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PubMed1998

Determination of proteins, phosphatidylethanolamine, and phosphatidylserine in organic solvent extracts of tissue material by analysis of phenylthiocarbamyl derivatives.

Amino acid analysis of organic solvent extracts of tissue material has been evaluated for determination of protein content. Conventional ninhydrin-based analysis does not allow determination of a large number of lipid-rich samples. Therefore, the hydrolyzed samples were treated with phenylisothiocyanate and the phenylthiocarbamyl (PTC) derivatives obtained were separated by reverse-phase HPLC. With this method, analysis of many lipid-rich samples is feasible. In addition, phosphatidylethanolamine and phosphatidylserine can then be determined together with the amino acid constituents. The PTC/reverse-phase HPLC method was used for analysis of chloroform/methanol extracts of spinal cord, lung, and bile after chromatography on Lipidex 5000 in methanol/ethylene chloride, 4:1 (v/v). The chromatography profiles show that in all tissue samples the proteins elute before the phospholipids. Consequently, a single step of Lipidex 5000 chromatography can be used to purify polypeptides present in organic solvent extracts. Using pulmonary surfactant extracts (with about 98% phospholipids and 1-2% proteins), we find that individual contents of surfactant proteins B and C can be determined by amino acid analysis.

Amino Acids↗
PubMed1975

An indirect radioimmunoassay for mouse casein using 125I-labeled antigen.

A new indirect radioimmunoassay was developed for detection of casein in mouse milk and in mammary tissue extract. Preincubation of rabbit gamma globulin to mouse milk casein (Ca2+-rennin precipitate) with unlabeled casein, milk, extracts of mammary tissues of late pregnancy and lactation, virtually blocked subsequent binding of 125I-labeled mouse milk casein to the antibody. Preincubation with mouse serum, bovine serum albumin, rennin, extracts of liver or immature mammary tissue had little effect on [125I]casein binding to the antibody. The inability of [125I]casein to bind to the antibody after preincubation with protein samples, which are likely to contain casein, is indicative of a specific antigen-antibody reaction. The assay is capable of detecting 0.2 mug casein, 1 mug milk proteins and 10 mug lactating mammary tissue extract. The application of the assay was also demonstrated using organ culture of the entire mammary gland. The glands treated with the lactogenic hormones, insulin + prolactin + cortisol, showed a saturation level of antibody-antigen reaction, indicating hormonal induction of casein; whereas, no reaction was observed with the non-treated gland.

Animals↗
PubMed1981

A case with glucagonoma syndrome--heterogeneity of glucagon and insulin.

The heterogeneity of glucagon and insulin in plasma and tissue extracts from a 57-year-old female with glucagonoma syndrome with surgically and autopsy verified islet-cell tumors was studied by Bio-Gel P-10 filtration. The preoperative plasma immunoreactive glucagon (IRG) level was 20.2 ng/ml, and plasma glucagon-like immunoreactivity(GLI) 25.8 ng/ml. The column chromatography of the preoperative plasma revealed three or four IRG components and four GLI components. Among these, peak II, the large glucagon immunoreactivity (LGI) peak, considered a candidate for proglucagon, was prominent, along with peak III. The resected metastatic liver tumor contained an enormous amount of IRG and an appreciable amount of immunoreactive insulin (IRI), indicating that the elevated plasma IRG was mainly of tumor origin. The IRG pattern of the tumor tissue extract revealed a small quantity of IRG in peaks I and II, and a large amount in peak III; control pancreatic tissue extract manifested a similar elution pattern. The IRI elution pattern of the tumor tissue extract revealed two major IRI peaks which migrated close to the elution volume of cytochrome C and insulin, respectively. This is a quite different pattern from the control pancreatic tissue extract in which the RI peak was localized in the elution volume of the insulin. We conclude that the present metastatic liver tumor produced not only enormous amounts of glucagon but heterogeneous peptides which contained immunological insulin determinants within their.

Adenoma, Islet Cell↗
PubMedDate not supplied

Molecular forms of alpha-melanocyte-stimulating hormone in the canine pituitary anterior and intermediate lobe.

