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PubMed1997

The cardiac ganglia: cellular and molecular aspects.

The cardiac ganglia are distributed in different regions of the atria of a number of mammalian species, surrounding the SA node, around the roots of the venae cava and pulmonary veins, interatrial septum, in the proximity of the AV node and in the auricular appendages. A typical cardiac ganglion consists of neurons, satellite cells and SIF cells. The mammalian cardiac ganglia contain unipolar, bipolar and multipolar neurons with differing dimensions and shapes. The neurons and satellite cells of the cardiac ganglia originate from neural crest cells that migrate to the heart. Upon arriving in the outflow tract the cells segregate into parasympathetic neurons and supporting cells to form the cardiac ganglia. The sympathetic innervation of the heart arises from the cervicothoracic sympathetic chains and is derived from the trunk neural crest. The ultrastructural development of the cardiac ganglia in the chicken can be divided into three phases: (1) migration and aggregation of neuroblasts on days 3.5-5; (2) differentiating ganglia, days 5-10; (3) maturing ganglia, days 11 to hatching. The development of cholinergic mechanisms precedes that of adrenergic mechanisms. As a consequence the parasympathetic-cholinergic control becomes functional and plays a role in cardiac function earlier than the sympathetic-adrenal neural control. The genes that may control the development of certain components of the cardiac ganglia are described.

Animals↗

PubMed2001

GDNF is a chemoattractant for enteric neural cells.

In situ hybridization revealed that GDNF mRNA in the mid- and hindgut mesenchyme of embryonic mice was minimal at E10.5 but was rapidly elevated at all gut regions after E11, but with a slight delay (0.5 days) in the hindgut. GDNF mRNA expression was minimal in the mesentery and in the pharyngeal and pelvic mesenchyme adjacent to the gut. To examine the effect of GDNF on enteric neural crest-derived cells, segments of E11.5 mouse hindgut containing crest-derived cells only at the rostral ends were attached to filter paper supports and grown in catenary organ culture. With GDNF (100 ng/ml) in the culture medium, threefold fewer neurons developed in the gut explants and fivefold more neurons were present on the filter paper outside the gut explants, compared to controls. Thus, in controls, crest-derived cells colonized the entire explant and differentiated into neurons, whereas in the presence of exogenous GDNF, most crest-derived cells migrated out of the gut explant. This is consistent with GDNF acting as a chemoattractant. To test this idea, explants of esophagus, midgut, superior cervical ganglia, paravertebral sympathetic chain ganglia, or dorsal root ganglia from E11.5-E12.5 mice were grown on collagen gels with a GDNF-impregnated agarose bead on one side and a control bead on the opposite side. Migrating neural cells and neurites from the esophagus and midgut accumulated around the GDNF-impregnated beads, but neural cells in other tissues showed little or no chemotactic response to GDNF, although all showed GDNF-receptor (Ret and GFRalpha1) immunoreactivity. We conclude that GDNF may promote the migration of crest cells throughout the gastrointestinal tract, prevent them from straying out of the gut (into the mesentery and pharyngeal and pelvic tissues), and promote directed axon outgrowth.

Animals↗

PubMed1992

[Molecular nature of beta-thalassemia in Tajikistan: a four base pair deletion in codons 41-42 of the beta-globin gene].

Thirty tajiks, whose relatives had beta-thalassemia traits (revealed in previous investigations by determination of the HbA-2 and HbF levels) were selected to screen beta-thalassemia mutations. DNA samples from each individual were subjected to the PCR (polymerase chain reaction) to amplify the 635 bp beta-globin gene fragment. One additional band was detected in three samples after the amplified fragment underwent electrophoresis in 2% agarose gel and the EtBr was stained, and two additional ones were revealed by 6% PAAGE and staining of the EtBr. All additional bands migrated more slowly than appropriate 635 bp fragment. It is supposed that additional bands are heteroduplexes formed from the wild type chains and mutated chains carrying a deletion or insertion. The 4 bp deletion of the 41-42 (-tctt) was detected after the direct sequencing of the amplified fragments. This mutation is common among Chinese but it was not revealed in the Middle Asia populations. The mutation can be easily screened using the PCR and electrophoresis in 2% agarose gel or PAAG of the amplified beta-globin gene fragments.

Base Composition↗

PubMed1990

Purification and characterization of platelet factor XI.

