低温对植物叶片中超氧物歧化酶、过氧化氢酶和过氧化氢水平的影响

番茄和鸡蛋果叶片中可提取的SOD活性不受低温的影响。在电泳谱带上SOD主同工酶带被氰化物而不被低温抑制,次同工酶带在低温下不稳定,且活性很低,它的变化不影响总的SOD活性。一些冷敏感植物叶片中CAT活性被低温抑制,而H_2O_3水平在低温下稳定或有增加,这可能使毒性更强的羟基离子(OH·)易于形成。     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Nuclear staining with alum hematoxylin

The hematoxylin and eosin stain is the most common method used in anatomic pathology, yet it is a method about which technologists ask numerous questions. Hematoxylin is a natural dye obtained from a tree originally found in Central America, and is easily converted into the dye hematein. This dye forms coordination compounds with mordant metals, such as aluminum, and the resulting lake attaches to cell nuclei. Regressive formulations contain a higher concentration of dye than progressive formulations and may also contain a lower concentration of mordant. The presence of an acid increases the life of the solution and in progressive solutions may also affect selectivity of staining. An appendix lists more than 60 hemalum formulations and the ratio of dye to mordant for each.     

beta-Catenin has sequential roles in the survival and specification of ventral dermis

The dermis promotes the development and maintains the functional components of skin, such as hair follicles, sweat glands, nerves and blood vessels. The dermis is also crucial for wound healing and homeostasis of the skin. The dermis originates from the somites, the lateral plate mesoderm and the cranial neural crest. Despite the importance of the dermis in the structural and functional integrity of the skin, genetic analysis of dermal development in different parts of the embryo is incomplete. The signaling requirements for ventral dermal cell development have not been established in either the chick or the mammalian embryo. We have shown previously that Wnt signaling specifies the dorsal dermis from the somites. In this study, we demonstrate that Wnt/beta-catenin signaling is necessary for the survival of early ventral dermal progenitors. In addition, we show that, at later stages, Wnt/beta-catenin signaling is sufficient for ventral dermal cell specification. Consistent with the different origins of dorsal and ventral dermal cells, our results demonstrate both conserved and divergent roles of beta-catenin/Wnt signaling in dermal development.     

A Forward Genetic Strategy Reveals Destabilizing Mutations in the Ebolavirus Glycoprotein That Alter Its Protease Dependence during Cell Entry

