2005年 · 第118卷第09期

Abstract：Background The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CLpro, following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain.Methods The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein.Results The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7 cells successfully. The result of sequencing and sequence comparison with other SARS-CoV strains showed that nsp2 gene was relatively conservative during the transmission and total five base sites mutated in about 100 strains investigated, three of which in the early and middle phases caused synonymous mutation, and another two base sites variation in the late phase resulted in the amino acid substitutions and secondary structure changes. The three-dimensional structure of the nsp2 protein was successfully constructed. Conclusions The results suggest that polymerase nsp2 is relatively stable during the phase of epidemic. The amino acid and secondary structure change may be important for viral infection. The fact that majority of single nucleotide variations (SNVs) are predicted to cause synonymous, as well as the result of low mutation rate of nsp2 gene in the epidemic variations, indicates that the nsp2 is conservative and could be a target for anti-SARS drugs. The three-dimensional structure result indicates that the nsp2 protein of GD strain is high homologous with 3CLpro of SARS-CoV urbani strain, 3CLpro of transmissible gastroenteritis virus and 3CLpro of human coronavirus 229E strain, which further suggests that nsp2 protein of GD strain possesses the activity of 3CLpro。

Abstract：Background Severe acute respiratory syndrome (SARS) is an infectious disease caused by SARS-CoV. There are no effective antiviral drugs for SARS although the epidemic of SARS was controlled. The aim of this study was to develop an RNAi (RNA interference) approach that specifically targeted the N gene sequence of severe acute respiratory syndrome associated coronavirus (SARS-CoV) by synthesizing short hairpin RNA (shRNA) in vivo, and to assess the inhibitory effect of this shRNA on SARS-CoV N antigen expression. Methods The eukaryotic expression plasmid pEGFP-C1-N, containing SARS-CoV N gene, was co-transfected into 293 cells with either the RNAi plasmid pshRNA-N or unrelated control plasmid pshRNA-HBV-C4. At 24, 48 and 72 hours post transfection, the green fluorescence was observed through a fluorescence microscope. The RNA levels of SARS-CoV N were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression of Green Fluorescent Protein (GFP) and protein N were detected using Western blot.Results The vector, pshRNA-N expressing shRNA which targeted the N gene of SARS-CoV, was successfully constructed. The introduction of RNAi plasmid efficiently and specifically inhibited the synthesis of protein N. RT-PCR showed that RNAs of N gene were clearly reduced when the pEGFP-C1-N was cotransfected with pshRNA-N, whereas the control vector did not exhibit inhibitory effect on N gene transcription.Conclusions Our results demonstrate that RNAi mediated silencing of SARS-CoV gene could effectively inhibit expression of SARS-CoV antigen, hence RNAi based strategy should be further explored as a more efficacious antiviral therapy of SARS-CoV infection。

Abstract：Background Illegal plasma collecting activities in mid 1990s caused a large number of human immunodeficiency virus (HIV) infections in rural areas of central-eastern China. Although most of these activities have been stopped, there were few reports on secondary transmission from infected former plasma donors to their spouses and from infected mothers to their children. This study was to determine the extent of HIV infections among young children in a rural community with a large proportion of plasma donors.Methods A survey was conducted among children aged under 7 years in a former plasma donating community in September 2000: finger blood was collected for HIV antibody testing. Another survey was repeated among children aged under 8 years and their families in the same community in April 2001: urine samples were collected for HIV testing. HIV positive children and samples of HIV negative children, whose mothers were positive based on 2001 survey, were followed up until September 2002 to investigate HIV seroconversion, disease progression and HIV strain analysis. Questionnaires were administered to collect information on children's delivery, breast feeding, medical history and their parents' commercial blood donation history and HIV status.Results Among 169 children surveyed in 2000, 10 (5.9%) were HIV positive. Of 224 children, 11 were positive in 2001. The overall prevalence rate in the two surveys was 5.0% (17/337) when counting 56 repeated children only once. Of children born to HIV positive mothers, 28.9% were infected. A seroconversion rate of 2.5 per 100 child-years was observed by following up 28 HIV negative children. No statistically significant associations were found between children's HIV infection and their histories of blood transfusion, surgery, immunization injection or medical injections. All infections were HIV-1 subtype B' strain, the average dispersion rate is 7.4%. DNA sequence analysis showed a close relationship between the seroconverted children and their infected mothers.Conclusions HIV vertical transmissions in the rural former plasma donating community was significant. Intervention measures should be taken to prevent further transmission. It was estimated that the HIV spread in this community occurred in 1994 or even earlier. Many infected people are developing AIDS now: treatment and care are urgently needed for these sick people。