Factor XI activity and antigen was purified about 300 fold from human platelets through chromatography on Con-A Sepharose, SP-Sephadex C-50, immobilized goat anti-factor XI, and SP-Sephadex. The partially purified platelet factor XI (Pt-XI) could be activated by activated factor XII generated in situ from single chain factor XI in a reaction requiring high molecular weight kininogen (HMWK) and a surface. Native Pt-XI migrated as a molecule of Mr = 245,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as identified by Western blotting. On reduction, Pt-XI appeared to have a Mr = 52,000. Neither form was affected by exposure to trypsin. Incubation of Pt-XI with purified factor XII, HMWK, and kaolin produced activated platelet factor XI clotting activity and, concomitantly, the generation over time of a new chain on reduced SDS-PAGE of Mr = 44,500. The coagulant activity of the activated form could be neutralized by diisopropyl flurophosphate (DFP). Incubation of the activated mixture with 3H-DFP followed by reduced SDS-PAGE showed the active site to be associated with a unit of Mr = 44,500. The adsorption domain as defined by adsorption to kaolin was localized to the Mr = 44,500 chain containing the active site. Hence, both active site and adsorption functions, properties of separate chains in plasma factor XI, reside in the same chain of Mr = 44,500 of platelet factor XI.

Blood Platelets↗

PubMed1996

Tepoxalin blocks neutrophil migration into cutaneous inflammatory sites by inhibiting Mac-1 and E-selectin expression.

Inflammation is characterized by the migration of polymorphonuclear leukocytes from the vasculature into the tissue causing profound injury. Adhesion and migration of neutrophils across the vascular bed are governed by a series of complex events including cytokine/chemokine production which in turn orchestrates the temporal expression of a cohort of adhesion molecules mediating the migration. Many of these adhesion molecules and their inducers are under the control of inflammatory response transcriptional factors such as NF kappa B and AP-1. Recently we showed tepoxalin, previously known as a dual cyclooxygenase/lipoxygenase (CO/LO) inhibitor, to be a potent inhibitor of NF kappa B-induced transcription in vitro. In this study, we demonstrated that when administered in vivo, tepoxalin but not naproxen (a nonsteroidal anti-inflammatory drug, NSAID) or zileuton (an LO inhibitor), effectively inhibits neutrophil migration into inflammatory sites in murine skin stimulated by either lipopolysaccharide (LPS) or tumor necrosis factor-alpha. Immunohistochemical analysis indicates that 10-50 mg/kg of tepoxalin inhibits neutrophil migration. It also effectively blocks the upregulation of Mac-1 (CD11b/CD18) on neutrophils. Quantitative polymerase chain reaction Mac-1 analysis shows that LPS-induced transcription of E-selectin mRNA was dramatically suppressed by both 25 and 50 mg/kg of tepoxalin, whereas the level of ICAM-1 was only affected by 50 mg/kg of tepoxalin. Since it has been documented that the expression of E-selectin and Mac-1 is regulated either directly or indirectly by the transcription factor NF kappa B, our studies provide in vivo evidence that tepoxalin is a potent inhibitor of NF kappa B-mediated events in animal models and this novel molecular mechanism clearly defines it as a new class of anti-inflammatory compounds.

Animals↗

PubMed1998

Capillary electrophoretic analysis of ginseng polypeptide.

A ginseng polypeptide (GPP) found in ginseng roots and its modified peptides were tested by capillary zone electrophoresis (CZE) under acidic as well as basic conditions. Modified peptides were synthesized for three purposes: (i) to analyze their functions in the first three acidic amino acid residues, (ii) to analyze their functions in three sequenced glycines, and (iii) to analyze the length of side chains in acidic amino acids. The roles of glycines, acidic amino acids and amino acid side chains in the binding of Mg2+ were studied at pH values less than 7.0. The migration times of GPP varied with the pH of various electrophoresis buffers, and electrophoresis patterns were significantly changed between pH 7.0-7.5. Based on the electrophoretic analysis, it was concluded that the binding mechanisms for Mg2+ or conformations of GPP changed between low pH and high pH.

Buffers↗

PubMedDate not supplied

Characterization of rabbit masseter muscle fibers.

