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Jian Kang

Publications and source records attributed to Jian Kang.

At least 19 recordsLinked to original sources
PubMed2026

RAD54L coordinates the nucleolar DNA damage response to maintain rDNA stability.

The nucleolus is organized around actively transcribed ribosomal RNA genes (rDNA), where high RNA polymerase I (Pol I) activity creates intrinsic susceptibility to replication stress and DNA damage. Here, we identify the DNA translocase RAD54L as a critical regulator of the nucleolar DNA damage response (nDDR) to rDNA double-strand breaks (DSBs) and replication stress. We show that RAD54L localizes to the nucleolus under basal conditions and is recruited to nucleolar caps following CRISPR-Cas9-induced rDNA-DSBs to promote repair. RAD54L loss results in persistent RAD51 foci, increased nucleolar γH2AX, and micronuclei formation, indicating defective resolution of rDNA lesions and genome instability. Under baseline conditions and replication stress induced by the Pol I transcription inhibitor CX-5461, RAD54L limits the accumulation of ssDNA and coordinates nDDR signaling. We further show that rDNA-DSBs induce RNA polymerase II-dependent RNA-DNA hybrids (R-loops) at intergenic rDNA regions, which facilitate nucleolar reorganization and cap formation and repair factor recruitment. Together, these findings establish RAD54L as a key regulator that coordinates replication stress response and rDNA repair, maintaining rDNA stability and genome integrity.

DNA, Ribosomal↗
PubMed2026

Alleviation of CO2-Induced Reductions in Tomato Photosynthesis Under Deficit Irrigation by Purple Nonsulfur Photosynthetic Bacteria.

The stimulatory effect of elevated CO2 (eCO2) on photosynthesis in most C3 crops under water deficit often declines over time due to photosynthetic acclimation. An exception occurs in plants inoculated with symbiotic nitrogen-fixing bacteria. Photosynthetic bacteria (PSB), specifically anoxygenic purple nonsulfur bacteria (Rhodopseudomonas palustris in this study), a group of nitrogen-fixing bacteria, are effective in enhancing crop photosynthesis. Therefore, this study investigated the synergistic effects of PSB and eCO2 in alleviating the effects of deficit irrigation and enhancing photosynthetic capacity in tomato plants during prolonged exposure. Our results showed that photosynthetic efficiency was significantly reduced in noninoculated plants under eCO2, and this reduction was more pronounced under water deficit. Proteomic analysis revealed that in eCO2-treated plants, the downregulation of cell wall proteins increased mesophyll resistance to CO2 diffusion, while the suppression of the photosynthetic apparatus impaired electron transport capacity, ultimately reducing CO2 assimilation efficiency. In contrast, these negative effects were alleviated by PSB inoculation. PSB promoted the upregulation of proteins involved in photosynthesis under deficit irrigation, as well as proteins related to chlorophyll biosynthesis, components of photosystem I and II, and light-harvesting complex proteins. These proteins contributed to improved photosynthetic efficiency during deficit irrigation and photosynthetic acclimation. Physiological analyses further confirmed that PSB inoculation enhanced nitrogen content, electron transport capacity, chlorophyll biosynthesis, and overall photosynthetic performance under eCO2 and deficit irrigation, resulting in improved plant growth. These findings suggest that PSB inoculation is a promising strategy to sustain and enhance the CO2 fertilization effect on crop productivity under water-limited conditions.

Photosynthesis↗
PubMed2005

Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells.

Human embryonic stem (hES) cells may generate all major somatic cell types, yet no neuronal subtype has yet been specifically generated in useful purity from hES culture. We report here the selective induction and isolation of functional spinal motor neurons (MNs) from human ES cells. hES cells of the H1 line were transfected with plasmids encoding GFP placed under the control of an MN-specifying enhancer within the 5'-regulatory region of the gene encoding the transcription factor Hb9 and treated with sonic hedgehog (Shh) and retinoic acid (RA). As MNs were induced under the influence of Shh and RA, they activated Hb9-driven GFP expression, permitting their isolation by fluorescence-activated cell sorting (FACS). The MNs thereby generated and isolated became cholinergic and achieved functional maturation in vitro, as evidenced by their fast sodium currents and action potentials on whole-cell patch-clamp and alpha-bungarotoxin-identified clustering of AChR receptors on co-cultured skeletal myoblasts. The serial combination of these two approaches, motor neuron phenotypic induction followed by Hb9 enhancer-based FACS, permitted the high-efficiency induction and isolation of functional motor neurons from hES cells. These results suggest the utility of promoter/enhancer-based FACS for the isolation of specific phenotypes from hES cell populations as a means of purifying clinically appropriate vectors for cell therapy.

