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PubMed1982

[Changes of acid phosphatase in the gastric mucosa during ulcerogenesis].

Experiments were performed on white rats with experimental acetate ulcer of the stomach and intestine. At the height of destruction of gastric mucosa tissue (60 minutes from the onset of ulcer formation) a consistent increase in acid phosphatase secretion was recorded. This process was accompanied, as a rule, by significant elevation of proteolytic activity (pH 6.5-7.0) in tissue extract obtained from the affected area. It is suggested that the increased proteolytic activity observed at pH 6.5-7.0 in the tissue extract from the ulcerous area is most likely related to the release of lysosomal proteases at the height of tissue destruction. It thus appears that ulcer of the gastric mucosa induced by acetic acid application is determined by interstitial proteolytic activity of cathepsins that are released during the destruction of lysosomal membranes.

Acid Phosphatase↗
PubMed2006

The effects of ginkgo biloba extract on tissue adenosine deaminase, xanthine oxidase, myeloperoxidase, malondialdehyde, and nitric oxide in cisplatin-induced nephrotoxicity.

This study was carried out to determine if Ginkgo Biloba Extract (GBE or Egb 761) exerts a beneficial effect against cisplatin-induced renal failure in rats. Sprague Dawley rats were divided into four groups. The first group (control) received orally 1 mL/kg/day of 0.9% saline by an oral carrier vehicle on days 1 to 10. The second group was injected with 7 mg/kg cisplatin intraperitoneally (i.p.) on the fourth day, once only. The third group (vit E+cisplatin) was administered 10 mg/kg/day i.p. vit E on 1 to 10 days with one dose of i.p. cisplatin (7 mg/kg) injection on the fourth day. The fourth group (GBE+cisplatin) was given GBE orally at 100 mg/mL/kg started on the first day up to the tenth day with one dose of cisplatin (7 mg/kg) injection on the fourth day. Cisplatin was found to lead a statistically significant increase in plasma BUN and creatinine levels, as well as urine micro total protein (MTP) levels, leading to acute renal failure (ARF) in rats. Renal xanthine oxidase (XO) activities increased in all groups (statistically significant in cisplatin + GBE-treated rats; P < 0.001). Adenosine deaminase (AD) activities were increased in cisplatin-treated rats, and decreased in cisplatin+GBE-treated (P < 0.041) and cisplatin+vit E-treated (P < 0.005) rats, compared to controls. Malondialdehyde (MDA), nitric oxide (NO) levels and myeloperoxidase (MPO) activities were increased in the kidney tissue of cisplatin-treated rats. Vit E improved plasma creatinine and urine MTP levels, together with tissue MDA, NO levels, and MPO activities. But GBE had no statistically significant effect on those parameters. These results indicate that increased XO, AD and MPO activities, as well as MDA and NO levels play a critical role in cisplatin nephrotoxicity. GBE has been shown to protect against cisplatin-induced nephrotoxicity.

Adenosine Deaminase↗
PubMed1994

Increased levels of lipid hydroperoxides in the parkinsonian substantia nigra: an HPLC and ESR study.

Previous studies examining the involvement of oxidative stress in the substantia nigra in Parkinson's disease have measured terminal products of lipid peroxidation or the function of antioxidant defense systems. We report a more specific early marker of lipid peroxidation, lipid hydroperoxides, in a high-performance liquid chromatography (HPLC) and electron spin resonance (ESR) investigation. HPLC-chemiluminescent detection revealed two classes of lipid hydroperoxides in brain tissue extracts--free fatty acid hydroperoxides and cholesterol lipid hydroperoxides. Only cholesterol lipid hydroperoxides were consistently detected in all tissue extracts. Cholesterol lipid hydroperoxides had a 10-fold increase in the Parkinson's disease substantia nigra compared to control subjects. ESR detection of radical degradation products, including those of lipid hydroperoxides, in nigral homogenates incubated with the spin trap N-t-butyl-alpha-phenyl nitrone (PBN) showed a marked variation in ESR signal between tissues. Despite the increased levels of lipid hydroperoxides in parkinsonian substantia nigra, there was no overall difference in ESR signal intensity between nigral tissues from controls and from patients with Parkinson's disease. The increased levels of an early component of the peroxidation chain in substantia nigra in Parkinson's disease support the hypothesis of a continuous toxic process involving oxygen radical activity. However, using previously frozen tissue, ESR evidence for increased radical formation could not be demonstrated.