Myosins of histochemically distinguishable single fibers of rabbit masseter muscle--type 1, 2A, 2B, and slow fibers--have been characterized by gel electrophoresis under dissociating (sodium dodecyl sulfate) and nondissociating (inorganic pyrophosphate) conditions, and by analysis of peptide maps of the heavy chains following limited proteolytic degradation. Type 2B fibers contain more LC3 homodimer than type 2A fibers; peptide maps of their heavy chain are different although the two myosins comigrate on pyrophosphate gel electrophoresis. Slow fiber myosin migrates more slowly than fast myosin and has a distinct peptide map. Differences were also found among fibers of the same histochemical type but originating in different muscles. In adductor magnus 2B myosin the LC1 + LC3 heterodimer band is the strongest, while in masseter 2B myosin the heterodimer is the weakest. Statistical considerations suggest that in masseter there is a mechanism preferentially forming the homodimers. More work is needed to determine the mechanism by which phenotypical differences occur among various fiber types in the same muscle and between corresponding fiber types in different muscles.

Adenosine Triphosphatases↗

PubMed2000

Evolution of the thyroid hormone-binding protein, transthyretin.

Transthyretin (TTR) belongs to a group of proteins, which includes thyroxine-binding globulin and albumin, that bind to and transport thyroid hormones in the blood. TTR is also indirectly implicated in the carriage of vitamin A through the mediation of retinol-binding protein (RBP). It was first identified in 1942 in human serum and cerebrospinal fluid and was formerly called prealbumin for its ability to migrate faster than serum albumin on electrophoresis of whole plasma. It is a single polypeptide chain of 127 amino acids (14,000 Da) and is present in the plasma as a tetramer of noncovalently bound monomers. The major sites of synthesis of TTR in eutherian mammals, marsupials, and birds are the liver and choroid plexus but in reptiles it is synthesised only in the choroid plexus. The observation that TTR is strongly expressed in the choroid plexus but not in the liver of the stumpy-tailed lizard and the strong conservation of expression in the choroid plexus from reptiles to mammals have been taken as evidence to suggest that extrahepatic synthesis of TTR evolved first. The identification and cloning of TTR from the liver of an amphibian, Rana catesbeiana, and a teleost fish, Sparus aurata, and its absence from the choroid plexus of both species suggest an alternative model for its evolution. Protein modelling studies are presented that demonstrate differences in the electrostatic characteristics of the molecule in human, rat, chicken, and fish, which may explain why, in contrast to TTR from human and rat, TTR from fish and birds preferentially binds triiodo-l-thyronine.

Amino Acid Sequence↗

PubMed1987

Separation of the myogenic and chondrogenic progenitor cells of undifferentiated limb mesenchyme.

Undifferentiated limb bud mesenchyme consists of at least two separate, possibly predetermined, populations of progenitor cells, one derived from somitic mesoderm that gives rise exclusively to skeletal muscle and one derived from somatopleural mesoderm that gives rise to the cartilage and connective tissue of the limb. In the present study, we demonstrate that the inherent migratory capacity of myogenic precursor cells can be used to physically separate the myogenic and chondrogenic progenitor cells of the undifferentiated limb mesenchyme at the earliest stages of limb development. When the undifferentiated mesenchyme of stage 18/19 chick embryo wing buds or from the distal subridge region of stage 22 wing buds is placed intact upon the surface of fibronectin (FN)-coated petri dishes, a large population of cells emigrates out of the explants onto the FN substrates and differentiates into an extensive interlacing network of bipolar spindle-shaped myoblasts and multinucleated myotubes that stain with monoclonal antibody against muscle-specific fast myosin light chain. In contrast, the cells of the explants that remain in place and do not migrate away undergo extensive cartilage differentiation. Significantly, there is no emigration of myogenic cells out of explants of stage 25 distal subridge mesenchyme, which lacks myogenic progenitor cells. Myogenic precursor cells stream out of mesenchyme explants in one or occasionally two discrete locations, suggesting they are spatially segregated in discrete regions of tissue at the time of its explantation. There are subtle overall differences in the morphologies of the myogenic cells that form in stage 18/19 and stage 22 distal subridge mesenchyme explants. Finally, groups of nonmyogenic nonfibroblastic cells which are fusiform-shaped and oriented in distinct parallel arrays characteristically are found along the periphery of stage 18/19 wing mesenchyme explants. Our observations provide support for the concept that undifferentiated limb mesenchyme consists of independent subpopulations of committed precursor cells and provides a system for studying the early determinative and regulatory events involved in myogenesis or chondrogenesis.

Animals↗

PubMed2000

Separation of bisphenol A and three alkylphenols by micellar electrokinetic chromatography.

Analytical conditions of pH, surfactants, and additives were investigated for the simultaneous separation of bisphenol A and alkylphenols by micellar electrokinetic chromatography. Reproducibility of migration time was improved at higher pH (pH 8.0). When five surfactants having linear alkyl chains or four bile salts were used, the separation of hydrophobic phenols and 4-nonylphenol isomers was not achieved. In order to improve the separation, the use of additives with sodium dodecyl sulfate solution was investigated. The separation of hydrophobic phenols was improved by the addition of organic solvents, however, isomers were not separated. Their separation was achieved by the addition of beta- or gamma-cyclodextrin.

