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At least 1,189 records · Page 66Linked to original sources
PubMed1999

Estrogen regulation of angiotensin-converting enzyme mRNA.

Estrogen replacement therapy is cardioprotective in postmenopausal women; however, the precise molecular mechanisms for this modulation are not fully elucidated. We previously showed that chronic estrogen replacement therapy reduced angiotensin-converting enzyme (ACE) activity in tissue extracts and serum with an associated reduction in plasma angiotensin II. A reverse transcriptase-polymerase chain reaction assay was developed to determine whether estrogen treatment regulates tissue ACE mRNA concentration. Total RNA was isolated from kidney cortex, kidney medulla, lung, and aorta of ovariectomized Sprague-Dawley rats after 21 days of chronic 17beta-estradiol replacement therapy (5 mg pellet per rat SC) or placebo. A marked decrease in densitometric intensity ratios of amplified ACE cDNA to elongation factor-1alpha control cDNA was observed in all tissues from placebo-treated rats compared with the estradiol-treated rats (renal cortex: 0.29+/-0.04 versus 0.14+/-0.02; renal medulla: 0. 37+/-0.04 versus 0.24+/-0.03; lung: 4.49+/-0.37 versus 2.49+/-0.59; and aorta: 0.41+/-0.04 versus 0.29+/-0.02; all P<0.05). A comparable reduction in ACE activity was detected in tissue extracts from kidney cortex, kidney medulla, and lung of hormone-treated animals. Incubation of purified rat lung ACE with 1 or 10 micromol/L 17beta-estradiol had no effect on enzyme activity. These results suggest that estrogen treatment regulates tissue ACE activity by reducing ACE mRNA concentrations. Thus, the beneficial cardiovascular effects of estrogen may be mediated in part by downregulation of ACE with a consequent reduction in the circulating levels of the vasoconstrictor angiotensin II, a decrease in the metabolism of the vasodilator bradykinin, and an increase in the production of the vasorelaxant angiotensin-(1-7).

Animals↗
PubMed1987

Measurement of opioid peptides with combinations of reversed phase high performance liquid chromatography, radioimmunoassay, radioreceptorassay, and mass spectrometry.

Novel state-of-the-art mass spectrometric methods have been developed and are now used to identify and to quantify enkephalins and other neuropeptides in biological tissue extracts. As the first step, RP-HPLC gradient elution is performed of a Sep-Pak treated peptide-rich fraction from a tissue extract, and the eluent is monitored by a variety of post-HPLC detectors. In an effort to maximize the structural information that can be obtained from the analysis, UV (200 nm) provides the analog absorption trace; receptorassay analysis (RRA) data of all (90) fractions that are collected are used to construct the profile of opioid-receptoractive peptides; radioimmunoassay (RIA) of selected HPLC fractions at retention times corresponding to the retention time of standards, or in some special cases of all 90-fractions, provides immunoreactivity information; and fast atom bombardment mass spectrometry (FAB-MS) in two modes - corroboration of the (M+H)+ of the expected peptide, or MS/MS to monitor an amino acid sequence-determining fragment ion unique to that peptide in the selected ion monitoring (SIM) mode - provides structural information. As a demonstration of the level of quantification sensitivity that can be attained by these novel MS methods, FAB-MS-MS-SIM of solutions of synthetic leucine enkephalin was sensitive to the 70 femtomole level. This paper discusses RIA versus RRA data, and recent MS measurements of peptides in human tissues.

Chromatography, High Pressure Liquid↗
PubMed1997

Measurement of myeloperoxidase in leukocyte-containing tissues.

An improved method is reported for measurement of myeloperoxidase (MPO) activity in tissues. While spectrophotometric methods based on oxidation of O-dianisidine or other dyes have been reported for MPO measurement in pure polymorphonuclear leukocytes (PMNs), these methods often fail to accurately assay MPO activity in tissues. We observe that tissue myoglobin or vascular hemoglobin markedly effects the spectrophotometric assay for MPO. Under optimal conditions of 0.53 mM O-dianisidine, 0.15 mM H2O2, pH 6.0, either myoglobin or hemoglobin produced absorbance at 460 nm in a concentration-dependent manner similar to that of MPO. In perfused heart tissue, myoglobin caused a major problem with the assay resulting in an inability to obtain accurate linear results as a function of MPO concentration and PMN number. To eliminate the effect of tissue myoglobin or vascular hemoglobin on the assay, one-step gel filtration chromatography of tissue extracts was introduced. MPO, myoglobin, and hemoglobin were easily separated using a Sephadex G-75 column according to the difference in their molecular weights. A linear relationship between the MPO activity and PMN number was observed only after processing the tissue extracts through the Sephadex G-75 column. Thus, MPO activity in PMN-containing tissues can be precisely quantitated after one-step purification on a molecular exclusion column. Enzyme purification with removal of myoglobin is essential for obtaining accurate measurement of MPO activity and quantitation of PMNs in muscle tissue.

