equipment and can be used for routine DNA isolation from various human tissues.

Brain↗
PubMed2000

An ELISA-based assay for detergent-solubilized cellular beta 1,4-galactosyltransferase activity. Use of a polyacrylamide derivative with GlcNAc-beta side chains as a solid phase acceptor substrate.

In our previous paper [Oubihi et al. (1998) Anal. Biochem., 257, 169-175], we have shown that a polyacrylamide-derived synthetic glycopolymer with GlcNAcbeta side chains, termed PAP(GlcNAcbeta), is useful as a solid phase acceptor substrate for the ELISA-based analyses of soluble beta1,4(-)galactosyltransferase (GalT) activity in milk. This method is now used to assay detergent-solubilized cellular GalT. The glycopolymer coated on polystyrene plates was shown to be highly stable against the non-ionic and ionic detergents tested (0 approximately 5% solutions of Triton X-100 and SDS). Such stability made it possible to incubate the ELISA plate with detergent-solubilized GalT and to wash the ELISA plate with SDS solution after the GalT reaction, leading to high accuracy and sensitivity of this assay. The GalT activity was assayed using this method for 1% Triton X-100 extracts of various tissue samples of mice and several cultured cell lines. The results showed that the specific GalT activity of tissue extracts was low in brain and intestine, and high in ovary, muscle, and kidney. As for the cultured cell lines, COS7, COMMA-1D and C2C12 cells showed high specific activity, while CHO and MDCK cells showed low activity. The myoblast C2C12 had a slight increase in GalT activity during starvation-induced cell differentiation. On the other hand, GaIT-I transcript estimated by RT-PCR rather decreased during C2C12 cell differentiation, suggesting a differentiation-dependent switch in GalT isozymes. Taken all together, the ELISA-based assay using PAP(GlcNAcbeta) as a solid phase acceptor substrate was demonstrated to be a useful method for the assay of membrane-bound galactosyltransferases.

Acrylic Resins↗
PubMedDate not supplied

Characteristics of tissue plasminogen activator from paranasal mucous membrane in chronic sinusitis.

Tissue extract of paranasal mucous membrane from patients with chronic sinusitis was found to show a large lysis area on plasminogen-rich fibrin plates, but not on plasminogen-free fibrin plates. This indicates the existence of tissue plasminogen activator in the tissue extract. Further studies by the gel filtration technique showed that two plasminogen activators of different molecular weights were present in the tissue extract. The existence of tissue plasminogen activator with a low molecular weight has not previously been reported. This activator is labile at neutral pH at 37 degrees C, but stable on fibrin under the above conditions. The molecular weight of this compound is lower than that of cytochrome c. It may be a compound which is proteolytically modified by proteases, ie, trypsin-like enzymes, existing in the paranasal mucous membrane tissue of patients with chronic sinusitis.

Chronic Disease↗
PubMed1977

[Relaxin and cervical dilatation during parturition (author's transl)].

Relaxin concentrations during pregnancy and parturition were measured in tissue specimens of the uterine cervix of 27 women in childbearing age. During pregnancy relaxin levels increase from 0.4 guinea pig unit (GPU) to 0.94 GPU/mg soluble tissue protein. Already during parturition (0.41 GPU) and immediately post partum (0.39 GPU) the hormone concentrations have dropped back to normal. It appears that the biochemical changes which occur in the connective tissue of the uterine cervix during pregnancy and parturition are influenced by relaxin. Experimental animals were taken to show if relaxin acts by activation of collagenolytic enzymes. In the mouse a rise in the activity of collagen-peptidase and of the inhibitor of collagen-peptidase in plasma was found after subcutaneous injections of relaxin. The addition of human cervical tissue extracts resulted in an increase of enzyme-activation (even higher than could be expected of the hormone concentrations of the individual extracts). The highest levels of the enzyme were found after injections of extracts from cervical tissue specimens of parturient women.