Ebolavirus (EBOV) entry into cells requires proteolytic disassembly of the viral glycoprotein, GP. This proteolytic processing, unusually extensive for an enveloped virus entry protein, is mediated by cysteine cathepsins, a family of endosomal/lysosomal proteases. Previous work has shown that cleavage of GP by cathepsin B (CatB) is specifically required to generate a critical entry intermediate. The functions of this intermediate are not well understood. We used a forward genetic strategy to investigate this CatB-dependent step. Specifically, we generated a replication-competent recombinant vesicular stomatitis virus bearing EBOV GP as its sole entry glycoprotein and used it to select viral mutants resistant to a CatB inhibitor. We obtained mutations at six amino acid positions in GP that independently confer complete resistance. All of the mutations reside at or near the GP1-GP2 intersubunit interface in the membrane-proximal base of the prefusion GP trimer. This region forms a part of the “clamp” that holds the fusion subunit GP2 in its metastable prefusion conformation. Biochemical studies suggest that most of the mutations confer CatB independence not by altering specific cleavage sites in GP but rather by inducing conformational rearrangements in the prefusion GP trimer that dramatically enhance its susceptibility to proteolysis. The remaining mutants did not show the preceding behavior, indicating the existence of multiple mechanisms for acquiring CatB independence during entry. Altogether, our findings suggest that CatB cleavage is required to facilitate the triggering of viral membrane fusion by destabilizing the prefusion conformation of EBOV GP.Filoviruses are enveloped, filamentous, nonsegmented negative-sense RNA viruses that can cause a deadly hemorrhagic fever with case fatality rates in excess of 90% (see references 4, 20, and 37 for recent reviews). All known filoviruses belong to one of two genera: Ebolavirus (EBOV), consisting of the five species Zaire (ZEBOV), Côte d''Ivoire, Sudan, Reston, and Bundibugyo (tentative); and Marburgvirus, consisting of the single Lake Victoria species (21, 62).Cell entry by filoviruses is mediated by their envelope glycoprotein, GP (60, 68). Mature GP is a trimer of three disulfide-linked GP1-GP2 heterodimers. GP1 and GP2 are generated by endoproteolytic cleavage of the GP0 precursor polypeptide by a furin-like protease during transport to the cell surface (31, 39, 63, 69). The membrane-distal subunit, GP1, mediates viral adhesion to host cells (10, 18, 38, 42, 56, 59) and regulates the activity of the transmembrane subunit, GP2, which catalyzes fusion of viral and cellular membrane bilayers (30, 39, 41, 64, 65). The consequence of membrane fusion is cytoplasmic delivery of the viral nucleocapsid cargo.Lee et al. (39) recently solved the crystal structure of a ZEBOV GP prefusion trimer lacking the heavily glycosylated GP1 mucin domain (Muc) and the GP2 transmembrane domain (see Fig. ​Fig.5).5). The three GP1 subunits together form a bowl-like structure encircled by sequences from the three GP2 subunits. The trimer is held together by GP1-GP2 and GP2-GP2 contacts; the hydrophobic GP2 fusion loop packs against the external surface of adjacent GP1 subunits, and each GP2 subunit contributes a strand to a trimeric α-helical coiled-coil stem. GP1 is organized into three subdomains. The base is intimately associated with GP2 and clamps it in its prefusion conformation. The head is proposed to mediate virus receptor binding during entry (10, 18, 38, 42). The glycan cap resides at the top of the trimer and is critical for GP assembly but must be removed during entry (see below) (31, 42). The base and glycan cap are connected by the β13-β14 loop, which was not visualized in the structure. The location and structure of the Muc domain are also unknown, but it is proposed to sheathe the top and/or sides of the prefusion GP trimer (39). Muc is dispensable for ZEBOV GP-dependent entry in tissue culture but may play roles in virus-cell adhesion and immune evasion in vivo (31, 42, 44, 56, 59).Open in a separate windowFIG. 5.CA074^R mutations localize at or near the GP1-GP2 interface in the GP prefusion crystal structure. In all diagrams, GP1 is depicted in blue, GP2 in red, GP1 CA074^R mutations in green, and GP2 CA074^R mutations in yellow. (A) Linear representation of the amino acid sequence of GPΔMuc. S-S indicates the intersubunit disulfide bond between C53 and C609. sp, signal peptide; fl, fusion loop; hr1 and hr2, heptad repeats; tm, transmembrane domain; N, N terminus; C, C terminus. (B) Structure of GP in a prefusion conformation (39). Cartoon representation of a GP1-GP2 monomer is shown. Remaining subunits are shown as a surface-shaded watermark. The boxed inset contains the membrane-proximal base of the trimer, in which the CA074^R mutations are located. The β13-β14 loop is modeled as a chain of blue circles. (C) Magnified view of the inset shown in panel B rotated by 90°. The side chains of D47, I584, K588, and their contacting residues are shown. Dashed pink lines connect atoms from different side chains separated by ≤3.9 Å. Other CA074^R residues are not shown for clarity. (D) View shown in panel C rotated by 90°. (E) Schematic diagram of the potential interactions made by residues mutated in the CA074^R viruses. Residues approaching ≤3.9 Å to each CA074^R residue are shown. Beige arcs, hydrophobic interactions; dashed lines, potential ionic interactions. Visualizations of the GP structures shown in panels B to D (Protein Data Bank accession no. 3CSY) were rendered in Pymol (Delano Scientific).Crystal structures of ZEBOV GP2 in its postfusion conformation indicate that filovirus GP is a “class I” viral membrane fusion protein (41, 65). Like the prototypic class I fusion proteins of human immunodeficiency virus and influenza virus, GP2 contains a hydrophobic fusion peptide near its N terminus and N- and C-terminal α-helical heptad repeat sequences (HR1 and HR2, respectively) (22, 28, 30, 39, 41, 64, 