Benzhydryl Compounds↗

PubMed2002

Prox1 is an early specific marker for the developing liver and pancreas in the mammalian foregut endoderm.

Although important progress has been made recently in the elucidation of the molecular mechanisms that regulate differentiation and morphogenesis of endoderm-derived tissues such as pancreas and liver, less is known about the preliminary steps of early regional specification. Recent evidence supports the proposal that the early endoderm contains a bipotential precursor cell type for pancreas and liver. We have also previously shown that the activity of the homeobox gene Prox1 controls hepatocyte migration during liver morphogenesis. Using detailed comparative analysis of whole embryos and reverse transcriptase polymerase chain reaction of dissected embryonic endoderm, we have now determined that in the early endoderm, Prox1 expression is restricted to regions giving rise to the mammalian pancreas and liver. This finding indicates that Prox1 is one of the earliest specific markers of this commonly fated region of the mammalian endoderm.

Activin Receptors, Type I↗

PubMed2002

Vascular endothelial growth factor stimulates chemotactic migration of primary human osteoblasts.

Recent studies have indicated a critical role for vascular endothelial growth factor (VEGF) during the process of endochondral ossification, in particular in coupling cartilage resorption with bone formation. Therefore, we studied the chemoattractive and proliferative properties of human VEGF-A on primary human osteoblasts (PHO) and compared these data with the effects of human basic fibroblast growth factor (bFGF) and human bone morphogenetic protein-2 (BMP-2). Furthermore, initial experiments were carried out to characterize VEGF-binding proteins on osteoblastic cells possibly involved in the response. For the first time, to our knowledge, we could demonstrate a chemoattractive effect of VEGF-A, but not VEGF-E, on primary human osteoblasts. The effect of VEGF-A was dose-dependent and did not reach a maximum within the concentration range tested (up to 10 ng/mL). The maximal effect observed was a chemotactic index (CI) of 2 at a concentration of 10 ng/mL. bFGF and BMP-2 exhibited maxima at 1.0 ng/mL with CI values of 2.5 and 2, respectively. In addition to its effect on cell migration, VEGF-A stimulated cell proliferation by up to 70%. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the expression of VEGF receptors VEGFR-1 (Flt-1), VEGFR-2 (Kdr), and VEGFR-3 (Flt-4), as well as neuropilin-1 and -2. An in vitro kinase assay failed to demonstrate activation of VEGFR-2 upon stimulation with either VEGF-E or VEGF-A, consistent with the idea that the effect of VEGF-A on primary human osteoblasts is mediated via VEGFR-1. Taken together, our data establish that human osteoblasts respond to VEGF-A, suggesting a functional role for this growth factor in bone formation and remodeling.

Aged↗

PubMedDate not supplied

PEI-g-chitosan, a novel gene delivery system with transfection efficiency comparable to polyethylenimine in vitro and after liver administration in vivo.

Polyethylenimine-graft-chitosan (PEI-g-chitosan) was synthesized by performing cationic polymerization of aziridine in the presence of water-soluble oligo-chitosan (M(n) = 3400). The absolute molecular weight and chemistry of the PEI-g-chitosan obtained were characterized using GPC, 13C and 1H NMR, respectively. The results indicated that all the amines of chitosan were grafted with oligo-PEI, and the average length of the oligo-PEI side chains was determined by the feed molar ratio of aziridne/amine in chitosan. PEI-g-chitosan of M(n) = 7400 with a polydispersity index (PDI) of 1.50, and PEI side chains of M(n) = 206 was prepared for gene delivery. Gel electrophoresis showed that DNA migration was retarded completely at a N/P ratio of 2.5/1, indicating good DNA condensation capability of PEI-g-chitosan. The sizes and the zeta-potentials of the complexes of PEI-g-chitosan/DNA were characterized. The cytotoxicity of PEI-g-chiotsan was evaluated, and the results reflected that PEI-g-chitosan had a lower cytotoxicity than PEI (25 K). Gene transfection efficiency of PEI-g-chitosan in HepG2, HeLa, and primary hepatocytes cells and after administration in the common bile duct of rat liver was determined. Remarkably, PEI-g-chitosan showed a higher transfection efficiency than that of PEI (25 K) both in vitro and in vivo. The systematic distribution and the distribution in liver of the gene expression of the complexes of PEI-chitosan/DNA were determined as well.

