Molecular basis of recognition of human osteopontin by 23C3, a potential therapeutic antibody for treatment of rheumatoid arthritis

Osteopontin plays an important role in the development and perpetuation of rheumatoid arthritis (RA). Antibodies targeting osteopontin have shown promising therapeutic benefits against this disease. We have previously reported a novel anti-RA monoclonal antibody, namely, 23C3, and shown it capable of alleviating the symptoms of RA in a murine collagen-induced arthritis model, restoring the cytokine production profile in joint tissues, and reducing T-cell recall responses to collagen type II. We describe here the crystal structure of 23C3 in complex with its epitope peptide. Analyses of the complex structure reveal the molecular mechanism of osteopontin recognition by 23C3. The peptide folds into two tandem β-turns, and two key residues of the peptide are identified to be critical for the recognition by 23C3: TrpP43 is deeply embedded into a hydrophobic pocket formed by AlaL34, TyrL36, LeuL46, TyrL49, PheL91, and MetH102 and therefore has extensive hydrophobic interactions with 23C3, while AspP47 has a network of hydrophilic interactions with residues ArgH50, ArgH52, SerH53, and AsnH56 of the antibody. Besides the complementarity-determining region loops, the framework region L2 of 23C3 is also shown to interact with the epitope peptide, which is not common in the antibody-antigen interactions and thus could be exploited in the engineering of 23C3. These results not only provide valuable information for further improvement of 23C3 such as chimerization or humanization for its therapeutic application, but also reveal the features of this specific epitope of osteopontin that may be useful for the development of new antibody drugs against RA.     

Kinesin KIF4 regulates intracellular trafficking and stability of the human immunodeficiency virus type 1 Gag polyprotein

Retroviral Gag proteins are synthesized as soluble, myristoylated precursors that traffic to the plasma membrane and promote viral particle production. The intracellular transport of human immunodeficiency virus type 1 (HIV-1) Gag to the plasma membrane remains poorly understood, and cellular motor proteins responsible for Gag movement are not known. Here we show that disrupting the function of KIF4, a kinesin family member, slowed temporal progression of Gag through its trafficking intermediates and inhibited virus-like particle production. Knockdown of KIF4 also led to increased Gag degradation, resulting in reduced intracellular Gag protein levels; this phenotype was rescued by reintroduction of KIF4. When KIF4 function was blocked, Gag transiently accumulated in discrete, perinuclear, nonendocytic clusters that colocalized with endogenous KIF4, with Ubc9, an E2 SUMO-1 conjugating enzyme, and with SUMO. These studies identify a novel transit station through which Gag traffics en route to particle assembly and highlight the importance of KIF4 in regulating HIV-1 Gag trafficking and stability.     

Superfluous role of mammalian septins 3 and 5 in neuronal development and synaptic transmission

The septin family of GTPases, first identified for their roles in cell division, are also expressed in postmitotic tissues. SEPT3 (G-septin) and SEPT5 (CDCrel-1) are highly expressed in neurons, enriched in presynaptic terminals, and associated with synaptic vesicles. These characteristics suggest that SEPT3 or SEPT5 might be important for synapse formation, maturation, or synaptic vesicle traffic. Since Sept5^−/− mice do not show any overt neurological phenotypes, we generated Sept3^−/− and Sept3^−/− Sept5^−/− mice and found that SEPT3 and SEPT5 are not essential for development, fertility, or viability. Changes in the expression of septins were noted in the absence of SEPT3, SEPT5, and both septins. SEPT5 association with other septins in brain tissue was unaffected by the removal of SEPT3. No abnormalities were observed in the gross morphology and synapses of the hippocampus. Similarly, axon development and synapse formation were unaffected in vitro. In cultured hippocampal neurons, the size of the recycling synaptic vesicle pool was unaltered in the absence of SEPT3. Furthermore, synaptic transmission at two different central synapses was not significantly affected in Sept3^−/− Sept5^−/− mice. These results indicate that SEPT3 and SEPT5 are dispensable for neuronal development as well as for synaptic vesicle fusion and recycling.     

应用分子生物学方法检测宫颈液基细胞残液中HPV16／18感染的对比研究

对比免疫蛋白印迹及斑点杂交检测人乳头瘤病毒HPV16/18的检出率,以期找到子宫颈癌早期筛查的最佳方法。采用免疫蛋白印迹与斑点杂交分别对173份宫颈液基细胞进行HPV16/18检测。150例经宫颈液基细胞学镜下筛查为宫颈上皮内病变及宫颈癌的患者,其中免疫蛋白印迹对低度病变检出率为47.6%(30/63),对高度病变检出率为57.9%(33/57),对浸润癌的检出率为86.7%(26/30),其中鳞癌为85%(17/20),腺癌为90%(9/10),采用斑点杂交对低度病变检出率为28.6%(18/63),对高度病变检出率为42.1%(24/57),浸润癌的检出率为80%(24/30),其中鳞癌为75%(15/20),腺癌为90%(9/10)。采用免疫蛋白印迹对23例正常对照的细胞HPV检出率为13.1%(3/23),斑点杂交的检出率为8.7%(2/23)。各级宫颈病变中HPV16/18的检出率均较对照组差异有统计学意义(P<0.05)。免疫蛋白印迹检测HPV16/18比斑点杂交的检出率要高,为发现HPV一过性携带者,达到早期诊断的目的。     

