Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans

The terminal process of xylogenesis, autolysis, is essential for the formation of a tubular system for conduction of water and solutes throughout the whole plant. Several hydrolase types are implicated in autolysis responsible for the breakdown of cytoplasm. Here, we characterize p48h-17 cDNA from in vitro tracheary elements (TEs) of Zinnia elegans which encodes a preproprotein similar to papain. The putative mature protein, a cysteine protease, has a molecular mass of 22,699 Da with a pI of 5.7. DNA gel blot analysis indicated that p48h-17 is likely encoded by one or two genes. The p48h-17 mRNA accumulated markedly in in vitro differentiating TEs, whereas it appeared not to be induced in response to senescence and wounding in the leaves or H₂O₂ challenge in the cultured mesophyll cells. In stems, the expression of the p48h-17 gene was preferentially associated with differentiating xylem. Activity gel assays demonstrated that a cysteine and a serine protease, which had apparent molecular masses of 20 kDa and 60 kDa, respectively, were markedly induced during in vitro TE differentiation. The cysteine protease activity was also preferentially present in the xylem of Zinnia stems. Transient expression of the p48h-17 cDNA in tobacco protoplasts resulted in the production of a 20 kDa cysteine protease. Taken together, the results indicate that the p48h-17 gene appears to be preferentially associated with xylogenesis, and both the cysteine and serine proteases might be involved in autolysis during xylogenesis.     

Sequence analysis of a cDNA containing the gag and prot regions of the soybean retrovirus-like element, SIRE-1

SIRE-1 is a family of several hundred dispersed copies of a very large DNA element from Glycine max that has features characteristic of retroviruses and retrotransposons. A 2.4 kb SIRE-1-specific fragment was recovered from a soybean cDNA library and sequenced. The sequence contains two ORFs. Theoretical translation of ORF1 produces a gag-prot-like polyprotein containing highly conserved motifs found in retroelement nucleocapsids (CX₂CX₄HX₄C) and aspartic proteases (LDSG). The second ORF is foreshortened. The cDNA also contains nearly 200 bp of a putative 5 LTR just upstream of a tRNA primer-binding site.     

The effect on yield in courgette and marrow of the mild strain of zucchini yellow mosaic virus used for cross-protection

The effects on yield in courgette and marrow (Cucurbita pepo) crops resulting from inoculation with the mild strain of zucchini yellow mosaic virus (ZYMV:WK), have been determined in polythene-house trials and in three years of outdoor, commercial field trials. In polythene-house trials ZYMV:WK inoculated plants were up to 10 days later in flowering than uninoculated plants and their cumulative yields were between 5% and 26% less than uninoculated plants depending on the cultivar. In most field trials cumulative yields from inoculated plants were between 4% and 38% less than uninoculated plants depending on the site and cultivar, but in one trial the yield was 7% higher from inoculated plants. In all experiments, courgette and marrow fruits harvested from ZYMV:WK inoculated plants were symptomless and indistinguishable from fruit harvested from uninoculated plants. The mild leaf symptoms induced by ZYMV:WK infection did not intensify to severe leaf symptoms and where there were natural outbreaks of severe ZYMV infection, fruits from inoculated plants remained symptomless whilst those from uninoculated plants were severely affected and unmarketable.     

Eubacterial proteasomes

Proteasomes are large, multisubunit proteases with highly conserved structures. The 26S proteasome of eukaryotes is an ATP-dependent enzyme of about 2 MDa, which acts as the central protease of the ubiquitin-dependent pathway of protein degradation. The core of the 26S complex is formed by the 20S proteasome, an ATP-independent, barrel-shaped protease of about 700 kDa, which has also been detected in archaebacteria and, more recently, in eubacteria. Currently, the distribution of 20S proteasomes in eubacteria appears limited to the actinomycetes, while most other eubacteria contain a related complex of simpler structure.     

IgA Protease Produced by Streptococcus sanguis and Antibody Production against IgA Protease in Patients with Behçet's Disease

The production of IgA protease in twelve strains of Streptococcus sanguis isolated from patients with Behçet's disease (BD) was examined. Protease activity was detected in 10 out of 12 strains. The protease was purified from one representative strain, S. sanguis 113–20, by employing Rotofor and DEAE-Sephacel chromatography. The molecular mass of the purified protease was approximately 100 kDa, and it cleaved the proline-threonine site of the IgA. Both IgG and IgA titers against the cells (113–20) and the purified IgA protease in the sera of BD patients and healthy controls, 36 each, were assayed. The IgG titers against the cells and protease were not significant in the BD patients or controls, but the IgA titers against the cells and protease in the BD patients were significantly higher than those of the controls. These data indicate that the BD patients are infected with IgA protease-producing S. sanguis strains, which cause an increase of IgA titer against these organisms and IgA protease antigen. Since the organisms can proliferate in BD patients for a long period of time (years), it seems that IgA antibodies cannot effectively eliminate the organisms.     