Abstract：Background Epstein-Barr virus (EBV) associated malignancies with a Type Ⅱ latency gene expression pattern, such as Hodgkin's disease, and nasopharyngeal carcinoma (NPC), frequently express the EBV antigen latent membrane protein 2A (LMP2A). We expected to establish a highly expressing LMP2A yeast cell strain and get the high quality LMP2A protein, which was used for detection, analysis and characterization of its antibodies in various patients' sera of EBV associated malignancies.Methods The plasmid pPICZαA-LMP2A containing the full length of LMP2A cDNA was constructed and transformed to Pichia pastoris GS115 to express LMP2A protein. After fermentation and purification, the LMP2A protein was used as an antigen to detect anti-LMP2A antibodies (Abs) in the sera of patients with EBV-associated malignancies in enzyme linked immunosorbent assay (ELISA) or Western-blot.Results LMP2A was expressed successfully with an expected molecular weight of approximately 54 kD and Abs to LMP2A were strikingly specific to NPC. Two-thirds or more sera from NPC patients were positive for anti-LMP2A immunoglobulin G (IgG) Abs. The antibodies were absent from the sera of other EBV-associated diseases except a small fraction of the gastric carcinoma. Comparing anti-viral capsid Ags (VCA) IgA and LMP2A IgA titers in the sera from 76 NPC patients, only 55% were positive for anti-LMP2A IgA Abs while 70% were positive for anti-VCA IgA. However, we found that 3 sera negative for VCA IgA were positive for LMP2A IgA.Conclusion The results suggested the potential significance of LMP2A specific Abs for the diagnosis of EBV-associated malignancies, especially NPC。

Abstract：Background Chronic heart failure (CHF) is associated with calcium transients and calcium handling proteins. Angiotensin converting enzyme (ACE) inhibitor has been demonstrated to have beneficial effect on CHF. Yet studies addressed to the relationship between ACE inhibitor and calcium transients in CHF are rare. The aim of this study was to investigate the influence of ACE inhibitor (perindopril) on the contractility and calcium transients and calcium handling proteins in ventricular myocytes from rats with experimental heart failure.Results The fraction of cell shortening (FS%) and [Ca2+]imax (nmol/L) were significantly reduced in group CHF-C compared with group PS (FS%: 7.51±1.15 vs 13.21±1.49;[Ca2+]imax:330.85±50.05 vs 498.16±14.07; both P<0.01), and restored at least partially in CHF-T group. In CHF-C group, the left ventricular mRNA of NCX1 and PLB were significantly upregulated in comparing with PS group (RNCX1/β-Actin: 0.51±0.12 vs 0.19±0.06, P<0.01; RPLB/β-Actin: 0.26±0.12 vs 0.20±0.08, P<0.05), while SERCA2 mRNA was downregulated (0.48±0.10 vs 0.80±0.11, P<0.01). The mRNA levels of NCX1 and SERCA2 in CHF-T group were between the CHF-C and PS group, and the differences of the latter two groups were significant (all P<0.05). In CHF-C and CHF-T groups, the protein expression of NCX1 were 1.141±0.047 and 1.074±0.081 times of that in PS group respectively (both P<0.05), and SERCA2 protein levels were 0.803±0.100 and 0.893±0.084 times of that in PS group respectively (both P<0.05). The protein expression of NCX1 and SERCA2 in the CHF-C and CHF-T groups is significantly different (both P<0.05).Conclusion ACE inhibitor could improve cardiac function of failing heart through directly enhancing the contractility of single cardiomyocyte, and these effects are probably mediated by its roles in preventing the deleterious changes of calcium transients and calcium handling proteins in CHF。

