庆大霉素产生菌23-18诱变育种研究

用青霉素、超声波对庆大霉素产生菌棘孢小单抱菌23-18的孢子进行预处理，以增加其通透性，再用钻60进行辐射诱变，结合耐自身代谢产物的筛选，获得数株高产菌株，其中79，167菌株摇瓶发酵数价比亲株23－18提高20％，发酵周期缩短10h。萌种连续传五代，生产能力稳定，其发酵液用高效液相色谱分析，C组分比例符合中国药典要求。     

Isolation and properties of yeast mutants affected in farnesyl diphosphate synthetase

Summary Two yeast mutant strains auxotrophic for ergosterol and blocked in farnesyl diphosphate synthetase (EC 2.5.1.1) were isolated. Genetic analysis has shown that these mutant strains carry additional mutations in the ergosterol pathway besides erg20-1 and erg20-2 which affect FPP synthetase. The novel feature of these mutants is their ability to excrete prenyl alcohols (farnesol and geraniol). As geraniol is toxic for yeast cells, the above leaky mutations in FPP synthetase have to be associated with others in the sterol pathway, in order to slow down geraniol synthesis.Abbreviations FPP farnesyl diphosphate - GPP geranyl diphosphate - IPP isopentenyl diphosphate - DMAPP dimethylallyl diphosphate - MVAP mevalonate phosphate - MVAPP mevalonate diphosphate - HMG CoA 3-hydroxy-3-methylglutaryl-CoA - MIC minimal inhibitory concentration     

Absence of Pneumocystis dihydropteroate synthase mutants in Brittany,France

Archival Pneumocystis jirovecii specimens from 84 patients monitored at Rennes University Hospital (Rennes, France) were assayed at the dihydropteroate synthase (DHPS) locus. No patient was infected with mutants. The results provide additional data showing that P. jirovecii infections involving DHPS mutants do not represent a public health issue in Brittany, western France.     

Effect of lipooligosaccharide mutations of Haemophilus influenzae on the middle and inner ears

Objective

The purpose of this study was to determine the virulence of nontypeable Haemophilus influenzae 2019 (NTHi 2019) and its two lipooligosaccharide (LOS) mutant strains, B29 (gene htrB) and DK1 (gene rfaD), and compare their effect on the middle ear, round window membrane, and inner ear.

Results

Fifteen chinchillas were divided into three equal groups and their bullas inoculated bilaterally with 0.5 ml of 10² CFU/ml of parent NTHi 2019, B29 or DK1 mutant strains. Two days after inoculation all animals had otitis media and inflamed middle ear mucosa. There was a trend of greater thickness and infiltration of the round window membrane in animals inoculated with the wild-type NTHi strain compared to the mutant strains and a significant increase in both inflammatory cell infiltration and bacteria presence in the scala tympani area of the inner ear. Strial edema was only observed in the wild-type-inoculated group.

Conclusions

LOS mutants of NTHi appear to have a reduced ability to pass through the round window membrane resulting in less inner ear inflammation and pathological changes.     

Impact of genetic changes to the CRPV genome and their application to the study of pathogenesis in vivo

The cottontail rabbit papillomavirus (CRPV)/rabbit model has been used to study oncogenicity and immunogenicity of different antigens from the papillomavirus genome and has therefore served as a preclinical model for the development of preventive and therapeutic vaccines against papillomavirus infections. One unique property of the CRPV model is that infection can be initiated using viral DNA. This property allows for the functional testing of viral mutants in vivo. We have introduced point mutations, insertions and deletions into all of the different coding and non-coding regions of the CRPV genome and have tested their infectivity in this model. We found that the majority of the mutant genomes retained viability and could induce papillomas in domestic rabbits. These data indicated that the CRPV genome is tolerant of many modifications without compromising its ability to initiate skin papillomas. In combination with our recently established HLA-A2.1 transgenic rabbit model, this plasticity allows us to extend the utility of the CRPV/rabbit model to the screening of HLA-A2.1 restricted epitopes from other human viral and tumor antigens.     

Relationships between trehalose metabolism and maltose utilization in Saccharomyces cerevisiae

Summary A specific deficiency in UDPG-linked trehalose-6-phosphate synthase in the yeast, Saccharomyces cerevisiae has been associated with a single nuclear gene, sst1. Strains bearing this abnormal allele lacked the capacity to accumulate trehalose during growth on glucose or galactose medium or when incubated with glucose in nonproliferating conditions. However, sst1 strains still exhibited trehalose accumulation during growth on maltose medium, provided they contained a gene for maltose fermentation (MAL gene). Introduction of a constitutive MAL ^c gene into an sst1 strain rendered the strain capable of accumulating trehalose during growth on glucose medium, but did not restore the normal capacity to convert glucose to trehalose in nonproliferating conditions. Different systems, I and II, of trehalose accumulation are proposed. System I would require the UPDG-linked synthase, whereas system II, which is normally specific for maltose, would utilize a different enzyme. It is unlikely that system II produces trehalose by trans-glucosylation, since it converted glucose to trehalose in MAL ^c sst1 strains. The results indicate that maltose specifically induces the production of the MAL gene-product, which, in turn, would stimulate the formation (or activation) of system II.     

