Objective To construct a simple method for the measurement of activity of S-homocysteine methyltransferase (HMT),and explore the best processing condition for HMT and the preservation of HMT.Methods HMT was expressed in pro-karyotic system by using genetic engineering technology,then was purified by using affinity and Sephadex G1 5 chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)was performed to identify the physicochemical and bio-logical properties of target protein.Based on the principle of 5 ,5′-disulfide-double (2- nitro benzoic acid)(DTNB)could react with sulfydryl compounds rapidly,the reduction of homocysteine was detect to evaluate the activity of enzyme,then the best processing conditions of HTM were determined.Activity of enzyme,preserved in preservation solution with or without glycerol and preserved under different temperatures,was detected.Activity remaining ratios were detected and compared between HMT preserved in pres-ervation solution with different protective agents of different concentration and preserved by cryodesiccation.Results The purity of recombined HMT was above 95%,with molecular weight of 36 000 and excellent catalytic activity,and the catalytic activity was 2 000 U/mg.The optimum condition for the detection of biological activity was using HEPES buffer of pH 7.4 at 37 ℃ and reac-tion for 25 min.Glycerol could significantly prolong the preserving time of HMT,and half activity of HMT could be remained for six months.The reservation rates of activity of HMT,preserved in preservation solution with mannitol and trehalose,were 104%and 100%,respectively.Conclusion HMT could be obtained through genetic engineering.A simple test method of HMT was es-tablished,and the best processing conditions and preserving methods of HMT were determined,which laid a foundation for clinical application.

Objective To investigate the clinical and neuroimaging features of adults with myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease at the first attack. Methods We retrospectively analyzed the clinical manifestations,radiological features,laboratory findings,and outcome of 14 adult patients with MOG antibody-associated disease at the first attack who were hospitalized in the departments of neurology of Jiangxi Provincial People's Hospital and Xinyu People's Hospital from January 2018 to January 2022. Results The 14 patients included six males and eight females,with a median age of 29.5 years. The most common initial symptoms were fever and headache (n=5),seizure (n=3),and dizziness (n=3). The supratentorial lesions were located in the thalamus (n=7),subcortical white matter (n=6),cortex (n=5),corpus callosum (n=2), and basal ganglia (n=2). The infratentorial lesions were frequently located in the brainstem:the pons (n=5),middle cerebellar peduncle (n=3),midbrain (n=2), and medulla (n=2). Three patients had spinal cord involvement,with one case of longitudinally extensive transverse myelitis. Thirteen patients had elevated cerebrospinal fluid cell counts,and seven had elevated cerebrospinal fluid protein levels. The serum MOG antibody titer ranged from 1∶3.2 to 1∶512. All the 14 patients received intravenous pulse glucocorticoid therapy. Only one patient had a relapse with optic neuritis. Conclusion In our study,MOG antibody-associated disease showed a slight female predominance and frequently presented as acute disseminated encephalomyelitis. The supratentorial lesions were often located in the thalamus and subcortical white matter,while the infratentorial lesions were frequently in the pons. Intravenous pulse steroid therapy was effecitve in the acute phase. The majority of the patients had a favorable outcome.

Transient receptor potential (TRP) channels affect several inflammatory and neoplastic conditions.and are widely distributed and activated by multiple stimuli.Mean while,as the sensor to the changes in cellular local environments,TRP channels have multiple functions,including thermal,mechanical and taste sensing,as well as ion homeostasis maintaining.They can regulate muscle contractions,transmitter release,cell multiplication,cell differentiation,genetic transcription,apoptosis and molecular death.TRPM8,a nonselective cation channel,was originally cloned as a prostate-specific protein.Now,TRPM8 is found in carcinoma of colon,lung cancer,mammary cancer,pancreatic cancer and cutaneum carcinoma.The aim of this review is to summarize data reported so far on the expression and functional role of TRPM8 channels in different types of cancers.

