Disruption of the gene encoding the secreted acid protease (ACP) in the yeast Candida tropicalis

The gene for the secreted acid protease (ACP), a potential virulence factor of Candida species, was inactivated in Candida tropicalis by gene disruption. The disruption was performed by cotransformation of an ade2 C. tropicalis mutant with a linear DNA fragment carrying a deletion in ACP, and the replicative vector pMK16 which carries a selectable ADE2 gene marker. Few of the transformants exhibited lower protease secretion levels and were shown to have one deleted and one unaffected ACP copy, since C. tropicalis is a diploid yeast. These transformants were rendered homozygotic for this deletion by mild UV-treatment. One of the homozygotic acp deletion mutants obtained was completely devoid of extracellular protease activity and grew poorly on bovine serum albumin-containing medium. This mutant could be complemented by an ACP fragment inserted in pMK16, but also by an acid protease gene isolated from C. parapsilosis.     

Interaction between monobactams and model d-alanyl-d-alanine-cleaving peptidases

Abstract Several monobactams reacted with the serine dd -peptidases of Streptomyces R61 and Actinomadura R39 in a manner similar to that of bicyclic penicillins and cephalosporins. The dissociation constants of the Michaelis complexes formed between the R61 enzyme and sulfazecin (32 μM) and between the R39 peptidase and SQ 26324 (0.35 μM) had the lowest values ever observed with any β-lactam compound, suggesting an excellent fit of these two monobactams with the active sites of the respective enzymes. Azthreonam had a very poor inactivating potency, confirming its high selective reactivity towards the penicillin binding protein No. 3 of Escherichia coli . The Zn²⁺ dd -peptidase (from Streptomyces albus G) had a high intrinsic resistance to β-lactam compounds whether they possessed a mono- or a bicyclic structure.     

Articular cartilage and changes in Arthritis: Matrix degradation

While many proteases in articular cartilage have been described, current studies indicate that members of two families of metalloproteases – MMPs and the ADAMTSs – are responsible for the degradation of the major components of this tissue. Collagenases (MMPs) make the first cleavage in triple-helical collagen, allowing its further degradation by other proteases. Aggrecanases (ADAMTSs), in conjunction with other MMPs, degrade aggrecan, a component of the proteoglycan aggregate. Anti-neoepitope antibodies that recognize the cleavage products of collagen and aggrecan generated by these enzymes are now available and are being used to detect the sites of action and to quantitate degradation products.     

Extreme variation in rates of evolution in the plastid Clp protease complex

Eukaryotic cells represent an intricate collaboration between multiple genomes, even down to the level of multi‐subunit complexes in mitochondria and plastids. One such complex in plants is the caseinolytic protease (Clp), which plays an essential role in plastid protein turnover. The proteolytic core of Clp comprises subunits from one plastid‐encoded gene ( clpP1 ) and multiple nuclear genes. The clpP1 gene is highly conserved across most green plants, but it is by far the fastest evolving plastid‐encoded gene in some angiosperms. To better understand these extreme and mysterious patterns of divergence, we investigated the history of clpP1 molecular evolution across green plants by extracting sequences from 988 published plastid genomes. We find that clpP1 has undergone remarkably frequent bouts of accelerated sequence evolution and architectural changes (e.g. a loss of introns and RNA ‐editing sites) within seed plants. Although clpP1 is often assumed to be a pseudogene in such cases, multiple lines of evidence suggest that this is rarely true. We applied comparative native gel electrophoresis of chloroplast protein complexes followed by protein mass spectrometry in two species within the angiosperm genus Silene , which has highly elevated and heterogeneous rates of clpP1 evolution. We confirmed that clpP1 is expressed as a stable protein and forms oligomeric complexes with the nuclear‐encoded Clp subunits, even in one of the most divergent Silene species. Additionally, there is a tight correlation between amino acid substitution rates in clpP1 and the nuclear‐encoded Clp subunits across a broad sampling of angiosperms, suggesting continuing selection on interactions within this complex.     

Excretion of two proteases by the ectomycorrhizal fungus Amanita muscaria

In the soils of mature forests, nitrogen availability is mainly the result of litter decomposition. Thus protein degradation is of major interest for nutrition. Two aspartic proteases were excreted in a pH‐dependent manner by the ectomycorrhizal fungus Amanita muscaria grown in liquid culture. AmProt1 with a molecular mass of approximately 45 kDa was mainly present at pH values up to 5·4, whereas the excretion of AmProt2 with a molecular mass of about 90 kDa was only detectable at pH values between pH 5·4 and 6·3. In addition, the pH optima of both enzymes differed significantly. AmProt1 had a narrow pH optimum around 3, whereas AmProt2 had a broad pH optimum between 3 and 5·5 and a higher affinity to the substrate methylcasein. One cDNA‐clone (AmProt1*) that presumably encodes AmProt1 was identified. Like AmProt1, this cDNA was expressed in a pH‐dependent manner. In addition, carbohydrate and to a lesser extent nitrogen depletion significantly enhanced AmProt1* expression. In fully developed Populus hyb./A. muscaria ectomycorrhizas the expression of AmProt1* was significantly higher in hyphae of the Hartig net compared with those of the fungal sheath. The role of AmProt1 and AmProt2 for fungal physiology and competitiveness is discussed.     