Animals↗

PubMed2004

Slow motion, trapping, and sorting of water- and chloroform-soluble porphyrins in nanowells.

A two-step self-assembly procedure on smooth, aminated silica particles established holey monolayers. At first, single, flat-lying porphyrin tetraamides (A) were bound covalently, followed by the build-up of a rigid monolayer made of diamido bolaamphiphiles (bolas) around the porphyrin islands. "Nanowells" around porphyrin (A) bottoms with a uniform diameter of 2.2 nm and varying depths of 0.6, 1.0, or 1.5 nm depending on the length of the applied bolas were thus obtained. Oligoethylene headgroups solubilized the particles in water, ethanol, and chloroform/ethanol, and two hydrogen bond chains between the secondary amide groups prevented swelling of the monolayer. Manganese(III) porphyrinates (B) migrated from the bulk solution to the bottom of the form-stable nanowells with a speed of about 1 pm/s and were trapped there above porphyrin (A). After isolation of the (A,B) particles by centrifugation or ultrafiltration, the particles were suspended in a chloroform solution of a chlorin (C), which was also fixated irreversibly on the bottom of the nanowells. The nanowells thus contained three different porphyrins A,B,C in a noncovalent stack. The reverse sequence A,C,B was built-up correspondingly, first in chloroform/ethanol, and then in water. The "sorting" of A,B,C and A,C,B systems was characterized by visible spectra, sequence-dependent fluorescence quenching, and cyclic voltammetry of the top component. The molecular sorting method is the first of its kind and should be generally useful for the production of noncovalent reaction systems on any smooth surface.

Journal Article↗

PubMed2002

Expression, regulation, and function of alpha V integrins in hepatocellular carcinoma: an in vivo and in vitro study.

The expression of alpha V integrins by neoplastic cells contributes to the promotion of local invasion and metastasis. The most characteristic extracellular ligands of alpha V integrins are vitronectin and fibronectin. Hepatocytes are the main source of vitronectin, and the capacity to synthesize and secrete vitronectin is usually retained in hepatocellular carcinoma. The aim of this study was to explore the expression, regulation, and functional role of alpha V integrins in hepatocellular carcinoma. We first analyzed the expression of alpha V integrins and their ligands fibronectin and vitronectin in 80 cases of hepatocellular carcinoma. alpha V integrin chain was detected in 44 cases and vitronectin in 50. Twenty-four of the 44 alpha V-positive tumors contained large amounts of vitronectin. These cases presented more frequently with adverse histoprognostic factors, including infiltrative growth pattern (62.5%), lack of capsule (71%), presence of capsular invasion (57%), and satellite nodules (50%). We then used HepG2 and Hep3B cell lines as in vitro models to study alpha V integrin regulation and function. HepG2 and Hep3B cells expressed alpha V integrin chain and used alpha V beta 1 and alpha V beta 5 for adhesion and migration on vitronectin. Tumor necrosis factor (TNF) alpha and transforming growth factor (TGF) beta significantly increased the expression levels of alpha V integrins and stimulated the adhesion and migration of both HepG2 and Hep3B cell lines on vitronectin. The effects of growth factors on cell adhesion and migration were reproduced by incubation with conditioned medium from rat liver myofibroblasts. In conclusion, our results support the existence of an alpha V integrin/vitronectin connection in hepatocellular carcinoma and suggest that this connection may be an adverse prognostic factor.

Adult↗

PubMed2000

Chlamydia pneumoniae DNA in non-coronary atherosclerotic plaques and circulating leukocytes.

