长效促胰岛素降糖酵母的构建及其对糖尿病模型小鼠的治疗效果

益生菌生物药物是指通过口服表达药用多肽(蛋白)的重组益生菌活细胞达到治疗疾病的新型口服给药系统。为了构建一种能有效防治2型糖尿病的酵母生物药物,文章首先构建了酿酒酵母(S.cerevisiae)整合型表达载体pNK1-PGK,并且通过绿色荧光蛋白(GFP)证明其表达功能正常,利用该载体将10×GLP-1 (Glucagon-like peptide-1)基因转化到酿酒酵母INVSc1中,通过营养缺陷型和Western blotting成功筛选出表达10×GLP-1的长效促胰岛素降糖酵母(Long-acting GLP-1 hypoglycemic yeast, LHY)。该酵母生长迅速,外源基因10×GLP-1表达稳定,表达量达到1.56 mg/g细胞湿重。通过链脲佐菌素和高脂高糖饮食联合诱导的方法构建了2型糖尿病小鼠模型,用LHY对其进行口服灌胃治疗,证明LHY具有较好疗效,明显降低血糖水平。     

Piwi-Interacting RNAs (piRNAs) Are Dysregulated in Renal Cell Carcinoma and Associated with Tumor Metastasis and Cancer-Specific Survival

Piwi-interacting RNAs (piRNAs) are a distinct group of small noncoding RNAs (sncRNAs) that silence transposable genetic elements to protect genome integrity. Because of their limited expression in gonads and sequence diversity, piRNAs remain the most mysterious class of small RNAs. Studies have shown piRNAs are present in somatic cells and dysregulated in gastric, breast and liver cancers. By deep sequencing 24 frozen benign kidney and clear cell renal cell carcinoma (ccRCC) specimens and using the publically available piRNA database, we found 26,991 piRNAs present in human kidney tissue. Among 920 piRNAs that had at least two copies in one specimen, 19 were differentially expressed in benign kidney and ccRCC tissues, and 46 were associated with metastasis. Among the metastasis-related piRNAs, we found three piRNAs (piR-32051, piR-39894 and piR-43607) to be derived from the same piRNA cluster at chromosome 17. We confirmed the three selected piRNAs not to be miRNAs or miRNA-like sncRNAs. We further validated the aberrant expression of the three piRNAs in a 68-case formalin-fixed and paraffin-embedded (FFPE) ccRCC tissue cohort and showed the up-regulation of the three piRNAs to be highly associated with ccRCC metastasis, late clinical stage and poor cancer-specific survival.     

Association of the SNP rs1800925(C/T) in the Interleukin-13 Gene Promoter with Pulmonary Function in Chinese Han Patients with Idiopathic Pulmonary Fibrosis

The present report studied potential association of the rs1800925(C/T) single nucleotide polymorphism (SNP) of the Interleukin (IL)-13 gene promoter with idiopathic pulmonary fibrosis (IPF) in patients of Chinese Han ethnicity. Seventy patients with IPF were enrolled and divided into three subgroups: group A (61–79 % pred. D_LCO; n = 22), group B (51–60 % pred. D_LCO; n = 20), and group C (≤50 % pred. D_LCO; n = 28). Control group consisted of 80 healthy individuals of Chinese Han ethnicity. The SNP rs1800925(C/T) was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) analysis. The IL-13 CC genotype was present in 28/70 (40.0 %), homozygous TT in 6/70 (8.6 %) and heterozygous CT in 36/70 (51.4 %) patients with IPF. In control group, these genotypes were present in 30/80 (37.5 %), 11/80 (13.75 %), 39/80 (48.75 %), respectively, indicating that the distribution of the above three genotypes was not significantly different between patients with IPF and healthy controls. When the patients were stratified according to their D_LCO and D_LCO/VA, the frequencies of genotypes CT and TT in the groups A, B, and C were, respectively, 40.9 % (9/22), 50 % (10/20), and 82.1 % (23/28). Thus, significant differences in the distribution of alleles at ?1112 region of IL-13 gene were observed among the study groups A, B, and C, with the highest frequency in group C (p < 0.05). In conclusion, the rs1800925 T allele of the IL-13 gene is associated with worse pulmonary function in patients with IPF of Chinese Han ethnicity.     

Details of the nucleic acid binding site of T4 gene 32 protein revealed by proteolysis and DNA Tm depression methods

The affinities and location of oligonucleotides bound to intact and truncated bacteriophage T4 gene 32 protein have been elucidated by two independent and sensitive methods. The nucleic acid binding site is located within the core domain of 32 protein, residues 22-253. Oligonucleotides protect the core domain against proteolysis catalyzed by mammalian endoproteinase Arg-C. Of the three cleavage sites, Arg111, within the internal "LAST" ((Lys/Arg)3(Ser/Thr)2) motif, is selectively protected. We have previously suggested that these LAST residues, Lys-Arg-Lys-Thr-Ser, residues 110-114, are involved in nucleic acid binding, and our results are also consistent with crystallographic studies. The inhibitory effects of oligonucleotides on the kinetics of core domain proteolysis were used to quantify binding affinities. In addition, affinities of oligonucleotides for both core domain and intact protein were obtained from their effect on the Tm-depressing activities of these proteins. For both core and intact protein, the degree of affinity increases with oligonucleotide length. The presence of a 5' terminal phosphate increases the affinity two- to fourfold. Placement of methylphosphonodiester (uncharged) linkages at alternating linkages vastly lowers binding affinity for the intact protein and core domain. We conclude that at least two and likely three adjacent phosphodiester linkages are a minimal requirement for binding, further defining the electrostatic component of the interaction. The length-dependence of binding affinity suggests that additional interactions, both ionic and non-ionic, likely occur with longer oligonucleotides.     