Animals↗
PubMed1999

Identification of cis-9,10-methylenehexadecanoic acid in submitochondrial particles of bovine heart.

Submitochondrial particles of bovine heart were hydrolyzed by phospholipase A2 and the products were analyzed by liquid chromatography electrospray ionization-mass spectrometry. We found a fatty acid with a molecular mass of 268 Da and a retention time longer than that of linoleic acid. Next, we synthesized organically cis-9,10-methylenehexadecanoic acid, which has a molecular mass similar to that of the extracted fatty acid, and characterized its high performance liquid chromatography and gas chromatography-mass spectrometry profiles. Using these data we were able to identify endogenous cis-9,10-methylenehexadecanoic acid in rat and human heart and liver tissues that had been hydrolyzed by phospholipase A2. This fatty acid was not detected in tissue extracts that had not been hydrolyzed by phospholipase A2. Similar amounts of cis-9, 10-methylenehexadecanoic acid were measured in tissue extracts after total hydrolysis. These results suggest that cis-9, 10-methylenehexadecanoic acid is a fatty acid component, in the sn-2 position, of phospholipids in some mammalian tissue.

Animals↗
PubMed1998

The effect of dietary flaxseed supplementation on organic anion and osmolyte content and excretion in rat polycystic kidney disease.

Progression of chronic renal failure in the Han:SPRD-cy rat polycystic kidney disease is associated with renal depletion of citric acid cycle metabolites and betaine. Amelioration of this disease by a soy protein diet is associated with retention of citric acid cycle anions, despite increased excretion, and preservation of tissue levels of betaine. As we have recently found that modest dietary supplementation with flaxseed preserves renal function and reduces histologic injury in the Han:SPRD-cy rat, we undertook a high-resolution 1H NMR spectroscopic study of urine and renal tissue extracts from Han:SPRD-cy rats to explore the renal biochemical consequences of a flaxseed diet. There was no significant dietary effect upon organic anion, methylamine, or osmolyte excretion in healthy animals. There was increased citrate excretion in Han:SPRD-cy rats fed flaxseed. Urinary ammonium excretion did not differ, suggesting that the observed increase in citrate excretion was not due to an alkaline effect of diet. Tissue extract studies revealed that disease amelioration was associated with tissue retention of succinate and betaine. Amelioration of Han:SPRD-cy rat polycystic kidney disease by diet is associated with alteration in the handling of citric acid cycle metabolites. Betaine may have a metabolic role in the reduction of chronic renal injury.

Animals↗
PubMed2003

Increased acetylcholinesterase activities in specimens of Sparus auratus exposed to sublethal copper concentrations.

The present study looks at possible changes in the activity of acetylcholinesterase (AChE) in tissues (brain and white muscle) of the Mediterranean bony fish Sparus auratus after a 20 days exposure to sublethal concentrations (0.1 or 0.5 ppm) of copper in the marine water and on control untreated animals. The trials also included measurements of Cu concentration in the tissues to evaluate possible metal accumulation. Moreover, sedimentation analysis as well as V(max) and K(m) determination were carried out in tissue extracts of Cu-exposed or control animals. V(max) and K(m) were also determined with or without addition of Cu(2+) in the assay. No Cu accumulation occurred in brain and muscle after Cu exposure. AChE showed in both tissues a molecular polymorphism with putative globular (G) and asymmetric (A) forms. Cu exposition led to an increased specific activity and improved catalytic efficiency of AChE in brain and muscle, seemingly regarding G forms. The increase in catalytic efficiency also resulted from the in vitro assay with tissue extracts and Cu(2+) addition. The higher AChE activity and catalytic efficiency in both tissues after Cu exposition and without metal accumulation, suggests an increase of free Cu aliquot into the cells, likely due to mechanisms of metal homeostasis.

