BACKGROUND: In recent years, foreign scholars have found that apolipoprotein E gene was associated with osteoporosis and its complicated hip fracture. National scholars adopted polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to primarily study the correlation of apolipoprotein E gene polymorphism with bone mineral density (BMD) and osteoporosis.OBJECTIVE: To observe the polymorphism of apolipoprotein E gene, and analyze its correlation with primary osteoporosis and osteoporotic fracture.SETTING: Department of Orthopaedics, Nanchang First Hospital.DESIGN: A clinical controlled observation.PARTICIPANTS: Sixty patients with primary osteoporosis (31 males and 29 females), were enrolled, including 30 cases of hip fracture, who were newly fractured and diagnosed according to radiogram, and 30 cases of simple osteoporosis; Thirty healthy subjects (16 males and 14 females)were also involved.METHODS: Sixty outpatients and inpatients with primary osteoporosis were selected from the Department of Orthopaedics, Nanchang First Hospital from January to December 2002, and they were divided into hip fracture group (n=30) and simple osteoporosis (n=30); Another 30 healthy physical examinees were taken as the healthy control group. All the subjects were fasted for more than 12 hours, and the samples of venous blood were collected on the next morning. DNA was extracted from the separated leucocytes. Gene site DNA amplification was analyzed with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The individual DNA templates (provided by Daan Gene Diagnosis Center of Sun Yat-sen University) of identified apolipoprotein E2/2, E3/3 and E4/4 homozygotes were analyzed with PCR-SSCP, and the band showed that E3 was in the front, E2 in the middle, followed by E4. They were in electrophoresis with DNA templates of the samples at the same time, and the apolipoprotein E genotype of the template could be identified by the migration rate of the band.MAIN OUTCOME MEASURES: Apolipoprotein E genotypes were observed.RESULTS: All the enrolled subjects were involved in the analysis of results. ① Six genotypes were detected with PCR-SSCP in the 60 patients with primary osteoporosis and 30 healthy controls, including apolipoprotein E2/2, E3/2, 3/3, E4/2, 4/3, 4/4. ② There were very significant differences in the allele frequency of apolipoprotein E4 among the three groups (χ2=17.520, P ＜ 0.01). The allele frequencies of apolipoprotein E4 in the simple osteoporosis group and osteoporotic fracture group were obviously higher that that in the normal control group (χ2=4.904, 16.681, P ＜ 0.01),it was also markedly higher in the osteoporotic fracture group than in the simple osteoporosis group (χ2=4.658, P ＜ 0.01).CONCLUSION: Apolipoprotein E4 is closely associated with primary osteoporosis and osteoporotic fracture. Apolipoprotein E is a useful marker for primary osteoporosis, especially for osteoporotic fracture.

Metallic cancellous screw is one of the fixation devices widely used for bone fractures. It has always been reported that many cases of internal fixation failed just because of screw loosening or pullout. The stability of fixation is associated with the pullout strength of screws. The pullout strength strongly depends on the geometrical design of the screw thread, the material and the insertion technique of the screw, and the bone mineral density of the insertion site of the screw.

BACKGROUND: Biological characteristics of porcine cardiovascular system are similar to that of human. The metabolism, immune system, mechanism of disease have 99% homology between pig and human. OBJECTIVE: To develop ventricular aneurysm animal models by plugging left anterior descending branch. DESIGN, TIME AND SETTING: The animal observational study was performed at the Laboratory of Functional Material and Animal Experimental Center, Tongji Hospital Affiliated to Tongji University from December 2005 to July 2007. MATERIALS: A total of 13 pigs, of both genders, weighing 30-40 kg, were used in this study. METHODS: After abdominal cavity and intravenous anesthesia, balloon with "WYW" stent was put into 13 pig left anterior descending coronary artery following No. 7 artery sheathing canal was implanted. The balloon was dilated and the stent was positioned into the distal point of the first diagonal branch to obstruct the artery under digital subtraction angiography. During and after the operation, electrocardiogram was monitored and recorded to maintain stable vital sign. MAIN OUTCOME MEASURES: Electrocardiogram, serum myocardial enzymes, myocardial radionuciide imaging, echocardiography and angiography and pathological changes were observed. RESULTS: One rat died of anesthetic accident before surgery. Six rats died from ventricular fibrillation during plugging, and the other 6 rats were survival. 4 weeks following surgery, coronary angiography showed blood flow at distal end was blocked 100%. Left ventricle angiography demonstrated that wall motion at apex of the heart and the left ventricle disappeared. Before embolism, electrocardio-monitoring displayed electrocardiogram was normal. After plugging, ST segment was raised continuously; amplitude of R wave was decreased; T wave was erected; ST-T fusion wave appeared; precordial leads were obvious. ST segment was reduced to basic level 2 week later, and pathological Q wave appeared 4 weeks later. Serum treponin was increased at 12 hears following surgery compared with before surgery (P < 0.01). Radioactive nuclide myocardial imaging revealed that cardiac apex and left ventdcle anterior wall showed radioactive nuclide perfusion filling defect, thin ventricular wall and ventricular aneurysm. Cardiac ultrasonic showed six pigs suffered from weaken segmentation contraction motor of local ventricular wall, especially at anterior wall of the left ventricle and cardiac apex. After hematoxylin-eesin staining, optical microscope exhibited that cardiac muscle fiber disappeared, which was replaced by collagen fiber, with the presence of some capillary at the infarct of cardiac apex. Pyknosis and lysis of residual cardiac muscle fiber were visible in infarct region of anterior wall of the right ventricle. Many inflammatory cells and capillary infiltration were detected besides collagen fiber filling. CONCLUSION: The new approach of "WYW" intervention embolization is able to develop the ventricular aneurysm animal model, which is similar to clinical pathophysiological variation. The outcome is precise and reliable.