Microelectrode studies of amphotericin B on Na+ and K+ conductance in bullfrog cornea

Addition of 10(-5) M amphotericin B to the tear solution of an in vitro preparation of the frog cornea increased the transepithelial conductance, gt, and decreased the apical membrane fractional resistance, f(R0), in the presence or absence of tear Na+ and Cl-. In the presence of tear Na+ and Cl-, amphotericin B increased the short-circuit current, Isc, from 3.9 to 8.8 microA.cm-2 and changed the intracellular potential, V0, from -48.5 to -17.9 mV probably due to a higher increase in the Na+ than in the K+ conductance. In the absence of tear Na+ and Cl-, amphotericin B decreased Isc from 5.5 to about 0 microA.cm-2 due to K+ (and possibly Na+) flux from cell to tear and changed V0 from -35.4 to -63.6 mV due to the increase in conductance of both ions. Increase in the tear K+ from 4 to 79 mM (in exchange for choline), in the presence of amphotericin B and absence of tear Na+ and Cl-, decreased f(R0) from 0.09 to 0.06, increased gt from 0.23 to 0.31 mS, increased Isc from 0.63 to 7.3 microA.cm-2, and changed V0 from -65.5 to -17.3 mV due to the change in EK in the presence of a high conductance in the tear membrane. Similar effects were observed with an increase of tear Na+. Results support the concept that the Na+ conductance opened by amphotericin B in the apical membrane is greater than the K+ conductance. Previously observed transepithelial effects of the ionophore may be explained mostly on the basis of its effect on the apical membrane.     

人染色体脆性位点部位的显微光谱学研究 Study of Microspectroscopy for the Position of the Fragile Sites in Human Chromosome

采用显微分光光度法,对染色体脆性位点的部位进行了显微光谱学研究。实验证明,带有脆点的染色体其DNA含量大多数趋向减少,少数略有增加,推测染色体脆性部位的产生是由于染色质DNA在高度凝缩形成中期染色体过程中超旋结构改变的结果。 The position of fragile sites in human chromosome was studied by means of the microspectroscopy. The results show that the amount DNA in chromosome with fragile sites decreases in most condition. We can suppose that the fragile sites of chromosome is caused by the superhelix structure changes of chromosome DNA during the formation of metaphase chromosome which is formed in high condensation.     

小立碗藓属在中国的记录

本文作者在湖南张家界采得小立碗藓属植物,经扫描电镜显示,其孢子具多数柱状疣的特征与小立碗藓加利福尼亚亚种相一致,可确定它为P．patens(Hedw．)B．S．G．subsp．californica(Crum et Anderson)Tan。此为小立碗藓属植物在我国的首次记录。     

Acetone-soluble cellulose acetates prepared by one-step homogeneous acetylation of cornhusk cellulose in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl)

Cellulose samples extracted from cornhusk have been successfully acetylated in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl). Without using any catalyst, cornhusk cellulose acetates (CCAs) with the degree of substitution (DS) in a range from 2.16 to 2.63 were prepared in one-step. Under the homogeneous state, the DS value of CCAs was easily controlled by the acetylation time. The obtained CCAs were characterized by means of FT-IR, ¹³C NMR, DSC, TGA, and a mechanical test. The NMR results showed that the distribution of the acetyl moiety among the three OH groups of the anhydroglucose unit shows a preference at the C₆ position. The CCAs exhibited good solubility in some organic solvents, such as acetone and DMSO. The cast CCA films from their acetone solutions had good mechanical properties. At the end of each acetylation of cornhusk cellulose, the ionic liquid AmimCl could be effectively recovered. Therefore, this study presents a promising approach and “green process” to make use of crop by-products.     

病毒衣壳结构亚单位的种类数与三角形剖分数关系的研究和探讨

本文报道了在正廿面体病毒衣壳中,当蛋白结构亚单位以“三聚体”的形式聚集在单位三角形(?)时,其亚单位的种类数应等于该病毒的三角形剖分数值。     

抗人PAI—1单克隆抗体制备,鉴定和应用

以重组PAI-(rPAI-1)为抗原,通过杂交瘤技术获得6株阳性杂交瘤细胞(AP1、AP2、AP3、AP4、AP5和AP6),并用SPA亲和层析纯化了抗PAI-1单克隆抗体(McAb)。所有McAb均能识别rPAI和天然PAI-1,腹水滴度均为10~6以上。6种McAb对PAI-1亲和常数在3.45×10~7—1.05×10~9mol/L之间。AP2、AP3 McAb能完全抑制PAI-1活性,AP4、AP和AP6只能部分抑制PAI-1活性,AP1则不抑制PAI-1活性。6种McAb中只有AP1、AP4和AP5能识别PAI-1/t-PA复合物。利用AP1、AP3、和AP4 McAb制备免疫亲和柱一步纯化HepG_2细胞分泌的PAI-1,纯度大于98％,回收率92％,纯化倍数51倍。利用抗PAI-1 McAb建立了夹心法ELISA,测定了人血浆PAI-1水平,正常人血浆PAI-1平均含量(±D)为24.7±7.75ng/ml。