Ca2+-independent,protein-mediated fusion of chromaffin granule ghosts with liposomes

We have investigated the interaction between isolated membrane vesicles from chromaffin granules and large unilamellar phospholipid vesicles (liposomes). Mixing of membrane lipids has been monitored continuously, utilizing the fluorescence resonance energy transfer assay described by Struck et al. ((1982) Biochemistry 20, 4093–4099). To demonstrate coalescence of the internal vesicle volumes the transfer of colloidal gold from the liposomes to the interior of the granule membrane vesicles has been examined. Efficient fusion of the liposomes with the granule membranes was observed. Significant fusion occurred in the absence of Ca²⁺, although the extent of interaction was enhanced in its presence. The sensitivity of the interaction to pretreatment of the granule membranes with trypsin showed the fusion reaction to be a protein-mediated process.     

Progress in plant protoplast research

During the past years plant regeneration from protoplasts was achieved for a number of important crops (maize, sorghum, rice, wheat, sugar beet). The use of embryogenic tissue for protoplast isolation greatly contributed to this success. There was also some progress in woody plant species and ornamentals. Fusion of protoplasts resulted in may fertile hybrid plants, especially in the Brassicaceae and Solanaceae. These somatic hybridization studies led to introduction of new agronomical traits from sexually incompatible species into the cultivar gene pool and to new nucleus-organelle compositions. The limitations of somatic hybridization, mainly imposed by the taxonomic distance of the parents, and expressed as chromosome loss and reduced fertility, are more clearly recognized now. Asymmetric hybridization with irradiated donor protoplasts resulted in cybrids with new cytoplasmic traits (e. g. intraspecific fusions in Brassica ), as well as in the transfer of only a few donor choromosomes (e. g. intrageneric fusions in Nicotiana ). Most intrageneric fusions, however, resulted only in a limited elimination of donor chromosomes (e. g. in Lycopersicon ), and polyploidization occurred (e. g. in Nicotiana ). Also some success on protoplast transformation was obtained in both monocots and dicots. Fertile transgenic rice plants (Japonica, Indica) were produced after direct gene transfer into protoplasts derived from embryogenic cell suspensions. Particle gun experiments using embryogenic cell suspension of maize resulted in fertile transgenic plants. Transformation of citrus and lettuce by direct gene transfer was also reported.     

Unusual Fatty Acids with Specific Odor from Mature Male Goat

Unusual five 4-ethyl-fatty acids were estimated as main constituents of sebaceous gland secretion of male goat which showed the releaser pheromone activity against the estrous female. These acids exist in both free state and ester form. The structures were confirmed by synthesis. Among the five acids, 4-ethyloctanoic acid showed the strong goaty odor at low concentration.     

一种真核细胞分泌型重组融合蛋白rPC的构建、表达与纯化

抑制性免疫检查点PD-1或CTLA-4靶向治疗药物已用于肿瘤的临床治疗,但单一靶点药物会有耐药发生,联合使用同时封闭多个靶点可提高疗效,因此拟构建一个可封闭多个靶点的新型重组蛋白。首先设计并合成了一个由人类PD-1和CTLA-4两个受体的胞外功能域组成并且C端带6×His标签的分泌型重组融合蛋白rPC编码序列,插入真核细胞表达载体pLVX-IRES-ZsGreen1,稳定转染HEK293细胞,收集细胞培养上清,以亲和方法纯化重组蛋白rPC,通过实时荧光定量PCR检测多个人类肿瘤细胞系中PD-1配体PD-L1、PD-L2和CTLA-4配体CD80、CD86的表达,以选择相对高表达的细胞,利用细胞免疫荧光染色方法检验rPC与肿瘤细胞的结合能力,并用CCK-8法检测rPC是否对肿瘤细胞的生长有影响。结果表明,重组融合蛋白rPC可由稳定转染表达载体的HEK293细胞表达并分泌,纯化后的rPC可以与PD-1和CTLA-4配体表达相对较高的肺癌细胞NCI-H226结合,并且rPC处理对其生长并无直接影响,与预期一致。成功获得的重组融合蛋白rPC可用于进一步的体内外功能研究,也为今后研发新型多靶点肿瘤免疫治疗蛋白药物打下了坚实的基础。     

Isolation and characterization of intergeneric somatic hybrids in the Apocynaceae family

Summary Protoplasts were isolated from callus cultures of Rauwolfia serpentina Benth., Rhazya stricta Decaisne, and Catharanthus roseus (L.) G. Don, or from leaves of Vinca minor L. Protoplast isolation, culture, and fusion techniques as well as hybrid screening systems were developed for these species, and hybrids were obtained. Hybrid combinations were Rauwolfia + Vinca, Rauwolfia + Catharanthus, Rauwolfia + Rhazya, and Catharanthus + Vinca. For hybrid isolation, the physiological complementation method was utilized. Analyses of the material obtained included a cytogenetic study of the chromosomes, a study of multiple molecular forms of transferases and esterases, and the blot hybridization of restricted nuclear DNA using ribosomal DNA as a probe. Hybrids were identified in all species' combinations tried. A ten-fold increase in the accumulation of raucaffricine (relative to the parental Rauwolfia strain) was observed in one cell line of the Rauwolfia + Vinca hybrid. Our studies indicated the genetic stability of the great majority of the hybrid cell lines over a period of more than 20 months of in vitro growth. No shoot morphogenesis has so far been observed in this material.     