Neuroprotection of aucubin in primary diabetic encephalopathy

Hippocampal neuronal apoptosis accompanied by impairment of cognitive function occurs in primary diabetic encephalopathy. In this study, we investigated the neuroprotective mechanism of the iridoid glycoside, aucubin, using rats (n=8). Diabetes mellitus was induced in the rats by intraperitoneal (i.p.) injection of streptozotocin (60 mg/kg body weight). After 65 d, half of the DM rats were administered aucubin (5 mg/kg; i.p.) for 15 d, yielding treatment DM+A. A third group of rats received no streptozotocin or aucibin, and served as controls (CON). Encephalopathy was assessed using Y-maze behavioral testing. Rats were euthanized on Day 87, and hippocampi were excised for visual (light and transmission electron microscopic) and immunochemical (Western blot; immunohistochemical) assessments of the CA1 subfield for apoptosis and expression of regulatory proteins Bcl-2 and Bax. Treatment responses to all the parameters examined (body weight, plasma glucose, Y-maze error rates, pyramidal cell ultrastructure, proportions of apoptotic cells, levels of expression of Bcl-2 and Bax, and survivability of neuronal cells) were identical: there were highly significant differences between DM and CON groups (P<0.001), but the effects were significantly moderated (P<0.01) in DM+A compared with DM. These findings confirm the association of apoptosis with the encephalopathic effects of diabetes mellitus, and suggest a major role of the expression levels of Bcl-2 and Bax in the regulation of apoptotic cell death. All of the results suggest that aucubin could effectively inhibit apoptosis by modulating the expressions of Bcl-2 and Bax genes.     

Fine mapping of a major quantitative trait loci, qSSP7, controlling the number of spikelets per panicle as a single Mendelian factor in rice

In our previous studies, one putative QTL affecting number of spikelets per panicle (SPP) was identified in the pericentromeric region of rice chromosome 7 using a recombinant inbred population. In order to define the QTL (qSPP7), RI50, a recombinant inbred line with 70% of genetic background same as the female parent of Zhenshan 97, was selected to produce near-isogenic lines for the target region in the present study. In a BC₂F₂ population consisting of 190 plants, the frequency distribution of SPP was shown to be discontinuous and followed the expected Mendelian ratios (1:2:1 by progeny test) for single locus segregation. qSPP7 was mapped to a 0.4 cM region between SSR marker RM3859 and RFLP marker C39 based on tests of the BC₂F₂ population and its progeny. Its additive and dominant effects on SPP were 51.1 and 24.9 spikelets, respectively. Of great interest, the QTL region also had effects on grain yield per plant (YD), 1,000 grain weight (GW), tillers per plant (TPP) and seed setting ratio (SR). Significant correlations were observed between SPP and YD (r = 0.66) and between SPP and SR (r = −0.29) in the progeny test. 1082 extremely small panicle plants of a BC₃F₂ population containing 8,400 individuals were further used to fine map the QTL. It turns out that qSPP7 co-segregated with two markers, RM5436 and RM5499 spanning a physical distance of 912.4 kb. Overall results suggested that recombination suppression occurred in the region and positional cloning strategy is infeasible for qSPP7 isolation. The higher grain yield of Minghui 63 homozygote as compared to the heterozygote suggested that Minghui 63 homozygote at qSPP7 in hybrid rice could further improve its yield. Y. Z. Xing and W. J. Tang contributed equally to this work.     

Development and characterization of microsatellite loci for lotus (Nelumbo nucifera)

This paper reports the development of microsatellite primers for Nelumbo nucifera Gaerten. By screening genomic libraries enriched with 10 kinds of probes, Seventeen polymorphic loci were isolated and primers were designed. Polymorphism of these 17 loci was assessed in 24 individuals. All the 17 loci are polymorphic and the number of alleles ranged from two to seven. Observed heterozygosity and expected heterozygosity ranged from 0.0000 to 0.9176 and from 0.2837 to 0.7917 respectively. These microsatellite loci should be useful for studying the genetic diversity of N. nucifera.     

Structural elucidation of in vivo metabolites of penehyclidine in rats by the method of liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry and isotope ion cluster

The structural elucidation of metabolites of penehyclidine in rats, a novel anti-cholinergic drug, by the method of liquid chromatography-electrospray ionization mass spectrometry, gas chromatography-mass spectrometry with electron impact ion source and stable isotope ion cluster was described. Identification and elucidation of the phase I and phase II metabolites were performed by comparing the daughter ion pairs of stable isotope cluster, changes of the protonated molecular masses, full scan MS(n) spectra and retention times with those of the parent drug, penehyclidine and penehyclidine deuterium-labeled. Penehyclidine was easily biotransformed by the pathways of oxidative, hydroxylated, methoxylated and phase II conjugated reactions to form several metabolites that retained the some features of the parent molecules. Twelve metabolites (penehyclidine monoxide, hydroxypenehyclidine, penehyclidine dioxide, hydroxypenehyclidine monoxide, dihydroxypenehyclidine, dihydroxypenehyclidine (position isomer), dihydroxypenehyclidine monoxide, trihydroxypenehyclidine, methoxypenehyclidine dioxide, dimethoxypenehyclidine, trihydroxymethoxypenehyclidine and glucuronide conjugated hydroxypenehyclidine) were identified. The results from electrospray ion and electron impact ion data with the stable isotope cluster showed the qualitative differences in the mass spectral patterns, suggesting that these technologies should be used in parallel to ensure comprehensive metabolites detection and characterization. The described method has wide applicability to rapidly screen and provide structural information of metabolites.