Application of hepatitis B virus replication mouse model

AIM:To evaluate the value of the hepatitis B virus(HBV) replication mouse model with regard to several aspects of the study of HBV biology.METHODS:To evaluate the HBV replication mouse model in detecting the efficacy of anti-HBV agents,the interferon inducer polyinosinic-polytidylin acid(polyIC) and nucleotide analogues adefovir and entecavir were administered to mice injected with wild type pHBV4.1,and the inhibiting effect of these agents on HBV DNA replication was evaluated.To identify the model‘s value ...     

Glycerol permeability of mutant aquaporin 1 and other AQP-MIP proteins: Inhibition studies

In a recent work, we showed that the aquaporins 1 (AQP1) are permeable to certain small solutes such as glycerol. Here, we have further investigated the permeation pathway of glycerol through human AQP1 (hAQP1) by the use of mutants (C189S, H180A, H209A) and inhibitors such asP-chloromercuribenzene sulphonate (pCMBS), CuSO₄ or phloretin, in comparison with other AQP-MIP (where MIP denotes major intrinsic protein) proteins: hAQP2, plant water channel TIP and bacterial glycerol permease facilitator, G1pF. Glycerol movements were measured inXenopus laevis oocytes. Apparent glycerol permeability coefficients (P _gly ) were calculated from the rates of oocyte swelling upon exposure to an isoosmotic medium containing an inwardly directed gradient of glycerol and from [³H]glycerol uptake measurements. SimilarP _gly values were obtained for hAQP1 and hAQP2, 6 to 8 times greater than control indicating that hAQP2 also transports glycerol. Pof hAQP2P injected oocytes waspCMBS and CuSO₄ sensitive. In contrast, theP _gly value of TIP was close to that of control, indicating that TIP does not transport glycerol. The hAQP1-C189S, -H180A and -H209A mutants gaveP _gly values similar to those obtained for wild hAQP1, indicating that these mutations did not affect glycerol movements. However, the H209A mutant has an osmotic water permeability coefficient (P _f) value decreased by 50%. The inhibitory effect ofpCMBS onP _gly was maintained for the 2 His mutants and, more interestingly, was also conserved for the C189S mutant. CuSO₄ significantly inhibitedP _gly of oocytes expressing hAQP1, hAQP1-C189S,-H180A, and -H209A mutants and had no effect onP _gly of G1pF-injected oocytes. Phloretin was shown to inhibit by around 80% the glycerol fluxes of wild and mutant hAQP1, hAQP2 and to fully inhibit glycerol uptake in G1pF-injected oocytes.     

pcDNA3.1/NT-GFP小凹蛋白1及突变体表达载体的构建及功能分析

目的 构建pcDNA3．1/NT-GFP小凹蛋白1及对突变体表达载体及表达蛋白生物活性进行分析。方法采用硫化修饰引物与Pfu酶结合的高度保真性聚合酶链反应体系，自行设计多对引物，分别扩增带His标签的小凹蛋白1全长目的片段、小凹蛋白1（缺失81～101位氨基酸）突变体1片段及小凹蛋白1（缺失143～156位氨基酸）突变体2片段；分别亚克隆入peDNA3．1／NT-GFP-TOPO真核表达栽体，转化TOP10E．coil大肠杆菌，在含氨苄的固体LB培养基上随机挑取6个克隆，分别提取质粒后，用PCR法筛选含正确插入阅读框的阳性克隆子并测序鉴定。用脂质体介导法瞬时转染入HepG2细胞，用MTT法、Western blot法初步鉴定GFP-His小凹蛋白1及突变体重组融合蛋白的生物学活性。结果筛选得到正确peDNA3．1／NT-GFP-His小凹蛋白1及突变体表达我体，测序结果无碱基突变及阅读框移码，GFP-His小凹蛋白1及突变体重组融合蛋白具有天然表达蛋白相似的生物学活性。结论成功构建具有生物学活性的pcDNA3．1／NT-GFP小凹蛋白1及突变体表达栽体，为小凹蛋白1的功能研究奠定基础。