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PubMed1999

The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance.

Vibrio cholerae is a facultative pathogen of humans that must survive exposure to inorganic and organic acids in the stomach and small intestine. To learn more about the mechanisms by which this pathogen colonizes the intestinal tract, we used a recombinase gene fusion reporter to identify transcripts induced during infection in an adult rabbit model of cholera. One of the genes identified was cadA, which encodes an inducible lysine decarboxylase. CadA was also induced during infections of the suckling and adult mouse intestines, and in vitro under conditions of low pH and high lysine concentration. We show that V. cholerae is capable of mounting an acid tolerance response (ATR) to both inorganic and organic acid challenges. Mutational analyses revealed a significant role for cadA, but not for speF, which encodes an ornithine decarboxylase, in both inorganic and organic ATR. Potential roles for toxR, toxT and rpoS in ATR were examined, and it was found that toxR plays a ToxT-independent role in mediating organic ATR, whereas rpoS played no detectable role in either ATR. Transcriptional analysis showed that the toxR defect in ATR is not caused by decreased cadA transcription. Despite induction of cadA in these animal models, competition assays revealed that neither cadA nor speF alone or together were required for colonization of suckling or adult mice. However, acid-adapted wild-type V. cholerae exhibited a major competitive advantage over unadapted cells during colonization of suckling mice.

Animals↗
PubMed2005

Isolation and characterization of a retinal pigment epithelial cell fluorophore: an all-trans-retinal dimer conjugate.

Several lines of investigation suggest that the nondegradable fluorophores that accumulate as lipofuscin in retinal pigment epithelium (RPE) cells contribute to the etiology of macular degeneration. Despite evidence that much of this fluorescent material may originate as inadvertent products of the retinoid cycle, the enzymatic pathway by which the 11-cis-retinal chromophore of rhodopsin is generated, the only fluorophores of the RPE to be characterized as yet have been A2E and its isomers. Here, we report the isolation and structural characterization of an additional RPE lipofuscin fluorophore that originates as a condensation product of two molecules of all-trans-retinal (ATR) dimer and forms a protonated Schiff base conjugate with phosphatidylethanolamine (PE), the latter conjugate (ATR dimer-PE) having UV-visible absorbance maxima at 285 and 506 nm. ATR dimer was found to form natively in bleached rod outer segments in vitro and when rod outer segments were incubated with ATR. HPLC analysis of eye-cups that included RPE and isolated neural retina from Abcr-/- mice and RPE isolated from human donor eyes revealed the presence of a pigment with the same UV-visible absorbance and retention time as synthetic ATR dimer-PE conjugate. Evidence that ATR dimer undergoes a photooxidation process involving the addition of oxygens at double bonds as well as an aromatic demethylation also may indicate a role for this molecule, or its derivatives, in the photoreactivity of RPE lipofuscin.

Animals↗
PubMed2001

The human decatenation checkpoint.

Chromatid catenation is actively monitored in human cells, with progression from G(2) to mitosis being inhibited when chromatids are insufficiently decatenated. Mitotic delay was quantified in normal and checkpoint-deficient human cells during treatment with ICRF-193, a topoisomerase II catalytic inhibitor that prevents chromatid decatenation without producing topoisomerase-associated DNA strand breaks. Ataxia telangiectasia (A-T) cells, defective in DNA damage checkpoints, showed normal mitotic delay when treated with ICRF-193. The mitotic delay in response to ICRF-193 was ablated in human fibroblasts expressing an ataxia telangiectasia mutated- and rad3-related (ATR) kinase-inactive ATR allele (ATR(ki)). BRCA1-mutant HCC1937 cells also displayed a defect in ICRF-193-induced mitotic delay, which was corrected by expression of wild-type BRCA1. Phosphorylations of hCds1 or Chk1 and inhibition of Cdk1 kinase activity, which are elements of checkpoints associated with DNA damage or replication, did not occur during ICRF-193-induced mitotic delay. Over-expression of cyclin B1 containing a dominant nuclear localization signal, and inhibition of Crm1-mediated nuclear export, reversed ICRF-193-induced mitotic delay. In combination, these results imply that ATR and BRCA1 enforce the decatenation G(2) checkpoint, which may act to exclude cyclin B1/Cdk1 complexes from the nucleus. Moreover, induction of ATR(ki) produced a 10-fold increase in chromosomal aberrations, further emphasizing the vital role for ATR in genetic stability.

