Synthesis of 2,3,4,5-tetra-O-methyl-D-glucono-1,6-lactone as a monomer for the preparation of copolyesters

2,3,4,5-tetra-O-methyl-D-glucono-1,6-lactone has been prepared as a crystalline compound in acceptable yield by two different routes. An initial assay of copolymerization with L-lactide by ring-opening polymerization was carried out. The incorporation of the carbohydrate monomer into the polymer chain was about 2%.     

VIP and PACAP receptors coupled to adenylyl cyclase in human lung cancer: a study in biopsy specimens

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are important neuropeptides in the control of lung physiology. Both of these commonly bind to specific G protein coupled receptors named VPAC(1)-R and VPAC(2)-R, and PAC(1)-R (with higher affinity for PACAP). VIP and PACAP have been implicated in the control of cell proliferation and tumor growth. This study examined the presence of VIP and PACAP receptors in human lung cancer samples, as well as the functionality of adenylyl cyclase (AC) stimulated by both peptides. Results from RT-PCR and immunoblot experiments showed the expression of VPAC(1)-, VPAC(2)- and PAC(1)-R in lung cancer samples. Immunohistochemical studies showed the expression of VPAC(1) and VPAC(2) receptors. These receptors were positively coupled to AC, but the enzyme activity was impaired as compared to normal lung. There were no changes in Galpha(s) or Galpha(i) levels. Present results contribute to a better knowledge of VIP/PACAP actions in lung cancer and support the interest for the development of VIP/PACAP analogues with therapeutic roles.     

CD40, but not CD154, expression on B cells is necessary for optimal primary B cell responses

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40(-/-) and CD154(-/-) mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.     

Does Optic Nerve Head Surface Topography Change Prior to Loss of Retinal Nerve Fiber Layer Thickness: A Test of the Site of Injury Hypothesis in Experimental Glaucoma

Purpose

To test the hypothesis that optic nerve head (ONH) deformation manifesting as changes in its mean surface height precedes thinning of the peripapillary retinal nerve fiber layer (RNFL) in experimental glaucoma (EG).

Methods

68 rhesus macaque monkeys each had three or more baseline imaging sessions under manometric intraocular pressure (IOP) control to obtain average RNFL thickness (RNFLT) and the ONH surface topography parameter mean position of the disc (MPD). Laser photocoagulation was then applied to the trabecular meshwork of one eye to induce chronic, mild-to-moderate IOP elevation and bi-weekly imaging continued. Event analysis was applied to determine for each parameter when an ‘endpoint’ occurred (signficant change from baseline) for eight different endpoint criteria. Specificity was assessed in the group of 68 fellow control eyes. Classical signal detection theory and survival analysis were used to compare MPD with RNFLT.

Results

Regardless of the endpoint criterion, endpoints were always more frequent for MPD than for RNFLT. The discriminability index (d’) was 2.7 ± 0.2 for MPD and 1.9 ± 0.2 for RNFLT (p<0.0001). Endpoints were reached by MPD an average of 1-2 months earlier than by RNFLT (p<0.01). At the onset of the first specific, detectable MPD change in EG eyes, there was still no significant change in RNFLT on average (p=0.29) and only 25% of individual eyes exhibited signficant reduction. In contrast, at onset of signficant RNFLT change, MPD had already changed an average of 101 µm from baseline (p<0.0001) and 71% of the individual eyes had exhibited significant change. The magnitude of MPD change was more than could be explained on the basis of axon loss alone.

Conclusions

This study demonstrates that the average surface height of the ONH changes prior to any detectable loss of average peripapillary RNFL thickness in non-human primate eyes with experimental glaucoma.     

Gene Copy-Number Variation in Haploid and Diploid Strains of the Yeast Saccharomyces cerevisiae

The kinetochore is the macromolecular protein complex that mediates chromosome segregation. The Dsn1 component is crucial for kinetochore assembly and is phosphorylated by the Aurora B kinase. We found that Aurora B phosphorylation of Dsn1 promotes the interaction between outer and inner kinetochore proteins in budding yeast.     

GST pull-down筛选冠突曲霉MAT的互作蛋白*

目的: 冠突曲霉(Aspergillus cristatus)是一种同宗结合菌,它的产孢受渗透压调控,与构巢曲霉的光调控产孢机制存在较大差异。冠突曲霉的有性生殖主要受MAT1-1-1和MAT1-2-1调控,但MAT基因对该菌有性生殖的调控机制仍不清楚。期望筛选得到冠突曲霉MAT的互作蛋白,为深入研究冠突曲霉有性产孢机制奠定基础。方法: 利用GST pull-down联合液相色谱-串联质谱(LC-MS/MS)技术筛选可能与冠突曲霉MAT1-1-1和MAT1-2-1互作的蛋白,结合ProteinPilot和冠突曲霉基因组注释结果进行互作蛋白的注释及GO分析,其中互作蛋白SI65_00917和SI65_03348利用RT-qPCR探索它们与有性发育的联系,并利用酵母双杂交技术初步验证它们与MAT蛋白的互作关系。结果: 成功构建了GST-MAT1-1-1、GST-MAT1-2-1表达载体,诱导表达纯化出目的诱饵蛋白,分别利用诱饵蛋白捕获冠突曲霉总蛋白中的互作蛋白,经分析、筛选共鉴定出与MAT1-1-1互作的蛋白56个,与MAT1-2-1互作的蛋白413个。GO分析表明,这些蛋白参与翻译调控、代谢过程、蛋白质转运及蛋白结合等生物学过程,具有核苷酸结合活性、催化活性、蛋白结合活性;RT-qPCR结果表明互作蛋白SI65_00917可能与有性发育相关。酵母双杂交结果表明,SI65_00917蛋白具有自激活作用,可能是转录因子;SI65_03348蛋白与MAT1-1-1、MAT1-2-1在酵母中均有互作。结论: MAT通过与其他蛋白直接或间接的相互作用调控其有性发育过程。     

Zipping and Unzipping of Adenylate Kinase: Atomistic Insights into the Ensemble of Open ↔ Closed Transitions

Adenylate kinase (AdK), a phosphotransferase enzyme, plays an important role in cellular energy homeostasis. It undergoes a large conformational change between an open and a closed state, even in the absence of substrate. We investigate the apo-AdK transition at the atomic level both with free-energy calculations and with our new dynamic importance sampling (DIMS) molecular dynamics method. DIMS is shown to sample biologically relevant conformations as verified by comparing an ensemble of hundreds of DIMS transitions to AdK crystal structure intermediates. The simulations reveal in atomic detail how hinge regions partially and intermittently unfold during the transition. Conserved salt bridges are seen to have important structural and dynamic roles; in particular, four ionic bonds that open in a sequential, zipper-like fashion and, thus, dominate the free-energy landscape of the transition are identified. Transitions between the closed and open conformations only have to overcome moderate free-energy barriers. Unexpectedly, the closed state and the open state encompass broad free-energy basins that contain conformations differing in domain hinge motions by up to 40°. The significance of these extended states is discussed in relation to recent experimental Förster resonance energy transfer measurements. Taken together, these results demonstrate how a small number of cooperative key interactions can shape the overall dynamics of an enzyme and suggest an “all-or-nothing” mechanism for the opening and closing of AdK. Our efficient DIMS molecular dynamics computer simulation approach can provide a detailed picture of a functionally important macromolecular transition and thus help to interpret and suggest experiments to probe the conformational landscape of dynamic proteins such as AdK.