BACKGROUND:More and more evidence have shown that autoimmune-induced inflammatory demyelinating mostly leads to optic neuritis that is quite an early manifestation of multiple sclerosis, but whether the pathogenesis of optic neuritis in experimental autoimmune encephalomyelitis (EAE) mice is correlated with helper T cellsubsets has rarely been reported.
OBJECTIVE:To analyze the correlation between pathogenesis of optic neuritis of mouse EAE model with helper T cellsubsets.
METHODS:The mice were injected intraperitoneal y Bordetel a pertussis to establish EAE models. Then, the animal models were subjected to immunization for 11, 15, 19 days, respectively. Mice undergoing intraperitoneal injection of normal saline served as controls (adjuvant group).
RESULTS AND CONCLUSION:Compared with the adjuvant group, the protein expression of interleukin 4 in the optic nerve decreased in the 19-day immunization group (P<0.05);the protein expression of interleukin 17 in the optic nerve increased in the 11-and 15-day immunization groups (P<0.05);the protein expression of interferonγin the optic nerve increased in the 15-and 19-day immunization groups (P<0.05);the protein expression of Foxp3 in the optic nerve decreased in the 11-, 15-and 19-day immunization groups (P<0.05). Real-time PCR results showed that compared with the adjuvant group, the mRNA expression of interferonγand Foxp3 in the optic nerve decreased (P<0.05), while mRNA expression of RORt increased in the 11-, 15-and 19-day immunization groups;the mRNA expression of interleukin 4, interleukin 17, T-beat increased in the 15-and 19-day immunization groups (P<0.05);the mRNA expression of GATA3 reduced in the 19-day immunization group (P<0.05). These results reveal that Foxp3 expression and helper T cellreduction have important influences on the development of optic neuritis in EAE mouse models, interleukin 17 may mediates inflammatory injury in the early stage, while interferon-γmakes inflammatory injury worse in the peak incidence of the disease.

Objective To observe a recombinant mutant of HBV core protein for dominant negative gene therapy against HBV encapsidation in vitro. Methods C gene and S gene of HBV were acquired through PCR and subcloned into pGEM T to construct pGEM T C and pGEM T S respectively. After digestion and ligation of these two plasmids, pGEM T CS was constructed. The cloned gene was inserted into pcDNA3.1 + to construct pcDNA3.1 + CS, which was identified by DNA sequencing. The recombinant plasmids were transformed into HepG2 cells, and screened with G418. The resistant HepG2 cell clones were chosen to test the expression of core surface protein by RT PCR, and the expressing HepG2 clones were cultured with 10% HBV DNA positive human serum for 72 hours. The intracellular HBV particles were extracted and the DNA was subjected to dot hybridization. Results The analysis showed that the HepG2 cells expressing mutant C protein had capabilities to resist HBV invasion in varied degrees. The mutant C protein had a dominant negative role in the encapsidation of HBV compared with the naive part of core protein. Conclusions The production of recombinant mutant core protein has a potential value for gene therapy against HBV infection.

Objective To study the changes in expression of osteoglycin (OGN) in the lung tissue in a rat acute pulmonary embolism (PE) model and its effects on the metabolism of collagen. Methods A rat acute PE model was reproduced by injecting 3-4 emboli into the left jugular vein. The lung tissue samples were collected at different time points as following: 1h, 8h, 24h and 48h, then the total RNA and total proteins of the lung tissue were extracted. Normal rats were used as control. The changes in mRNA level in OGN were assayed by semi-quantitative RT-PCR, and the changes in protein level were determined by Western blot method. The immunohistochemical method was employed to study the distribution and expression changes in OGN in the lung tissue after PE. Masson staining was employed to observe the deposition of collagen in the lung tissue 4 weeks after acute PE. Results t different time points, the mRNA levels and the protein levels of OGN were lowered gradually in the lung tissue in rat acute PE models. The immunohistochemical study indicated that OGN was distributed beneath the bronchial epithelium, and in the periphery of cartilaginous tissue and the lung alveoli. It also could be observed beneath the arterial endothelium and in the adventitia of pulmonary arteries. In pulmonary veins, OGN accumulated in the adventitia, media, and intima. The deposition of collagen in the lung tissue increased obviously 4 weeks after acute PE. Conclusion The expression of OGN is down-regulated after acute PE. It facilitates the deposition of collagen in the lung tissue.