Acetylcholinesterase↗
PubMed2005

Astrocytic glutamate release-induced transient depolarization and epileptiform discharges in hippocampal CA1 pyramidal neurons.

A paroxysmal depolarization shift (PDS) has been suggested to be a hallmark for epileptic activity in partial-onset seizures. By monitoring membrane potentials and currents in pairs of pyramidal neurons and astrocytes with dual patch-clamp recording and exocytosis of vesicles from astrocytes with two-photon laser scanning microscopy in hippocampal slices, we found that infusion of inositol 1,4,5-trisphosphate (IP(3)) into astrocytes by patch pipettes induced astrocytic glutamate release that triggered a transient depolarization (TD) and epileptiform discharges in CA1 pyramidal neurons. The TD is due to a tetrodotoxin (TTX)-insensitive slowly decaying transient inward current (STC). Astrocytic glutamate release simultaneously triggers both the STC in pyramidal neurons and a transport current (TC) in astrocytes. The neuronal STC is mediated by ionotropic glutamate receptors leading to the TD and epileptiform discharges; while the astrocytic TC is a glutamate reuptake current resulting from transporting released glutamate into the patched astrocyte. Fusion of a large vesicle in astrocytes was immediately followed by an astrocytic TC, suggesting that the fused vesicle contains glutamate. Both fusion of large vesicles and astrocytic TCs were blocked by tetanus toxin (TeNT), suggesting that astrocytic glutamate release is via SNARE-dependent exocytosis of glutamate-containing vesicles. In the presence of TTX, the epileptogenic reagent, 4-AP, also induced similar neuronal STCs and astrocytic TCs, suggesting that astrocytic glutamate release may play an epileptogenic role in initiation of epileptic seizures under pathological conditions. Our study provides a novel mechanism, astrocytic release of glutamate, for seizure initiation.

2-Amino-5-phosphonovalerate↗
PubMed2005

An astrocytic basis of epilepsy.

Hypersynchronous neuronal firing is a hallmark of epilepsy, but the mechanisms underlying simultaneous activation of multiple neurons remains unknown. Epileptic discharges are in part initiated by a local depolarization shift that drives groups of neurons into synchronous bursting. In an attempt to define the cellular basis for hypersynchronous bursting activity, we studied the occurrence of paroxysmal depolarization shifts after suppressing synaptic activity using tetrodotoxin (TTX) and voltage-gated Ca(2+) channel blockers. Here we report that paroxysmal depolarization shifts can be initiated by release of glutamate from extrasynaptic sources or by photolysis of caged Ca(2+) in astrocytes. Two-photon imaging of live exposed cortex showed that several antiepileptic agents, including valproate, gabapentin and phenytoin, reduced the ability of astrocytes to transmit Ca(2+) signaling. Our results show an unanticipated key role for astrocytes in seizure activity. As such, these findings identify astrocytes as a proximal target for the treatment of epileptic disorders.

Action Potentials↗
PubMed2005

[Serum response factor participates in RhoA-induced endothelial cell F-actin rearrangements].