65, 67) (see Fig. ​Fig.5).5). GP2 drives membrane fusion by undergoing large-scale conformational changes; the prefusion HR1 helix-loop-helix rearranges to an unbroken α-helix, projecting the fusion loop into the endosomal membrane, and GP2 jackknifes on itself to form a hairpin-like structure in which the HR2s pack against grooves in the trimeric HR1 coiled coil (41, 65).The available GP structures make clear that the transition of GP2 from prefusion to postfusion conformation requires its release from its binding groove in the GP1 base subdomain. For all known class I fusion proteins, this transition is controlled by priming and triggering events. Priming typically involves a single endoproteolytic cleavage of the glycoprotein mediated by a cellular protease within the secretory pathway of the virus-producer cells (e.g., human immunodeficiency virus ENV → SU + TM by furin [27]). This cleavage is essential because it liberates an N-terminal fusion peptide and allows the glycoprotein to rearrange during fusion. Unusually for a class I fusion glycoprotein, however, ZEBOV GP does not require cleavage to GP1 and GP2 by a furin-like protease, even though this cleavage occurs efficiently (46, 69). Instead, the GP trimer is primed by extensive proteolytic remodeling during entry. This process is mediated by cysteine cathepsins, a class of papain superfamily cysteine proteases active within the cellular endosomal/lysosomal pathway (14, 54).The cysteine cathepsins B (CatB) and L (CatL) play essential and accessory roles, respectively, in ZEBOV entry into Vero cells (14). The functions of these enzymes in viral entry can be recapitulated in vitro. Incubation of vesicular stomatitis virus (VSV) pseudotypes bearing ZEBOV GP (VSV-GP) with a mixture of purified human CatL and CatB, or with the bacterial protease thermolysin (THL), results in the cleavage and removal of GP1 Muc and glycan cap sequences, leaving a stable ∼17-kDa N-terminal GP1 fragment and intact GP2 (see Fig. ​Fig.5)5) (18, 54). VSV particles containing this GP_17K intermediate no longer require CatB activity within cells, strongly suggesting that this protease plays a critical role in generating a related primed species during viral entry (54). Strikingly, incubation of VSV-GP with CatL alone (14, 54) or with bovine chymotrypsin (CHT) (this study) (Fig. ​(Fig.1;1; see also Fig. ​Fig.7)7) generates a similar but distinct GP_18K intermediate (containing a slightly larger ∼18-kDa GP1 fragment) that cannot bypass the requirement for CatB during entry. Therefore, the removal of a few residues from GP_18K by CatB is crucial for viral entry. The reason for this requirement is unknown. Finally, VSV-GP_17K particles cannot infect cells completely devoid of cysteine cathepsin activity, indicating the existence of at least one additional cysteine protease-dependent step during entry (34, 54; present study). The signal that acts on a fully primed GP intermediate to trigger membrane fusion remains unknown.Open in a separate windowFIG. 1.CatB activity is required for entry of ZEBOV GP-dependent entry, whereas CatL activity is dispensable. Vero cells were pretreated for 4 h with 1% (vol/vol) DMSO (vehicle), 0.5 μM FYdmk (CatL-selective inhibitor), 80 μM CA074 (CatB-selective inhibitor), 0.5 μM FYdmk plus 80 μM CA074, or 300 μM E64 (pan-cysteine cathepsin inhibitor). (A) The cells were then challenged with VSV-GPΔMuc, CHT-derived VSV-GP_18K (CHT-GP_18K), THL-derived VSV-GP_17K (THL-GP_17K), or VSV-G pseudotypes at a low MOI (0.02 to 0.1 eGFP-positive infectious units [iu] per cell) in the presence of drug, and viral titers (iu/ml) were determined at 18 h postinfection. CatB and CatL activities in extracts prepared from a parallel set of pretreated cells were measured by fluorogenic peptide turnover and are shown (bottom). Averages ± standard deviations (SD) for six trials from three independent experiments are shown. CatL activity below the detection threshold is indicated as zero without an accompanying SD. (B) Vero cells pretreated with protease inhibitors were challenged with VSV-GPΔMuc, cathepsin L-derived VSV-GP_18K (CatL-GP_18K), or cathepsin B-derived VSV-GP_17K (CatB-GP_17K), and viral infectivity was measured as described above. Averages ± SD for three trials from a representative experiment are shown.Open in a separate windowFIG. 7.rVSV-GPΔMuc mutants resemble the WT in cleavage to GP_18K and GP_17K intermediates. WT or mutant rVSV-GPΔMuc was incubated with the indicated protease(s) as described in Materials and Methods and then deglycosylated with PNGaseF (except for N40K and T42A, which lack the N40 glycan and do not require deglycosylation at this position). The resulting GP1 proteolytic fragments were resolved by SDS-PAGE and detected by Western blotting. Shorter protease incubation times were necessary to obtain cleavage intermediates for some mutants (see text for details). Positions of uncleaved GP1 and the ∼18-kDa and ∼17-kDa cleavage fragments are indicated on the left. *, partially cleaved GP fragment of unknown composition. Experimental samples shown on each gel (bold labels) were flanked by WT samples cleaved with CHT (WT 18K) or THL (WT 17K) to provide markers of band mobility.In this study, we used a forward genetic strategy to investigate the CatB-dependent step in ZEBOV entry. Specifically, we engineered and rescued a recombinant VSV (rVSV) encoding a mucin domain-deleted ZEBOV GP in place of the VSV glycoprotein G and used it to select viral mutants resistant to the CatB inhibitor CA074. Analysis of these viruses identified mutations in both GP1 and GP2 that allow CatB-independent cell entry. We found that GP_18K and/or GP_17K intermediates derived from some but not all of the mutant GPs are conformationally distinct from the wild type (WT), suggesting the existence of multiple mechanisms for CA074 resistance. Taken together, our results indicate that ZEBOV GP→GP_17K cleavage by CatB promotes fusion triggering and viral entry by destabilizing the prefusion conformation of GP.     

