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PubMed1984

Morphological changes induced in mouse mammary gland by porcine and human relaxin.

The effects of pure porcine relaxin and of human decidual extracts with relaxin-like activity on the mammary gland of virgin mice primed with estrogen have been studied by the light microscope. Porcine relaxin enhanced the changes induced by estrogen alone; the effect was different in the various mammary tissues. In the stroma, relaxin only slightly increased the loosening of connective tissue, the extent of the adipose tissue and of the capillary bed, as well as the degranulation of the mast cells. The changes in the parenchyma, such as elongation and branching of ducts, are strikingly enhanced. Moreover, relaxin seems to promote differentiation of the cells forming the walls of distal ducts, and of the myoepithelial cells. Tissue extracts of human decidua with relaxin-like activity induce changes in the mammary gland similar to those due to porcine relaxin. Such data indicate that relaxin synergizes with estrogen to cause growth of ducts of the mammary gland and that tissue extracts of human decidua have a similar effect, thus providing further evidence that decidua may be a source of relaxin in humans.

Adipose Tissue↗
PubMed1989

Characterization of beta-endorphin in human pituitary by fast atom bombardment mass spectrometry of trypsin-generated fragments.

A novel mass spectrometric method possessing a high level of structural specificity is described for characterization in biological fluids and tissues of endogenous beta-endorphin of the human amino acid sequence (beta h-EP). The method is based upon purification of tissue extracts by an RP-HPLC gradient, followed by trypsinolysis of that particular HPLC fraction corresponding to the elution time of synthetic beta h-EP. The tryptic digest of that endogenous beta h-EP fraction was purified further by a second RP-HPLC gradient. A unique tryptic fragment selected from the second gradient was analyzed by fast atom bombardment mass spectrometry and B/E linked-field scan MS/MS techniques to provide molecular weight and amino acid sequence-determining fragment ion information, respectively, of that fragment. Collectively, these independent analytical methodologies provided unequivocal structure evidence for the presence of endogenous beta h-EP in human pituitary. The method was established first by utilizing synthetic beta h-EP to optimize experimental parameters, and then applied to the analysis of beta h-EP in post-mortem human pituitary extracts. The suitability of the present method for semi-quantitation of tissue extracts is also demonstrated. The corresponding detection limit of the synthetic beta h-EP was 90 fmol, and human pituitary contained 1.5 pmol of beta h-EP mg-1 protein. The method can be extended readily to the analysis of beta-endorphin derived from other species and tissues.

Amino Acid Sequence↗
PubMed1993

Single-pass gill extraction and tissue distribution of atrial natriuretic peptide in trout.

A new method is described in the present experiments to quantify atrial natriuretic peptide (125I-ANP) extraction from plasma in a single transit through the gill vasculature of an unanesthetized trout. Tissue distribution of 125I relative to an inert extracellular space marker, 58Co-EDTA, was also measured 1 h after injection of 125I-ANP. Single-pass extraction of 125I-ANP by the gill was 60% in control fish; it fell to 18% in fish previously injected with saline and became negative (relative to 58Co-EDTA) after treatment with the clearance (C-type) receptor inhibitor SC 46542. Approximately 90% of a 125I-ANP bolus injected into control trout is removed from the circulation within 2 min. 125I concentration in gill tissue was nearly seven times greater than that predicted by the 58Co-EDTA space. 125I was also concentrated in brain (2X), gallbladder, gastrointestinal tract, and eye (all 1X). SC 46542 decreased gill 125I binding from seven times to one times the 58Co-EDTA space and increased 125I accumulation in heart, kidney, fat, and skeletal muscle. Extraction of 125I-ANP by the isolated perfused gill was 53%, similar to that observed in vivo. These results show that the gill is a major site for ANP removal from trout plasma and that C-type receptors are predominant in this process.

Animals↗
PubMed1999

A simple, rapid method of nucleic acid extraction without tissue homogenization for detecting viroids by hybridization and RT-PCR.