Abstract：Background γD-crystallin plays an important role in human cataract formation. Being highly stable, γD-crystallin proteins are composed of two domains. In this study we constructed and analyzed protein models of the mutant γD-crystallin gene, which caused a special fasciculiform congenital cataract affecting a large Chinese family. Methods γD-crystallin protein structure was predicted by Swiss-Model software using bovine γD-crystallin as a template and Prospect software using human βb2-crystallin as a template. The models were observed with a Swiss-Pdb viewer.Results The mutant γD-crystallin structure predicted by the Swiss-Model software showed that proline23 was an exposed surface residue and P23T change made a decreased hydrogen bond distance between threonine23 and asparagine49. The mutant γD-crystallin structure predicted by the Prospect software showed that the P23T change exerted a significant effect on the protein's tertiary structure and yielded hydrogen bonds with aspartic acid21, asparagine24, asparagine49 and serine74.Conclusion The mutant γD-crystallin gene has a significant effect on the protein's tertiary structure, supporting that alteration of γ-crystallin plays an important role in human cataract formation。

Abstract：Background At present, the therapy for patients with lung cancer that achieves a high rate of cure is surgical resection at an early stage of the disease. The aim of this study is to evaluate quantitative computed tomography (QCT) for predicting postoperative pulmonary function in patients with lung cancer. Methods The data of thirty-one patients with lung cancer who underwent both pulmonary functional tests and QCT scan before operations were collected. A CT program was used to quantify the volume of whole lung parenchyma with attenuation of -910 HU to -600 HU, which was defined as total functional lung volume (TFLV). Similarly, the volume of lung (lobes or segments) with attenuation of -910 HU to -600 HU was defined as regional functional lung volume (RFLV). Forced vital capacity (FVC), forced expiratory volume in first second (FEV1), FVC% and FEV1% (ratio to reference values of the matched population) were obtained from preoperational pulmonary functional tests. According to the formula: predicted FVC (pre-FVC)=preoperative FVC×[1-(RFLV/TFLV)]; predicted FEV1 (pre-FEV1)=preoperative FEV1×[1-(RFLV/TFLV)], we obtained values of predicted FVC, predicted FEV1, predicted FVC% (pre-FVC/reference values of the matched population), and predicted FEV1% (pre-FEV1/reference values of the matched population). The paired t test and Pearson correlation test were used to assess significance of differences and correlations between CT predicted values and postoperative measured results of FVC, FEV1, FVC% and FEV1%. Results QCT predicted values correlated well with postoperative FVC, FEV1, FVC% and FEV1% (r=0.873, 0.809, 0.849 and 0.801 respectively, all P<0.01).Conclusions QCT is an effective and accurate way to predict postoperative pulmonary function in patients undergoing pulmonary resection, regardless of the patients' preoperative pulmonary functional status。

Abstract：Background Numerous studies from Europe and North America have provided a wealth of information regarding the epidemiological and clinical characteristics of inflammatory bowel disease (IBD) in Caucasians. Previous studies in mainland China have been limited by small patient numbers or by lack of detailed information about clinical subgroups of the disease. This study was carried out to assess the demographic and clinical characteristics of IBD in Chinese patients. Methods In the Sir Run Run Shaw Hospital between 1994 and 2003, 379 patients were diagnosed as IBD. Demographic and clinical data were collected and analysed. Conclusions This study shows similar caracteristics of IBD to that in the West but there are some differences with respect to severity and extraintestinal manifestations. The ethnic and geographic differences may give important clues to the aetiology of IBD。

Abstract：Background The YIGSR is a pentapeptide, from the laminin-1 of the β1 chain, which can mediate cell adhesion and bind the 67 kD laminin receptor. The purpose is to evaluate the usefulness of 99mTc-YIGSR, a novel tumour radiotracer, in the receptor imaging of Ehrlich ascites tumour.Methods Using S-Acetly-NH3-MAG3 as chelate, YIGSR, a pentapeptide from laminin, was tagged with 99mTc. 99mTc-YIGSR was detected in the tumour group bearing Ehrlich ascites tumour and blocked group. Tumour, normal, inflammatory and blocked groups were imaged. Results Through reverse phase Sep-Pak C18 chromatogram, it was revealed that YIGSR could conjugate with S-Acetly-NH3-MAG3, and be radiolabelled at room temperature and neutral pH with a radiolabelling yield of 62%, and of 4% without chelate. 99mTc-YIGSR was rapidly cleared from kidney, then liver. The imaging findings showed tumour tissue accumulated initial radioactivity at fifteen minutes after injection in the tumour group, and the uptake increased to peak at three hours with a tumour/muscle ratio (T/M) of 11.36, then cleared slowly to a T/M of 7.50 at eight hours. The tumour uptake of radiotracer in blocked group was significantly lower with T/M of 4.61 at three hours and 0.89 at eight hours. The T/M was only 3.72 at three hours and 1.29 at eight hours after injection in inflammatory group. Compared with inflammatory group and control obstructive group, the ratio of T/M in tumour group was significantly different (P<0.001). Conclusions Using S-Acetly-NH3-MAG3, we radiolabelled YIGSR with 99mTc. 99mTc-YIGSR possesses many merits of tumour imaging: rapid visualization, high sensitivity and specificity, and satisfactory target/nontarget ratio. Our data suggest 99mTc-YIGSR is a promising tumour radiotracer.