Chromatography, High Pressure Liquid↗
PubMed1976

Identification of a butanol-extractable human placenta-specific antigen with alkaline phosphatase activity.

N-Butanol extracts of whole-term placenta from different individuals were prepared, and used as immunogens to raise heterologous hyperimmune sera in rabbits. Upon immunoelectrophoresis the anti-placenta antisera could recognize at least six antigenic components in the placental extract even after they had been completely absorbed with pooled male serum proteins. However, the antisera so absorbed, designated (-PMS) antisera, could still react strongly with several normal adult tissue extracts including kidney. Systematic and quantitative absorptions of the (-PMS) antisera were thus further carried out with individual butanol extracts of normal adult liver, lung, intestine, stomach, kidney, bone, pancreas, spleen, heart, cerebrum, cerebellum, breast, and packed red cells, as well as a composite extract containing equal amounts of each of the 13 adult tissue extracts. Of the six antigenic components in the placental extracts reacting with the (-PMS) antisera the only one which retained its reactivity with the antisera throughout exhaustive absorptions was associated with alkaline phosphatase activity. This immunologic and enzymologic identity was confirmed with homogeneous placental alkaline phosphatase. Extracts from each of three placentae injected into three pairs of rabbits all produced an identical antibody reaction with the unique determinant(s) of placental alkaline phosphatase. The same identity of precipitin reaction was also found with extracts of 14 other placentae against each of these antisera. It thus firmly establishes that placental alkaline phosphatase is a characteristic placenta-specific fetal protein.

Absorption↗
PubMed2001

Measurement of tissue oxygen extraction ratios from venous blood T(2): increased precision and validation of principle.

It has recently been shown that parenchymal oxygen extraction ratios (OERs) can be quantified using the absolute T(2) of venous blood draining from this tissue (Oja et al., J Cereb Blood Flow Metab 1999;19:1289-1295). Here, a modified Carr-Purcell-Meiboom-Gill (CPMG) multiecho experiment was used to increase the efficiency and precision of this approach and to test the applicability of the two-compartment exchange model for spin-echo BOLD effects in pure venous blood. Relaxation measurements on bovine blood as a function of CPMG interecho spacing, oxygen saturation, and hematocrit provided the baseline relaxation and susceptibility shift parameters necessary to directly relate OER to T(2) of venous blood in vivo. Using an interecho spacing of 25 ms, the results on visual activation studies in eight volunteers showed T(2)(CPMG) values increasing from 128 +/- 9 ms to 174 +/- 18 ms upon activation, corresponding to local OER values of 0.38 +/- 0.04 and 0.18 +/- 0.05 during baseline activity and visual stimulation, respectively. These OER values are in good agreement with literature data on venous oxygenation and numbers determined previously using a single-echo approach, while the measured T(2)s are about 20-40 ms longer.

Adult↗
PubMed1991

Rapid increase in inositol phosphate levels in norepinephrine-stimulated vascular smooth muscle.

We examined the correlation between agonist-stimulated increases in inositol phosphates and force development in vascular smooth muscle. Segments of rat tail artery were preincubated with [3H]inositol and treated with norepinephrine (10(-5) M) for 3-10 s. Tissue levels of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) were measured. IP and IP2 increased significantly after 3 s of norepinephrine treatment. IP3 increased significantly after 5 s of norepinephrine treatment. Analysis of tissue extracts by high-pressure liquid chromatography demonstrated that the only isomer of IP3 present in any tissue extract was the 1,4,5-isomer [Ins(1,4,5)P3]. Contractile response to norepinephrine stimulation showed that the increase in inositol phosphates coincides well with the time course of force development. This is the first report demonstrating such an early increase in Ins(1,4,5)P3 in agonist-stimulated vascular smooth muscle. These results are consistent with the hypothetical role of Ins(1,4,5)P3 as a mediator linking agonist-receptor activation to increased intracellular calcium and force development in norepinephrine-stimulated vascular smooth muscle.

Animals↗
PubMed2005

Contribution of hMTH1 to the maintenance of 8-oxoguanine levels in lung DNA of non-small-cell lung cancer patients.

