Objective. To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT pro-tein for futher study. Methods. The gene for encoding hTRT catalytic domain was cloned based on RT-PCR amplification from HeLa cells and sequenced. The cloned hTRTcDNA was in-frame inserted into His-tag fusion expression vector pEK318. The His-tag hTRT fusion proteins were purified by Ni-NTA chromatography and stained by westerm blotting. Results. An approximately 620bp fragment was generated and cloned into pBluescript SK + between Sail and BamHI sites. DNA sequencing showed the isolated fragment was consistem to those reported. SDS-PAGE present that a 17kDa protein was expressed stably in E. coli JM109 harboring pEKTRTM4 containing 6 × His-tag and hTRT 150aa, and the expression level of the protein was about 26% of the total bacterial proteins, while the expression of pEKTRT containing 6 × His-tag and hTRT 243aa was only detectable as 27 kDa band in western blotting. Both of fu-sion proteins were purified by Ni-NTA chromatography and showed single band( ＞ 95% purifity) in Coomassie Bril-liant staining. Westem-blotting confirmed that two proteins could be recognized by the Ni-NTA AP conjugate. Conclusions. The hTRT catalytic domain was highly conserved. The expressed hTRT protein contained recogniz-able His-tag, telomerase-specific and strong antigenic epitops, which may be convenient for further investigation.

Objective. The aim of this study is to investigate the functional relationship between filamin, a known actin bind-ing protein, and myosin and the effects of filamin on the interaction between myosin and actin. Methods. Ultra-centrifugation method was used to investigate the binding of filamin to both phosphorylated and unphosphorylated myosins. Mg-ATPase activities of both phosphorylated and unphosphorylated myosins in the presence and absence of aefn were measured to observe the effects resulted from fdamin-actn and filamin-myosin interactions. Results. It was found that fiiamin is also a myosin binding protein. Filamin inhibited the actin activated Mg-ATPase activity of phosphorylated myosin and stimulated Mg-ATPase of phosphorylated myosin in the absence of actin;in addition, filamin stimulated Mg-ATPase activity of unphosphorylated myosin in both the presence or absence of actin.

Objective. The anti-atherosclerotic effect of fluvastatin at doses insufficient to lower serum cholesterol on the catheter-induced intimal thickening and possible mechanism were investigated in abdominal aorta of rabbits. Methods. Fifty-six rabbits were randomly divided into eight groups(n = 7, each). Fluvastatin was given mixed with food at daily dose of 8mg/kg starting 5 days before catheterization, Light microscope, immunohistochemistry, transmis-sion electron microecope and RT-PCR assay were applied to assess vascular smooth muscle cell (VSMC)proliferation and apoptosis, as well as oncogene expression in vascular wall. Results. At day 10 and day 15 after catheter induced denudation intima/media(I/M)thickness ratio was obviously higher, and also the percentage of PCNA-positive cells and TUNEL-positive cells in media was significantly higher compared with controls. The intimal hyperplasia was mostly composed of α-SM-actin-positive cells. In rabbits given flu-vastatin I/M ratio and the percentage of these positive cells significantly decreased compared with those without fluvas-tatin. The overexpression of proto-oncogene H-ras mRNA and decreased expression of anti-oncogene p53 mRNA were found after vascular injury, whereas fluvastatin significantly reduced H-ras mRNA and increased p53 mRNA expres-sion. Conclusion. Proliferation of VSMC in the media and the migration to the intima can be inhibited, and apoptosis of VSMC be induced by short-term use of fluvastatin after balloon catheter denudation, independent of serum lipid change. The underlying mechanism is presumably associated with the influence of fluvastatin on oncogene expression in the injured vascular wall.

Objective. To construct ScFv and Fab from murine anti-gastric cancer monoclonal antibody( mAb)3H11. Methods. At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FRI. The bacterial expressed 3H11 Ab fragments showed no antigen binding activity. Then, phage antibody library andrandom mutated library were constructed from 3H1 1 hybridoma cells and panning selection was performed. Again the i-dentification of positive clone was failed. Finally the N-terminal sequences of V regions were resumed to 3H1 1 original sequences by site-directed mutagenesis via PCR. Results. Binding activity to gastric cancer cells was detected only from N-terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fiagments was not affected. Correction of either VL or VH N-terminal se-quences could partially resume the antigen binding activity. Conclusion. Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody.Received for publication Oct. 26,1998.