Ataxia Telangiectasia↗
PubMed2001

Phosphorylation of serines 635 and 645 of human Rad17 is cell cycle regulated and is required for G(1)/S checkpoint activation in response to DNA damage.

ATR [ataxia-telangiectasia-mutated (ATM)- and Rad3-related] is a protein kinase required for both DNA damage-induced cell cycle checkpoint responses and the DNA replication checkpoint that prevents mitosis before the completion of DNA synthesis. Although ATM and ATR kinases share many substrates, the different phenotypes of ATM- and ATR-deficient mice indicate that these kinases are not functionally redundant. Here we demonstrate that ATR but not ATM phosphorylates the human Rad17 (hRad17) checkpoint protein on Ser(635) and Ser(645) in vitro. In undamaged synchronized human cells, these two sites were phosphorylated in late G(1), S, and G(2)/M, but not in early-mid G(1). Treatment of cells with genotoxic stress induced phosphorylation of hRad17 in cells in early-mid G(1). Expression of kinase-inactive ATR resulted in reduced phosphorylation of these residues, but these same serine residues were phosphorylated in ionizing radiation (IR)-treated ATM-deficient human cell lines. IR-induced phosphorylation of hRad17 was also observed in ATM-deficient tissues, but induction of Ser(645) was not optimal. Expression of a hRad17 mutant, with both serine residues changed to alanine, abolished IR-induced activation of the G(1)/S checkpoint in MCF-7 cells. These results suggest ATR and hRad17 are essential components of a DNA damage response pathway in mammalian cells.

Amino Acid Sequence↗
PubMed2000

Diffusible ligand all-trans-retinal activates opsin via a palmitoylation-dependent mechanism.

In rhodopsin's function as a photoreceptor, 11-cis-retinal is covalently bound to Lys(296) via a protonated Schiff base. 11-cis/all-trans photoisomerization and relaxation through intermediates lead to the metarhodopsin II photoproduct, which couples to transducin (G(t)). Here we have analyzed a different signaling state that arises from noncovalent binding of all-trans-retinal (atr) to the aporeceptor opsin and enhances the very low opsin activity by several orders of magnitude. Like with metarhodopsin II, coupling of G(t) to opsin-atr is sensitive to competition by synthetic peptides from the COOH termini of both G(t)alpha and G(t)gamma. However, atr does not compete with 11-cis-retinal incorporation into the Lys(296) binding site and formation of the light-sensitive pigment. Blue light illumination fails to photorevert opsin-atr to the ground state. Thus noncovalently bound atr has no access to the light-dependent binding site and reaction pathway. Moreover, in contrast to light-dependent signaling, removal of the palmitoyl anchors at Cys(322) and Cys(323) in the rhodopsin COOH terminus impairs the atr-stimulated activity. Repalmitoylation by autoacylation with palmitoyl-coenzyme A restores most of the original activity. We hypothesize that the palmitoyl moieties are part of a second binding pocket for the chromophore, mediating hydrophobic interactions that can activate a large part of the catalytic receptor/G-protein interface.

Acylation↗
PubMed1983

Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells.

The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using 125I-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 125I and gold colloid. Using 125I-transferrin we have shown that, whereas at 37 degrees C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37 degrees C, the intracellular half-life of the most rapidly recycled transferrin was 7.5 min. 125I-ATR displayed the same kinetics of uptake but following its internalization at 37 degrees C, it was partially degraded. At 22 degrees C and below, the intracellular degradation of 125I-ATR was selectively inhibited and as a result it accumulated intracellularly. Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR-gold colloid complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22 degrees C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22 degrees C, no other compartments became labeled but if they were warmed to 37 degrees C the gold complexes were transferred to lysosome-like elements. Extracting ATR-gold complexes with Triton X after a 30-min chase at 22 degrees C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.