BACKGROUND: The level of 8-oxoguanine (8-oxoG), a general marker of oxidative DNA damage, in DNA is the result of both an equilibrium between the rates of its formation and removal from DNA by DNA repair enzymes and the removal of 8-oxodGTP from the cellular nucleotide pool by hydrolysis to 8-oxodGMP, preventing its incorporation into DNA. To determine the contribution of each component to the level of 8-oxoG in DNA, we compared 8-oxoG-excising activity (encoded by hOGG1), 8-oxodGTPase activity (encoded by hMTH1), and 8-oxoG levels in DNA from tumors and surrounding normal lung tissues from non-small-cell lung cancer patients. METHODS: We measured the level of 8-oxoG in DNA of 47 patients by high-performance liquid chromatography/electrochemical detection (HPLC/ECD), hOGG1 activity in tissue extracts of 56 patients by the nicking assay using an oligodeoxynucleotide containing a single 8-oxoG, and hMTH1 activity in tissue extracts of 33 patients by HPLC/UV detection. All statistical tests were two-sided. RESULTS: The 8-oxoG level was lower in tumor DNA than in DNA from normal lung tissue (geometric mean: 5.81 versus 10.18 8-oxoG/10(6) G, geometric mean of difference = 1.75; P<.001). The hOGG1 activity was also lower in tumor than in normal lung tissue (geometric mean: 8.76 versus 20.91 pmol/h/mg protein, geometric mean of difference = 2.39; P<.001), whereas the hMTH1 activity was higher in tumor than in normal lung tissue (geometric mean: 28.79 versus 8.94 nmol/h/mg protein, geometric mean of difference = 0.31; P<.001). The activity of hMTH1 was three orders of magnitude higher than that of hOGG1 (nanomoles versus picomoles per hour per milligram of protein, respectively). CONCLUSIONS: Several different components contribute to the maintenance of 8-oxoG levels in human DNA, with the greatest contributor being the removal of 8-oxodGTP from the cellular nucleotide pool by hMTH1.

Adult↗
PubMed2003

Visualizing metabolic changes in breast-cancer tissue using 1H-NMR spectroscopy and self-organizing maps.

In-vitro NMR spectroscopic examinations of tissue extracts can be combined with appropriate pattern-recognition and visualization techniques in order to monitor characteristic metabolic differences between tissue classes. In the present study, such techniques are applied to a set of 88 breast-tissue samples with the intention of identifying typical differences between various tissue classes. The set contains 49 breast-tumor samples of various tumor grades and 39 samples of healthy tissue. The metabolite compositions of the tissue extracts were investigated using a dual extraction technique and high-resolution (1)H-NMR spectroscopy. The spectra of the hydrophilic and the lipophilic compounds were assigned to three groups according to different malignancy grades of the respective tissue samples. The group characteristics were analyzed using the k-nearest-neighbor method and self-organizing-map visualizations. The results show an increase of UDP-hexose, phosphocholine and phosphoethanolamine concentrations according to the tumor grade. Higher concentrations of taurine were detected in the malignant samples. Myo-inositol and glucose content were elevated in control samples compared with malignant tissue. Both compounds also characterized different subgroups in the pool of unaffected tissue samples depending upon fat content or fibrosis. Several lipid metabolites showed a characteristic elevation with high malignancy.

Algorithms↗
PubMedDate not supplied

Immunological specificity of beta-glucuronidase from human seminal plasma and its immunolocalization in the human reproductive tract.

Beta-glucuronidase (beta-glucuronidase) in human seminal plasma consists of two forms of isozymes (major and minor forms). Antiserum prepared against the purified major form of beta-glucuronidase in rabbits was used for studies of immunological specificity and immunohistochemical localization of beta-glucuronidase in the human male reproductive tract. By ouchterlony immunodiffusion technique, anti-beta-glucuronidase serum showed reaction of identity with human tissue extracts such as liver, spleen, lungs, testis, epididymis, prostate, and seminal vesicles. Though monkey testis and epididymis extracts completely reacted with human seminal beta-glucuronidase antiserum, no cross-reaction was observed with mouse and rat tissue extracts. Immunofluorescent studies revealed that beta-glucuronidase is localized in the early stages of spermatogenesis in testis, in columnar cells of the epididymis, and in glandular cells of the prostate and seminal vesicles. Consistent absence of fluorescence in the lumens of testis and epididymis as well as negative cross-reaction between antiserum and sperm extracts suggested that major form of beta-glucuronidase in human seminal plasma is not immunologically identical with human spermatozoa. It is concluded that major form of beta-glucuronidase in seminal plasma is not tissue-specific although it appears to have limited species specificity.