Objective. Patients with Graves' disease (GD) have marked lymphocytic infiltration in their thyroid glands. We examined the gene for the variable regions of the α-chain of the Chinese T-cell receptor( Vα gene) in intrathyroidal Tcells to determine the role of T cells in the pathogenesis of GD and offer potential for the development of immunothera-peutic remedies for GD. Methods. We used the reverse transcription and polymerase chain reaction(RT-PCR) to amplify complementary DNA(cDNA) for the 18 known families of the Vα gene in intrathyroidal T cells from 5 patients with Graves' disease.The findings were compared with the results of peripheral blood T cells in the same patients as well as those in normalsubjects. Results. We found that marked restriction in the expression of T cell receptor Vα genes by T cells from the thyroidtissue of Chinese patients with GD(P ＜ 0.001). An average of only 4.6 ± 1.52 of the 18 Vα genes were expressed insuch samples, as compared with 10.4 ± 2.30Vα genes expressed in peripheral blood T cells from the same patients.The pattem of expressed Vα genes differed from patient to patient with no clear predominance. Condusions. Expression of intrathyroidal T cell receptor Vα genes in GD is highly restricted suggesting the prima-cy of T cells in causing the disorders.

Objective. To investigate the effects of coxsackievims B3 (CVB3) on ion channel currents in rat ventricular my-Methods. Rat hearts were isolated with collagenase to acquire single ventricular myocytes, L-type voltnge-depen-dent calcium channel( VDCC)current (Ica), Na + current (INa), outward potassium current (Iout), inwardly rectifying potassium current(IKI) were recorded using whole cell patch clamp techniques. Results. CVB3 infection increased Ica and Iout, while decreased IKI; but it had no obvious effect on INa. Conclusion. Tne effects of CVB3 on Ica、 Iout、 IKI may be one of the mechanisms of myocytes damage and the oc-currence of abnormal electroactivities induced by CVB3 infection.

Objective. To investigate the dynamic changes of serum vascular endothelial growth factor(VEGF) levels in a rat model of acute myocardial infarction. Materials and methods. Eighty-eight adult male Sprague-Dawley rats weighing approximately 270 g were used in this study. Eighty rats were subjected to left coronary artery ligation, with 8 rats for each different duration of infarct.Eight sham-operated animals in which the left coronary artery was surgically exposed without ligation were used as con-trols. Blood samples were drawn from the right atrium before ( sham animals ) and 1,3,6,12,24 h and 2,3,5,7,14 dafter myocardial infarction. The concentrations of serum VEGF were measured by a sensitive enzyme-linked im munosorbent assay with a rabbit polyclonal antibody specific for VEGF. Results. In the 8 control animals, the mean concentration of serum VEGF was 66.99 ± 17.83 pg/ml. Six hours after myocardial infarction, the level of serum VEGF significantly increased to 125.68 ± 28.07 pg/ml ( P ＜0.01 vs.sham controls), and reached a peak (240.61 ± 70.63 pg/ml. P ＜0.01 vs. sham animals) at 24 h after ligation andthen decreased gradually over the remaining 2 weeks. However, the level remained significantly elevated for 14 d(107.64±30.13pg/ml, P＜0.01 vs. sham controls). Condusion. The present study shows that the levels of serum VEGF are markedly increased until 14 d in the ratmodel of acute myocantial infarction. The increased serum VEGF level may play an important role in the angiogencsisassociated with myocardial infarction.