A simple, rapid method of nucleic acid extraction on a microcentrifuge tube scale for detecting viroids is presented. Five distinct citrus viroids (CVds), chrysanthemum stunt viroid (CSVd), hop stunt viroid (HSVd), hop latent viroid (HLVd) and potato spindle tuber viroid (PSTVd) were detected in their natural host plants by hybridization using cRNA probes and reverse transcription-polymerase chain reaction (RT-PCR). Nucleic acids (NA) were liberated from tissues by incubation in a buffer containing potassium ethyl xanthogenate (PEX) without tissue homogenization, and then precipitated with ethanol (NA-PEX). All the viroids except CVd-IV could be detected clearly in NA-PEX by hybridization. HSVd, HLVd and PSTVd could also be detected in NA-PEX by RT-PCR. Although CVds and CSVd could not be detected in NA-PEX by RT-PCR, they were detected after further purification: differential precipitation with 2-butoxyethanol and HCl treatment followed by ethanol-precipitation. In addition, PCR in the presence of tetramethylammonium chloride specifically amplified the cDNA of all five distinct CVds under the same temperature and cycle conditions. Since all the viroids could be detected in NA liberated by PEX, the amount of NA extracted by the method described here is sufficient for detecting viroids, enabling the processing of a large number of samples.

DNA, Viral↗
PubMed2001

Characterization of O(6)-methylguanine-DNA-methyltransferase (O(6)-MGMT) activity in Xiphophorus fishes.

We utilized a custom-synthesized double-strand oligonucleotide containing a single O(6)-methylguanine (O(6)-MG) residue within a restriction endonuclease recognition site to determine O(6)-methylguanine-DNA-methyltransferase (O(6)-MGMT) activity in various tissue extracts prepared from Xiphophorus fish. The results suggest Xiphophorus fish O(6)-MGMT activity has many of the same characteristics as Escherichia coli and mammalian O(6)-MGMT's including rapid reaction kinetics consistent with stoichiometric removal of methyl groups, but exhibits a temperature optimum of 23 degrees C. Results from protein extract activity assays indicate O(6)-MGMT activity patterns among four Xiphophorus tissues followed the order: brain> or =testes>gill> or =liver. In mammals, O(6)-MGMT activity is high in liver, while activity in brain is minimal (i.e. approximately 9% of liver); however, we report that in the Xiphophorus fishes examined, brain tissue extracts exhibited much higher (approximately six-fold) O(6)-MGMT activity levels than liver. Comparison of O(6)-MGMT activity between Xiphophorus species employed in tumor induction experiments did not indicate significant differences in ability to clear the pre-mutagenic O(6)-MG from the oligonucleotide substrate.

Animals↗
PubMed2005

Tetanus toxin fragment C fusion facilitates protein delivery to CNS neurons from cerebrospinal fluid in mice.

To improve protein delivery to the CNS following intracerebroventricular administration, we compared the distribution of a human Cu/Zn superoxide dismutase:tetanus toxin fragment C fusion protein (SOD1:TTC) in mouse brain and spinal cord with that of tetanus toxin fragment C (TTC) or human SOD1 (hSOD1) alone, following continuous infusion into the lateral ventricle. Mice infused with TTC or SOD1:TTC showed intense anti-TTC or anti-hSOD1 labeling, respectively, throughout the CNS. In contrast, animals treated with hSOD1 revealed moderate staining in periventricular tissues. In spinal cord sections from animals infused with SOD1:TTC, the fusion protein was found in neuron nuclear antigen-positive (NeuN+) neurons and not glial fibrillary acidic protein-positive (GFAP+) astrocytes. The percentage of NeuN+ ventral horn cells that were co-labeled with hSOD1 antibody was greater in mice treated with SOD1:TTC (cervical cord = 73 +/- 8.5%; lumbar cord = 62 +/- 7.7%) than in mice treated with hSOD1 alone (cervical cord = 15 +/- 3.9%; lumbar cord = 27 +/-4.7%). Enzyme-linked immunosorbent assay for hSOD1 further demonstrated that SOD1:TTC-infused mice had higher levels of immunoreactive hSOD1 in CNS tissue extracts than hSOD1-infused mice. Following 24 h of drug washout, tissue extracts from SOD1:TTC-treated mice still contained substantial amounts of hSOD1, while extracts from hSOD1-treated mice lacked detectable hSOD1. Immunoprecipitation of SOD1:TTC from these extracts using anti-TTC antibody revealed that the recovered fusion protein was structurally intact and enzymatically active. These results indicate that TTC may serve as a useful prototype for development as a non-viral vehicle for improving delivery of therapeutic proteins to the CNS.

Animals↗
PubMed1999

Matrix metalloproteinase-2 in a murine model of infantile-type polycystic kidney disease.