RhoA is one of the main members of RhoGTPase family involved in cell morphology, smooth muscle contraction, cytoskeletal microfilaments and stress fiber formation. It has been demonstrated that RhoA modulates endothelial cell permeability by its effect on F-actin rearrangement, but the molecular mechanism of rearrangement of actin cytoskeleton remains unclear. Recent studies prove that RhoA/Rho kinase regulates smooth muscle specific actin dynamics by activating serum response factor (SRF)-dependent transcription. To further investigate the molecular mechanism of the rearrangement of vascular endothelial cell actin cytoskeleton, we explored the relationship between the activation of SRF and F-actin rearrangement induced by RhoA in human umbilical vein endothelial cells (HUVECs). HUVECs were infected with the constitutively active forms of RhoA (Q63LRhoA) or the dominant negative forms of RhoA(T19NRhoA) using retrovirus vector pLNCX-Q63LRhoA or pLNCX-T19NRhoA, the positive clone was obtained by G418 selection. The expression and distribution of SRF in normal and infected cells were evaluated by immunohistochemistry and Western blot in complete medium and in serum-free medium. The effect of F-actin polymerization was detected by Rhodamine-Phalloidine staining. Infection of PLNCX-Q63LRhoA induced F-actin rearrangement and stress fiber formation in HUVECs, as well as enhanced the expression of SRF in the nuclei. In contrast, the cells infected with T19NRhoA showed no distinct changes. With serum deprivation, the expression of SRF increased obviously in both normal and infected HUVECs, but the subcellular localization of SRF was evidently different. In HUVECs, the localization of SRF was in the nuclei after 3 d with serum deprivation, but it was redistributed outside the nuclei after 5 d with serum deprivation. In cells infected with Q63LRhoA, the immunolocalization of SRF was always in the nuclei compared with HUVECs infected with T19NRhoA, which was almost always localized in the cytoplasm. In HUVECs, the rearrangement of F-actin and formation of stress fiber increased after 3 d with serum deprivation, but appeared decreased and unpolymerized after 5 d with serum deprivation. The polymerization of F-actin and the formation of stress fiber in HUVECs infected with Q63LRhoA kept during the period of serum-free culture, whereas the rearrangement of F-actin in cells infected with T19NRhoA was not found. These results suggest that RhoA influences endothelial F-actin rearrangement in part by regulating the expression and subcellular localization of SRF.

Actins↗
PubMed2005

Activity-dependent long-term potentiation of intrinsic excitability in hippocampal CA1 pyramidal neurons.

The efficiency of neural circuits is enhanced not only by increasing synaptic strength but also by increasing intrinsic excitability. In contrast to the detailed analysis of long-term potentiation (LTP), less attention has been given to activity-dependent changes in the intrinsic neuronal excitability. By stimulating hippocampal CA1 pyramidal neurons with synaptic inputs correlating with postsynaptic neuronal spikes, we elicited an LTP of intrinsic excitability (LTP-IE) concurring with synaptic LTP. LTP-IE was manifested as a decrease in the action potential threshold that was attributable to a hyperpolarized shift in the activation curve of voltage-gated sodium channels (VGSCs) rather than activity-dependent changes in synaptic inputs or A-type K+ channels. Cell-attached patch recording of VGSC activities indicated such an activity-dependent change in VGSCs. Induction of LTP-IE was blocked by the NMDA receptor antagonist APV, intracellular BAPTA, the CaM kinase inhibitors KN-62 and autocamtide-2-related inhibitory peptide, and the protein synthesis inhibitors emetine and anisomycin. The results suggest that induction of LTP-IE shares a similar signaling pathway with the late phase of synaptic LTP and requires activation of the NMDA glutamate receptor subtype, Ca2+ influx, activity of CaM kinase II, and function of the protein synthesis. This new form of hippocampal neuronal plasticity could be a cellular correlate of learning and memory besides synaptic LTP.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2005

[Expression of cellular repressor of E1A-stimulated genes in vascular smooth muscle cells of different phenotypes].

OBJECTIVES: The phenotypic modulation of vascular smooth muscle cells (VSMC) plays a central role in the pathogenesis of arteriosclerosis. The purpose of this study was to investigate the expression of the cellular repressor of E1A-activated genes (CREG) at the transcriptional and protein level in human internal thoracic artery smooth muscle cells (HITASY), which express different patterns of differentiation markers after serum withdrawal. METHODS: After cloning and recombining the CREG vector, the antiserum against the CREG protein was produced from the rabbits immunized by the purification CREG protein. The specificity of purified polyclonal antibody was detected by Western blot assay. The DNA synthesis of HITASY cultured in serum-free and serum-supplemented medium was measured by the [(3)H]-thymidine incorporation. Western blot analysis detected the expression of smooth muscle-specific markers (smooth muscle alpha-actin, calponin). The localization of CREG in cells was examined with immunohistochemistry staining and expression of CREG mRNA and protein were analyzed by RT-PCR and Western blot in HITASY after serum withdrawal. RESULTS: The high specificity of polyclonal antibody against CREG obtained from rabbits was confirmed by Western blot assay. In response to serum withdrawal, cultured HITASY cells exhibited phenotypic conversion from synthetic into contractile one as evidenced by the data of [(3)H]-thymidine incorporation and Western blot. The DNA synthesis of HITASY precipitously dropped to background levels after serum withdrawal and nearly restored after reintroduction of serum to culture medium 2 days later. Western blot revealed a reversible upregulation of smooth muscle alpha-actin and calponin in HITASY after serum deprivation. Moreover, serum withdrawal also induced a prominent increase of CREG mRNA and protein expression which reached a peak on 3 days and decreased gradually on 5 approximately 7 days after serum withdrawal. Immunohistochemistry stain indicated the CREG protein mainly localizes in a perinuclear pattern in HITASY cells. CONCLUSIONS: Those data provide evidence that the coordinated changes in CREG gene and protein expression as well as smooth muscle-specific markers may take place in connection with the process of phenotypic modulation of VSMC in vitro.