Reverse-phase high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) were used to determine the distribution of naturally occurring forms of alpha-melanocyte-stimulating hormone (alpha-MSH) in acid extracts of pars intermedia (PI) and anterior lobe (AL) tissue from canine and rat pituitary. Similarly, intracellular and secreted forms of alpha-MSH were determined using cultured canine PI and AL cells. Rat PI tissue contained predominantly diacetyl-alpha-MSH, while monoacetyl-alpha-MSH was the most abundant form in canine PI. In both canine and rat AL tissue extracts desacetyl-alpha-MSH was the major form of alpha-MSH. The profile of alpha-MSH contained in and secreted into culture medium by canine PI cells was found to be very similar to that in PI tissue extracts. The proportion of monoacetyl-alpha-MSH and diacetyl-alpha-MSH secreted by cultured canine AL cells and contained in extracts of AL cells in culture, however, was much higher than that in tissue extracts. These results indicate that in the dog, as in all other mammalian species studied, acetylated forms of alpha-MSH predominate in PI tissue, while nonacetylated alpha-MSH is the major form in AL tissue. It appears, however, that acetylation of alpha-MSH may occur in cultured canine AL cells, possibly as a result of the absence of factors that normally inhibit acetyltransferase in vivo or as a consequence of culture conditions.

Animals↗
PubMed1985

Regulation of tissue oxygen extraction is disturbed in adult respiratory distress syndrome.

To test the hypothesis that regulation of tissue oxygen (O2) extraction is disturbed in adult respiratory distress syndrome (ARDS), the relationship between O2 availability and O2 extraction ratio (O2ER) was studied in 36 patients with pulmonary edema who met the criteria for either ARDS (n = 21) or acute congestive heart failure (CHF) (n = 15). We found that in response to changes in the levels of O2 availability there was a significantly (p = 0.01) greater negative correlation of change in O2ER for the CHF (r = -0.67) than for the ARDS group (r = -0.41). Whereas patients with CHF eventually died of pump failure and low O2 delivery, the ARDS group developed multiorgan failure, especially renal failure, despite significantly higher ratios of O2 availability to O2 consumption. These findings suggest that patients with ARDS have lost their ability to regulate their tissue O2 extraction to compensate for changes in the availability of O2 in order to meet demand. Pulmonary capillary endothelial injury, through its role on whole-body metabolism, may be responsible for this observation.

Adult↗
PubMed1982

Absorption of NPH-insulin from subcutaneous tissue: a methodological study in pigs.

To elucidate the validity of the indirect method of following insulin absorption from the subcutaneous tissue, i.e. external counting of injected 125-I insulin, the disappearance of 125-I NPH insulin from subcutaneous tissue was measured by a biotelemetric method (external measurement of radioactivity) and by direct determination of radioactivity and insulin in subcutaneous tissue extracted one to 300 min. after injection of 8 units 125-I NPH porcine insulin, 5 mm beneath the surface of the skin in 6 anaesthesized pigs. Furthermore, the appearance of insulin in arterial plasma was measured after inhibiting the endogenous insulin secretion of the pigs by epinephrine and propanolol. The disappearance of radioactivity measured continuously for 5 hours was of first order kinetics with t1/2=462+/-36 min. (mean+/-S.E.M.). The half-life of the injected depot of radioactivity (t1/2) demonstrated a large intra- as well as interindividual variation as seen in diabetics. In tissue extracts a highly significant (2P less than 0.001) correlation was found between radioactivity and insulin (r=0.93), indicating the radioactivity to be representative of insulin. During gel chromatography on Sephadex G 50 M only one peak of radioactivity was seen in all tissue extracts. 300 min. after NPH-insulin injection, however, the specific radioactivity of insulin was significantly higher (2P less than 0.05) than in tissue extracts sampled one min. after NPH insulin injection, indicating a slight accumulation of non-immunoreactive insulin degradation products. A fair correlation (r=0.75, 2 P less than 0.05) was seen between the disappearance of externally measured radioactivity or insulin from tissue extracts and the appearance of insulin in plasma. It is concluded that the insulin absorption coefficient is a relevant and biologically sensible expression of insulin absorption.

Absorption↗
PubMed1984

Cardiovascular effects of atrial extracts in anesthetized rats.