Acetylcholinesterase↗
PubMed1995

Protein oxidation and aging. II. Difficulties in measuring alkaline protease activity in tissues using the fluorescamine procedure.

A current hypothesis explaining the aging process implicates the accumulation of oxidized protein in animal tissues. This is primarily based on a series of reports showing an age-dependent increase in protein carbonyl content and an age-dependent decrease in the activities of enzymes, especially of alkaline proteases, which preferentially degrade oxidatively modified protein. Recently, this hypothesis was strongly supported by the report of a novel effect of the spin-trapping compound N-tert-butyl-alpha-phenylnitrone (PBN) in reversing these age-dependent changes. However, we found that the reactive protein carbonyls could not be reliably measured in tissues by using the 2,4-dinitrophenylhydrazine procedure described in the PBN study. We now focus on the alkaline protease activity assay and show that alkaline protease activity cannot be reliably measured in crude tissue extracts by using the fluorescamine procedure also described in the PBN study. We were, however, able to reliably measure a protease activity in crude tissue extracts at alkaline pH by using a synthetic fluorogenic peptide substrate, but no effect of aging or PBN treatment was found on the protease activity in rat brain cortexes. Thus, the reported age-dependent changes in protein carbonyl formation and alkaline protease activity remain to be confirmed.

Aging↗
PubMed1987

Developmental forms of human skeletal muscle AMP-deaminase.

Chromatography on phosphocellulose revealed the existence of two well-separable forms of skeletal muscle AMP-deaminase in the tissue extracts of 11- and 16-week-old human fetuses. One of these forms elutes from the column at the same salt concentration as the muscle isozyme found in the skeletal muscle extract from adult man, and seems to have similar kinetic properties. The second form, which was found only in vestigial amounts in adult human tissue extract, represents different kinetic properties and seems to be a form characteristic for the fetal period of ontogenesis.

AMP Deaminase↗
PubMed2003

Analysis of the mouse CSF-1 gene promoter in a transgenic mouse model.

CSF-1 stimulates monocyte and osteoclast populations. However, the molecular mechanisms involved in regulating CSF-1 gene expression are unclear. To identify regulatory regions that control normal CSF-1 gene expression, a -774/+183-bp fragment of the murine CSF-1 promoter was analyzed in vitro and in vivo. Transcriptional activity was high in cultured osteoblasts that express CSF-1 mRNA compared to ARH-77 B cells that lack CSF-1 gene expression. Transient transfection of osteoblasts with promoter deletion constructs showed that the -774-bp fragment conferred the highest transcriptional activity and contained activator and repressor sequences. To assess the ability of the CSF-1 promoter to confer normal tissue expression of CSF-1, transgenic mice containing the -774/+183-bp region driving the E. coli beta-galactosidase (lacZ) reporter gene were generated. beta-Gal analysis of whole tissue extracts showed transgene expression in all tissues tested except liver and kidney. At the cellular level, the pattern of beta-gal expression in the spleen, thymus, bone, lung, and testes of adult transgenic mice mimicked normal endogenous CSF-1 mRNA expression in non-transgenic littermates detected by in situ hybridization. This region also directed appropriate transgene expression to sites in other tissues known to synthesize CSF-1, with the exception of the liver and kidney. These findings indicate that the -774-bp fragment contains cis-acting elements sufficient to direct CSF-1 gene expression in many tissues. CSF-1 promoter/lacZ mice may be useful for studying the transcriptional mechanisms involved in regulating CSF-1 gene expression in tissues throughout development.

Animals↗
PubMed1995

Assessment of cathepsin L activity by use of the inhibitor CA-074 compared to cathepsin B activity in human lung tumor tissue.