Objective To evaluate the role of inositol triphosphate receptor (IP3 R) in the fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial cells.Methods BV-2 microglial cells were seeded in 3.5 cm diameter dishes (5 ml/dish),50 ml culture flasks (8 ml/flask) or 24-well plates (1 ml/hole) with a density of 1 × 105/ml and randomly divided into 5 groups (n =25 each) ∶ control group (group C),fractalkinegroup (group F),CX3C chemokine receptor 1 (CX3CR1) antibody anti-CX3CR1 + fractalkine group (group CF),IP3R antagonist 2-APB + fractalkine group (group AF) and p38 mitogen-activated protease (p38MAPK) inhibitor SB203580 + fractalkine group (group SF).Fractalkine 10 nmol/L was added to the culture medium in groups F,CF,AF and SF.The anti-CX3CR1 15 μmol/L,2-APB 50 μmol/L and SB203580 10 μmol/L were added to the culture medium in groups CF,AF and SF,respectively,1 h before addition of fractalkine.The cells were then cultured for 24 h.The intracellular Ca2+ concentration ([Ca2+]i) was measured during the 10 min incubation with fractalkine.The phosphorylation of p38MAPK was measured at 0,30,60,120 and 240 min of incubation with fractalkine.The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in theculture medium were determined at 24 h of incubation with fractalkine.Results Compared with group C,[Ca2+]i,and the phosphorylation of p38MAPK and concentrations of IL-1β and TNF-α were significantly increased in groups F,CF,AF and SF (P ＜ 0.05).[Ca2+]i was significant lower in groups AF and CF and phosphorylation of p38MAPK and concentrations of IL-1β and TNF-α were significantly lower in groups CF,AF and SF than in group F (P ＜ 0.05).Conclusion IP3 R is involve in the fractalkine-induced activation of p38MAPK signaling pathway in BV-2 microglial cells.

Objective To determine whether p38 mitogen-activated protein kinase (p38MAPK) signaling pathway is involved in cerebral fractalkine-induced hyperalgesia in mice.Methods Two hundred and twenty-five male Kunming mice weighing 30-40 g were randomly divided into 4 groups:group control ( group C,n =55 ) ;group fractalkine (group F,n =60); group anti-CX3CR1 + fractalkine (group CF,n =55) and group SB203580 (p38MAPK inhibitor) + fractalkine (group SF,n =55).Fractalkine 100 ng was injected into cerebral lateral ventricle (i.c.v.) in groups F,CF and SF.Anti-CX3CR1 1 μg and SB203580 1 μg were injected i.c.v.at 1 h before fractalkine injection in groups CF and SF respectively.Paw withdrawal latency to a thermal nociceptive stimulus (PWL) was measured at 30 min before the drugs were injected into cerebral lateral ventricle and 30,60,120 and 240 min after fractalkine injection.Five animals were sacrificed after PWL measurement at each time point and their brains were removed for determination of phosphorylated p38MAPK protein expression (by Western blot analysis).Five animals were sacrificed at 30 min before the drugs were injected into cerebral lateral ventricle and 6,12 and 24 h after fractalkine injection for determination of IL-1β and TNF-α contents in the brain (by ELISA) in all the 4 groups.In group F 5 animals were sacrificed at 4 h after fractalkine injection for determination of action of fractalkine on microglia or astrocyte (by immunofluorescence).Results Fractalkine i.c.v.injection significantly reduced PWL and increased phosphorylated 38MAPK,IL-1β and TNF-α levels in group F as compared with group C.Pretreatment with anti-CX3CR1 or SB203580 significantly decreased fractalkine-induced hyperalgesia and phosphorylated-p38MAPK,IL-1β and TNF-α levels in groups CF and SF as compared with group F.Fractalkine was localized at microglia.Conclusion p38MAPK signal transduction pathway is involved in cerebral fractalkine-induced hyperalgesia in mice.

Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or developed by ourselves. Totally, 21 open reading frames (ORFs) of genes or putative uncharacterized proteins (PUPs) were predicted. Seven PUPs had not been reported previously, and two of them were predicted to contain transmembrane regions. Eight ORFs partially overlapped with or embedded into those of known genes, revealing that the SARS-CoV genome is a small and compact one with overlapped coding regions. The most striking discovery is that an ORF locates on the minus strand. We have also annotated non-coding regions and identified the transcription regulating sequences (TRS) in the intergenic regions. The analysis of TRS supports the minus strand extending transcription mechanism of coronavirus. The SNP analysis of different isolates reveals that mutations of the sequences do not affect the prediction results of ORFs.
Amino Acid Substitution ; Base Composition ; Base Sequence ; Computational Biology ; methods ; Genome, Viral ; Isoelectric Point ; Models, Genetic ; Molecular Sequence Data ; Molecular Weight ; Open Reading Frames ; SARS Virus ; genetics ; Sequence Analysis ; Transcription, Genetic

The Coronaviridae family is characterized by a nucleocapsid that is composed of the genome RNA molecule in combination with the nucleoprotein (N protein) within a virion. The most striking physiochemical feature of the N protein of SARS-CoV is that it is a typical basic protein with a high predicted pI and high hydrophilicity, which is consistent with its function of binding to the ribophosphate backbone of the RNA molecule. The predicted high extent of phosphorylation of the N protein on multiple candidate phosphorylation sites demonstrates that it would be related to important functions, such as RNA-binding and localization to the nucleolus of host cells. Subsequent study shows that there is an SR-rich region in the N protein and this region might be involved in the protein-protein interaction. The abundant antigenic sites predicted in the N protein, as well as experimental evidence with synthesized polypeptides, indicate that the N protein is one of the major antigens of the SARS-CoV. Compared with other viral structural proteins, the low variation rate of the N protein with regards to its size suggests its importance to the survival of the virus.
Amino Acid Motifs ; genetics ; Amino Acid Sequence ; Antigens, Viral ; immunology ; Base Composition ; Base Sequence ; Cluster Analysis ; Computational Biology ; DNA Primers ; Enzyme-Linked Immunosorbent Assay ; Genetic Variation ; Molecular Sequence Data ; Nucleocapsid Proteins ; genetics ; immunology ; metabolism ; Phosphorylation ; SARS Virus ; genetics ; Sequence Analysis, DNA

The E (envelope) protein is the smallest structural protein in all coronaviruses and is the only viral structural protein in which no variation has been detected. We conducted genome sequencing and phylogenetic analyses of SARS-CoV. Based on genome sequencing, we predicted the E protein is a transmembrane (TM) protein characterized by a TM region with strong hydrophobicity and alpha-helix conformation. We identified a segment (NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH) in the carboxyl-terminal region of the E protein that appears to form three disulfide bonds with another segment of corresponding cysteines in the carboxyl-terminus of the S (spike) protein. These bonds point to a possible structural association between the E and S proteins. Our phylogenetic analyses of the E protein sequences in all published coronaviruses place SARS-CoV in an independent group in Coronaviridae and suggest a non-human animal origin.
Amino Acid Sequence ; Base Sequence ; Cluster Analysis ; Codon ; genetics ; Gene Components ; Genome, Viral ; Membrane Glycoproteins ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Phylogeny ; Protein Conformation ; SARS Virus ; genetics ; Sequence Alignment ; Sequence Analysis, DNA ; Sequence Homology ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; genetics ; metabolism

Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.
Genome, Viral ; Haplotypes ; Humans ; Mutation ; Open Reading Frames ; Phylogeny ; SARS Virus ; genetics

We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.
Base Sequence ; China ; Cluster Analysis ; Gene Components ; Genetic Variation ; Genome, Viral ; Genotype ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; Sequence Analysis, DNA ; Severe Acute Respiratory Syndrome ; genetics