Actins↗
PubMed2005

Automated assessment of blood pressure using BpTRU compared with assessments by a trained technician and a clinic nurse.

OBJECTIVE: To determine the accuracy and reproducibility of a new automated blood pressure manometer (BpTRU) relative to auscultatory blood pressure assessed by a research nurse and to that assessed by a clinic nurse. METHODS: Firefighters in a cohort study had blood pressure assessed on up to five occasions with BpTRU and by a trained research technician. Patients in an internal medicine clinic had blood pressure assessed by the clinic nurse and by BpTRU. The absolute values of blood pressure, reproducibility and effect on hypertension classification were compared with the different methods. RESULTS: The research technician readings were higher than the BpTRU readings at visit 1 (3.0/2.7 mmHg, P<0.0001) but the readings converged by visits 4-5 because of a greater reduction in the research nurse readings. The BpTRU readings had similar reproducibility and classification of hypertension as the research technician but did not exhibit terminal digit preference while the research technician readings did. The BpTRU had substantially lower readings (8/7 mmHg) and fewer hypertensive readings than those of the nurse in the internal medicine clinic. CONCLUSIONS: This preliminary study found that the BpTRU had desirable characteristics that suggest that it would be a suitable replacement for auscultatory assessment of blood pressure in clinical practice. A large confirmatory study performed in a usual clinic setting is required.

Adult↗
PubMed2005

Diffuse panbronchiolitis in China.

OBJECTIVE: Diffuse panbronchiolitis (DPB) is a progressive inflammatory disease, well recognized in Japan, that is characterized by chronic sinusitis and obstructive small airway disease. A total of 40 patients with DPB in mainland China were reviewed in order to describe the epidemiology of DPB in China and to compare their clinical characteristics with those of cases reported in Japan. METHODOLOGY: A systematic search was conducted of the Chinese language literature published in mainland China from 1996 to 2003. The 40 subjects who fulfilled the diagnostic criteria for DPB were retrospectively analysed for clinical, physiological, radiological, and pathological features and their geographic distribution. RESULTS: The 40 patients included in this study (31 male and nine female) were distributed in 12 provinces and three other cities in China. All presented with chronic cough, sputum production, and 32 had exertional dyspnoea. All but three had a history of sinusitis. Lung function assessment showed a mixed obstructive-restrictive pattern in 30 cases. CXR revealed diffuse fine nodular shadows in all patients. Human leukocyte antigen-B54 was positive in five patients (n = 12). A total of 30 (75%) patients had been misdiagnosed prior to their diagnosis of DPB being made. CONCLUSIONS: DPB is not rare in China but its incidence is relatively low. Poor recognition and/or genetic factors may be the reason. The clinical, radiographic and histological features of Chinese patients resemble those described in Japanese patients.

Adult↗
PubMed2005

Numerical modeling of the sound fields in urban squares.

This paper studies the basic characteristics of sound fields in urban squares surrounded by reflecting building façades and the effectiveness of architectural changes and urban design options. A radiosity model and an image source model are developed, and a parametric study is carried out in hypothetical squares. The results show that the reverberation time (RT) is rather even in a square, whereas the early decay time (EDT) is very low in the near field, and then becomes close to RT after a rapid increase. Compared to diffuse boundaries, with geometrical boundaries the RT and EDT are significantly longer and the sound pressure level (SPL) attenuation with distance is generally smaller unless the height/side ratio is high. With a boundary diffusion coefficient of 0.2, the sound field is already close to that resulting from purely diffusely reflecting boundaries. The SPL in far field is typically 6-9 dB lower if the square side is doubled; 8 dB lower if the height of building façades is decreased from 50 m to 6 m (diffuse boundaries); 5 dB (diffuse boundaries) or 2 dB (geometrical boundaries) lower if the length/width ratio is increased from 1 to 4; and 10-12 dB lower if the boundary absorption coefficient is increased from 0.1 to 0.9.

Journal Article↗
PubMed2005

Functional interaction of H2AX, NBS1, and p53 in ATM-dependent DNA damage responses and tumor suppression.

