Sequence Polymorphism and Evolution of Three Cetacean MHC Genes

Sequence variability at three major histocompatibility complex (MHC) genes (DQB, DRA, and MHC-I) of cetaceans was investigated in order to get an overall understanding of cetacean MHC evolution. Little sequence variation was detected at the DRA locus, while extensive and considerable variability were found at the MHC-I and DQB loci. Phylogenetic reconstruction and sequence comparison revealed extensive sharing of identical MHC alleles among different species at the three MHC loci examined. Comparisons of phylogenetic trees for these MHC loci with the trees reconstructed only based on non-PBR sites revealed that allelic similarity/identity possibly reflected common ancestry and were not due to adaptive convergence. At the same time, trans-species evolution was also evidenced that the allelic diversity of the three MHC loci clearly pre-dated species divergence events according to the relaxed molecular clock. It may be the forces of balancing selection acting to maintain the high sequence variability and identical alleles in trans-specific manner at the MHC-I and DQB loci.     

RESPONSES TO SOLAR UV RADIATION OF THE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE) GROWN AT DIFFERENT Zn2+ CONCENTRATIONS1

Zn availability in the ocean has been suggested to limit primary production by affecting CO₂ acquisition processes for photosynthesis, therefore influencing the global carbon cycle. Also, UV radiation (UVR, 280–400 nm) is known to affect primary production in different ways. It remains to be ascertained whether Zn availability and UVR can act synergistically, antagonistically, or independently on oceanic primary production. We cultured the cosmopolitan diatom Skeletonema costatum (Grev.) Cleve under different radiation treatments with or without UVR (only photosynthetically active radiation), at 0, 3, and 10 pmol · L^?1 Zn²⁺. Specific growth rate, photosynthetic carbon assimilation, external carbonic anhydrase (eCA) activity, and estimated cell abundance increased with increasing concentrations of Zn²⁺ from 0 to 3 and 10 pmol · L^?1, irrespective of the radiation treatment. Higher eCA activity was observed in the cells grown at the high level of Zn²⁺ in the presence of UVR. An approximately linear relationship between μ and the daily dose of PAR was observed at 3 and 10 pmol · L^?1 Zn²⁺ concentrations. However, the dependency of μ on the daily PAR dose disappeared when the cells were grown in the presence of UVR, which overall depressed both μ and photosynthetic carbon assimilation. The inhibitory effect of UVR was inversely related to Zn²⁺ concentrations. The ultraviolet‐B (UVB)‐related inhibition of growth and photosynthesis decreased with time, reflecting a faster acclimation of the cells to UVR at replete Zn²⁺ levels. Overall, growth in the presence of higher Zn²⁺ concentrations reduced the sensitivity to UV radiation in Skeletonema costatum.     

Analysis of gene expression in granulosa cells of ovine antral growing follicles using suppressive subtractive hybridization

Follicular growth, development and ovulation are highly ordered processes that involve the expression of many genes under precise temporal and spatial regulation. However, information on stage-specific gene expression during the antral follicle phase in sheep is not well understood. In the present study, suppressive subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the granulosa cells between large follicles (LF, >5 mm) and small follicles (SF, 3–5 mm), and subtractive cDNA library was constructed. Furthermore, with dot-blot analysis, a total of 90 clones randomly selected from the library were proven to be differentially expressed in the granulosa cells. Among these, 38 exhibited high homology to known genes, 14 sequences were corresponding to novel expressed sequence tags (ESTs). Four ESTs, LAPTM4A, SERPINE2, GSTA1, and INHBA, were further examined the reproducibility of the SSH data by the real-time quantitative PCR. Results confirmed an increase expression of respective mRNA in granulosa cells of large follicles compared with that of small follicles. It is concluded that we have identified several genes (known or unknown) that may effect follicular growth, dominance or ovulation in ewes.     

