Induced expression of sarcotoxin IA enhanced host resistance against both bacterial and fungal pathogens in transgenic tobacco

We demonstrate here that induced expression of sarcotoxin IA, a bactericidal peptide from Sarcophaga peregrina, enhanced the resistance of transgenic tobacco plants to both bacterial and fungal pathogens. The peptide was produced with a modified PR1a promoter, which is further activated by salicylic acid treatment and necrotic lesion formation by pathogen infection. Host resistance to infection of bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tabaci was shown to be dependent on the amounts of sarcotoxin IA expressed. Since we found antifungal activity of the peptide in vitro, transgenic seedlings were also inoculated with fungal pathogens Rhizoctonia solani and Pythium aphanidermatum. Transgenic plants expressing higher levels of sarcotoxin were able to withstand fungal infection and remained healthy even after 4 weeks, while control plants were dead by fungal infection after 2 weeks.     

Analysis of a gene cluster for sarcotoxin II, a group of antibacterial proteins of Sarcophaga peregrina.

Sarcotoxin II is a group of antibacterial proteins of Sarcophaga peregrina (flesh fly) with related primary structures. We have cloned three genes in this family. These genes formed a tandem array with about 2-kb intervals, and one of them was present in the opposite strand. The putative amino acid sequences of the proteins encoded by these genes were very similar except for a deletion in one of them. All of the genes were found to be activated transiently in the same way when the larval body wall was injured, suggesting that the encoded proteins are acute-phase-responsive proteins for protecting the insect from bacterial infection.     

Cloning of a housekeeping-type gene (MER5) preferentially expressed in murine erythroleukemia cells

DNA complementary to mRNA preferentially produced in murine erythroleukemia (MEL) cells was cloned from a cDNA library of anemic mouse spleen mRNAs. An open reading frame was noted in the cloned DNA, and was tentatively designated MER5. The MER5 mRNA is abundant in three MEL cell lines, but less in other tissues or cell lines. The levels of the MER5 mRNA changed periodically during MEL cell differentiation and decreased as globin mRNA accumulated. The MER5 promoter region contained no typical TATA-like sequence, but possible target sequences for AP1, AP2, SP1 and octamer-binding protein. More interestingly, this promoter contained the duplicated CACCC boxes, which are common in the adult beta-globin promoter from many species, but uncommon for promoters of other eukaryotic genes.     

Expression of tumor necrosis factor at a specific developmental stage of mouse embryos

We investigated the expression of the tumor necrosis factor (TNF) gene during development of mouse embryos, and observed its transient expression on Days 9 and 10 of gestation. We also detected a 25-kDa protein showing immunological cross-reactivity with mouse TNF antibody in an extract of 10-day embryos. These results suggest that TNF plays a role in mammalian ontogenesis.     

Integrated flow-through purification for therapeutic monoclonal antibodies processing

An integrated all flow-through technology platform for the purification of therapeutic monoclonal antibodies (mAb), consisting of activated carbon and flow-through cation and anion exchange chromatography steps, can replace a conventional chromatography platform. This new platform was observed to have excellent impurity clearance at high mAb loadings with overall mAb yield exceeding 80%. Robust removal of DNA and host cell protein was demonstrated by activated carbon and a new flow-through cation exchange resin exhibited excellent clearance of mAb aggregate with high monomer recoveries. A ten-fold improvement of mAb loading was achieved compared to a traditional cation exchange resin designed for bind and elute mode. High throughput 96-well plate screening was used for process optimization, focusing on mAb loading and solution conditions. Optimum operating windows for integrated flow-through purification are proposed based on performance characteristics. The combination of an all flow-through polishing process presents significant opportunities for improvements in facility utilization and process economics.     

Induction of selective phosphorylation of a fat body protein of Sarcophaga peregrina larvae by 20-hydroxyecdysone.

20-Hydroxyecdysone was shown to induce selective phosphorylation of a fat body protein of Sarcophaga peregrina larvae with a molecular mass of 30 kDa. This phosphorylation was not associated with synthesis of new protein. Fractionation of 32P-labelled fat body by differential centrifugation showed that this protein was mainly present in the membrane-rich fraction, although we could not specify the membrane. Thus, 20-hydroxyecdysone may modify the function of the fat body by inducing phosphorylation of a specific membrane protein.     

Identification of storage-protein messenger RNA of the fleshfly Sarcophaga peregrina.

Storage-protein mRNA was found to be abundant in poly(A)-containing RNA extracted from the fat-body of third-instar larvae of Sarcophaga peregrina (fleshfly). This RNA sedimented at the position of 19S on sucrose-density-gradient centrifugation and the product of its translation in vitro was 75K protein (protein of mol.wt. 75 000), which was precipitated specifically with antibody against storage protein. This product was suggested to contain a signal sequence that is missing in mature storage protein. The poly(A)-containing RNA was also found to contain much of another mRNA coding for 25K protein (protein of mol.wt. 25 000), but the function of this protein is unknown.     

Craniometrical variations among eastern Brazilian marmosets and their systematic relationships

The craniometrical variations among marmosets on the eastern coast of Brazil or theCallithrix jacchus group (C. jacchus, C. penicillata, C. kuhli, C. geoffroyi, C. flaviceps, andC. aurita) were analyzed to test various hypotheses for the arrangement of the species and phylogenetic relationships ofC. kuhli andC. flaviceps. Q-mode correlation coefficients and principal component analysis were employed to extract important shape factors from the craniometrical data. On this basis, the shape distances among the eastern Brazilian marmosets were found to be larger than those between the marmosets which are recognized as distinct species. This evidence supports the hypothesis that the members of theC. jacchus group are distinct species.C. kuhli shows the strongest resemblance toC. geoffroyi within theC. jacchus group.C. flaviceps is the most similar toC. aurita in its cranial shape. These findings indicate thatC. kuhli is most closely related toC. geoffroyi, whileC. flaviceps is toC. aurita.