Tissue extracts derived from atria or ventricles of Sprague-Dawley rats were injected into Inactin-anesthetized assay rats. Compared with ventricular extracts, atrial extracts produced a 20 mmHg (1 mmHg = 133.322 Pa) fall in mean arterial blood pressure. This fall resulted from failure to increase cardiac output in compensation for peripheral vasodilation. Two factors were responsible: depression of heart rate (by 25 beats/min) and failure to increase cardiac performance. The time patterns and magnitudes of changes in cardiovascular parameters after cardiac extracts were not changed by prior atropinization. However, assay rats that were vagotomized showed no cardiac slowing after atrial extract and showed a significantly smaller decrease in mean arterial blood pressure than did sham-vagotomized or intact rats. Another group of assay rats was vagotomized as well as carotid-sinus-denervated before extract injection. In these rats the degree of hypotension caused by atrial extract was significantly greater than that observed after vagotomy alone and was not significantly different from that observed in rats with intact innervation. The results suggest that the hypotension that is caused by atrial extract, but not by ventricular extracts, results in part from the reflex effects of direct stimulation of chemosensitive cardiopulmonary receptors with vagal afferents and partly from the reflex effects of baroreceptor unloading. Ventricular extract had no hypotensive effect in any group of assay rats.

Animals↗
PubMed1988

Affinity chromatography of naturally occurring folate derivatives.

In a previous report (1980, in Methods in Enzymology, Vol. 66, p. 686, Academic Press, New York) we described the preparation and use of purified milk folate-binding protein covalently linked to a Sepharose matrix. The present study was undertaken to test the capacity of this preparation to purify quantitatively folates from tissue extracts. In experiments that employed tissue extracts containing biologically synthesized [3H]folates, recovery of radioactivity from columns of the immobilized folate-binding protein (affinity columns) was consistently 90-95%. Folates eluted from the affinity column were practically pure. Anion-exchange chromatography of the purified fraction from rat liver extract yielded a number of uv-absorbing peaks which corresponded to the elution profile based on 3H counts, with respect to both the position and the area of the peaks. Treatment of the original extracts with folylpolyglutamate hydrolases resulted in the shift of these peaks to lower retention times, which corresponded to mono- or diglutamyl folate derivatives. Similar results were obtained with extracts from Lactobacillus casei. The purification of tissue folates by affinity chromatography allows determination of folate activity by direct physiochemical methods, which is particularly useful for analysis of folate composition in tissues.

Animals↗
PubMed1989

Isolation, separation and analysis in neurochemistry: trace amines and acids as an illustrative example.

In this brief overview various neurochemical isolation procedures that can be adopted for the analysis of several monoamine neurotransmitters/neuromodulators and their principal oxidatively deaminated metabolites are outlined. With respect to the trace amines, they can be identified and quantitated as their so-called dansyl derivatives after thin-layer chromatographic separation by mass spectrometric (MS) electron-impact (EI) ionisation followed by selected-ion monitoring (SIM) of their molecular ions. Deuterated homologues are added as internal standards at the start of the analytical procedure. The MS-EI-SIM procedure offers a tissue extract or releasate sensitivity of about 100 pg/g of tissue or fluid. In the case of tryptamine or phenylethylamine, by utilising different derivatives (N-acetylpentafluoropropionyl or N-acetylpentafluorobenzoyl), which cyclise to form perfluorinated spirocyclic compounds, it is possible using MS negative chemical ionisation techniques coupled with monitoring of the (M-HF) ions to achieve sensitivities for tissue extracts of 1 pg/g or less. Acidic and neutral metabolites (up to twelve of them can be assayed simultaneously) can be detected and quantitated in tissue extracts, releasates or biological fluids as their methylpentafluoropropionyl or trifluoroethyl-pentafluoropropionyl derivatives in the 100-1000 pg range using gas chromatographic-MS-SIM procedures.

Amines↗
PubMed1979

Binding of the Cain quinolinium, NSC 113089, to rat tissue lipid extracts.