It was previously found that elevated levels of matrix metalloproteinase (MMP)-2 (gelatinase A) and -9 (gelatinase B) were synthesized and secreted into the medium by cultured kidney tubules derived from cystic C57BL/6J-cpk mice. To determine whether increased synthesis and secretion occur in vivo in this mouse model of polycystic kidney disease, kidney protein extracts, mRNA, and tissue sections were compared for expression and activity of MMP-2 and -9. Although both MMP were detected in tissue extracts, the differences in expression levels and activity in normal and cystic kidneys were far greater for MMP-2. High levels of MMP-2 seemed to result from increased expression by the cystic kidneys predominantly in the second and third postnatal weeks (a time when the kidneys are undergoing rapid cystic enlargement). Much of the increased MMP was present in the inactive zymogen form, although active enzyme was readily detected by sodium dodecyl sulfate-polyacrylamide gel zymography and in situ zymography. MMP-2 was abnormally localized to the interstitium and to foci between cysts, suggesting that MMP-2 may regulate collagen accumulation at those sites, thus allowing cyst enlargement and limiting the severity of interstitial fibrosis.

Animals↗
PubMed1977

Immunological and ultrastructural studies of neurofilaments isolated from rat peripheral nerve.

Neurofilaments were isolated from desheathed and minced segments of rat peripheral nerve by osmotic shock into 0.01 M Tris-HCI buffer, pH 7.2. Freshly isolated neurofilaments were observed to undergo disassembly by progressive fragmentation upon exposure of dilute tissue extracts to this buffer. Low- and high-speed centrifugations of these tissue extracts separated membranous and particulate constituents and produced a progressive enrichment of 68,000-dalton polypeptide band in successive supernates, as determined by analyses of soluble proteins by SDS-polyacrylamide electrophoresis. The final high-speed supernatant fractions (S3) of nerve extracts, which were predominantly composed of 68,000-dalton polypeptide, were used to raise a specific experimental antisera in rabbits. Utilizing techniques of immune electron microscopy, experimental rabbit antisear was shown to contain antibodies against neurofilaments. Intact neurofilaments isolated from rat nerves and attached to carbon-coated grids became decorated when exposed to experimental rabbit antisera or purified gamma globulin (IgG) derivatives. The decoration of neurofilaments closely resembled the IgG coating seen in immune electron microscopy. Antibody absorption techniques were used to identify the biochemical constituency of neurofilamentous antigenic determinants. The decoration of neurofilament by experimental IgG was not altered by additions of tubulin or bovine serum albumin, but was prevented by additions of S3 fractions as well as the 68,000-dalton polypeptide of this fraction which was eluted and recovered from polyacrylamide gels. These findings are indicative that a 68,000-dalton polypeptide is a constituent subunit of rat peripheral nerve neurofilaments.

Animals↗
PubMed1992

One-dimensional thin-layer chromatographic separation of phospholipids and lysophospholipids from tissue lipid extracts.

A modified one-dimensional thin-layer chromatographic procedure is presented for the separation from tissues of five phospholipids (phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine and sphingomyelin) and three lysophospholipids (lysophosphatidylserine, lysophosphatidylethanolamine and lysophosphatidylcholine). This is achieved by simple involvement of 0.4% ammonium sulphate in silica gel H and of acetone in a developing solvent as chloroform-methanol-acetic acid-acetone-water (40:25:7:4:2, v/v). The procedure is simple and the separation is reproducible. The weakness of this method is the partial degradation of phosphatidylethanolamine to lysophosphatidylethanolamine, but a method to prevent this degradation is also presented.

Chromatography, Thin Layer↗
PubMed1977

Mouse brain extract with "fibroblast growth factor"-activity.

Crude tissue extracts prepared by homogenization and ammonium sulfate precipitation of mouse brains as described by Gospodarowicz et al. (1975) for brains and hypophyses of cattle have also been found to contain fibroblast growth factor-activity. In the presence as well as in the absence of calf serum in the medium the survival and proliferation of primary mouse embryo fibroblasts in culture were promoted as documented by dose-dependent increase of cell number and protein content during an incubation period of 48 hours. In the case of serum-free incubation also a significant rise of thymidine incorporation induced within 24 hours by the extract was noted. In contrast, the proliferation of primary chicken embryo fibroblasts or transformed mouse cells of the line "L" was not stimulated. Some possibilities for a practical use of such extracts are discussed.

Animals↗
PubMed2006

Regulation of the Atm promoter in vivo.

