点状气单胞菌引起虹鳟感染症及其病原学机制的初步研究

对山西省朔县省虹鳟试验场1987年8月在亲鱼中爆发的一次流行病进行了病原菌分离、培养、毒力试验和详细的生理生化测定,确定点状气单胞菌为其病原菌。并对该菌胞外产物的致病性进行了研究,通过测定其溶血活性、酪蛋白酶活性、对鱼致死性以及对其引起的组织病理学变化的观察,初步确定了胞外产物与该菌侵袭力的相关性。     

Characterization of an RNase with two catalytic centers. Human RNase6 catalytic and phosphate-binding site arrangement favors the endonuclease cleavage of polymeric substrates

Background

Human RNase6 is a small cationic antimicrobial protein that belongs to the vertebrate RNaseA superfamily. All members share a common catalytic mechanism, which involves a conserved catalytic triad, constituted by two histidines and a lysine (His15/His122/Lys38 in RNase6 corresponding to His12/His119/Lys41 in RNaseA). Recently, our first crystal structure of human RNase6 identified an additional His pair (His36/His39) and suggested the presence of a secondary active site.

Human eosinophil-derived neurotoxin: involvement of a putative non-catalytic phosphate-binding subsite in its catalysis

Human eosinophil-derived neurotoxin (EDN) or RNase 2, found in the non-core matrix of eosinophils is a ribonuclease belonging to the Ribonuclease A superfamily. EDN manifests a number of bioactions including neurotoxic and antiviral activities, which are dependent on its ribonuclease activity. The core of the catalytic site of EDN contains various base and phosphate-binding subsites. Unlike many members of the RNase A superfamily, EDN contains an additional non-catalytic phosphate-binding subsite, P₋₁. Although RNase A also contains a P₋₁ subsite, the composition of the site in EDN and RNase A is different. In the current study we have generated site-specific mutants to study the role of P₋₁ subsite residues Arg³⁶, Asn³⁹, and Gln⁴⁰ of EDN in its catalytic activity. The individual mutation of Arg³⁶, Asn ³⁹, and Gln⁴⁰ resulted in a reduction in the catalytic activity of EDN on poly(U) and poly(C). However, there was no change in the activities on yeast tRNA and dinucleotide substrates. The study shows that the P₋₁ subsite is crucial for the ribonucleolytic activity of EDN on polymeric RNA substrates. Deepa Sikriwal and Divya Seth contributed equally to this work.     

孟鲁司特钠联合布地奈德混悬液对哮喘急性发作患儿血清EOS、ECP水平和肺功能的影响

目的:探索孟鲁司特钠联合布地奈德混悬液对哮喘急性发作患儿血清嗜酸性粒细胞(EOS)、嗜酸性粒细胞阳离子蛋白(ECP)水平和肺功能的影响。方法:选择自2015年10月至2016年10月我院收治的200例支气管哮喘急性发作患儿,按照随机数表法分成观察组和对照组各100例。对照组患儿口服孟鲁司特钠,观察组患儿在对照组基础上给予布地奈德气雾剂进行雾化治疗,两组均治疗1周。统计分析两组患儿的临床有效率,肺功能指标,包括第1秒用力呼气容积(FEV1)、用力肺活量(FVC)及FEV1/FVC,对比治疗前后两组患儿血清中EOS、ECP水平的变化。结果:治疗后,观察组的总有效率为97.00%,显著高于对照组的81.00%(P0.05);经治疗后两组患儿肺功能指标均较治疗前明显改善,且观察组患儿优于对照组(P0.05);两组患儿治疗后EOS、ECP水平均低于治疗前,而观察组患儿低于对照组,差异均有统计学意义(P0.05)。结论:孟鲁司特钠联合布地奈德对于小儿哮喘的急性发作具有良好的临床疗效,能显著改善患儿肺功能和血清中炎症因子的水平,减轻患儿体内的炎症反应,值得在临床上推广应用。     

Ribonucleases and their antitumor activity

The antitumor effect of ribonucleases was studied with animal ribonucleolytic enzymes, bovine pancreatic RNase A, bovine seminal RNase (BS-RNase), onconase and angiogenin. While bovine pancreatic RNase A exerts a minor antitumor effect, BS-RNase and onconase exert significant effects. Angiogenin, as RNase, works in an opposite way, it initiates vascularization of tumors and subsequent tumor growth. Ribonunclease inhibitors are not able to inhibit the antitumor effectiveness of BS-RNase or onconase. However, they do so in the case of pancreatic RNases. Conjugation of BS-RNase with antibodies against tumor antigens (preparation of immunotoxins) like the conjugation of the enzyme with polymers enhances the antitumor activity of the ribonuclease. After conjugation with polymers, the half-life of BS-RNase in blood is extended and its immunogenicity reduced. Recombinant RNases have the same functional activity as the native enzymes. The synthetic genes have also been modified, some of them with gene sequences typical for the BS-RNase parts. Recent experimental efforts are directed to the preparation of ‘humanized antitumor ribonuclease’ that would be structurally similar to human enzyme with minimal immunogenicity and side effects. The angiogenesis of tumors is attempted to be minimized by specific antibodies or anti-angiogenic substances.     

