Expression and characterization of the active molecular forms of choline/ethanolamine kinase-α and -β in mouse tissues, including carbon tetrachloride-induced liver

2002 ◽  
Vol 363 (3) ◽  
pp. 777-784 ◽  
Author(s):  
Chieko AOYAMA ◽  
Akiko OHTANI ◽  
Kozo ISHIDATE
Keyword(s):  
Carbon Tetrachloride ◽  
Mammalian Cells ◽  
Molecular Form ◽  
Polyclonal Antibodies ◽  
Active Form ◽  
Peptide Sequence ◽  
Molecular Forms ◽  
Native Form ◽  
Ethanolamine Kinase ◽  
Mouse Tissues

Choline/ethanolamine kinase (ChoK/EtnK) exists as at least three isoforms (α1, α2 and β) in mammalian cells. The physiological significance for the existence of more than one form of the enzyme, however, remains to be determined. In the present study, we examined the expression and distribution of the isoforms in mouse tissues using isoform-specific cDNA probes and polyclonal antibodies raised against each N-terminal peptide sequence. Both Northern- and Western-blot analyses indicated that either the α (α1 plus α2) or the β isoform appeared to be the ubiquitously expressed enzyme. The mRNA abundance for the α isoform was highest in testis, whereas that for the β isoform was relatively high in heart and liver. While the native form of each isoform was reported to consist of either homodimers or homotetramers, our immunotitration studies clearly indicated that a considerable part of the active form of the enzyme consists of α/β hetero-oligomers, with relatively small parts of activity expressed by α/α and β/β homo-oligomers. This is the first experimental evidence for the presence of heteromeric ChoK/EtnK in any source. Thus our results strongly suggested that the activity of ChoK/EtnK in the cell is controlled not only by the level of each isoform but also by their combination to form the active oligomer complex. Carbon tetrachloride (CCl4) was shown to induce ChoK activity 2–4-fold in murine liver. Our analysis for the mechanism involved in this induction revealed that the responsible isoform for CCl4 was α, not β. The level of α mRNA was strongly induced in mouse liver, which resulted in a sustained increase in the amount of the α isoform. Consequently, the composition of α/α homo-oligomers came to represent up to 80% of the total active molecular form of ChoK in CCl4-induced liver, whereas it was less than 20% in normal uninduced liver.

The active molecular form of plasma adrenomedullin is extracted in the pulmonary circulation in patients with mitral stenosis: possible role of adrenomedullin in pulmonary hypertension

2000 ◽  
Vol 100 (1) ◽  
pp. 61-66 ◽  
Author(s):  
Toshio NISHIKIMI ◽  
Seiki NAGATA ◽  
Tatsuya SASAKI ◽  
Fumiki YOSHIHARA ◽  
Noritoshi NAGAYA ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Pulmonary Artery ◽  
Left Atrium ◽  
Pulmonary Circulation ◽  
Mitral Stenosis ◽  
Molecular Form ◽  
Femoral Vein ◽  
Active Form ◽  
Molecular Forms ◽  
Main Site