Earlier studies have associated atherosclerosis with Chlamydia pneumoniae infection. C. pneumoniae may circulate via monocytes and migrate into plaques by leukocyte infiltration; however, detection is difficult. We developed a novel polymerase chain reaction (PCR) method to test the hypothesis that C. pneumoniae DNA in circulating leukocytes is correlated with C. pneumoniae DNA in plaque material and that C. pneumoniae copy number is associated with disease severity. We obtained plaques from 130 patients who underwent surgery for carotid stenosis, aneurysm, or peripheral vascular disease. From 60 patients and 51 normal control subjects we also obtained circulating leukocytes. The C. pneumoniae 16 S rRNA gene was amplified with a highly specific quantitative PCR protocol relying on the TaqMan technology. Immunohistochemistry was performed with antibody against the C. pneumoniae outer membrane protein. C. pneumoniae DNA was present in 25% of atherosclerotic plaques and 20% of circulating leukocytes from patients. The copy number was not correlated with disease severity. C. pneumoniae DNA was more common in younger patients and smokers. C. pneumoniae antibody titers, C-reactive protein, fibrinogen, leukocyte count, cholesterol, and diabetes were not associated with C. pneumoniae DNA. Although immunostaining of plaque and PCR results were highly correlated, we found no relationship between C. pneumoniae DNA in plaques and that in circulating leukocytes. Finally, 13% of normal control subjects had positive leukocytes; however, their copy number was significantly lower than that of the patients. C. pneumoniae DNA is frequent in atherosclerotic plaques and is correlated with positive immunohistochemistry. C. pneumoniae DNA may also be found in circulating leukocytes; however, infected leukocytes and plaques do not coincide. Serology is unreliable in predicting C. pneumoniae DNA. Smoking increases the risk of harboring C. pneumoniae DNA. Our results do not suggest that either test for antibodies or C. pneumoniae DNA from leukocytes in blood is of value in predicting infected plaques.

Aged↗

PubMed2003

Clustering of immunoreceptor tyrosine-based activation motif-containing signalling subunits in CD4(+) T cells is an optimal signal for IFN-gamma production, but not for the production of IL-4.

CD4(+) T cells with pre-defined MHC-unrestricted specificity to type II collagen (CII) were engineered for cell-based anti-inflammatory gene therapy of autoimmune arthritis. To this end, recombinant chimeric immunoreceptors, C2gamma or C2zeta, were expressed in primary mouse keyhole limpet hemocyanin (KLH)-specific T(h)1 and T(h)2 cells using retrovirus vector-based somatic cell gene transfer. The ectodomain of these tyrosine-based activation motif (ITAM)-containing immunoreceptors is a single-chain IgG variable domain of an anti-CII mAb. The engineered cells might arrest migration when they encounter CII in articular cartilage. Up to 19 and 55% of transduced CD4(+) T cells expressed respectively C2gamma and C2zeta. The expression of C2gamma or C2zeta on the surface of CD4(+) T cells was down-regulated upon binding CII, and cells activated in such a way proliferated, up-regulated CD25 expression and produced cytokines. Comparison of cytokine levels normalized by the number of producer cells revealed that C2gamma and C2zeta were as potent as TCR in the induction of IFN-gamma, but induced lower levels of IL-4. It appears that the reason why CD4(+) T cells stimulated through C2gamma and C2zeta produce low levels of IL-4 is a lack of integration between co-stimulatory signals required for the optimal production of this cytokine and the ITAM-dependent signals generated by the immunoreceptors. The significance of these data for the development of anti-inflammatory gene therapy based on CD4(+) T cells targeted to a tissue-specific protein is discussed.

Animals↗

PubMed1997

Biosynthesis of renin in mouse kidney tumor As4.1 cells.

As4.1, a renin-expressing cell line isolated from a mouse renal tumor, was characterized for synthesis, processing, storage and secretion of renin polypeptides. Metabolic labeling, immunoprecipitation and SDS/PAGE analysis revealed that renin was secreted into the culture supernatant predominantly in the form of prorenin which migrated as products of 42-47 kDa. The predominant intracellular renin was processed into two chains, of 33-34 and 5 kDa. N-glycanase treatment removed N-linked oligosaccharides and yielded products of 41 kDa for prorenin and 31-32 kDa for the heavier chain of two-chain renin. The N-terminus of the constitutively secreted prorenin was determined by automated Edman degradation to be Leu22 while the N-terminus of the heavy chain was Ser72. Renin polypeptides constituted 3.1 +/- 1.4% (mean percentage of total precipitable radioactivity +/- SD) of de-novo-synthesized protein secreted into the medium and 0.2 +/- 0.17% retained intracellularly. Extrapolation of renin activity assays suggest that a single cell stores approximately 680 fg of active renin. A slow incremental release into the medium of processed renin heavy chain was detected by immunoprecipitation and SDS/PAGE. Renin activity assays confirmed the release of approximately 4 fg prorenin and 0.32 fg active renin cell(-1) h(-1). Indirect immunofluorescence demonstrated intracellular renin to be distributed in a punctate pattern. Renin was found to be colocalized with the lysosomal marker, beta-glucuronidase, by double-fluorescent labeling. These cells have enabled characterization of glycosylated mouse renin-1 and may prove a valuable tool for studying intracellular trafficing of renin and associated processing enzymes.

Animals↗