Biological Transport, Active↗
PubMed2005

Dimerization of the ATRIP protein through the coiled-coil motif and its implication to the maintenance of stalled replication forks.

ATR (ATM and Rad3-related), a PI kinase-related kinase (PIKK), has been implicated in the DNA structure checkpoint in mammalian cells. ATR associates with its partner protein ATRIP to form a functional complex in the nucleus. In this study, we investigated the role of the ATRIP coiled-coil domain in ATR-mediated processes. The coiled-coil domain of human ATRIP contributes to self-dimerization in vivo, which is important for the stable translocation of the ATR-ATRIP complex to nuclear foci that are formed after exposure to genotoxic stress. The expression of dimerization-defective ATRIP diminishes the maintenance of replication forks during treatment with replication inhibitors. By contrast, it does not compromise the G2/M checkpoint after IR-induced DNA damage. These results show that there are two critical functions of ATR-ATRIP after the exposure to genotoxic stress: maintenance of the integrity of replication machinery and execution of cell cycle arrest, which are separable and are achieved via distinct mechanisms. The former function may involve the concentrated localization of ATR to damaged sites for which the ATRIP coiled-coil motif is critical.

Adaptor Proteins, Signal Transducing↗
PubMed1995

The stationary-phase sigma factor sigma S (RpoS) is required for a sustained acid tolerance response in virulent Salmonella typhimurium.

The acid tolerance response (ATR) of log-phase Salmonella typhimurium is induced by acid exposures below pH 4.5 and will protect cells against more extreme acid. Two systems are evident: a transiently induced system dependent on the iron regulator Fur that provides a moderate degree of acid tolerance and a more effective sustained ATR that requires the alternate sigma factor sigma S encoded by rpoS. Differences between the acid responses of virulent S. typhimurium and the attenuated laboratory strain LT2 were attributed to disparate levels of RpoS caused by different translational starts. The sustained ATR includes seven newly identified acid shock proteins (ASPs) that are dependent upon sigma S for their synthesis. It is predicted that one or more of these ASPs is essential for the sustained system. The sustained ATR also provided cross-protection to a variety of other environmental stresses (heat, H2O2 and osmolarity); however, adaptation to the other stresses did not provide significant acid tolerance. Therefore, in addition to starvation, acid shock serves as an important signal for inducing general stress resistance. Consistent with this model, sigma S proved to be induced by acid shock. Our results also revealed a connection between the transient and sustained ATR systems. Mutations in the regulator atbR are known to cause the overproduction of ten proteins, of which one or more can suppress the acid tolerance defect of an rpoS mutant. One member of the AtbR regulon, designated atrB, was found to be co-regulated by sigma S and AtbR. Both regulators had a negative effect on atrB expression. The results suggest AtrB serves as a link between the sustained and transient ATR systems. When sigma S concentrations are low, a compensatory increase in AtrB is required to engage the transiently induced, RpoS-independent system of acid tolerance. Results also suggest different acid-sensitive targets occur in log-phase versus stationary-phase cells.

Acids↗
PubMed2007

Dopaminergic toxicity associated with oral exposure to the herbicide atrazine in juvenile male C57BL/6 mice.

The herbicide atrazine (ATR) is a very commonly used pesticide in the United States. and a major ground water contaminant. It has also been recently implicated as a potential basal ganglia toxicant. In the present study, our objective was to determine the effects of ATR exposure on striatal neurochemistry, on the number of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and, as a reference, in the ventral tegmental area (VTA) of male juvenile C57BL/6 mice. Oral exposure to ATR for 14 days dose-dependently decreased the levels of dopamine (DA) and its metabolites in the striatum for up to a week post-treatment. ATR exposure also time- and dose-dependently decreased the number of tyrosine hydroxylase-positive (TH+) dopaminergic neurons in both SNpc and VTA (with effects being slightly more prominent in SNpc), such that the decreases were most evident at 7 weeks post-cessation of exposure to ATR. Together, these data indicate that, in the juvenile male C57BL/6 mouse, the neurotoxic effects of ATR appear to cause transient neurochemical alterations, whereas the loss of TH+ neurons appears to be persistent, possibly confined to basal ganglia dopaminergic neurons, but not exclusive to the SNpc.