The binding characteristics of the cancer chemotherapeutic Cain's quinolinium 6-amino-1-ethyl-4-[p-[[p-[(1-ethylpyridinium-4-yl) amino] phenyl] carbamoyl]-anilino]-quinolinium dibromide (NSC 113089) to lipid extracts from rat kidney, liver, heart and skeletal muscle has been studied. Such binding is saturable with an apparent KD congruent to 1.6 microM. Drug binding to the lipid extracts is displaceable by spermine, spermidine, calcium ions and protons. Spermine is the best displacing agent, achieving half drug displacement from the lipid extracts at approximately 6.3 microM regardless of tissue. The inability of the displacing agents to displace all the NSC 113089 bound to the lipid extracts as well as differences in the amount of agent bound to as compared to amount of drug displaced from the lipid extracts indicate that a number of drug binding sites may be present in the lipid extracts. The similarities of drug binding by rat tissue lipids to similar lipids extracted from normal animal and tumor tissues is discussed.

Animals↗
PubMed1993

Influence of hematocrit on tissue O2 extraction capabilities during acute hemorrhage.

This study was performed to test the hypothesis that tissue O2 extraction capabilities during hemorrhage may be greater when hematocrit (Hct) is initially reduced. Twenty-four anesthetized and splenectomized dogs were randomly assigned in three groups of eight dogs each: group 1 (Hct 40), 40-45% Hct; group 2 (Hct 30), 30-35% Hct; and group 3 (Hct 20), 20-25% Hct. In each animal, the desired Hct was obtained by normovolemic hemodilution using hydroxyethyl starch 450/0.7 and maintained throughout the experiment. O2 delivery (DO2) was progressively reduced by hemorrhage. At each step, DO2 and O2 consumption (VO2) were measured separately. Critical DO2 obtained from a plot of VO2 vs. DO2 was lower in the Hct 30 and Hct 20 groups than in the Hct 40 group [(in ml.min-1.kg-1) Hct 30, 7.9 +/- 2.2; Hct 20, 7.8 +/- 1.0; Hct 40, 10.4 +/- 1.1; P < 0.05]. Critical DO2 obtained from blood lactate levels was also significantly lower in the Hct 30 and Hct20 groups than in the Hct 40 group. Critical O2 extraction ratio was also greater in the Hct 30 and Hct 20 groups than in the Hct 40 group (Hct 30, 73.0 +/- 13.9%; Hct 20, 70.1 +/- 9.6%; Hct 40, 57.1 +/- 11.5%; P < 0.05). In the conditions of our study, moderate hemodilution was associated with an improvement of the O2 extraction capabilities of the body, probably related to the reduction in blood viscosity.

Acute Disease↗
PubMed1998

Tissue distribution, turnover, and glycosylation of the long and short growth hormone receptor isoforms in rat tissues.

Two isoforms of the GH receptor, the full-length receptor (GHRL) and a short isoform (GHRS) that lacks the transmembrane and intracellular domains of GHRL, have been analyzed in rat tissue extracts by Western blotting and immunoprecipitation. Although quantitative estimates of GHRS and GHRL based on coprecipitation of [125I]GH indicated similar amounts of both isoforms in tissue extracts, the 110 kDa band corresponding to GHRL was generally not detected on Western blots without enrichment by immunoprecipitation. Two bands with electrophoretic mobilities corresponding to 38 and 42 kDa were present in extracts prepared from liver, muscle, and adipocytes. Western blots of the GH binding protein in rat serum also revealed two bands, but these had electrophoretic mobilities corresponding to 44 and 52 kDa. After digestion by endoglycosidase F, a single band with an electrophoretic mobility corresponding to 31 kDa was detected in samples from adipocytes, liver or serum, indicating that GHRS retained in tissues is glycosylated less extensively than that in rat serum. Digestion with neuraminidase indicated that the smaller glycoproteins in tissue extracts lack sialic acid residues that are present in serum samples. Furthermore, endoglycosidase H degraded GHRS in liver extracts to a 31 kDa band but did not degrade serum samples, suggesting that tissues retain a high mannose form of GHRS. The abundance of GHRS or GHRL in tissues from male, virgin female, and pregnant rats was estimated from the amount of 125I-GH that was bound to each isoform after immunoprecipitation. Liver contained more than 10 times as much GHRS per gram of tissue as fat or muscle. In liver, muscle, and fat, the amount of GHRS exceeded that of GHRL, sometimes by as much as 6-fold. GHBP levels in serum of females exceeded those in males, and rose even higher in pregnant females. The abundance of GHRS in all tissue extracts paralleled serum levels. In muscle and fat, the levels of GHRL did not differ in male, female and pregnant rats, whereas in liver, the pattern was similar to the GHRS pattern. In all tissues, pools of GHRS exceeded those of GHRL by a factor that grew larger as tissue and serum levels increased. The half life of GHBP in serum was estimated to be 2.4 h in rats treated with cycloheximide, whereas that of GHRS was 20 min in liver and 8.5 h in fat. These results suggest that GHRS is synthesized in liver 8 times faster than it is released into serum, whereas synthesis in fat is less than 30% of the rate at which it is released into serum by all tissues. Therefore, liver appears to be the major source of GHBP in serum. Although secretion into the circulatory system accounts for little or perhaps none of its turnover in some tissues, GHRS pools in tissues do appear to be regulated, suggesting that GHRS may function primarily in the cells in which it is synthesized.