Adrenomedullin (AM), a novel hypotensive peptide, preferentially dilates pulmonary vessels rather than systemic vessels. This suggests the possibility that AM is a circulating hormone which participates in regulation of the pulmonary circulation. A recent study revealed that two molecular forms of AM, i.e. a mature, active form of AM (AM-m) and an intermediate, inactive, glycine-extended form of AM (AM-Gly), circulate in human plasma. In the present study we investigated the production and clearance sites and pathophysiological significance of the two molecular forms of AM in the pulmonary circulation in patients with mitral stenosis. We measured the plasma levels of AM-m and total AM (AM-T; AM-m+AM-Gly) using a recently developed specific immunoradiometric assay, and thus calculated plasma AM-Gly levels, in blood samples obtained from the femoral vein, pulmonary artery, left atrium and aorta of 28 consecutive patients with mitral stenosis (20 females and eight males; age 53±10 years). Patients with mitral stenosis had significantly higher venous concentrations of AM-T, AM-Gly and AM-m than age-matched normal controls (AM-T, 15.9±2.5 and 10.6±2.1 pmol/l respectively; AM-Gly, 14.0±2.1 and 9.8±1.9 pmol/l respectively; AM-m, 1.9±0.6 and 1.1±0.3 pmol/l respectively; each P < 0.001). There was a significant decrease in the concentrations of AM-m and AM-T between the pulmonary artery and the left atrium (AM-T, 16.1±2.7 and 14.0±2.4 pmol/l respectively; AM-m, 2.0±0.6 and 0.7±0.2 pmol/l respectively; each P < 0.001); however, there were no differences in plasma AM-Gly levels between the pulmonary artery and the left atrium (14.1±2.3 and 13.5±2.3 pmol/l respectively). The venous concentrations of AM-m, AM-Gly and AM-T showed similar correlations with mean pulmonary artery pressure (AM-T, r = 0.67; AM-Gly, r = 0.63; AM-m, r = 0.59; each P < 0.001) and total pulmonary vascular resistance (AM-T, r = 0.77; AM-Gly, r = 0.70; AM-m, r = 0.75; each P < 0.001). These results suggest that the plasma concentration of AM-m is increased in parallel with those of AM-Gly and AM-T, and that the main site for clearance of AM-m from the plasma is the lung; the extracted AM-m in the lungs may help to attenuate the increased pulmonary arterial resistance in secondary pulmonary hypertension due to mitral stenosis.

scholarly journals Novel cardiac peptide hormone in several teleosts

2000 ◽  
Vol 166 (2) ◽  
pp. 407-418 ◽  
Author(s):  
V Tervonen ◽  
H Ruskoaho ◽  
O Vuolteenaho
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Natriuretic Peptide ◽  
Fish Species ◽  
Molecular Form ◽  
Biologically Active ◽  
Endocrine Function ◽  
Peptide Sequence ◽  
Molecular Forms ◽  
Low Molecular Weight

To find out the significance of the newest member of the natriuretic peptide family, salmon cardiac peptide (sCP), we have determined the distribution of the peptide and its mRNA as well as the tissue and plasma molecular forms in several teleosts. Using probes based on the salmon sCP cDNA in Northern blot analysis we found mRNA homologous to that of sCP to be present in the heart of 15 fish species representing nine different genera. We developed a specific RIA for the salmon 29 amino acid peptide to be able to study the distribution of the peptide in the heart and plasma of different fish species. Despite the probable interspecies differences in the peptide sequence, large quantities of immunoreactive sCP were found in the atrium, ventricle and plasma of most of the fish species studied, suggesting that a cardiac hormone homologous to sCP has an endocrine function in a large variety of teleost species. The molecular form of the hormone secreted and stored in the tissue was determined by gel filtration high pressure liquid chromatography. In salmon, as in most of the other fish species studied, the predominant immunoreactive sCP in plasma corresponded to the low molecular weight form, with a size similar to that of the biologically active 29 amino acid sCP (sCP-29), whereas the form stored in the heart corresponded to the high molecular weight pro-sCP-sized material. The form secreted by isolated perfused salmon ventricle, in the basal state as well as when mechanically loaded, was the sCP-29-sized peptide, thus ruling out the possibility that the conversion from high to low molecular weight material is caused by plasma proteases. In conclusion, sCP-like peptides are produced and secreted from the heart of a large number of different fish species. Their post-translational processing appears to be remarkably similar to that of mammalian atrial natriuretic peptide.