Administration, Oral↗
PubMed2003

Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.

The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2002

The relationship of the anterior drawer sign to the shape of the tibial plafond in chronic lateral instability of the ankle.

To determine what factors correlate with values for tested anterior drawer (AD) sign, we investigated stress radiographs in 71 patients with severe chronic lateral instability of the ankle as well as 320 normal controls. We found no correlation between AD and talar tilt (TT), but measurement of the anterior tip ratio (ATR) (ATR=tanA x 100) demonstrated a link of ATR with AD (r=0.74, p<0.001). The posterior tip ratio (PTR) (PTR=tanP x 100) was not correlated with AD (r=0.34, p=0.16). The ankle mortise angle (AMA) (the anterior opening of the lateral ankle mortise) was also not correlated with AD (r=0.27, p=0.23). Investigation of 320 normal ankles revealed a mean ATR of 9.1%+/-1.4%. The ATR in patients was 7.6%+/-1.7%, significantly lower than in the control patients. The values in female patients with chronic lateral instabilities were significantly less than the values in the females in the control group. But values for male patients were not different from the control group. Thus the anterior tip of the lateral tibial plafond, as it affects the ATR, may influence instability in anterior drawer function, especially in female patients with chronic lateral ligamentous instability of the ankle.

Adult↗
PubMed2002

LCR-regulated transgene expression levels depend on the Oct-1 site in the AT-rich region of beta -globin intron-2.

Human beta-globin transgenes regulated by the locus control region (LCR) express at all integration sites in transgenic mice. For such LCR activity at ectopic sites, the 5'HS3 element requires the presence of the AT-rich region (ATR) in beta-globin intron-2. Here, we examine the dependence of 5'HS3 LCR activity on transcription factor binding sites in the ATR. In vitro DNaseI footprint analysis and electrophoretic mobility shift assays of the ATR identified an inverted double Gata-1 site composed of 2 noncanonical sequences (GATT and GATG) and an Oct-1 consensus site. Mutant Oct-1, Gata-1, or double mutant sites were created in the ATR of the BGT50 construct composed of a 5'HS3 beta/gamma-globin hybrid transgene. Transgenes with double mutant sites expressed at all sites of integration, but mean expression levels in transgenic mice were reduced from 64% per copy (BGT50) to 37% (P <.05). Mutation of the inverted double Gata-1 site had no effect at 61% per copy expression levels. In contrast, mutation of the Oct-1 site alone reduced per-copy expression levels to 31% (P <.05). We conclude that the ability of 5'HS3 to activate expression from all transgene integration sites is dependent on sequences in the ATR that are not bound at high affinity by transcription factors. In addition, the Oct-1 site in the ATR is required for high-level 5'HS3 beta/gamma-globin transgene expression and should be retained in LCRbeta-globin expression cassettes designed for gene therapy.

AT Rich Sequence↗
PubMed1988

Immunocytochemical and pharmacological evidence for an intrinsic cholinomimetic system modulating prolactin and growth hormone release in rat pituitary.