Antigen-induced blastogenesis of salmon, Salmo saluv L., head kidney leucocytes to modified Aeromonas salmonicida antigens: a preliminary evaluation method for potential vaccine antigens

Various Aeromonas salmonicida antigen preparations were tested in an antigen-induced blasto-genesis assay on the kidney leucocytes of Atlantic salmon. The ECP antigens that had been particularized onto polystyrene beads were the most effective in control fish, and this was further enhanced in fish thymectomized 1 week prior to the assay. The use of this assay for screening potential vaccine antigens is discussed.     

Bacterial invasion of eukaryotic cells can be mediated by actin‐hydrolysing metalloproteases grimelysin and protealysin

Earlier, we have shown that spontaneously isolated non‐pathogenic bacteria Serratia grimesii and Serratia proteamaculans invade eukaryotic cells, provided that they synthesize thermolysin‐like metalloproteases ECP32/grimelysin or protealysin characterized by high specificity towards actin. To address the question of whether the proteases are active players in entry of these bacteria into host cells, in this work, human larynx carcinoma Hep‐2 cells were infected with recombinant Escherichia coli expressing grimelysin or protealysin. Using confocal and electron microscopy, we have found that the recombinant bacteria, whose extracts limitedly cleaved actin, were internalized within the eukaryotic cells residing both in vacuoles and free in cytoplasm. The E. coli‐carrying plasmids without inserts of grimelysin or protealysin gene did not enter Hep‐2 cells. Moreover, internalization of non‐invasive E. coli was not observed in the presence of protealysin introduced into the culture medium. These results are consistent with the direct participation of ECP32/grimelysin and protealysin in entry of bacteria into the host cells. We assume that ECP32/grimelysin and protealysin mediate invasion being injected into the eukaryotic cell and that the high specificity of the enzyme towards actin may be a factor contributed to the bacteria internalization.     

Knockdown expression of eukaryotic initiation factor 5 C-terminal domain containing protein extends lifespan in Drosophila melanogaster

Inhibition of translation by mutations of a growing number of genes involved in protein synthesis could extend healthy lifespan in yeast, worm, fly and mouse as well. These genes vary from translation initiation factors to structural components of ribosomes and ribosomal RNA processing factors. ECP is a novel ribosome associated protein. Previous data supports the involvement of this gene in long term memory formation and exon guidance in Drosophila probably through its still unconfirmed functions in protein synthesis. However, the exact molecular function of ECP is still largely unknown. Our findings here show that fly lifespan could be significantly extended in ECP RNAi flies. Meanwhile, the locomotion ability of elder ECP RNAi flies was also improved remarkably. Further studies revealed an increase of mitochondria Complex IV activity in these ECP RNAi flies. A decrease of AKT and S6K phosphorylation level in contrast to an increase of AMPK phosphorylation level could also be detected in these flies. Together, these findings support a positive effect of ECP on longevity and delaying age-related impairment in locomotor behavior probably through activation of AMPK and enhancement of mitochondrial function via insulin/IGF-1 and TOR pathway.     

Modification of extracorporeal photopheresis technology with porphyrin precursors. Comparison between 8-methoxypsoralen and hexaminolevulinate in killing human T-cell lymphoma cell lines in vitro

Background

Extracorporeal photopheresis that exposes isolated white blood cells to 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light is used for the management of cutaneous T-cell lymphoma and graft-versus-host disease. 8-MOP binds to DNA of both tumor and normal cells, thus increasing the risk of carcinogenesis of normal cells; and also kills both tumor and normal cells with no selectivity after UV-A irradiation. Hexaminolevulinate (HAL)-induced protoporphyrin-IX is a potent photosensitizer that localizes at membranous structures outside of the nucleus of a cell. HAL-mediated photodynamic therapy selectively destroys activated/transformed lymphocytes and induces systemic anti-tumor immunity. The aim of the present study was to explore the possibility of using HAL instead of 8-MOP to kill cells after UV-A exposure.

Methods

Human T-cell lymphoma Jurkat and Karpas 299 cell lines were used to evaluate cell photoinactivation after 8-MOP and/or HAL plus UV-A light with cell proliferation and long term survival assays. The mode of cell death was also analyzed by fluorescence microscopy.

Results

Cell proliferation was decreased by HAL/UV-A, 8-MOP/UV-A or HAL/8-MOP/UV-A. At sufficient doses, the cells were killed by all the regimens; however, the mode of cell death was dependent on the treatment conditions. 8-MOP/UV-A produced apoptotic death exclusively; whereas both apoptosis and necrosis were induced by HAL/UV-A.

Conclusion

8-MOP can be replaced by HAL to inactivate the Jurkat and Karpas 299 T-cell lymphoma cells after UV-A irradiation via apoptosis and necrosis. This finding may have an impact on improved efficacy of photopheresis.