Adipocytes↗
PubMed1990

Non-specific inhibition of imipramine binding argues against an endogenous ligand.

We have attempted to characterize the product(s) inhibiting [3H]imipramine binding from rat cortical membranes. Fractions inhibiting [3H]imipramine binding were analysed by gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography-mass spectrometry (HPLC-MS). Tissue extracts showed marked inhibitory activity (two different peaks); however, extracts containing no brain tissue (blanks) also exhibited inhibitory activity (one peak after HPLC pre-purification). The chemical composition of the compound that inhibited [3H]imipramine binding was identical in both cases (tissue and blank extracts) and consisted of zinc chelates, probably formed by the use of zinc sulfate during the extraction procedure. The second inhibitory peak in the tissue extracts was 5-hydroxytryptamine (serotonin, 5-HT). These results suggest that the inhibitory activity on [3H]imipramine binding attributed to a low-molecular weight putative endacoid is non-specific and due to ions introduced in the sample during purification.

Animals↗
PubMed2001

Tumor necrosis factor-alpha expression in human uterine leiomyoma and its down-regulation by progesterone.

Although tumor necrosis factor-alpha (TNFalpha) has been shown mainly to inhibit proliferation and induce apoptosis in a variety of cells, no information is available regarding whether human leiomyoma cells express TNFalpha. In the present study, we examined the expression of TNFalpha in leiomyomas, in comparison with that in the adjacent normal myometrium, using immunohistochemical staining and Western immunoblot analysis with a polyclonal antibody to human TNFalpha. Furthermore, we investigated the effect of sex steroid hormones on TNFalpha expression in leiomyoma cells cultured under serum-free, phenol red-free conditions. Immunohistochemical staining showed that TNFalpha expression in leiomyoma cells was higher than that in the adjacent normal myometrial cells, being more abundant in the proliferative phase than in the secretory, progesterone (P4)-dominated, phase of the menstrual cycle. TNFalpha expression in leiomyoma cells in pregnant uterus was scarce. Western immunoblot analyses of leiomyoma and normal myometrial tissue extracts revealed that TNFalpha, with a molecular mass of 17.3 kDa, was abundantly present in leiomyoma tissue extracts, relative to normal myometrial tissue extracts, and that TNFalpha expression in leiomyoma cells was most abundant in the proliferative phase of the menstrual cycle, less abundant in the secretory phase, and least abundant in pregnant uterus; whereas no such changes in TNFalpha expression were noted in the normal myometrium. In monolayer cultures of uterine leiomyoma cells under serum-free conditions, addition of P4 (3.18 x 10(-7) mol/L) resulted in a decrease in TNFalpha expression in the cells, relative to that in control cultures, whereas treatment with 17beta-estradiol (3.67 x 10(-8) mol/L) did not affect the TNFalpha expression in the cells. The concentrations of sex steroids used were within the physiological tissue concentrations noted in leiomyoma and myometrium. The present results suggest that the abundant expression of TNFalpha may be a molecular basis characteristic of leiomyomas in the human uterus and that P4 may play a vital role in down-regulating the expression of TNFalpha in human uterine leiomyoma.

Adult↗
PubMed1976

The autoimmune response to male reproductive tissues of rabbits. IV. Induction of autosensitization to central accessory tissue by the intraabdominal release of endogenous secretions.