Pituitary cells were cultured as three-dimensional reaggregates in serum-free chemically defined medium supplemented with different concentrations of dexamethasone. Immunostaining of the cells using a polyclonal antiserum and three monoclonal antibodies raised against choline acetyl transferase (CAT), revealed the presence of CAT immunoreactivity in 4-10% of anterior pituitary cells depending on the antibody used. CAT immunoreactivity was also found in freshly dispersed anterior pituitary cells. CAT-immunoreactive cells could be enriched on BSA and Percoll gradients and codistributed with ACTH-immunoreactive cells in these gradients. Perifusion of the aggregates with the potent muscarinic receptor antagonist atropine (Atr) resulted in a dose-dependent (0.1-100 nM) increase in both basal PRL and GH secretion; the response was dependent on the dexamethasone concentration in the culture medium. A similar response to Atr was observed in organ-cultured pituitaries. The specificity of the Atr effect was supported by the findings that the potent and highly specific muscarinic receptor blocker dexetimide showed a similar action, whereas its inactive enantiomer levetimide and the nicotinic receptor blocker hexamethonium failed to do so. Two other muscarinic antagonists, benzatropine and pirenzepine, showed a dose-dependent hormone-releasing action similar to that of Atr, but were less potent than the latter. Pirenzepine was only effective at high molar concentrations, suggesting that an M2 muscarinic receptor subtype was mediating the present phenomenon. Atr also potentiated GH release stimulated by the beta-adrenergic agonist isoproterenol and PRL release stimulated by vasoactive intestinal peptide, but had no effect on GRF-stimulated GH release. The choline uptake blocker hemicholinium abolished the effect of Atr on GH and PRL release. These data suggest that certain pituitary cells can express CAT activity and that these cells exert a tonic inhibitory activity on GH and PRL release which is mediated by a cholinomimetic substance, possibly acetylcholine, through a muscarinic receptor.

Acetylcholine↗
PubMed1978

[Pharmacological actions of timepidium bromide on the motility of visceral smooth muscles and the secretion of digestive juice in experimental animals (author's transl)].

Effects of timepidium bromide (TB) on the motility of various smooth muscles and the secretion of digestive juice were examined and the activity was compared with those of hyoscine-N-butylbromide (HB) and atropine sulfate (Atr). In dogs, TB inhibited the spontaneous and bethanechol-induced motility of various regions of the gastrointestinal tract, the activity being almost the same as that of Atr but stronger than that of HB. All these drugs exhibited a similar inhibitory effect on the motility of the gallbladder. On the contrary, the inhbitory effect of TB on the spontaneous motion of urinary bladder was somewhat weaker than those of HB and Atr, while this effect on the bethanechol-induced motility was nearly equal the effects of HB and Atr. It was also evident that TB inhibited the gastric acid secretion induced by bethanechol: the activity was stronger than that of HB, while it was weaker than that of Atr. Similar effects on the gastric acid secretion were also found in rats. In the case of salivary secretion, however, TB and HB produced much weaker inhibitory actions than Atr. TB even at a higher dose produced little significant effect on the pancreatic and biliary secretion of rats. The uterine contraction induced by methacholine was inhibited by all these drugs. The mydriatic activity of TB in mice was found to be the weakest among the drugs tested.

Animals↗
PubMed2006

Gestational exposure to nonylphenol causes precocious mammary gland development in female rat offspring.

This study examined whether or not exposure to 4-nonylphenol (NP) during late gestation affects reproductive and mammary development in the offspring of female rats. Time pregnant Long Evans rats were gavaged with NP (10 or 100 mg/kg), atrazine (ATR, 100 mg/kg), or corn oil on gestation days 15-19. The uterus weights of the NP (100 mg/kg/d)-exposed pups were higher than those of the controls but the weights of the other organs were unchanged. Delayed mammary gland (MG) development was detected in the ATR pups on PND 4 and persisted through to PND 66. The high dose NP pups had advanced lobular development of their MG on PND 22, while the glands from the low dose NP pups were no different morphologically from the controls. Immunohistochemical comparisons of the mammary sections from PND 41 demonstrated low levels of estrogen receptor (ER) staining in the control gland stroma and epithelium but higher levels in the tissue of the pups exposed to NP and ATR. ATR also elevated ER in the stroma surrounding the epithelial layer of the terminal end buds. The level of progesterone receptor (PR) staining was markedly lower in the epithelium of the 100 mg/kg NP glands vs. the control glands. However, PR was present at high levels in the epithelium of the 10 mg/kg NP glands and was even more prominent in the ATR-exposed ductal epithelium and fat cell nuclei. The level of prolactin staining was only elevated in glands containing lobule areas (NP-exposed) compared with the control levels. These results suggest that NP and ATR have opposite effects on the development of MG after gestational exposure. Exposure to them during the critical period of epithelial outgrowth altered the receptor levels of mammary progesterone and prolactin and might contribute to the differences in the mammary morphology at PND 41.