Objective. This study characterized the activation of platelet integrin aⅡbβ3 induced by two anti-human platelet te-traspanin monoclonal antibodies(mAbs),HI117 and SJ9A4. Methods. Using 125I-labeled human fibdnogen(Fg), specific Fg binding to human platelets induced by HI117 and SJ9A4 was measured as indication of activation of platelet integrin αⅡbβ3 by the two mAbs. Results. H1117 and SJ9A4( 10μg/ml and 20μg/ml) induced evident specific Fg binding to human platelets, sug-gesting that the two mAbs evoked activation of platelet integrin αⅡbβ3. Further study indicated that HI117 and SJ9A4 induced integrin t Ⅱ 1β3 activation independent of platelet Fc-receptors, and that HI117 and SJ9A4-induced integrin αⅡbβ3 activation was inhibited by sphingosing, aspirin, apyrase, and/or PGI2. Conclusion. The anti-platelet tetraspanin(CD9)mAbe,HI117 and SJ9A4, can induce platelet integrin αⅡbβ3 act-vation independent of Fc-receptors. Three signaling pathways,i.e.thromboxane,secreted ADP, and cAMP pathways may be involved in the process, with protein kimse C activation presumably being the comtmon step of the three pathways.

Ain. To study the effect of kanglemycin C (KC) on production and gene transcription of lymphokins. Methods. Cell proliferation and lymphokin activities were quantified with MTT colorimetry and ELISA, and genetranscripdons of lymphokins semi-quantified with RT-PCR. Restlts. Suppression of KC on proliferation of enriched T-and B-cell respectively mediated by Con A and LPS was declined by addition of exogenous IL-1, IL-2, and IL-6. KC 80 nmol/L markedly inhibited IL-2 and IL-6 pro-duction and mRNA transcription of incubated mouse splenocytes induced by Con A. Additionally, KC had some sup-pression on IL-1β and IL-6 productions of peritoneal macrophage stimulated by LPS (5 μg/mL), whereas cyclosporine(CS) had not. Condusion. Immunosuppression of KC came true partially through the decrease of IL-1β, -2 and -6 productions,especially of IL-2. However, CS's immunosuppression was mainly through the decrease of IL-2 procduction.

Objective. To evaluate the results of operative treatment of spinal fracture-dislocation without neurologic deficits. Methods. Eighteen patients with spinal fracture-dislocation were neurologically intact at the time of injury, and all were treated operatively. The fracture sites were:8 cases in cervical spine, 3 cases in thoracic spine, and 7 cases in lumbar spine. Eight patients with cervical injuries had variant degrees of forward slide and kyphotic deformity. Of the 10 thoracic and lumbar fracttwes, one had lateral dislocation, 4 cases with kyphotic deformities, 5 cases with spinal canal compromise averaged 50% (ranging from 40% to 70%). Results. The average period of follow-up was 4.4 years with a range of 11 months to 13 years. All the patientsretumed to full-time work. No patient developed neurologic deterioration. Kyphotic deformity was corrected in the 4cases, and no progressive kyphosis was noted. There was no operation-related complication. The averaged post-opera-tive hospitalization time was 13 days. Conelusions. Despite the rare incidence of spinal fracture-dislocation without neumlogic deficits, we suggested that kind of fracture be considered unstable fracture because of its potential risk of delayed neurologic deterioration and kyphotic deformity, and be treated operatively to restore the sagittal ali~ment and the stability of the spine.

Objective. To observe the effects of closed reduction and pereutaneous K-wires fixation of displacd supracondylar humnerus fracture in children. Methods. Retrospective review of fourteen patients who sustained displaced supmcondylar fracture of distal humerus treated by closed reduction and percutaneous K-wires fixation. Results. All patients' K-wires were removed at 4 weeks post-operation. Their elbow function regained at 8weeks. The average period of followed up was 10 month (varies from 6 to 18 month), all fractmes healed very well without any permanent complications. Two transient nerves palsy, ulnar and radial nerve each, recovered completely at12 weeks and 16 weeks post-operation respectively. Conclusion. Closed reduction and percutaneous K-wires fixation is a safe and efficient treatment for displaced humerus surpracondylar fracture in children.