Animals↗
PubMed1997

False positive immunostaining of Locusta neurosecretory cells with a variety of preimmune sera.

A large number of antisera directed against vertebrate neuropeptides have been reported to yield positive staining when applied to insect brains. In most cases, the preimmune serum of the same animal in which the antiserum was developed is not available for testing in control experiments. We have experienced that a large percentage of preimmune sera, as well as a culture medium for hybridomas, stain cell populations and fibers in the central nervous system of the insect Locusta migratoria. Purification of these preimmune sera on a Protein A and Protein G support indicates that the reaction is due to preexisting antibodies of the IgG class. Western analysis of brain and nervous tissue extracts indicates the presence of two immunoreactive 27-kDa bands. These bands could also be visualized in other tissue extracts such as muscle, midgut, Malpighian tubules, and fat body of Locusta. The brain of other insect species, such as Periplaneta americana, Leucophaea maderae, and Neobellieria bullata were devoid of the false immunopositive reaction. There is no easy way to eliminate this type of immunoreaction. It follows that when affinity chromatographic purification of the antibody is not feasible, it is essential to include in the control procedure, the preimmune serum of the animal that was used for the production of the antiserum. This means that it should become common practice to sell or exchange sera together with their corresponding preimmune sera.

Animals↗
PubMed1986

Reflex splanchnic nerve stimulation increases levels of proenkephalin A mRNA and proenkephalin A-related peptides in the rat adrenal medulla.

The effect of reflex splanchnic nerve stimulation on proenkephalin A biosynthesis was investigated in the rat adrenal medulla. Tissue levels of native [Met5]enkephalin-like immunoreactivity (IR) (measured by direct RIA of tissue extracts), cryptic [Met5]enkephalin-like IR (calculated as the increase in [Met5]enkephalin-like IR detected in tissue extracts after sequential digestion with trypsin and carboxypeptidase B), and proenkephalin A mRNA were determined in adrenal medulla from rats sacrificed at various times after a period of insulin-induced hypoglycemia. Two hours of insulin hypoglycemia, which produced intense reflex stimulation of the splanchnic nerves as evidenced by a 55% decrease in the adrenal medulla catecholamine levels, resulted in a 3-fold increase in proenkephalin A mRNA levels in this tissue. The proenkephalin A mRNA levels reached a maximum 15-fold increase over control values 24 hr after this period of hypoglycemic stress and then gradually declined with an approximate half-life of 4 days. Native and cryptic [Met5]enkephalin-like IR had increased 9-fold and 12-fold, respectively, 24 hr after this period of hypoglycemia, and both demonstrated maximum increases of 130-fold and 50-fold, respectively, after 96 hr. Combined pretreatment (i.p. administration) with the ganglionic and muscarinic blocking agents chlorisondamine (5 mg/kg of body weight) and atropine (1 mg/kg) blocked the increase in levels of proenkephalin A mRNA seen in the rat adrenal medulla following insulin hypoglycemia. These data indicate that reflex splanchnic nerve discharge stimulates proenkephalin biosynthesis, probably at the level of gene expression.

Adrenal Medulla↗
PubMed1985

Extraction of tissue long-chain acyl-CoA esters and measurement by reverse-phase high-performance liquid chromatography.