Alterations in lipid turnover in developing muscle

The incorporation and turnover of [³H] glycerol into skeletal muscle cell cultures derived from embryonic chickens was studied. Both rates of incorporation and turnover of specific lipids were dependent on culture age and lipid species. The pattern of glycerol incorporation showed that prefusion myoblasts primarily synthesized both phosphatidylcholine and triglycerides whereas postfusion myotubes primarily synthesized phosphatidyl choline. This pattern could be modified in postfusion but not prefusion cells by briefly incubating the cells with unilammelar phosphatidyl choline vesicles. Analysis of major lipid species revealed that muscle triglycerides and phospholipids turned over at a higher rate in prefusion cultures compared to the postfusion state. These findings are discussed in light of the marked shift in lipid metabolism which occurs during myogenesis.     

Developmental Nutrition of Nematodes: the Biochemical Role of Sterols,Heme Compounds,and Lysosomal Enzymes

Attempts to develop defined in vitro culture systems for the growth, reproduction and development of free-living nematodes have yielded much basic information about their nutritional requirements and biochemistry. Requirements for sterol and heme have been identified suggesting that some nematodes lack de novo synthesis of these molecules. Possible pathways of metabolism of these nutritional requirements can be derived from in vitro experiments that use a variety of sterol and heine sources as supplements to the culture mediuin. These pathways are reviewed as well as the possible role of sterol and heme in the biology of free-living and parasitic nematodes, Since these molecules must be acquired dietarily, the possible involvement of lysosomal enzymes in digestion is discussed. Also considered is the possibility that lysosomal enzymes change when nematodes are fed on a heine protein source.     

Preliminary estimation of chemoattractant activity of staphylococcal serine proteinase in vitro

Human polymorphonuclear leukocytes isolated from peripheral blood of healthy donors migrated toward the staphylococcal serine proteinase.     

大鼠SUMO特异性蛋白酶1催化区蛋白制备及功能鉴定*

目的: 制备大鼠SUMO特异性蛋白酶1(sentrin-specific protease,SENP1)催化结构域(SENP1C)蛋白,并鉴定其酶活性。方法: 分别以大鼠SENP1-pcDNA3.1和EGFP-pcDNA3.1重组体为模板,PCR扩增目的基因,克隆入pGEM-T载体;酶切鉴定后,再亚克隆入原核表达载体pET-28a;阳性重组体导入原核表达细胞BL-21,异丙基硫半乳糖苷(IPTG)诱导蛋白质表达;SDS-PAGE及考马斯亮蓝染色鉴定蛋白质的表达。Ni-NTA吸附纯化蛋白质并透析处理,SDS-PAGE及考马斯亮蓝染色鉴定蛋白质的纯度;1 μmol/L及5 μmol/L Tat-EGFP分别孵育HT22细胞不同时间,荧光显微镜下观察细胞转染情况。采用5 μmol/L Tat-SENP1C预孵育HT22细胞10 h,免疫印迹检测整体蛋白质的SUMO化水平;用5 μmol/L Tat-SENP1C预孵育HT22细胞或过表达Myc-Akt1和HA-SUMO1的HT22细胞10 h后,免疫沉淀和免疫印迹检测内源性和外源性Akt1与SUMO1的结合(SUMO化)。结果: Tat-SENP1C-pET-28a和Tat-EGFP-pET-28a重组原核表达载体成功构建,IPTG可以诱导蛋白质高表达;采用Ni-NTA纯化和透析可获得较高纯度的蛋白质;5 μmol/L Tat-EGFP孵育HT22 细胞10 h后,蛋白质穿膜效率较高;Tat-SENP1C重组蛋白可以显著降低HT22细胞中整体蛋白质的SUMO化以及内源性和外源性Akt1 SUMO化。结论: Tat-SENP1C-pET-28a和Tat-EGFP-pET-28a重组原核表达载体构建成功,且被IPTG诱导后可高效表达蛋白质;纯化的Tat-SENP1C蛋白具有较强的穿膜能力及酶活性。