A study is described in which tissue- and secretion-specific autosensitization was induced in adult male rabbits through intraabdominal cannulation of one or more of their central accessory glands. Both primary and secondary humoral antibodies were elicited by this procedure, as determined by tanned cell hemagglutination and passive hemolysis, i.e., complement fixation. The secondary antibody responses were elicited by (i) cannulation of animals which had been previously stimulated by cryosurgery or combined cryosurgery and injection of pooled accessory tissue extract, and (ii) isoinjection of previously cannulated animals with accessory tissue extract. Primary antibody responses were less in animals castrated at the time of cannulation compared to those in noncastrated animals; but, paradoxically, subsequent challenges with tissue extract induced higher secondary antibody titers in castrated animals. These observations may be explained in castrates by (i) reduced uptake of antibodies--a result of the sparing effect on circulating antibodies in castrated animals because of accessory tissue atrophy, or (ii) increased production of antibodies through postcastration immune enhancement. In noncastrates secondary antibody depression may be related to (i) increased absorption of antibodies by an accessory gland complex which is much larger than that found in castrates, or (ii) tolerance, due to exposure of the antibody-producing system to excess accessory tissue antigen.

Abdomen↗
PubMed2003

Elevated expression level of survivin protein in soft-tissue sarcomas is a strong independent predictor of survival.

PURPOSE: Survivin is a member of the inhibitor-of-apoptosis gene family and is known to be overexpressed in a number of tumor types. The aim of this study was to evaluate the prognostic value of survivin protein expression in tumor tissue extracts in a group of well-characterized soft-tissue sarcoma (STS) patients. EXPERIMENTAL DESIGN: In this investigation, malignant tissue samples from 63 STS patients as well as from a panel of tumor cell lines were investigated, with nonmalignant tissues serving as controls. The survivin protein level was quantified by a novel ELISA and by Western blot analysis. Results obtained by both methods were compared with clinicopathological parameters regarding tumor grade and tumor entity, and they were then correlated to survival in a multivariate Cox regression model. RESULTS: High survivin levels were detected by ELISA and Western blot analysis in tumor tissue extracts and in lysates of tumor cell lines. None or only weak expression of survivin protein was found in nonmalignant cells and tissues. When comparing survivin values obtained by ELISA or Western blot, we found a significant correlation between both methods (P = 0.013, Pearson test). Our findings revealed that, in multivariate Cox regression analyses, survivin levels measured by ELISA and Western blot were significantly associated with tumor-related death in STS patients (P = 0.001, RR = 19.8, and P = 0.004, RR = 5.1, respectively). However, in a direct comparison of both survivin protein detection assays, we found a higher sensitivity and a stronger correlation to prognosis in survivin ELISA as compared with the Western blot assays. Furthermore, a higher tumor grade and more aggressive STS entity showed an elevated survivin protein expression level. CONCLUSION: Altogether, an elevated survivin content in tumor tissue extracts has a significant and independent negative predictive value on the survival-rate of STS patients. This finding corresponds well to data obtained for the mRNA level of survivin, as shown previously (M. Kappler et al., Int. J. Cancer, 95: 360-363, 2001).

Blotting, Western↗
PubMedDate not supplied

[Immunochemical detection of phenylalanine hydroxylase in extracts of various human organs].

Phenylalanine hydroxylase was found in extracts from autoptates of human liver, kidney, myocardium, small intestine, lung and spleen. In all the tissues studied, except of spleen, the antigen was detected by immunoelectrophoresis with monospecific antiserum against human liver phenylalanine hydroxylase. As shown by immunoblotting carried out after electrophoresis under denaturating conditions, the antigen, observed in various tissues, exhibited similar electrophoretic mobility which was very close to that of the enzyme purified from human liver tissue. Molecular mass of revealed antigen was estimated 55-57 kDa. Coincidence of immunochemical and chemical properties of the protein suggested that the antigen, detected in the tissue extracts, was a product of phenylalanine hydroxylase gene expression. The antigen concentration did not correlate with the content of albumin in tissue extracts, thus demonstrating that the revealed antigen did not occur in these preparations with blood contaminations. Content of the antigen in the tissue extracts studied was (ug per g): liver - 1500-1900, kidney - 300-575, brain - 20-40, myocardium - 85-105, lung - 40-125, small intestine - 45-70, spleen - 0-12.

Adult↗