Immunological Relationship Between Plasminogen Activator Inhibitors from Different Sources

1987 ◽  
Vol 57 (01) ◽  
pp. 029-034 ◽  
Author(s):  
Göran Urdén ◽  
Joanna Chmielewska ◽  
Tomas Carlsson ◽  
Björn Wiman
Keyword(s):  
Plasminogen Activator ◽  
Polyclonal Antibodies ◽  
Active Form ◽  
Polyclonal Antiserum ◽  
The Other ◽  
Hep G2 ◽  
Inhibitor Activity ◽  
Tissue Plasminogen ◽  
Immunological Relationship ◽  
Different Sources

SummaryPolyclonal antibodies have been raised against the inhibitor moiety in the purified complex between tissue plasminogen activator and its fast inhibitor (PA-inhibitor) in human plasma/ serum. A radioimmunoassay for quantitation of PA-inhibitor antigen was developed. The polyclonal antiserum and a previously described monoclonal antibody against the PA-inhibitor (14) have been used to study the immunological relationship between PA-inhibitors from plasma, serum, platelets, placenta extract and conditioned media from Hep G2 and HT 1080 cells. It was demonstrated that the ratio between PA-inhibitor activity and antigen varied considerably between the different sources. In the plasma samples studied, similar activity and antigen concentrations were found, suggesting that the PA-inhibitor in these samples mainly was in an active form. On the other hand the other sources seemed to contain variable amounts of inactive PA-inhibitor forms. Immunoadsorption experiments revealed that the PA-inhibitor (activity and antigen) from all the sources were specifically bound to the insolubilized antibodies (polyclonal and monoclonal). In no case, however, could active PA-inhibitor be eluted from the immunoadsorption columns. Also the competitive radioimmunoassays suggested that the PA-inhibitors from the different sources studied, were closely immunologically related.

scholarly journals Cells Expressing Murine RAD52 Splice Variants Favor Sister Chromatid Repair

2006 ◽  
Vol 26 (10) ◽  
pp. 3752-3763 ◽  
Author(s):  
Peter H. Thorpe ◽  
Vanessa A. Marrero ◽  
Margaret H. Savitzky ◽  
Ivana Sunjevaric ◽  
Tom C. Freeman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Splice Variants ◽  
Protein A ◽  
Single Stranded Dna ◽  
Culture Cells ◽  
Dominant Phenotype ◽  
Mouse Tissues

ABSTRACT The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.

Centrosomal components immunologically related to tektins from ciliary and flagellar microtubules

1994 ◽  
Vol 107 (8) ◽  
pp. 2095-2105 ◽  
Author(s):  
W. Steffen ◽  
E.A. Fajer ◽  
R.W. Linck
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Immunoblot Analysis ◽  
Surf Clam ◽  
Alpha Tubulin ◽  
Sea Urchin Sperm

Centrosomes are critical for the nucleation and organization of the microtubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule proteins, we characterize here the presence and behavior of certain components associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella and used to produce monoclonal antibodies, 16 of which were specific- or cross-specific for the major polypeptides associated with this microtubule fraction: tektins A, B and C, acetylated alpha-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylamide gel electrophoresis the tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal antibodies against a given tektin, indicating that the different polypeptide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain centrosomes of S. solidissima oocytes and CHO and HeLa cells, by immunofluorescence microscopy. In particular, the centrosome staining of one monoclonal antibody specific for tektin B (tekB3) was cell-cycle-dependent for CHO cells, i.e. staining was observed only from early prometaphase until late anaphase. By immuno-electron microscopy tekB3 specifically labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal material throughout the cell cycle. Finally, by immunoblot analysis tekB3 stained polypeptides of 48–50 kDa in isolated spindles and centrosomes from CHO cells.

scholarly journals Molecular forms of Anopheles subpictus and Anopheles sundaicus in the Indian subcontinent

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Ankita Sindhania ◽  
Manoj K. Das ◽  
Gunjan Sharma ◽  
Sinnathamby N. Surendran ◽  
B. R. Kaushal ◽  
...  
Keyword(s):  
Sibling Species ◽  
Molecular Form ◽  
Indian Subcontinent ◽  
Phylogenetic Analyses ◽  
Coastal Areas ◽  
Molecular Forms ◽  
Nominal Species ◽  
Anopheles Subpictus ◽  
Species Complexes ◽  
Molecular Phylogenetic