Constitutive Endocytosis of the Chemokine CX3CL1 Prevents Its Degradation by Cell Surface Metalloproteases

CX₃CL1, a chemokine with transmembrane and soluble species, plays a key role in inflammation by acting as both chemoattractant and adhesion molecule. CX₃CL1 is the only chemokine known to undergo constitutive internalization, raising the possibility that dynamic equilibrium between the endocytic compartment and the plasma membrane critically regulates the availability and processing of CX₃CL1 at the cell surface. We therefore investigated how transmembrane CX₃CL1 is internalized. Inhibition of dynamin using a nonfunctional allele or of clathrin using specific small interfering RNA prevented endocytosis of the chemokine in CX₃CL1-expressing human ECV-304 cells. Perusal of the cytoplasmic domain of CX₃CL1 revealed two putative adaptor protein-2 (AP-2)-binding motifs. Accordingly, CX₃CL1 co-localized with AP-2 at the plasma membrane. We generated a mutant allele of CX₃CL1 lacking the cytoplasmic tail. Deletion of the cytosolic tail precluded internalization of the chemokine. We used site-directed mutagenesis to disrupt AP-2-binding motifs, singly or in combination, which resulted in diminished internalization of CX₃CL1. Although CX₃CL1 was present in both superficial and endomembrane compartments, ADAM10 (a disintegrin and metalloprotease 10) and tumor necrosis factor-converting enzyme, the two metalloproteases that cleave CX₃CL1, localized predominantly to the plasmalemma. Inhibition of endocytosis using the dynamin inhibitor, Dynasore, promoted rapid metalloprotease-dependent shedding of CX₃CL1 from the cell surface into the surrounding medium. These findings indicate that the cytoplasmic tail of CX₃CL1 facilitates its constitutive clathrin-mediated endocytosis. Such regulation enables intracellular storage of a sizable pool of presynthesized CX₃CL1 that protects the chemokine from degradation by metalloproteases at the plasma membrane.Inflammation is marked by the migration of circulating leukocytes into sites of injury, a process that occurs via a series of coordinated interactions between leukocytes and endothelial or epithelial cells. Central to this process are chemokines, a family of low molecular weight proteins that can attract leukocytes bearing the complementary receptors. When engagement of the chemokine receptor occurs, the leukocyte becomes activated and is induced to firmly adhere to the inflamed endothelium. These initial steps culminate in diapedesis of the leukocyte across the endothelium and migration into the injured tissue. The local complement of chemokines elaborated is organ-specific and varies with the type of inflammation present. In addition, specific leukocyte subsets also bear distinct chemokine receptors. In this way, chemokines and chemokine receptors confer organ specificity to leukocyte migration and help to “fine-tune” the nature of the observed inflammatory response.Among the 40 chemokines identified so far, CX₃CL1 is one of only two that have a transmembrane structure (1, 2). The chemokine domain of CX₃CL1 binds to its complementary receptor, CX₃CR1, through two distinct amino acid residues (3). The mucin stalk of CX₃CL1 allows efficient presentation of the chemokine to circulating leukocytes that express CX₃CR1, thereby allowing these leukocytes to be captured by the underlying endothelium (4, 5). CX₃CL1 also possesses a cytoplasmic tail 37 amino acids in length. However, the specific functions of the cytoplasmic tail have been left completely unexplored.Accumulating evidence demonstrates a critical role for CX₃CL1 in the pathogenesis of diverse inflammatory diseases, including atherosclerosis, systemic lupus erythematosus, and rejection of transplanted organs (6–15). Cell surface expression of CX₃CL1 is known to be regulated by proteolytic cleavage, or shedding, from the plasma membrane (16–18). Constitutive cleavage of CX₃CL1 occurs at low levels and is mediated by ADAM10 (a disintegrin and metalloprotease 10) (17). In response to inflammatory stimulation with lipopolysaccharide or to protein kinase C activation using phorbol 12-myristate 13-acetate, proteolytic cleavage of CX₃CL1 is markedly enhanced. Inducible cleavage of CX₃CL1 is mediated by tumor necrosis factor-α converting enzyme (TACE; ADAM17),² a related protease of the metzincin family (16, 18).In addition to proteolytic cleavage, surface expression of CX₃CL1 is also regulated by subcellular trafficking. We recently demonstrated that cell surface CX₃CL1 rapidly recycles to and from a specialized endocytic compartment, raising the possibility that the intracellular pool serves as a storage depot and that dynamic equilibrium between the endocytic compartment and the plasma membrane determines the availability and processing of transmembrane CX₃CL1 (19). In the current study, we explored whether the unique cytoplasmic tail of CX₃CL1 is important for this novel mode of regulation of the chemokine and whether it affects susceptibility of the chemokine to surface proteases. Our data suggest that plasmalemmal CX₃CL1 undergoes constitutive clathrin-mediated endocytosis (CME), facilitating storage of an intracellular pool of chemokine that is protected from cell surface metalloproteases.     

