El Tor型霍乱弧菌及其细胞壁缺陷型分子遗传学背景的研究

El Tor 型霍乱弧菌(以下简称 El Tor 弧菌)可以在人工培养条件下长期存活。当微环境改变时可形成细胞壁有不同程度缺陷的菌株如抗噬菌体突变株或 L 型菌株。我们以 DNA 酶切图谱和 El Tor 弧菌溶血素、神经氨酸酶基因探针杂交图谱为参数对 El Tor 弧菌的野生型及其细胞壁缺陷型变异株在遗传背景上进行了比较分析研究。结果提示细胞壁缺陷型菌株与其野生型在DNA 水平上高度同源。此外,文中还介绍了一种从 L 型菌株中制备 DNA 的方法。     

A SPOROPHYTE CELL WALL PROTEIN OF THE RED ALGA PORPHYRA PURPUREA (RHODOPHYTA) IS A NOVEL MEMBER OF THE CHYMOTRYPSIN FAMILY OF SERINE PROTEASES1

A complementary DNA (cDNA) clone from a Porphyra purpurea (Roth) C. Agardh sporophyte-specific subtracted cDNA library was found to encode a protein similar to serine proteases of the chymotrypsin class. The encoded protein contains a typical signal peptide and is particularly similar to chymotrypsins in the regions surrounding the active site residues and the activation site where cleavage of the propeptide occurs. In addition, the six cysteine residues characteristic of chymotrypsins are conserved. However, two of the three residues of the active site His/Asp/Ser charge relay triad have been replaced, indicating that the protein is unlikely to have peptidase activity. Northern hybridization confirmed that this cDNA is derived from an abundant, sporophyte-specific messenger RNA (mRNA). The presence of signal peptide on the encoded protein and the abundance of its mRNA suggested that this protein might be localized in the cell wall. Consequently, sporophyte cell walls were isolated and a major protein having a molecular weight similar to that estimated for the encoded protein was purified. N-terminal sequence analysis indicated that this cell wall protein is identical to that encoded by the cDNA with the amino terminus of the mature protein beginning at the activation site. This cell wall structural protein appears to have evolved from a chymotrypsin-like progenitor but has been adapted to bind cell wall proteins and/or polysaccharides rather than to cleave proteins.     

chs-4, a class IV chitin synthase gene fromNeurospora crassa

InSaccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by theCSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized aCSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomyceteNeurospora crassa and have used a reverse genetics approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of aN. crassa gene (designatedchs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those ofS. cerevisiae andCandida albicans. N. crassa strains in whichchs-4 had been inactivated by the Repeat-Induced Point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in thechs-4 ^RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme inN. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.A. Beth Din and C. A. Specht contributed equally to this work     

Immunocytochemical localization of arabinoxylans in the cell wall of maize apical internode after microbial degradation in the rumen

Summary— Arabinoxylans were localised by immunocytochemistry using polyclonal antibodies in the cell walls of the apical internode of maize after degradation in the rumen. In order to understand the significance of arabinoxylan in digestibility property, two lines of maize differing in digestibility were used. Wide variations in the intensity of labelling were observed in the four tissues studied (sclerenchyma, fibres, xylem and parenchyma) from the first hours of incubation in the rumen. Incubation time in the rumen greatly influences the intensity of labelling.     

Local renin-angiotensin systems

The existence of a local cardiovascular renin-angiotensin system (RAS) is often invoked to explain the long-term beneficial effects of RAS inhibitors in heart failure and hypertension. The implicit assumption is that all components of the RAS are synthesized in situ, so that local angiotensin II formation may occur independently of the circulating RAS. Evidence for this assumption however is lacking. The angiotensin release from isolated perfused rat hearts or hindlimbs depends on the presence of renal renin. When calculating the in vivo angiotensin production at tissue sites in humans and pigs, taking into account the extensive regional angiotensin clearance by infusing radiolabeled angiotensin I or II, it was found that angiotensin production correlated closely with plasma renin activity. Moreover, in pigs the cardiac tissue levels of renin and angiotensin were directly correlated with their respective plasma levels, and both in tissue and plasma the levels were undetectably low after nephrectomy. Similarly, rat vascular renin and angiotensin decrease to low or undetectable levels within 48 h after nephrectomy. Aortic renin has a longer half life than plasma renin, suggesting that renin may be bound by the vessel wall. In support of this assumption, both renin receptors and renin-binding proteins have been described. Like ACE, renin was enriched in a purified membrane fraction prepared from cardiac tissue. Binding of renin to cardiac or vascular membranes may therefore be part of a mechanism by which renin is taken up from plasma. It appears that the concept of a local RAS needs to be reassessed. Local angiotensin formation in heart and vessel wall does occur, but depends, at least under normal circumstances, on the uptake of renal renin from the circulation. Tissues may regulate their local angiotensin concentrations by varying the number of renin receptors and/or renin-binding proteins, the ACE level, the amount of metabolizing enzymes and the angiotensin receptor density.Abbreviations RAS renin-angiotensin system - ANG angiotensin - ACE angiotensin-converting enzyme - PRA plasma renin activity     