Abstract Background Anopheles subpictus and Anopheles sundaicus are closely related species, each comprising several sibling species. Ambiguities exist in the classification of these two nominal species and the specific status of members of these species complexes. Identifying fixed molecular forms and mapping their spatial distribution will help in resolving the taxonomic ambiguities and understanding their relative epidemiological significance. Methods DNA sequencing of Internal Transcribed Spacer-2 (ITS2), 28S-rDNA (D1-to-D3 domains) and cytochrome oxidase-II (COII) of morphologically identified specimens of two nominal species, An. subpictus sensu lato (s.l.) and An. sundaicus s.l., collected from the Indian subcontinent, was performed and subjected to genetic distance and molecular phylogenetic analyses. Results Molecular characterization of mosquitoes for rDNA revealed the presence of two molecular forms of An. sundaicus s.l. and three molecular forms of An. subpictus s.l. (provisionally designated as Form A, B and C) in the Indian subcontinent. Phylogenetic analyses revealed two distinct clades: (i) subpictus clade, with a single molecular form of An. subpictus (Form A) prevalent in mainland India and Sri Lanka, and (ii) sundaicus clade, comprising of members of Sundaicus Complex, two molecular forms of An. subpictus s.l. (Form B and C), prevalent in coastal areas or islands in Indian subcontinent, and molecular forms of An. subpictus s.l. reported from Thailand and Indonesia. Based on the number of float-ridges on eggs, all An. subpictus molecular Form B were classified as Species B whereas majority (80%) of the molecular Form A were classified as sibling species C. Fixed intragenomic sequence variation in ITS2 with the presence of two haplotypes was found in molecular Form A throughout its distribution. Conclusion A total of three molecular forms of An. subpictus s.l. and two molecular forms of An. sundaicus s.l. were recorded in the Indian subcontinent. Phylogenetically, two forms of An. subpictus s.l. (Form B and C) prevalent in coastal areas or islands in the Indian subcontinent and molecular forms reported from Southeast Asia are members of Sundaicus Complex. Molecular Form A of An. subpictus is distantly related to all other forms and deserve a distinct specific status.

scholarly journals Activation of platelet heparitinase by tumor cell-derived factors

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 789-796 ◽  
Author(s):  
A Haimovitz-Friedman ◽  
DJ Falcone ◽  
A Eldor ◽  
V Schirrmacher ◽  
I Vlodavsky ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Mammalian Cells ◽  
Human Platelets ◽  
Native Form ◽  
Heat Stable ◽  
Inactive Form ◽  
Cell Lining ◽  
Mouse Lymphoma

Abstract The nature of the cooperation between platelets and tumor cells during the process of blood-borne metastasis is essentially unknown. In previous in vitro studies we showed that platelets participated in the formation of gaps in the endothelial cell lining, and that concomitantly heparan sulfate glycosaminoglycans were degraded by the platelet heparitinase, released on activation of platelets. In the current study we show that the ability to degrade proteoheparan sulfate derived from endothelial extracellular matrix is gradually eliminated when the number of human platelets is decreased from 5 x 10(7) to 10(6) cells/mL. When aliquots of conditioned media or lysates of either Eb or heat-inactivated ESb mouse lymphoma cells (both of which showed no heparanase activity) were added to freeze-thawed lysates of 10(6) platelets, a reappearance of platelet heparitinase activity was observed. A similar activation was not elicited by lysates of several normal mammalian cells. These data suggest that in its native form, a fraction of the platelet heparitinase is stored in an inactive form that can be activated by a factor secreted by lymphoma, but not by normal cells. Partial characterization of the heparitinase-activating factor showed that it is a heat-stable polyanionic molecule, devoid of proteolytic activity and resistant to both proteolytic and chondroitinase digestions. Activation of platelet heparitinase was also observed on coincubation with chondroitinases ABC and AC, suggesting that the inactive form of platelet heparitinase could result from a complex formation with a chondroitinase-sensitive proteoglycan. The lymphoma-derived heparitinase activating factor itself is, however, not a chondroitinase, because activity of chondroitinase could not be detected in Eb and ESb cells. A possible mechanism by which tumor cells recruit and regulate the activity of platelet heparitinase, and its relevance to the progression of blood borne metastasis, is discussed.

