Prevalence and evolution of drug resistance HIV-1 variants in Henan, China

To understand the prevalence and evolution of drug resistant HIV strains in Henan China after the implementation of free antiretroviral therapy for AIDS patients. 45 drug naive AIDS patients, 118 AIDS patients who received three months antiretroviral therapy and 124 AIDS patients who received six months antiretroviral treatment were recruited in the southern part of Henan province. Information on general condition, antiretroviral medicines, adherence and clinical syndromes were collected by face to face interview. Meanwhile, 14ml EDTA anticoagulant blood was drawn. CD4/CD8 T cell count, viral load and genotypic drug resistance were tested. The rates of clinical improvement were 55.1% and 50.8% respectively three months and six months after antiretroviral therapy. The mean CD4 cell count after antiretroviral therapy was significantly higher than in drug naive patients. The prevalence rate of drug resistant HIV strains were 13.9%, 45.4% and 62.7% in drug naive patients, three month treatment patients and six month treatment patients, respectively.The number of resistance mutation codons and the frequency of mutations increased significantly with continued antiretroviral therapy. The mutation sites were primarily at the 103, 106 and 215 codons in the three-month treatment group and they increased to 15 codon mutations in the six-month treatment group. From this result, the evolution of drug resistant strains was inferred to begin with the high level NNRTI resistant strain, and then develop low level resistant strains to NRTIs. The HIV strains with high level resistance to NVP and low level resistance to AZT and DDI were highly prevalent because of the AZT DDI NVP combination therapy. These HIV strains were also cross resistant to DLV, EFV, DDC and D4T. Poor adherence to therapy was believed to be the main reason for the emergence and prevalence of drug resistant HIV strains. The prevalence of drug resistant HIV strains was increased with the continuation of antiretroviral therapy in the southern part of Henan province. Measures, that could promote high level adherence,provide new drugs and change ART regimens in failing patients, should be implemented as soon as possible.     

桐花树五环三萜化学成分的研究

从桐花树树皮的乙醇提取物的乙酸乙酯部分分离到3个化合物,利用波谱技术分别鉴定为protoprimulagenin(1),embelinone(2)和aegicerin(3),其中化合物1和2是首次从该植物中分离到。     

TEC酪氨酸激酶基因是一种与肝再生调控相关的早期反应基因

 肝再生过程中立即早期反应基因的表达在成熟肝细胞由G0 期向G1期的转变中起着关键作用 .为探讨肝再生早期基因表达的变化 ,利用表达性差异显示分析 (RDA)技术研究了 2 3肝部分切除后 1h再生肝选择性基因表达 ,发现一株TEC酪氨酸激酶同源序列存在于差减产物中 ,RNA狭缝杂交证实确为差异表达基因 .从大鼠肝cDNA文库中分离其全长cDNA ,序列分析结果表明 ,该基因为小鼠 人TEC酪氨酸激酶的同源体 ,进而以该cDNA为探针 ,用Northern杂交证实 2 3肝部分切除后TEC酪氨酸激酶基因在 1h内呈现瞬间表达增加 ,其表达水平较基础水平增高 2 5倍 ;在原代培养大鼠肝细胞体系中 ,EGF可迅速诱导TEC基因表达 ,且不被蛋白合成抑制剂阻断 .结果表明 ,TEC基因是一种与肝再生调控密切相关的早期反应基因 .     

Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL)is a rare hereditary cerebrovascular disease caused by a NOTCH3 mutation.However,the underlying cellular and molecular mechanisms remain unidentified.Here,we generated non-integrative induced pluripotent stem cells(iPSCs)from fibroblasts of a CADASIL patient harboring a heterozygous NOTCH3 mutation(c.3226C>T,p.R1076C).Vascular smooth muscle cells(VSMCs)differentiated from CADASIL-specific iPSCs showed gene expression changes associated with disease phenotypes,including activation of the NOTCH and NF-kB signaling pathway,cytoskeleton disorganization,and excessive cell proliferation.In comparison,these abnormalities were not observed in vascular endothelial cells(VECs)derived from the patients iPSCs.Importantly,the abnormal upregulation of NF-kB target genes in CADASIL VSMCs was diminished by a NOTCH pathway inhibitor,providing a potential therapeutic strategy for CADASIL.Overall,using this iPSCbased disease model,our study identified clues for studying the pathogenic mechanisms of CADASIL and developing treatment strategies for this disease.     

重组人钙调素的制备及对细胞增殖作用影响的研究

The gene coding for human CaM was amplified by PCR in which pUC/hCaM3 cDNA was usd as template. After inserting the hCaM III cDNA into the expression plasmid pBV220, we constructed the hCaM3 cDNA-recombinant expression vector(hCaM3/pBV220). The recombinant plasmid was then transformed into E. coli DH5 alpha. After heat induction, a high level expression of CaM protein was obtained. SDS-PAGE analysis showed that the recombinant E. coli could express a 17 kD protein which accounted for about 20% of the total cellular protein. Western blot analysis showed that anti-CaM monoclonal antibody(McAb) specifically bound to the 17 kD band of expression product. rhCaM was purified by Phenyl-sepharose CL-4B affinity chromatography from recombinant bacterial lysate. 3-4 mg of the purified protein were obtained from 1 liter of bacterial culture. The rhCaM was able to activate NAD kinase to the same extent as the standard human brain CaM (Sigma). K562 cells and SP2/0 cells were seeded in 24-well or 96-well plate and cultured for 48 h with rhCaM and CaM-antagonist trifluoperazine(TFP). Cell proliferation rates was determined by MTT assay. There was a significant positive correlation between the concentrations of rhCaM and the cell proliferation rates. CaM-antagonist TFP had an inhibitory effect on cell proliferation rate. The inhibition could be corrected by the addition of extracellular rhCaM.     

Monocyclic Aromatic Hydrocarbon Degradation by Rhodococcus sp. Strain DK17

Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.     

Chromosomal integration and expression of green fluorescent protein in Zymomonas mobilis

An integrative vector was constructed to allow expression of heterologous proteins into the adhB locus of Zymomonas mobilis. As a reporter gene, the ORF of a bright variant of green fluorescent protein from Aequorea victoria (GFPuv) was fused to the adhB strong promoter from Z. mobilis by using a two-step PCR strategy. Z. mobilis recombinant strains that were stably marked by precise gene replacement at adhB locus with a single chromosomal copy of gfpuv. Protein expression was confirmed by fluorescence microscopy and measured by fluorescence spectroscopy, showing high expression levels (12 to 30 times higher than those obtained in E. coli) without affecting the host growth.