Objective The calcium oxalate stone is the most common type of the kidney stones.By building the rat renal calcium oxalate stone model, preliminary study the function of CaSR-Claudin14 regulating pathways on renal calcium oxalate stone for-mation model in rats. Methods 30 Male S-D rats were randomly divided into control group (n=15) and model group (n=15). Adult male S-D rats were given ethylene glycol and ammonium chloride to induce urolithiasis.Application of full automatic biochemical analyzer to test rat renal function and the changes of urine biochemical index.Immunohistochemistry was used to detect the expression of CaSR protein;RT-PCR was used to detect the Claudin-14 mRNA expression;Western Blotting was used to detect the expression of CaSR and Claudin-14 protein respectively. Results By observing model group has large stones crystallization under light microsco-py;model group rats 24 h urine calcium are significantly higher than control group([9.66 ±1.10]mmol vs [3.26 ±0.60]mmol, P<0.01); and model group 24 h urine volume are significantly higher than control group ([21.27 ±1.08]mL vs [13.2 ±0.55]mL, P<0.01 ); and urinary PH has no significant difference between the groups( P >0.05 ) .Expression of Claudin-14 mRNA in the model group is significantly higher than normal control group([0.150 ± 0.004] vs [0.047 ±0.008], P<0.01); Expression of Claudin-14 protein in the model group is significantly higher than normal control group([1.526 ±0.089] vs 0, P<0.01).Expression of CaSR protein in the model group is significantly higher than normal control group([6.697 ±0.051] vs [5.016 ±0.053], P<0.05). Conclusion CaSR can raise the expression of Claudin-14, increase re-nal tubular urinary calcium excretion to promote renal calcium oxalate stone formation.

Background and purpose:The expression of ERβin triple negative breast cancer(TNBC) might be associated with good prognosis in TNBC patients. ERβand ERαhave considerable homology. FOXA1 plays an important role in ERαexpression and function. The aim of this study was to analyze the expression of FOXA1 and ERβin TNBC and the relationship between them and the clinicopathologic characteristics and prognosis. Methods:The breast cancer samples in Peking Union Medical College Hospital were collected from Nov. in 2011 to Dec. in 2013, and TNBC were screened out based on the expression of ERα, PR and HER-2. Thirty ERβ-negative samples and 30 ERβ-positive samples were selected randomly according to the ERβexpression. We used immunohistochemical method to detect the expression of FOXA1. Finally, 48 TNBC samples were obtained to analyze the results. Results:The total positive rate of FOXA1 was 35.4%(17/48). In the ERβ-positive group, the positive rate of FOXA1 was 35.7%(10/28),and in the ERβ-negative group, the positive rate of FOXA1 was 35% (7/20). The expression of FOXA1 in these 2 groups had no signiifcant difference (P=0.83), which indicated that there was no relation between ERβand FOXA1. The FOXA1 positive group and FOXA1 negative group also showed no signiifcant difference in age, tumor size, and lymphatic metastasis number in axilla, tumor grade, tumor stage, NPI and DFS. However, Ki-67 showed negative correlation with FOXA1 expression (P<0.01). Conclusion:FOXA1 expression had no relationship with ERβexpression in TNBC. Ki-67 showed negative correlation with FOXA1 expression, which might hint that the proliferation of tumor cell was lower in FOXA1 positive TNBC.

Objective:To clone and express the full length cDNA of human CD38. Methods:The full length cDNA of the human CD38 antigen was amplified from total RNA of Daudi cell by RT-PCR, and it was inserted into pGEM-T. The validity on the sequences was confirmed by automatic DNA sequencing. Inserting the valid CD38 gene into pcDNA3.1(+) plasmid to obtain recombinant mammalian expression vector pcDNA3.1(+)/CD38Z; Using lipofectin gene transfer technique system, recombinant expression vector containing CD38 gene was transfected into COS7 cells. The expression of CD38 molecules on the surface of COS7 cells was detected by FACS and immunohistochemical technique. Results:DNA sequencing showed that the cloned full length cDNA sequence was identical with reported. The result of FACS and immunohistochemical technique indicated that CD38 molecules were expressed on the surface of COS7 cells. Conclusion:The full length cDNA of human CD38 is obtained, recombinant mammalian expression vector pcDNA3.1(+)/CD38Z is successfully constructed, and the CD38 molecules is expressed on the surface of COS7 cells,this may facilitate studies on the biochemistry and function of CD38 antigen.

Objective To prepare and identify the monoclonal antibody against asymmetric dimethylarginine (ADMA) and to do further research on clinical application .Methods The short immune peptide antigen was synthetized by solid phase technology . Splenocytes from the Balb /c mice immunized by synthetized antigen were fused with myeloma cells SP 2/0 for producing hybrido-ma .Hybridoma cell line secreting antibodies against ADMA was sifted by indirect ELISA and limiting dilution assay .The specificity of monoclonal antibodies against human ADMA were evaluated with Western blot and ELISA .Results Two hybridoma cell lines stably secreting monoclonal antibodies against ADMA were developed and named 22-1-1and 38-1-6 respectively .By applying West-ern blot and ELISA ,the results indicated that all monoclonal antibodies raised could specifically react with ADMA .Conclusion The success in the production of ADMA monoclonal antibody establishes foundation for application in the practice of clinical labora-to ry .