scholarly journals Spatial swarm segregation and reproductive isolation between the molecular forms of Anopheles gambiae

2009 ◽  
Vol 276 (1676) ◽  
pp. 4215-4222 ◽  
Author(s):  
Abdoulaye Diabaté ◽  
Adama Dao ◽  
Alpha S. Yaro ◽  
Abdoulaye Adamou ◽  
Rodrigo Gonzalez ◽  
...  
Keyword(s):  
Reproductive Isolation ◽  
Anopheles Gambiae ◽  
Assortative Mating ◽  
Molecular Form ◽  
Mate Recognition ◽  
Spatial Segregation ◽  
Molecular Forms ◽  
Ecological Niches ◽  
Ecological Criteria ◽  
Underlying Mechanisms

Anopheles gambiae , the major malaria vector in Africa, can be divided into two subgroups based on genetic and ecological criteria. These two subgroups, termed the M and S molecular forms, are believed to be incipient species. Although they display differences in the ecological niches they occupy in the field, they are often sympatric and readily hybridize in the laboratory to produce viable and fertile offspring. Evidence for assortative mating in the field was recently reported, but the underlying mechanisms awaited discovery. We studied swarming behaviour of the molecular forms and investigated the role of swarm segregation in mediating assortative mating. Molecular identification of 1145 males collected from 68 swarms in Donéguébougou, Mali, over 2 years revealed a strict pattern of spatial segregation, resulting in almost exclusively monotypic swarms with respect to molecular form. We found evidence of clustering of swarms composed of individuals of a single molecular form within the village. Tethered M and S females were introduced into natural swarms of the M form to verify the existence of possible mate recognition operating within-swarm. Both M and S females were inseminated regardless of their form under these conditions, suggesting no within-mate recognition. We argue that our results provide evidence that swarm spatial segregation strongly contributes to reproductive isolation between the molecular forms in Mali. However this does not exclude the possibility of additional mate recognition operating across the range distribution of the forms. We discuss the importance of spatial segregation in the context of possible geographic variation in mechanisms of reproductive isolation.

scholarly journals Laminin γ3 Chain Binds to Nidogen and Is Located in Murine Basement Membranes

2005 ◽  
Vol 280 (23) ◽  
pp. 22146-22153 ◽  
Author(s):  
Nikolaus Gersdorff ◽  
Eddie Kohfeldt ◽  
Takako Sasaki ◽  
Rupert Timpl ◽  
Nicolai Miosge
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Self Assembly ◽  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Basement Membranes ◽  
Electron Microscopic ◽  
Immunogold Staining ◽  
Adult Mice ◽  
Epidermal Growth

Recently a novel laminin γ3 chain was identified in mouse and human and shown to have the same modular structure as the laminin γ1 chain. We expressed two fragments of the γ3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3–5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The γ3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the β2 VI/V fragment. The γ3 III3–5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the γ1 III3–5 fragment. These data suggested that laminins containing the γ3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against γ3 VI/V and γ3 III3–5 showed no cross-reaction with homologous fragments from the γ1 and γ2 chains of laminin and allowed the establishment of γ chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20–100-fold lower content of the γ3 chain compared with the γ1 chain in various tissue extracts of adult mice. The expression of γ3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the γ3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the γ3 chain to basement membranes of these tissues.

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