The earliest investigations of parasitic diseases in China occurred in the 1870s and began with the work done by some foreign doctors studying Chinese customs, e.g., Monson ' s discovery in Xiamen of China in 1877, reporting for the first time that the mosquito was the intermediate host and vector of Wuchereria bancrofti。

Tuberculosis (TB) is the most common cause of death in infectious diseases; it is estimated that approximately 2 million people per year die of TB. The present available TB vaccine is a live attenuated strain, Mycobacterium bovis Bacillus Calmette Guérin (BCG). However, it has been shown that BCG has variable protective efficacy, ranging from 0 to 85% in different clinical experiments.1 Therefore, a new TB vaccine is urgently needed。

Haemorrhagic fever with renal syndrome (HFRS) is a worldwide epidemic plaguing over thirty nations. This disease has spread across 26 provinces, cities and autonomous regions of China with an annual onsets number of more than a hundred thousand and a mortality rate of 5% to 15%, accounting for over 80% of cases in the world, and threatening the safety and health of Chinese people.1 Analysis of serum samples over recent years indicates that the plagued areas are expanding。

Lung cancer is the leading cause of cancer-related death in the world as well as in China. It is estimated that approximately 429 000 Chinese individuals may die from lung cancer in 2005, and the mortality rate for lung cancer will double in the next century.1 Currently, chemotherapy is the a main treatment of advanced and recurrent lung cancer. However, the recent development of tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib may change the therapeutic approaches for this disease。

Umbilical-cord blood (UCB) is a source of hematopoietic stem cells that has been successfully used for transplantation, primarily in children. But the low cell dose severely limits the application of unrelated umbilical-cord blood transplantation (UCBT) in adult patients, particularly in those with high body weight. We hypothesized that the combined transplantation of two partially matched UCB units would improve engraftment without crossed immunological rejection. Since May 2000, six adult patients with high risk leukemia have been transplanted in this study。

Mechanical ventilation (MV) is a dual blade sward which if misused could lead to lung injury, called ventilator induced lung injury (VILI). Pathogenesis of VILI is very complex with various manifestations, which is the focus in MV field in recent years.1 In our research, the rats were ventilated with different tidal volume, then the pathological changes of the lungs were observed under macroscopy, light and electronic microscope, and various laboratory tests in blood and bronchoalveolar lavage fluid (BALF) were also carried out in order to probe further the pathologic characteristics and the pathogenesis of VILI。

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis.1 The histopathology of IgAN is characterized by abundance of mesangial matrix and proliferation of mesangial cells. IgA1 deposition in the mesangium plays an important role in the inflammatory process in this disease。

The internal fixation failure for treatment of femoral neck fracture is mainly due to improper fixation, loss of fixation stability and premature ambulation. Bone nonunion and avascular osteonecrosis of the femoral head caused by the internal fixation failure for femoral neck fracture are always the complex topics in orthopedics. With regard to patients who sustain these complications, total hip arthroplasty is a proper choice for elderly patients, but is not acceptable by young patients. We report nine patients with the failure of internal fixation for femoral neck fracture who were treated with free vascularized fibular grafting and internal fixation with cannulated screw from November 2001 to October 2003. All of them achieved good results。

Dyskeratosis congenita (DC) is a severe inherited disease characterized by a triad of clinical manifestations including abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia.1 Bone marrow failure is the principal cause of early mortality, together with an increased predisposition to malignancy and fatal pulmonary complications. According to the dyskeratosis congenita registry, a peripheral blood cytopenia of one or more lineages is reported in 93% of patients, with 51% developing pancytopenia before the age of 10 years.2 In patients with DC, bone marrow failure or bone marrow failure treatment-associated complications account for 67% of total mortality.3 Therefore, management of bone marrow failure syndrome is crucial in patients with DC。