Background. Our previous studies indicated that the increased arginine vasopressin(AVP) in ischemic brain regions of gerbils could exacerbate the ischemic brain edema. This experiments is further clarify the relation between AVP and cerebral ischemia at the molecular level. Methods. The contents of AVP, AVP mRNA, AVP immunoreactive(ir) neurons in supraoptic nucleus(SON)and paraventricular nucleus(PVN) after cerebral ischemia and reperfusion were respectively determined by radioim-munoassay(RIA), immunocytochemistry( Ⅱ C), situ hybridization and computed image pattem analysis. Results. The contents of AVP in SON, PVN were increased, and the AVP ir positive neurons in SON and PVN were also significantly increased as compared with the controls after ischemia and reperfusion. And there were very light staining of AVP ir positive neurons in the other brain areas such as suprachiasmatic nucleus (SC) and periven-tricular hypothalamic nucleus (PE), but these have no significant changes as compared with the controls. During dif-ferent periods of cerebral ischemia (30～ 120 min) and reperfusion (30 min), AVP mRNA expression in SON and PVN were more markedly increased than the controls. Condusions. The transcription of AVP gene elevated, then promoting synthesis and release of AVP in SON,PVN. Under the specific condition of cerebral ischemia and repeffusion, the activity and contents of central AVP in-creased abnormally is one of the important factors which causes ischemia brain damage.

Objective. To undertake the pilot experiments of prevention of disability (POD) in 14 different geographical areas to serve as examples for future development of rehabilitation work in China and in Asia. Methods. According to the principles and national criterion, 27 000 people aflected by leprosy were selected and assessed using disability record forms at beginning and followed up regularly for observing changes of different indica-tors. Results. A total of 197 neuritis cases were detected and treated with prednisolone out of 1 407 new or active cas-es. Self-care training of eyes, hands and feet were conducted for 10 500 disabled people affected by leprosy. Compre-hensive therapy was given to 1 804 cases having complicated ulcers of which 1 055 cases have got their ulcers healed.Out of 706 prostheses, 613 were given to patients with satisfactory results. Surgical treatment was given to 269 cases and 251 have shown good progress. Conclusion. Most of patients have got benefit from the project in function or appearance which is very helpful for their going back to the society and agreed by foreign experts during the final evaluation. The experiences from the pro-ject can be implemented in the whole country.

Objective. To investigate the cold preservation effect of rat livers by modified storage method with self-made HYD solution. Methods. The modified method was that the vascular bed of rat livers was expanded with an additional 20 to 40ml self-made HYD solution/100g liver. After removing the liver, the extra HYD solution expressed as % liver weight was entrapped via portal infusion by tying off the supra-and infra hepatic inferior vena cava. According to the amount of extra HYD solution, 40 rats were randomly divided into four groups including: control group with conventional storage method, 20% group, 30% group and 40% group. The preservation effect of modified storage method with that of conventional storage method by using isolated perfused rat liver model was compared. Results. Bile production and all the indices of hepatic microcirculation including portal perfusion pressure, en-dothelin-1 in the effluent, trypan blue distribution time and histology in modified method groups were significantly su-perior to those in control group ( P ＜ 0.05). The liver enzymes in 30% group were markedly lower than those in con-trol group (P＜ 0.05). The preservation effect of rat hver in 30% group was the best among the modified methodgroups. Conclusion. The modified cold storage method is effective and may have potential for clinical apphcation for hverpreservation.

The purpose of this study was to determine whether this normal ambulatory blood pressure (ABP) criterion deter-mined by JVC-VI was suitable for Chinese patients with es-sential hypertention (EH), in order to decrease target organ damage (TOD). 24-hour ABP monitoring (ABPM) results in 1 325 cases with reliable data according with the selected criteria were investigated. 106 normotensives (65 men, 41women, mean age 52 years), 498 untreated simple hyper-tensives (288 men, 210 women, mean age 54 years) and 722 recently untreated hypertensives with TOD (490 men,232 women, mean age 58 years) including 53% cardiac damage, 16% cerebral damage, 9% renal damage and 22 % more than one organ damage were studied.

This study assesses the safety and efficacy of direct in-tracoronary stenting in acute phase of myocardial infarction.