Animals↗
PubMed1999

Real-time extraction of tissue impedance model parameters for electrical impedance spectrometer.

This paper presents a new algorithm for real-time extraction of tissue electrical impedance model parameters from in vivo electrical impedance spectroscopic measurements. This algorithm was developed as a part of a system for muscle tissue ischemia measurements using electrical impedance spectroscopy. An iterative least square fitting method, biased with a priori knowledge of the impedance model was developed. It simultaneously uses both the real and imaginary impedance spectra to calculate tissue parameters R0, R infinity, alpha and tau. The algorithm was tested with simulated data, and during real-time in vivo ischemia experiments. Experimental results were achieved with standard deviations of sigma R0 = 0.80%, sigma R infinity = 0.84%, sigma alpha = 0.72%, and sigma tau = 1.26%. On a Pentium II based PC, the algorithm converges to within 0.1% of the results in 17 ms. The results show that the algorithm possesses excellent parameter extraction capabilities, repeatability, speed and noise rejection.

Algorithms↗
PubMed1984

Deoxyribose 1-phosphate: radioenzymatic and spectrophotometric assays.

A method has been developed to measure deoxyribose 1-phosphate in the presence of ribose 1-phosphate and other sugar phosphates. The specificity of the method is based on the observation that only deoxyribose 1-phosphate is hydrolyzed by heating at pH 7.4, while both deoxyribose 1-phosphate and ribose 1-phosphate remain unchanged when heated at pH 10. A tissue extract is heated at pH 10. The amount of deoxyribose 1-phosphate plus ribose 1-phosphate is determined from that of deoxyinosine plus inosine formed in a coupled enzymatic reaction, based on the following two-stage transformation: deoxyribose 1-phosphate (ribose 1-phosphate) + adenine in equilibrium deoxyadenosine (adenosine) + inorganic phosphate, catalyzed by adenosine phosphorylase; deoxyadenosine (adenosine) + H2O----deoxyinosine (inosine), catalyzed by adenosine deaminase. By taking advantage of its unique heat lability, deoxyribose 1-phosphate is eliminated by heating the tissue extract at pH 7.4, and ribose 1-phosphate is determined as above. The amount of deoxyribose 1-phosphate stems from the difference between the amount of deoxyinosine plus inosine measured in the tissue extract heated at pH 10 and that of inosine measured in the tissue extract heated at pH 7.4. Free deoxyribose 1-phosphate has been found in rat tissues, as well as in Bacillus cereus during stationary phase of growth.

Animals↗
PubMed2001

Effect of garlic (Allium sativum L.) extract on tissue lead level in rats.

The prophylactic efficacy of garlic (Allium sativum L.) extract to reduce tissue lead (Pb) concentration was evaluated experimentally in rats. Thirty female rats were divided into five groups, keeping group A as a healthy control. Rats of groups B, C, D and E received lead acetate orally at the rate of 5 mg per kg body weight daily for 6 weeks. The garlic extract was tried in three doses, viz. 100 (low), 200 (medium) and 400 mg (high) per kg body weight orally and given simultaneously with lead salt to the rats of group C, D and E, respectively. Mean blood lead concentrations in lead-exposed rats ranged between 0.13+/-0.02 and 0.96+/-0.06 microg/ml, whereas in garlic-treated rats, the range was between 0.16+/-0.01 and 0.80+/-0.05; 0.13+/-0.01 and 0.71+/-0.06 and 0.14+/-0.01 and 0.60+/-0.05 microg per ml in low, medium and high dose groups, respectively. The mean lead concentration in liver, kidneys, brain and bone of lead exposed rats was 2.943+/-0.206, 4.780+/-0.609, 1.019+/-0.100 and 44.075+/-2.60 microg per ml, respectively. Concomitant use of garlic extract at the three different doses was found to reduce lead concentration considerably indicating the potential therapeutic activity of garlic against lead.

Animals↗