The role of gap junction membrane channels in development

In most developmental systems, gap junction-mediated cell-cell communication (GJC) can be detected from very early stages of embryogenesis. This usually results in the entire embryo becoming linked as a syncytium. However, as development progresses, GJC becomes restricted at discrete boundaries, leading to the subdivision of the embryo into communication compartment domains. Analysis of gap junction gene expression suggests that this functional subdivision of GJC may be mediated by the differential expression of the connexin gene family. The temporal-spatial pattern of connexin gene expression during mouse embryogenesis is highly suggestive of a role for gap junctions in inductive interactions, being regionally restricted in distinct developmentally significant domains. Using reverse genetic approaches to manipulate connexin gene function, direct evidence has been obtained for the connexin 43 (Cx43) gap junction gene playing a role in mammalian development. The challenges in the future are the identification of the target cell populations and the cell signaling processes in which Cx43-mediated cell-cell interactions are critically required in mammalian development. Our preliminary observations suggest that neural crest cells may be one such cell population.     

Immunolocalization of extensin and potato tuber lectin in carrot, tomato and potato

Tissue-specific expression of two members of the cell wall hydroxyproline-rich glycoprotein (HRGP) family, extensin and potato tuber lectin, was examined by immunolocalization at the light microscope level in various organs (leaves, stems, roots, fruit, tuber) of carrot ( Daucus carota cv. Thumbelina), tomato ( Lycopersicon esclentum cv. Pixie Hybrid II), and potato ( Solanum tuberosum cv. Kennebec). Extensin was prominently expressed in vascular tissue, particularly xylem and also phloem, although virtually all cells displayed some degree of staining which varied as a function of the tissue, organ, and plant under study. Antibodies against potato tuber lectin (PTL) displayed a localization pattern similar to that observed for extensin; notably PTL did not stain cambium but did stain epithelial cells lining secretory cavities. These distribution patterns are consistent with a role for extensin, and possibly PTL, in providing mechanical support in tissues subjected to compression or torsional stress imparted by vascular growth, or by similar stress brought about by transport of vascular fluids.     

Substrate subsite recognition of the xyloglucan endo-transglycosylase or xyloglucan-specific endo-(1→4)-β-d-glucanase from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds

We have investigated the substrate subsite recognition requirement of the xyloglucan endo-transglycosylase/xyloglucan-specific endo-(14)--d-glucanase (NXET) from the cotyledons of nasturtium seedlings. Seed xyloglucans are composed almost entirely of the Glc₄ subunits XXXG, XLXG, XXLG and XLLG, where G represents an unsubstituted glucose residue, X a xylose-substituted glucose residue and L a galactosyl-xylose-substituted glucose residue. Thus in the xyloglucan sequence shown below, the xylose (Xyl) residues at the backbone glucose (Glc) residues numbered — 3,— 2, + 2 and + 3 may be galactose-substituted, and NXET cleaves between the unsubstituted glucose at — 1 and the xylose-substituted glucose at + 1, which never carries a galactosyl substituent. We have isolated the xyloglucan oligosaccharides XXXGXXXG and XLLGXLLG from NXET digests of tamarind seed xyloglucan, have modified them enzymatically using a pure xyloglucan oligosaccharide-specific -xylosidase from nasturtium seeds to give GXXGXXXG and GLLGXLLG, and have identified and compared the products of NXET action on XXXGXXXG, GXXGXXXG, XLLGXLLG and GLLGXLLG. We have also compared the molar proportions of XXXG, XLXG, XXLG and XLLG in native tamarind and nasturtium seed xyloglucans with those in NXET digests of these polysaccharides. Using these and existing data we have demonstrated that NXET action does not require xylosesubstitution at glucose residues — 4, — 2, + 1 and + 3 and that xylose substitution at + 2, is a requirement. There may also be a requirement for xylose substitution at — 3. We have demonstrated also that galactosyl substitution of a xylose residue at + 1 prevents, and at — 2 modifies, chain-cleavage. A partial model for the minimum substrate binding requirement of NXET is proposed.Abbreviations G unsubstituted glucose residue - X xylose-substituted glucose residue - L galactosylxylose-substituted glucose residue - F fucosyl-galactosylxylose-substituted glucose residue - Gal galactose - Glc glucose - HPAE high-performance anion-exchange chromatography - NXET nasturtium xyloglucan endo-transglycosylase or xyloglucan-specific endo-(14)--d-glucanase - Xyl xylose This work was funded jointly by Unilever UK and the Department of Trade and Industry (UK) via the LINK initiative Agro-Food Quality.     