Cav-1 participates in the development of diabetic neuropathy pain through the TLR4 signaling pathway

This study aims to determine whether caveolin-1 (Cav-1) participates in the process of diabetic neuropathic pain by directly regulating the expression of toll-like receptor 4 (TLR4) and the subsequent phosphorylation of N-methyl-D-aspartate receptor 2B subunit (NR2B) in the spinal cord. Male Sprague-Dawley rats (120–150 g) were continuously fed with high-fat and high-sugar diet for 8 weeks, and received a single low-dose of intraperitoneal streptozocin injection in preparation for the type-II diabetes model. Then, these rats were divided into five groups according to the level of blood glucose, and the mechanical withdrawal threshold and thermal withdrawal latency values. The pain thresholds were measured at 3, 7, and 14 days after animal grouping. Then, eight rats were randomly chosen from each group and killed. Lumbar segments 4–6 of the spinal cord were removed for western blot analysis and immunofluorescence assay. Cav-1 was persistently upregulated in the spinal cord after diabetic neuropathic pain in rats. The downregulation of Cav-1 through the subcutaneous injection of Cav-1 inhibitor daidzein ameliorated the pain hypersensitivity and TLR4 expression in the spinal cord in diabetic neuropathic pain (DNP) rats. Furthermore, it was found that Cav-1 directly bound with TLR4, and the subsequent phosphorylation of NR2B in the spinal cord contributed to the modulation of DNP. These findings suggest that Cav-1 plays a vital role in DNP processing at least in part by directly regulating the expression of TLR4, and through the subsequent phosphorylation of NR2B in the spinal cord.     

A preliminary method for estimating the age of brown kiwi (Apteryx mantelli) embryos

Morphological features of a collection of unknown-age wild kiwi (Apteryx mantelli) embryos from early development to point of hatch are described. Using these features, we assign developmental stages to each embryo and compare the progress of development to similar-staged ostrich (Struthio camelus) and chicken (Gallus gallus) embryos. Two ageing schemes for the kiwi embryos are developed by comparing measurements of their hindlimb segments, bills and crown–rump lengths with those of ostrich and chicken embryos at various stages of development. One of the 20 kiwi embryos was of known age. Both the ostrich model and the chicken model gave identical predictions for the marker and four other embryos. Developmental timing of some features differed between all three species, most markedly in the bill, with growth in the kiwi bill being relatively faster to achieve its larger relative and absolute size at hatch.