In a series of pairs of lung tumor tissue and non-tumor lung parenchyma from 50 patients, the activity of cathepsin L was measured with Z-Phe-Arg-AMC using the inhibitor CA-074 to delimitate from cathepsin B activity also present in the tissue extracts. Cathepsin B was assessed in the same samples with its specific substrate Z-Arg-Arg-AMC. It was found that in tumor tissue the median activities of cathepsin L and cathepsin B were increased 1.6-fold and 4.9-fold, respectively. The levels of activity of both enzymes did not correlate with TNM stages nor with cell differentiation of bronchial carcinomas. Cathepsin L activity was found to be insignificantly higher in adenocarcinoma compared to squamous cell carcinoma, while cathepsin B activity did not vary across the histologies. The activities of both enzymes were low in pulmonary carcinoids, which are known to be low-grade malignant neoplasms. The amount of cathepsin B activity exceeded by far that of cathepsin L activity as proven by measurement with Z-Phe-Arg-AMC in the presence of the inhibitor Z-Phe-Phe-CHN2:95-98% of cathepsin B activity vs 2-5% of cathepsin L activity were determined. By SDS-PAGE separation and immunoblot analysis, it could be demonstrated that significant amount of cathepsin L is complexed with the cysteine proteinase inhibitor kininogen. This explains the rather low cathepsin L activity values in the tissue extracts.

Adenocarcinoma↗
PubMed1993

Structure and significance of human luteinizing hormone-beta core fragment purified from human pituitary extracts.

A fragment of the hCG beta-subunit is present in high concentrations in the urine of pregnant women and the urine from individuals with ovarian or other cancers. The utility of immunoreactive measurement of this fragment to monitor therapy of such cancers is compromised, however, because high concentrations of the molecule are also detected in the urine of healthy postmenopausal women. It has been suggested that the latter observations may be due to a cross-reacting human (h) LH beta core fragment, presumably of pituitary origin, but no such fragment had ever been isolated. We have now isolated a hLH beta core fragment from a pituitary tissue extract. Its structure is exactly analogous to that of the hCG beta core fragment. The finding of a discrete hLH beta core fragment in a tissue extract suggests that it may be produced within pituitary tissue, rather than by a peripheral degradation process. We have also found that the same immunoaffinity method used to extract the hLH beta core fragment from the pituitary extract purified several protein fragments from postmenopausal urine, none of which was related to hLH or hCG. The availability of the pituitary hLH beta core fragment may allow development of assays that distinguish it from its hCG analog.

Amino Acid Sequence↗
PubMed1990

Organ distribution of the Ro (SS-A) antigen in the guinea pig.

Using an anti-Ro (SS-A)-specific "sandwich" enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting, we determined the Ro (SS-A) content (both quantitative and qualitative) in saline-perfused organs of the guinea pig. All tissue extracts contained substantial concentrations of Ro (SS-A) antigen and could be grouped into 3 categories based on quantitative reactivity in the sandwich ELISA. The 60-kd and 52-kd molecular forms of Ro (SS-A) present in tissue extracts were similar to those described in Wi-L2 extracts, and the 54-kd molecular form of Ro (SS-A) in guinea pig erythrocytes was similar to that found in human erythrocytes. The tissue distribution of the isoforms of Ro (SS-A) was shown by Western immunoblotting to vary in different tissues, and the reactivity to the 60-kd Ro (SS-A) was correlated with the activity seen in the ELISA. Both the 60-kd and 52-kd Ro (SS-A) bands in guinea pig liver extracts were very weak on Western immunoblots, in contrast to the high concentration of Ro (SS-A) antigen in the ELISA. Other data suggest the possible existence of a unique form of Ro (SS-A) in the liver. Guinea pig tissues have 4 Y RNA that are equivalent to the 4 human RNA--hY1, hY3, hY4, and hY5--present in HeLa cells, while guinea pig red blood cells have only one Y RNA, which is equivalent in size to human hY4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Cytokines that activate proteolysis are increased in abdominal aortic aneurysms.

BACKGROUND: Abdominal aortic aneurysm (AAA) disease is characterized by an increase in proteolysis and loss of matrix components. The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), products of activated macrophages and T cells, are known to increase the production of matrix-degrading enzymes in some pathological states. METHODS AND RESULTS: Seven AAA and five control aortic tissue extracts were assayed for TNF-alpha and IL-1 beta with ELISA. TNF-alpha was elevated significantly in AAA extracts compared with controls (86 +/- 34 pg/mg of total protein versus 1 +/- 1 pg/mg of total protein; P < .001). IL-1 beta concentration also was significantly increased in the AAA specimens (48 +/- 14 pg/mg of total protein versus 12 +/- 5 pg/mg of total protein; P < .05). Immunoblotting demonstrated secreted forms of TNF-alpha in the AAA extracts, and possible membrane-bound forms were observed when the tissues were detergent-extracted. Known forms of IL-1 beta also were observed on immunoblots of AAA tissue extracts. CONCLUSIONS: The presence of TNF-alpha and IL-1 beta in AAA tissue underscores the importance of the infiltrating inflammatory cells present in the media and adventitia of aneurysmal aortic wall and further implicates an inflammatory process in the pathogenesis of AAA.