蓖麻“哲蓖三号”主要数量性状遗传参数研究

蓖麻“哲蓖三号”主要数量性状遗传参数研究张春华包红霞（内蒙古哲里木盟农业科学研究所,通辽０２８０１５）ＳｔｕｄｉｅｓｏｎｔｈｅＨｅｒｅｄｉｔａｒｙＰａｒａｍｅｔｅｒｓｏｆＭａｊｏｒＱｕａｎｔｉｔａｔｉｖｅＣｈａｒａｃｔｅｒｓｏｆＣａｓｔｏｒ“ＺＨＥＢ...     

链霉菌SO1菌株几丁质酶的纯化及性质

由链霉菌（Streptomycessp.）SO1菌株产生的几丁质酶（Citinase）经硫酸铵盐析、OEAE纤维素柱层析、SephadexG－100柱层桥分离纯化后,得到SDS－PAGE均一样品。用SDS－PAG测得纯化后的几丁质酶分子量为41Ku,用PAGEIEF测得等电点PI为5.4。酶反应的最适pH值为6．0,最适温度为50℃,在pH5．0～9．0、温度30～50℃时酶活性比较稳定。在相当于0.1mol／LNaCl的离子强度下酶活性最高。金属离子中的Mg²⁺、Ca     

cDNAs induced by ozone from Atriplex canescens (saltbush) and their response to sulfur dioxide and water-deficit

Ozone effects on plant water relations have been reported to be similar to those of water-deficit. The objective was to identify ozone-inducible (OI) clones from Atriplex canescens (saltbush) and determine if they were also responsive to water-deficit as well as SO₂. cDNA clones derived from four different polyA RNAs which accumulate in 8-month-old shrub leaves exposed to ozone (0.2 μl I⁻¹, 6 h day⁻¹, 7 days) were isolated by differential screening, analyzed by northern blots, sequenced, and gene product homologies with other plant genes were determined. Clone OI12A-3 has homology with wound-inducible proteinase inhibitors, whereas clone OI8–3 protein is homologous to thiol proteases. Clones OI2–2 and OI14–3 putatively code for glycine-rich proteins with repeated motifs (Gly-Gly-Gly-Tyr-Gly-His)_n and putative cell-wall-targeting signal peptides. Clone OI2–2 and particularly clone OI14–3 were also induced by both SO₂ and water-deficit. These data indicate that woody plant genes associated with cell wall protein production and whose expression is induced by several stress factors may be responding to common oxidative stress pathways.     

Proteolytic cleavage of recombinant two-chain factor VIII during cell culture production is mediated by protease(s) from lysed cells. The use of pulse labelling directly in production medium

During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium. The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of ³⁵S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII against cleavage, probably by binding to rFVIII. This revised version was published online in July 2006 with corrections to the Cover Date.     

Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to α-lytic protease

-Lytic protease, a bacterial serine protease of 198 aminoacids (19800 Da), has been used as a model system for studies of catalyticmechanism, structure–function relationships, and more recently forstudies of pro region-assisted protein folding. We have assigned thebackbones of the enzyme alone, and of its complex with the tetrahedraltransition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boroVal, usingdouble- and triple-resonance 3D NMR spectroscopy on uniformly¹⁵N- and ¹³C/¹⁵N-labeled protein.Changes in backbone chemical shifts between the uncomplexed and inhibitedform of the protein are correlated with distance from the inhibitor, thedisplacement of backbone nitrogens, and change in hydrogen bond strengthupon inhibitor binding (derived from previously solved crystal structures).A comparison of the solution secondary structure of the uninhibited enzymewith that of the X-ray structure reveals no significant differences.Significant line broadening, indicating intermediate chemical exchange, wasobserved in many of the active site amides (including three broadened toinvisibility), and in a majority of cases the broadening was reversed uponaddition of the inhibitor. Implications and possible mechanisms of this linebroadening are discussed.