Ataxia-telangiectasia (A-T) mutated (ATM) kinase signals all three cell cycle checkpoints after DNA double-stranded break (DSB) damage. H2AX, NBS1, and p53 are substrates of ATM kinase and are involved in ATM-dependent DNA damage responses. We show here that H2AX is dispensable for the activation of ATM and p53 responses after DNA DSB damage. Therefore, H2AX functions primarily as a downstream mediator of ATM functions in the parallel pathway of p53. NBS1 appears to function both as an activator of ATM and as an adapter to mediate ATM activities after DNA DSB damage. Phosphorylation of ATM and H2AX induced by DNA DSB damage is normal in NBS1 mutant/mutant (NBS1m/m) mice that express an N-terminally truncated NBS1 at lower levels. Therefore, the pleiotropic A-T-related systemic and cellular defects observed in NBS1m/m mice are due to the disruption of the adapter function of NBS1 in mediating ATM activities. While H2AX is required for the irradiation-induced focus formation of NBS1, our findings indicate that NBS1 and H2AX have distinct roles in DNA damage responses. ATM-dependent phosphorylation of p53 and p53 responses are largely normal in NBS1m/m mice after DNA DSB damage, and p53 deficiency greatly facilitates tumorigenesis in NBS1m/m mice. Therefore, NBS1, H2AX, and p53 play synergistic roles in ATM-dependent DNA damage responses and tumor suppression.

Animals↗
PubMed2005

Human patient simulation is effective for teaching paramedic students endotracheal intubation.

OBJECTIVES: The primary purpose of this study was to determine whether the endotracheal intubation (ETI) success rate is different among paramedic students trained on a human patient simulator versus on human subjects in the operating room (OR). METHODS: Paramedic students (n = 36) with no prior ETI training received identical didactic and mannequin teaching. After randomization, students were trained for ten hours on a patient simulator (SIM) or with 15 intubations on human subjects in the OR. All students then underwent a formalized test of 15 intubations in the OR. The primary outcome was the rate of successful intubation. Secondary outcomes were the success rate at first attempt and the complication rate. The study was powered to detect a 10% difference for the overall success rate (alpha = 0.05, beta = 0.20). RESULTS: The overall intubation success rate was 87.8% in the SIM group and 84.8% in the OR group (difference of 3.0% [95% confidence interval {CI} = -4.2% to 10.1%; p = 0.42]). The success rate on the first attempt was 84.4% in the SIM group and 80.0% in the OR group (difference of 4.4% [95% CI = -3.4% to 12.3%; p = 0.27]). The complication rate was 6.3% in the SIM group and 4.4% in the OR group (difference of 1.9% [95% CI = -2.9% to 6.6%; p = 0.44]). CONCLUSIONS: When tested in the OR, paramedic students who were trained in ETI on a simulator are as effective as students who trained on human subjects. The results support using simulators to teach ETI.

Adolescent↗
PubMed2005

[Cell apoptosis and the change of Fas/FasL gene in experimental pulmonary fibrosis].

OBJECTIVE: To investigate the changes and significance of cell apoptosis and Fas/FasL gene in pulmonary fibrosis. METHODS: Forty mice were divided into two groups randomly, each group contained twenty mice. TUNEL, immunohistochemistry and in situ hybridization were used to detect the change of cell apoptosis, Fas/FasL mRNA and protein in mice with pulmonary fibrosis caused by bleomycin. RESULTS: The apoptosis index of lung cells in the pulmonary fibrosis group (55.3 +/- 12.2) was higher than that of control group (4.7 +/- 1.0, t = 13.06, < 0.01). The expression of Fas/FasL mRNA (175.8 +/- 21.6, 5.2 +/- 1.6) and protein (956 +/- 96, 285 +/- 76) in the pulmonary fibrosis group was higher than that of control group (mRNA: 26.6 +/- 1.9, 0.5 +/- 0.4, t = 21.7, 8.79, all < 0.01; protein: 491 +/- 96, 100 +/- 18, t = 5.03, 12.81, < 0.01). CONCLUSION: The apoptosis index of lung cells, Fas/FasL genes and protein were up-regulated in lung tissue of pulmonary fibrosis, which may play an important role in the development of disease.

Animals↗
PubMed2005

[Evaluation of the clinical efficacy and the safety of salmeterol/fluticasone propionate accuhaler compared to budesonide turbuhalar in the control of adult asthma].