Long-chain acyl-CoA esters were extracted from freeze-clamped livers of fed and fasted rats according to the method of Mancha et al. [M. Mancha, G. B. Stokes, and P. K. Stumpf (1975) Anal. Biochem. 68, 600-608] and analyzed on a radially compressed C18, 5 microns, reverse-phase column using a gradient system consisting of acetonitrile and 25 mM KH2PO4, pH 5.3, at 254 nm. Total analysis time was 25 min. Eight peaks in the extract with carbon chain lengths of 12 to 18, which subsequently disappeared on alkaline hydrolysis, were identified. The major acyl-CoA peaks in the extract in order of increasing retention times were 14:0, 16:1, 18:2, 16:0, 18:1, and 18:0. Total liver long-chain acyl-CoA esters were 108 +/- 11 and 248 +/- 19 nmol/g protein for fed and fasted rats, respectively. On fasting (48 h) the levels of 18:2, 16:0, and 18:1 increased two-to threefold and that of 18:0 sixfold. The advantages of this method are that it not only provides a more direct determination of total tissue long-chain acyl-CoA esters, in that no decomposition of the CoA ester is involved, but it also detects the constituent molecular species.

Acyl Coenzyme A↗
PubMed2006

Cytoplasmic domain of protocadherin-alpha enhances homophilic interactions and recognizes cytoskeletal elements.

Cell adhesion molecules of the protocadherin-alpha (pcdh-alpha), -beta, and -gamma families have been proposed to be synaptic specifiers. Pcdh-alpha and -gamma family members localize in part to synapses, and deletion of all pcdh-gammas in mouse affects synaptogenesis. Little is known, however, about the binding specificities and intracellular signaling of protocadherins. Using heterologous expression of tagged constructs, immunostaining, and biotinylation of surface components followed by Western blots we demonstrate that pcdh-alphas undergo homophilic interactions that are significantly enhanced by the cytoplasmic domain. Pcdh-alphas cloned from chick ciliary ganglion have one of two cytoplasmic constant regions (A- and B-types). Screening a yeast two-hybrid library of ciliary ganglion cDNA with the A-type domain yielded a fragment of neurofilament M (NFM); screening with B-type domain yielded a fragment of the actin-bundling protein fascin. Cotransfection of HEK cells with the constructs indicated that the NFM and A-type fragments codistributed as did the fascin and B-type fragments, and the latter could be coimmunoprecipitated. Antibody-induced clustering of full-length pcdh-alphas on the surface of transfected HEK cells induced coclustering of the interacting NFM fragment. Native full-length NFM in tissue extracts bound specifically to the A-type domain on beads, while native full-length fascin in tissue extracts specifically coimmunoprecipitated with pcdh-alpha. Immunostaining neurons demonstrated codistribution of full-length pcdh-alpha with both NFM and actin filaments. These findings suggest cytoskeletal links for pcdh-alphas and identify candidate targets. They also demonstrate homophilic interactions for pcdh-alphas as described for classical cadherins.

Actin Cytoskeleton↗
PubMedDate not supplied

[Detection of 1,4-benzodiazepine derivatives in liver tissues by immunochemical methods].

The possibility of using immunochemical methods in general forensic chemical analysis of cadaveric material for narcotics is demonstrated. Conditions of isolation of 1,4-benzodiasepines from tissues and analysis of the resultant tissue extracts by polarization fluoroimmunoassay (PFIA) and solid-phase enzyme immunoassay are described. Time course of concentrations in tissue extracts stored for a long time has been studied by PFIA. Characteristics of Benzodiazepines Serum kits (Abbott) for PFIA of liver extracts are determined.

Benzodiazepines↗
PubMed1985

Presence of corticotropin-releasing factor-like immunoreactivity in human tumors.

Corticotropin-releasing factor (CRF)-like immunoreactivity was measured by radioimmunoassay in human organs and tumors associated with and without ectopic adrenocorticotropic hormone (ACTH) syndrome. It was found to be distributed widely in the stomach, pancreas, adrenal gland, and various tumors (e.g., medullary thyroid carcinoma, small cell carcinoma of the lung, pheochromocytoma, and adenocarcinoma of the gastrointestinal tract and pancreas) in a concentration less than one tenth of that of the hypothalamus. Dilution curves of CRF-like immunoreactivity in tissue extracts paralleled that of synthetic rat (human) CRF. Sephadex G-50 gel filtration showed that a major CRF-like immunoreactivity in tissue extracts coeluted with synthetic rat (human) CRF. Results suggest that a material(s) closely related immunologically to CRF is present widely in normal and tumor tissues outside of the central nervous system.

ACTH Syndrome, Ectopic↗
PubMed1990

Immunocytochemical localization of follicle stimulating hormone in normal human stomach.