伊乐藻对浮游动物群落结构的影响

浮游动物是淡水生态系统的重要组成部分,一方面浮游动物主要以浮游植物作为食物^[1],同时又能摄食细菌^[2、3]、原生动物^[4、5];另一方面浮游动物又是一些鱼类优良的食物^[6、7]。国内外一些研究发现,有大量沉水植物存在的湖区,浮游动物的种类数、数量、生物量和多样性都比存在少量或没有沉水植物的湖区要高^[8-13]。     

Dinuclear gold(III) pyrazolato complexes - Synthesis, structural characterization and transformation to their trinuclear gold(I) and gold(I/III) analogues

The reaction of AuCl₃py with Na(pz∗) (pz∗ = pyrazolato, or substituted pyrazolato anion) yields stable dinuclear [cis-Au^IIICl₂(μ-pz∗)]₂ complexes. In the presence of a base, the latter undergo reduction with concomitant transformation of the dinuclear -structure to trinuclear Au^I, Au^III (containing trans Au^IIICl₂-centres) and species.     

Chiral separation performance of micrometer‐sized monodispersed silica spheres with high protein loading

Micrometer‐sized monodispersed silica spheres (～360 μm) with high loading of bovine serum albumin (BSA) (～450 mg/g) were prepared as an adsorbent for large‐scale chiral separation. The new adsorbent was characterized with scanning electron microscopy (SEM), nitrogen adsorption‐desorption isotherms, the mercury intrusion method, infrared spectroscopic analysis, and elementary analysis. The extent of chiral separation was tested with rac‐tryptophan (rac‐Trp) and rac‐phenylalanine (rac‐Phe) as solutes. The results showed that the absorbent exhibited a high surface area, large pore volume, and bimodal macromesoporous structures, enabling fast mass transfer and high separation efficiency. A fast adsorption rate, reaching equilibrium in less than 6 min, and a high degree of chiral separation, with the enantiomer excess (e.e.) value reaching as high as 100% in the first 10 min, was observed in a small (5 cm in height, 0.46 cm in internal diameter) packed column that could be regenerated with a pH 5.0 HAc‐NaAc buffer. The results show that monodispersed silica spheres with a high BSA loading have great potential for applications in large‐scale chiral separation processes. Chirality, 2009. © 2008 Wiley‐Liss, Inc.     

Effect of oleoyl-chitosan nanoparticles as a novel antibacterial dispersion system on viability,membrane permeability and cell morphology of Escherichia coli and Staphylococcus aureus

A novel chitosan antibacterial dispersion system was prepared by oleoyl-chitosan (O-chitosan) nanoparticles (OCNP) and the bactericidal activity against Escherichia coli and Staphylococcus aureus was evaluated by the enumeration of viable organisms at different incubation times. We further investigated the antimicrobial mode of OCNP using a combination of approaches, including cell integrity measurements, outer membrane (OM) and inner membrane (IM) permeabilization assays, SDS–PAGE and transmission electron microscopy (TEM). Results showed that when treated with OCNP, release of intracellular components quickly increased for both E. coli and S. aureus. OCNP also rapidly increased the 1-N-phenylnaphthylamine (NPN) uptake and the release of cytoplasmic β-galactosidase via increasing the permeability of OM and IM. Besides, SDS–PAGE indicated the content of cellular soluble proteins decreased significantly in OCNP-treated bacteria. TEM observations demonstrated adsorption behaviors of OCNP on bacteria and extensive cell surface alterations of OCNP-treated bacteria. OCNP has potential value in the determination of antibacterial mechanism of chitosan.     

烟草花粉管生长信号转导研究中一种简单的RNA提取方法(简报)

液氮研磨是一种从具有坚韧细胞壁的植物细胞中提取RNA的常规方法.然而,对于那种不易得到的少量的植物材料如花粉,这种方法的操作显得尤为困难.针对这一问题.本论文描述了一种不涉及液氮研磨的一种简单的RNA提取方法:改变萌发花粉管的渗透压使其破裂而释放RNA.这一方法减少了RNA抽提材料的用量并且提高了RNA的抽提效率,用于RNA抽提的花粉的量可以少至一个花药.后续的RNA分析需要的花粉可以少至1O毫克,这一用量相当于从50个烟草花药中得到的量.这一RNA提取方法的可靠性和可操作性为研究花粉管生长过程中对细胞外信号响应的基因时空表达提供了有益的参考.