While ATM, the protein defective in the human genetic disorder ataxia-telangiectasia (A-T), is primarily activated as a preexisting protein by radiation, there is also evidence that expression of the protein can be regulated at the transcriptional level. Activation of the ATM promoter by ionizing radiation has been reported only in quiescent cells in culture. To investigate how the Atm promoter is regulated in vivo, we generated transgenic mice that express the luciferase reporter gene under the control of the murine Atm promoter. Using a biophotonic imaging system luciferase activity was monitored in vivo. Strong promoter activity was detected throughout the transgenic animals with particularly high signals from the thymus, abdominal region, and reproductive organs. This activity further increased in response to both ionizing radiation and heat stress in a time dependent manner. Luciferase activity, measured in vitro in extracts from different tissues, showed highest activities in testes, ovaries, and cerebellum. Subjecting these mice to a single dose of 4 Gy total body radiation led to a time-dependent activation of the promoter with the strongest response observed in the peritoneal membrane, skin, and spleen. For most tissues tested, maximal promoter activity was reached 8 hr after radiation. The observed changes in promoter activity largely correlated with levels and activity of Atm protein in tissue extracts. These results demonstrate that, in addition to activation by autophosphorylation, Atm can also be regulated in vivo at the transcriptional level possibly ensuring a more sustained response to radiation and other stimuli.

Animals↗
PubMed2004

Interleukin-8 expression is increased in senescent prostatic epithelial cells and promotes the development of benign prostatic hyperplasia.

BACKGROUND: Benign prostatic hyperplasia (BPH) is an extremely common disease of older men characterized by increased growth of prostatic epithelial and stromal cells. Previously we showed that senescent epithelial cells accumulate in the prostate of aging men and secrete interleukin-1 alpha (IL-1 alpha). IL-8 is also present at increased levels in BPH tissues and induces expression of FGF2, a potent stromal growth factor. Therefore, we sought to determine if IL-8 is also expressed at increased levels by senescent epithelial cells and if this secreted IL-8 plays a role in the pathogenesis of BPH. METHODS: Expression of IL-8 in human BPH tissue and primary cultures of prostatic epithelial cells was analyzed using an enzyme-linked immunoabsorption assay (ELISA). Tissue senescence was assessed by a quantitative assay for senescence-associated beta galactosidase (SA-beta gal). Proliferation of primary and immortalized prostatic epithelial cells in response to IL-8 was determined by counting of cells at intervals after addition of IL-8. RESULTS: Expression of IL-8 is significantly increased in vitro when cultured prostatic epithelial cells undergo senescence. Quantitative assay of BPH tissue extracts revealed that tissue IL-8 levels are correlated with both SA-beta gal activity and prostate weight. IL-8 promotes proliferation of primary and immortalized prostatic epithelial cells in culture. CONCLUSIONS: Senescence of prostatic epithelial cells results in increased expression of IL-8, which can promote proliferation of non-senescent epithelial and stromal cells by direct and indirect mechanisms, and in this manner contributes to the increased tissue growth seen in BPH.

Cellular Senescence↗
PubMed2005

Quantitative determination of endogenous sorbitol and fructose in human nerve tissues by atmospheric-pressure chemical ionization liquid chromatography/tandem mass spectrometry.

Attachment of anions to sorbitol and fructose has been shown to enhance sensitivity in both electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) mass spectrometry. The post-column addition of CHCl3 produced Cl-adducts of sorbitol and fructose but their signals were suppressed due to the elevated background. Different chlorinated compounds and different additive methods were systematically investigated to form more abundant Cl-adduct precursor ions and deprotonated product ions. The major causes of the high background were explored and effective methods were developed to improve the signal-to-noise ratios and reproducibility. The compositions of mobile phase, percentages of organic modifiers (MeCN, MeOH and water), columns, oven temperature, flow rates and different gradients were investigated to separate sorbitol from fructose along with their isomers including glucose, galactose, mannose, sorbose, mannitol, and dulcitol. The optimized separation was achieved on a Luna 5 mu NH2 100A column (150 x 4.6 mm) using a mobile phase containing MeCN with 0.1% of CH2Cl2 and 50% MeOH in water at a flow rate of 800 microL/min and an oven temperature of 40 degrees C using a gradient liquid chromatography (LC) system. Human nerve tissue samples were extracted by protein precipitation followed by mixed-mode solid-phase extraction. The LC/ESI-MS/MS method produced higher peak intensities than LC/APCI-MS/MS. However, there were matrix effects from extracted tissues in LC/ESI-MS/MS but not in LC/APCI-MS/MS. Consequently, APCI proved to be the more effective method of ionization. Then the LC/APCI-MS/MS method was fully validated and successfully applied to analysis of clinical samples. The concentrations of endogenous sorbitol and fructose were determined using calibration curves employing sorbitol-13C6 and fructose-13C6 as surrogate analytes. The method has provided excellent intra- and inter-assay precision and accuracy with linear ranges of 0.2-80 ng/mg for sorbitol and 1-400 ng/mg for fructose in human nerve tissues.