Immunoreactive follicle-stimulating hormone (IR-FSH) is detected in sections of formalin-fixed and paraffin-embedded gastric mucosal tissue of normal men, using the immunoperoxidase staining technique and specific antisera to hFSH (NIDDK, NIH). Positive staining for IR-FSH was detected in the parietal cells lining the gastric glands of the intermediate zone. The staining was intracytoplasmic and distributed throughout the cytoplasm. IR-FSH was also found to be present in the basal part of the foveolar epithelium. Stromal tissue and nuclei were devoid of the stain. The zymogen cells in the deeper region of the mucosa did not show any detectable staining for IR-FSH. The presence of IR-FSH in gastric mucosa was also detected by radioimmunoassay. Gel chromatography of the gastric tissue extract showed a single peak of FSH immunoreactivity that coeluted with the 125I-labeled highly purified FSH preparation (NIDDK, NIH). Furthermore, the FSH in the pituitary tissue extract had a chromatographic profile similar to that of IR-FSH from gastric tissue, and 125I-FSH labeled highly purified FSH, indicating a close resemblance in their molecular sizes. These results demonstrate that IR-FSH is present in the normal human gastric mucosa. The role of this regulatory petpide in gastric tissue, if any, needs to be investigated.

Chromatography↗
PubMed2001

C-type natriuretic peptide system in rabbit oviduct.

C-type natriuretic peptide (CNP), a third member of the natriuretic peptide family, is known to be distributed mainly in brain and vascular endothelium and is considered to act as a local regulator in many tissues. The purpose of this study was to determine the presence of CNP system and its biological function in rabbit oviduct. The serial dilution curve of tissue extracts was parallel to the standard curve of CNP((1-22)) and a major peak of molecular profile of tissue extracts by HPLC was CNP((1-53)). mRNA of CNP which was the same size as positive control was also detected by Southern blot analysis. CNP increased the production of 3',5'-cyclic guanosine monophosphate (cGMP) in the purified membrane of oviduct, which was more in membranes derived from the isthmic portion than in the ampullar portion. The presence of mRNAs of natriuretic peptide receptor-A (NPR-A) and NPR-B was demonstrated by RT-PCR. Synthetic CNP((1-22)) inhibited both frequency and amplitude of basal motility of oviduct in a dose-dependent manner. The inhibitory effect of CNP on the basal motility was more potent in the isthmic portion than in the ampullar portion. These results demonstrate the presence of CNP system in the oviduct and regional differences in motility inhibition by CNP between isthmic and ampullar portions. Therefore, these findings suggest the possible existence of a CNP system that may exert a local regulator of basal motility, either alone or in concert with other hormones.

Animals↗
PubMed1997

Multiple forms of alkaline phosphatase in horse bone, liver, kidney, duodenum, caecum and serum separated by agarose gel electrophoresis with and without neuraminidase pretreatment.

Horse bone, liver, duodenum, caecum and kidney alkaline phosphatases were separated by a commercial agarose gel electrophoresis method with and without neuraminidase pretreatment, following the manufacturer's directions. Tissue extracts were obtained in saline solution and ALP extracted from cell membranes by the butanol method. Electrophoresis was performed using a TRIS/barbital/sodium barbital buffer with detergent, pH 8.6 to 9.0, at 250 V for 30 minutes. Bone, liver and kidney untreated extracts showed two ALP bands each, but with different relative migration (compared to albumin migration). When they were preincubated with neuraminidase, the two bone bands showed a marked decrease in their migration, followed by the kidney ALP bands and the most anodic band of liver Both intestinal untreated extracts showed three bands but with different mobilities. After preincubation with neuraminidase, the three bands of caecum mucosa decreased in their migration, and the most anodic duodenum band disappeared, overlapping the second one. When tissue extracts were incubated with wheat germ-lectin (WGL), 74.5% of bone extract ALP and 67.2% of caecum extract ALP precipitated, which demonstrated that the ALP band of both tissues have similar groups in the carbohydrate side chains. Horse serum showed two electrophoretic bands, which increased to three bands when treated with neuraminidase. ALP from hepatocytes was the dominant isoform, followed by a caecum band. Because the electrophoretic mobilities of some of the tissue bands studied were identical, the neuraminidase agarose electrophoretic method appeared to be a satisfactory alternative to separate them.

Journal Article↗