Variations in the structure of neutral sugar chains in the pectic polysaccharides of morphologically different carrot calli and correlations with the size of cell clusters

Carrot (Daucus carota L.) embryogenic callus (EC) loses its embryogenic competence and becomes nonembryogenic callus (NC) during long-term culture. With the loss of embryogenic competence, the cell clusters become smaller and the extent of intercellular attachments is reduced. Pectic fractions prepared from EC and NC were separated into two subfractions by gel filtration. A difference in sugar composition between EC and NC was found only in the high-molecular-mass (ca. 1300 kDa) subfraction, and the ratio of the amount of arabinose to that of galactose (Ara/Gal) was strongly and positively correlated with the size of cell clusters in several different cultures. From the results of sugar-composition and methylation analyses, and the results of treatment with exo-arabinanase, models of the neutral sugar chains of pectins from EC and NC are proposed. Both neutral sugar chains are composed of three regions. The basal region is composed of linearly linked arabinan 5-Ara_f> moieties in both types of callus. The middle galactan region is composed of 6-linked galactose, some of which branches at the 3 and 4 positions, and this region is larger and more frequently branched in NC than in EC. Finally, the terminal arabinan region is composed of 5-linked arabinose, branched at the 3 position, and the size of the terminal arabinan is larger in EC than in NC. The significance of the neutral sugar chains of pectins in the interaction of cell wall components and intercellular attachment is discussed.Abbreviations Ara/Gal ratio (w/w) of the amount of arabinose to that of galactose - EC embryogenic callus - NC non-embryogenic callus - T-Ara_f terminal arabinose The authors are grateful to Dr. Naoto Shibuya of the National Institute of Agrobiological Resources for his gift of exo-arabinanase.     

The sporophyte-gametophyte junction in the moss Acaulon muticum (Pottiaceae): EARLY STAGES OF DEVELOPMENT

The sporophyte-gametophyte junction in Acaulon muticum is composed of the sporophyte foot, the surrounding gametophyte vaginula, and an intervening placental space. At an early stage of development the foot has a large basal cell, characterized by extensive wall ingrowths beginning at the lowermost tip of the basal cell and extending along its tangential walls. Sporophyte cells in contact with the basal cell develop ingrowths on their outer tangential walls and on radial walls in contact with the basal cell. All sporophyte cells at this stage are characterized by numerous mitochondria, strands of endoplasmic reticulum, and dictyosomes, particularly in the cytoplasm adjacent to areas of extensive wall development. Plastids typically contain abundant starch reserves. As development proceeds, wall ingrowths become more extensive on all walls in the sporophyte foot but are never found on the upper wall of the basal cell in contact with the remainder of the sporophyte. Plastids in the foot contain fewer starch reserves later in development. Wall ingrowths are not visible in the cells of the gametophyte vaginula until well after extensive development has occurred in the sporophyte foot. Stacks or layers of endoplasmic reticulum are characteristic of the cells of the gametophyte vaginula, along with numerous mitochondria, dictyosomes, and well-developed plastids. Starch reserves typically are less abundant in cells of the gametophyte. The early development of extensive wall elaborations in the cells of the sporophyte foot, and particularly in the basal cell, may favor the rapid movement of water and nutrients from the gametophyte into the sporophyte at a time when rapid development in this minute, ephemeral moss is critical.