Aorta, Abdominal↗
PubMed1999

Aflatoxin B1 is an inhibitor of cyclic nucleotide phosphodiesterase activity.

Aflatoxin B1 (AFB1) action on cyclic nucleotide phosphodiesterase (PDE) activity has been tested on tissue extracts of various organs. In the presence of 100 microM AFB1 a significant inhibition of cAMP and cGMP hydrolytic activity is observed in all tested tissue extracts. However, cGMP hydrolytic activity appears more sensitive to AFB1 inhibition than cAMP hydrolytic activity and a considerably higher inhibition is observed in lung and spleen, than in liver, brain, kidney, and heart. When cGMP is used as substrate, the inhibitory response reaches 72% in lung and spleen extracts. We have also tested AFB1 effects on lung and liver PDE activity peaks separated by DEAE-cellulose chromatography. These data confirm the poor sensitivity to the toxin of all PDE activities present in liver, while the lung peak (where PDE V in present) shows a higher sensitivity to AFB1. In order to establish whether PDE V is in fact more sensitive to AFB1, we have used mouse neuroblastoma cells, in which cGMP hydrolytic activity has been shown to be due to PDE V only. In this case, the calculated IC50 is 24 microM and Dixon plot analysis shows a competitive inhibitory effect with a Ki of 16.7 microM. We have also used aflatoxin B2 and M2, and they proved to be much less effective than AFB1: AFB2 inhibits PDE V with an IC50 of 117 microM, while AFM2 does not show any effect. These results provide the first evidence of a competitive inhibition of AFB1 on an enzymatic activity and suggest that an alteration of cellular cyclic nucleotide levels may play a role in the mechanism of aflatoxin action.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Effect of neuroactive material on neurosecretion in adult cockroach. Periplaneta americana (L).

The neurosecretory material is found depleted in the medial neurosecretory cells in adult cockroaches poisoned with insecticide. It is, however, scanty in the neurosecretory pathway but abundant in the corpora cardiaca of these insects. The administration of toxicated haemolymph in the normal cockroach results in scanty material in medial neurosecretory cells, but the accumulation of the neurosecretory material in corpora cardiaca. The toxicated tissue extracts have also caused depletion of neurosecretory material from the medial neurosecretory cells as well as from the axonal tract and its accumulation in the corpora cardiaca. Thus effect is comparatively more pronounced by the extract from the Malpighian tubules and nerve cord than than of brain extract. Thus the effect produced by insecticide on the stimulation--secretion coupling mechanism of neurosecretion is comparable to the produced by the toxicated haemolymph and tissue extract. The possibility of involvement of the adrenergic system in regulating the action of neuroactive material on neurosecretory cells is suggested in the insects.

Animals↗
PubMed1977

Electrophoretic studies on alkaline phosphatases in normal and zinc intoxicated rats.

Polyacrylamide gel electrophoresis was used to characterize the isoenzymes of serum alkaline phosphatase in the rat. The electrophoresis of serum from normal rats resulted in two bands of alkaline phosphatase activity. A prominent band in serum corresponded in electrophoretic mobility to the alkaline phosphatase from bone and intestinal tissue extracts and also a slower migrating liver isoenzyme. A less prominent, fast migrating band in serum had a mobility similar to a faster migrating liver tissue extract isoenzyme. This band only represented 1-2% of the total alkaline phosphatase present in the serum of normal rats but approximately 15% of the total alkaline phosphatase in the serum of rats fed excess levels of zinc. The study also revealed an alteration in the electrophoretic mobility of alkaline phosphatase in bone homogenate by the addition of deactivated serum to the homogenate. The addition of deactivated serum did not alter the electrophoretic mobility of the liver and intestinal alkaline phosphatases in rats.

Alkaline Phosphatase↗