OBJECTIVE: To evaluate the efficacy and the safety of low dose salmeterol/fluticasone (SM/FP) combination therapy as compared to morate dose of budesonide (BUD) in the management of adult asthma. METHODS: A multicentre, randomised, open-label, parallel-group, 6-week treatment study was conducted. 398 patients (18 - 70 years) were given SM/FP (50/100 microg) twice daily via Accuhaler or BUD 400 microg twice daily via Turbuhaler. RESULTS: The morning and the evening peak expiratory flow (PEF) measurements both increased significantly (P < 0.01) in the SM/FP group, and the increase was greater than that in the BUD group. The significant benefit of SM/FP was evident from the first week. SM/FP led to a more significant reduction in the use of rescue medication and in the day- and night-time asthma symptom scores, as compared to budesonide. Both treatments were well tolerated, and the adverse reactions showed no significant difference between the two groups. CONCLUSIONS: Combination use of low doses of SM/FP is a better choice for the control of asthma. The addition of a low-dose long-acting beta(2) agonist is superior to the simple increase of the dosage of inhaled corticosteroids.

Adolescent↗
PubMed2005

[Serum resistin and leptin in patients with chronic obstructive pulmonary disease and their relationship to nutritional state].

OBJECTIVE: To investigate the serum resistin and leptin levels, their relationship to nutritional state and the associated factors in patients with chronic obstructive pulmonary disease (COPD). METHODS: The serum resistin and leptin levels in 57 stable COPD patients and 31 healthy controls were measured by enzyme-linked immunosorbent assay (ELISA) and radio-immunoassay respectively. Correlated factors of serum resistin and leptin were analyzed. RESULTS: The serum resistin and leptin levels in COPD patients [(2.1 +/- 1.2), (0.65 +/- 0.41) microg/L] were significantly lower than those in the healthy controls [(3.6 +/- 2.3), (1.03 +/- 0.71) microg/L, all P < 0.01]. The serum resistin and leptin levels in patients with malnutrition [(1.7 +/- 0.7), (0.43 +/- 0.16) microg/L] were significantly lower than those in patients without malnutrition [(2.2 +/- 1.2), (0.73 +/- 0.48) microg/L, all P < 0.05]. The serum resistin level in the patients was correlated with the serum leptin level, forced expiratory volume in one second (FEV(1)), and FEV(1)/forced vital capacity (FVC) (r = 0.426 - 0.531, all P < 0.01). The serum leptin level in the patients was correlated with serum resistin, FEV(1)/FVC, body mass index (BMI), percentage of ideal body weight (IBW%), chest circumference and abdominal circumference (r = 0.371 - 0.580, all P < 0.01). CONCLUSION: The serum resistin and leptin levels in stable COPD patients were significantly lower, especially in patients with malnutrition.

Adult↗
PubMed2005

[Expression, antibody production and bioactivity detection of cellular repressor of E1A-stimulated gene].

AIM: To obtain the human cellular repressor of E1A-stimulated gene (hCREG) protein and the polyclonal antibody against the hCREG, and to further observe the expression and localization of hCREG protein in human internal thoracic artery cells (HITASY). METHODS: The hCREG cDNA was amplified by PCR and cloned into the pGEX-4T-1. Glutathione-S-transferase (GST)-hCREG fusion protein was expressed in E.coli BL21 and was used to immunize rabbits to obtain anti-hCREG serum, which was purified by protein A and GST immobilized on glutathione-Sepharose beads. The titer and specificity of polyclonal antibodies were determined by ELISA and Western blot analysis. The expression and localization of hCREG protein were detected with immunofluorescence staining in HITASY cells after serum removal. The proliferation of HITASY cells affected by hCREG protein was examined by means of BrdU stain. RESULTS: It was confirmed that hCREG cDNA was correctly inserted into the vector by endonuclease digesting and DNA sequencing. The expressed GST-hCREG protein was purified by the gel-filtration and its purity was up to about 90%. The anti-hCREG polyclonal antibody was specific with high titer (>1:10(5)) and the hCREG protein was expressed in a perinuclear pattern in HITASY cells after serum deprivation. It was also observed that the proliferation of HITASY cells was obviously inhibited by hCREG protein. CONCLUSION: The hCREG protein was highly expressed in HITASY cells and inhibited HITASY cell proliferation, suggesting that it may be involved in the process of proliferation and differentiation of vascular smooth muscle cells.

Blotting, Western↗