Animals↗
PubMed1997

Trunk rotation in scoliosis. The influence of curve type and direction in 150 children.

We compared the angle of trunk rotation (ATR) from scoliometer readings with Cobb angle measurements of the lateral deviation of the spine in 150 children referred to hospital for evaluation of scoliosis. The mean Cobb angle in thoracic curves was 16 degrees. In thoracolumbar curves 17 degrees and in lumbar curves 20 degrees. In thoracic curves and in right convex curves no patient with a Cobb angle of 25 degrees or more had an ATR below 9 degrees. In thoracolumbar and lumbar and in left convex curves, 7 degrees ATR was occasionally associated with scoliosis of 25 degrees or more. The correlation coefficient between the ATR and Cobb angle in right convex curves was 0.65 compared to 0.57 in left convex curves. We conclude that a criterion of > 7 degrees ATR for thoracic or right convex curves and one of > 6 degrees ATR for thoracolumbar and lumbar or left convex curves seem adequate for identification of patients with Cobb angles of 25 degrees or more, which reduces the need for spinal radiography and follow-up outside the school screening programs.

Adolescent↗
PubMed2005

Inhibition of the ATR/Chk1 pathway induces a p38-dependent S-phase delay in mouse embryonic stem cells.

Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases, family members of the PI-3 kinase related proteins, play a key role in checkpoint activation and maintenance of genomic stability following DNA damage. We have used wild type (WT) and p38alpha-deficient mouse embryonic stem (ES) cells to investigate the role of ATR and ATM kinases during embryonic cell cycle. We have found that inhibition of ATR and ATM kinases with caffeine or Chk1 with UCN-01, results in activation of a p38-dependent intra-S-phase checkpoint and activation of apoptosis in ES cells. However, wortmannin at a concentration, that inhibits ATM kinase but not ATR kinase, did not affect cell cycle progression. Furthermore, the presence of caffeine results in activation of p38 kinase, accumulation of p21/Waf1 in a complex with Cdk2 and decrease of Cdk2 kinase activity. In contrast, caffeine-treated p38alpha-/- ES cells show less apoptosis, and fail to trigger an effective S-phase checkpoint and accumulation of p21/Waf1. We conclude that ATR kinase activity is essential for normal cell cycle progression of exponentially proliferating mouse ES cells even in the absence of exogenous DNA damage, and ATR deregulation triggers p38alpha-dependent cell-cycle checkpoint and apoptotic responses.

Animals↗
PubMed1999

[The inhibiting effect of antisense thrombin receptor gene on the proliferation of pig vascular smooth muscle cell].

OBJECTIVE: To search for an effective approach to prevent the formation of restenosis after angioplasty. METHODS: A recombinant eukaryotic expression plasmid vector containing partial antisense thrombin receptor (ATR) gene named pcDNA3/ATR was constructed using recombinant DNA technique. Mini-pig aorta injury model was established by over-sized balloon catheter combined with high cholesterol diet feeding and its aortic smooth muscle cells(ASMC) were cultured. The effect of ATR gene expression in mini pig ASMC proliferation and growth factor gene expression were studied by 3H-TdR incorporation and Northern blot. RESULTS: The DNA synthesis in pig ASMC could be inhibited by ATR gene expression (The DNA synthesis in normal ASMC was lowered by 41.8%, and that in ASMC from injured artery was lowered by (50.3%). The mRNA and protein synthesis of TR could be down regulated by ATR gene expression. The mRNA expression of PDGF-A chain and bFGF stimulated by fetal calf serum (FCS) with thrombin were both downregulated in pig ASMC with expressed ATR gene. CONCLUSION: ATR gene expression can inhibit the proliferation of pig ASMC, and this is induced by its inhibiting effect on TR and finaly the signal transduction in ASMC.

Animals↗