Molecular insights into development of Trichoderma interfusants for multistress tolerance enhancing antagonism against Sclerotium rolfsii Sacc

The current study aimed at developing diverse Trichoderma fusants for fungicides, drought, and salt tolerance with enhanced antagonistic activity against Sclerotium rolfsii Sacc. Trichoderma virens NBAII Tvs12 (mycoparasitic) and Trichoderma koningii MTCC796 (multistress tolerant) were used as parental strains for development of interspecific protoplast fusants. A total of 36 stable fusants were used for mycoparasitism, fungicides, and abiotic stresses (drought and salt) tolerance. The results revealed 20 homozygous progenies showing characteristics of either one parental strain and 14 heterozygous mutants depicting traits of both parental strains. A novel concept of inhibition coefficient was established using growth-related key parameters that represent the pathogen biology and the biocontrol-related biophysics of Trichoderma fusants. The results indicated a differential inhibition coefficient of the test pathogen and the highest (92.88%) inhibition coefficient of S. rolfsii was observed by interstable fusant Fu21. It also grew better under fungicides and abiotic stress (drought and salt) conditions. The molecular characterization and heterozygosity analysis evidenced the highest observed heterozygosity (0.5441) and gene flow (0.3872) in stable heterozygous Fu21. Principal coordinates analysis exhibited 62.7% of total variability. The ecofriendly heterozygous Trichoderma fusant (Fu21) might be useful for biocontrol of stem rot disease under adverse conditions or as a part of integrated disease management.     

Fosfomycin enhances the expression of penicillin-binding protein 2 in methicillin-sensitive and methicillin-resistant Staphylococcus aureus strains

The effects of fosfomycin on penicillin-binding proteins (PBPs) were studied on the methicillin-resistant Staphylococcus aureus strain CIP (Collection de l'Institut Pasteur, Paris, France) 65-25 and on a methicillin-susceptible S. aureus strain CIP 65-6. The combinations of fosfomycin and oxacillin were synergistic, additive or antagonistic, depending on antibiotic concentrations. Fosfomycin induced modifications of the PBP profile of the two strains studied. In particular, it increased the expression of PBP2. This suggested that this protein is inducible; the only PBP not affected by fosfomycin was PBP3.     

Deletion or alteration of hydrophobic amino acids at the first and the third transmembrane domains of hepatitis B surface antigen enhances its production in Escherichia coli

To investigate the failure of high-level production of hepatitis B viral (HBV) surface antigen (HBsAg), including three authentic forms, large (L), middle (M) and major/small (S) HBsAg, in Escherichia coli, we employed the high-expression vector pGEX containing the glutathione S-transferase-encoding gene (GST) to study HBsAg production. Different fragments of HBV DNA containing the entire pre-S1/pre-S2/S region (for L protein), or partial pre-S1, pre-S2, pre-S1/pre-S2 and pre-S2/S region (for M protein), were fused downstream from the GST gene, in order to obtain five plasmids which encode GST-HBsAg fusion proteins. SDS-PAGE analyses revealed that cells containing plasmids with a full-length S region (pGLS and pGMS) produced undetectable GST-HBsAg fusion proteins, in contrast to those cells harboring plasmids without the S region (pGS1, pGS2 and pGS1S2), which synthesized fusion proteins in 3–10% of the total cellular protein. Using an immunoblot method to screen HBsAg production in cells which harbored plasmids derived from exonuclease BAL 31-digested pGLS, we obtained eight positive clones. Nucleotide sequence analyses of plasmids from the positive clones revealed that termination, deletion or frameshift occurred at the regions encoding either the first or the third transmembrane domain of the major HBsAg. Correlation between the production level of GST-HBsAg fusion proteins and their constituent and arrangement of amino acids (aa) at the last 20 as among 15 clones suggested that the fusion protein ended with a longer stretch of or a higher ratio of hydrophobic as had a lower production in E. coli.     

Cytogenetic studies of Aotus from Eastern Amazonia. Y/Autosome rearrangement

Twenty-one specimens of Aotus were captured on both sides of the Tocantins river when the hydroelectric reservoir of Tucuruí, Brazil, was filled. The males had a diploid number of 49 chromosomes, and the females had 50. The observed difference is a consequence of the fusion of the Y chromosome with an autosome. The karyotype is similar to that of the Bolivian Aotus (A. azarae boliviensis). It differs, however, in the G- and C-banding patterns of the chromosome resulting from the Y/autosome fusion. The nucleolar organizing region is located on the secondary constriction of a pair of submetacentric chromosomes. Considerations are presented on the classification of A. infulatus as a separate species.