Atmospheric Pressure↗
PubMed1997

Vascular endothelial growth factor is the major angiogenic factor in omentum: mechanism of the omentum-mediated angiogenesis.

Omentum has been used clinically to promote wound healing and to stimulate the revascularization of ischemic tissues. The biologic mechanism responsible for these effects has, however, not yet been defined. A number of polypeptide growth factors that possess potent angiogenic properties have recently been identified, and we therefore sought to determine whether one of these growth factors might be responsible for the angiogenic properties of the omentum. The levels of vascular endothelial growth factor (VEGF) protein in a number of rat tissues and organs were analyzed by Western and enzyme immunoassay analysis. Because omentum was found to have the greatest VEGF concentrations of the tissues examined, antibody neutralization, transcription inhibition assays, and Northern blot analysis were performed under hypoxic and normoxic conditions on tissues extractions and primary tissue cultures of omentum to further characterize the functional significance of VEGF expression in these tissues. The omentum demonstrated the highest VEGF secretion rate as well as the highest concentration of VEGF protein of the various rat tissues and organs examined. Fractionation studies of the omentum furthermore demonstrated that omental adipocytes, rather than the stromal-vascular cells, were the primary source of VEGF protein. An endothelial cell mitogenic assay showed that a major portion of the mitogenic activity of heparin-binding proteins and conditioned media derived from omentum was abolished by VEGF antibody. Additional studies with the transcription inhibitor actinomycin-D furthermore demonstrated that the VEGF gene was continuously transcribed in the rat omental adipocytes. Incubation of the omental adipocytes under hypoxic conditions induced approximately a 1.7-fold increase in VEGF protein expression, which was abolished by actinomycin-D. Northern blot analysis demonstrated that hypoxia resulted in upregulation of the VEGF mRNA in the hypoxia-cultured omental adipocytes, suggesting that the augmentation of VEGF expression in omental adipocytes by hypoxia occurs at the transcriptional level. These data suggest that VEGF is the major angiogenic factor produced by omentum and possibly underlies the mechanism of omentum-induced angiogenesis. Augmented expression of VEGF by omental cells under hypoxic conditions may furthermore reflect the mechanism responsible for enhancing the angiogenic activity of omentum in the setting of ischemia.

Adipose Tissue↗
PubMed1993

Tissue variation of two large chondroitin sulfate proteoglycans (PG-M/versican and PG-H/aggrecan) in chick embryos.

PG-M and PG-H, chick large chondroitin sulfate proteoglycans corresponding to versican (fibroblast-type proteoglycan) and aggrecan (cartilage-characteristic proteoglycan), respectively, which are found in mammals, have been characterized in various tissues of chick embryos. Their distribution and the compositions of the core molecules were analyzed by immunofluorescence staining and immunoblotting, respectively, using various monospecific antibodies. Molecules reactive to a monoclonal antibody to the PG-M core protein (designated MY-174) were distributed in various tissues, including aorta, lung, cornea, brain, skeletal muscle and dermis. Immunoblotting with MY-174 of the chondroitinase ABC-digested tissue extracts showed a tissue variation of MY-174-reactive core molecules (550-kD, 500-kD, 450-kD, and 350-300-kD). In contrast, PG-H, besides massive occurrence in cartilage, was only found in a few tissues such as aorta and brain. In addition, PG-H in aorta, cornea, and skin was atypical in structure, because it lacked keratan sulfate. The expression of PG-M in developing chick embryos was then examined. PG-M was found in some developmentally active areas, such as the perinotochordal mesenchyme between notochord and neural tube, the basement membranes facing neuroepithelial cells, and condensing mesenchymal cells in limb buds, suggesting some functions distinctive of the developing tissues.

Aggrecans↗