Interleukin-2 mutants with enhanced alpha-receptor subunit binding affinity

Stimulation of T-cells by IL-2 has been exploited for treatment of metastatic renal carcinoma and melanoma. However, a narrow therapeutic window delimited by negligible stimulation of T-cells at low picomolar concentrations and undesirable stimulation of NK cells at nanomolar concentrations hampers IL-2-based therapies. We hypothesized that increasing the affinity of IL-2 for IL-2Ralpha may create a class of IL-2 mutants with increased biological potency as compared with wild-type IL-2. Towards this end, we have screened libraries of mutated IL-2 displayed on the surface of yeast and isolated mutants with a 15-30-fold improved affinity for the IL-2Ralpha subunit. These mutants do not exhibit appreciably altered bioactivity at 0.5-5 pM in steady-state bioassays, concentrations well below the IL-2Ralpha equilibrium binding constant for both the mutant and wild-type IL-2. A mutant was serendipitously identified that exhibited somewhat improved potency, perhaps via altered endocytic trafficking mechanisms described previously.     

Characterizing rice lesion mimic mutants and identifying a mutant with broad-spectrum resistance to rice blast and bacterial blight

Many plant mutants develop spontaneous lesions that resemble disease symptoms in the absence of pathogen attack. In several pathosystems, lesion mimic mutations have been shown to be involved in programmed cell death, which in some instances leads to enhanced disease resistance to multiple pathogens. We investigated the relationship between spontaneous cell death and disease resistance in rice with nine mutants with a range of lesion mimic phenotypes. All nine mutations are controlled by recessive genes and some of these mutants have stunted growth and other abnormal characteristics. The lesion mimics that appeared on the leaves of these mutants were caused by cell death as measured by trypan blue staining. Activation of six defense-related genes was observed in most of the mutants when the mimic lesions developed. Four mutants exhibited significant enhanced resistance to rice blast. One of the mutants, spl11, confers non-race-specific resistance not only to blast but also to bacterial blight. The level of resistance in the spl11 mutant to the two pathogens correlates with the defense-related gene expression and lesion development on the leaves. The results suggest that some lesion mimic mutations in rice may be involved in disease resistance, and cloning of these genes may provide a clue to developing broad-spectrum resistance to diverse pathogens.     

DOH1, a class 1 knox gene, is required for maintenance of the basic plant architecture and floral transition in orchid

We report here the isolation and identification of an orchid homeobox gene, DOH1, from Dendrobium Madame Thong-In. Analyses of its sequence and genomic organization suggest that DOH1 may be the only class 1 knox gene in the genome. DOH1 mRNA accumulates in meristem-rich tissues, and its expression is greatly downregulated during floral transition. In situ hybridization analysis demonstrates that DOH1 is also expressed in the incipient leaf primordia and is later detected in the same region of the inflorescence apex, as in DOMADS1. Overexpression of DOH1 in orchid plants completely suppresses shoot organization and development. Transgenic orchid plants expressing antisense mRNA for DOH1 show multiple shoot apical meristem (SAM) formations and early flowering. In addition, both the sense and antisense transformants exhibit defects in leaf development. These findings suggest that DOH1 plays a key role in maintaining the basic plant architecture of orchid through control of the formation and development of the SAM and shoot structure. Investigations of DOMADS1 expression in the SAM during floral transition reveal that the precocious flowering phenotype exhibited by DOH1 antisense transformants is coupled with the early onset of DOMADS1 expression. This fact, together with the reciprocal expression of DOH1 and DOMADS1 during floral transition, indicates that downregulation of DOH1 in the SAM is required for floral transition in orchid and that DOH1 is a possible upstream regulator of DOMADS1.     

Direct effects of varying doses of oestradiol on early embryonic development in in vitro culture of rat's two-cell embryos

Hyperstimulation in the rat using pregnant mares' serum gonadotrophin (PMSG) has been known to cause death in pre-implantation embryos, as well as enhancement of oestradiol production. This study examines the effect of oestradiol, in levels that are found in hyperstimulated pregnant rats, on pre-implantation embryonic development. Using a simplified in vitro system, 2-cell embryos retrieved from rats on the 2nd day of pregnancy were cultured in rat two-cell embryo culture medium (R2ECM) containing pharmacological doses of oestradiol for 96 h and scored daily in the morning. Three ngxmL(-1) oestradiol reduced the incidence of >8-cell embryos to morulae on the 5th day and blastocysts on the 6th day of development. Most embryos were retarded at the lower cell stages on the 5th day and degenerated by the 6th day. None of the blastocysts expanded on the last day of culture. Fifteen ngxmL(-1) oestradiol accelerated embryo development on the 3rd day but retarded development on the 4th day, and increased the incidence of degenerated embryos by the 5th and 6th day of development. These results suggest that the elevated oestradiol may constitute a mechanism by which PMSG induces death in pre-implantation rat embryos, possibly via a direct action on the embryos.     

The genes for benzene catabolism in Pseudomonas putida ML2 are flanked by two copies of the insertion element IS1489, forming a class-I-type catabolic transposon, Tn5542

Two directly repeated sequences of the IS elements IS1489v1 and IS1489v2 flank the benzene dioxygenase (bedC1C2BA) and the cis-benzene dihydrodiol dehydrogenase (bedD) genes on the catabolic plasmid pHMT112 in Pseudomonas putida ML2, forming a Class-I-type composite transposon, Tn5542. Both IS1489v1 and IS1489v2 contain an identical 1371-bp open reading frame, tnpA, that is preceded by a possible ribosome binding site. The tnpA gene of IS1489v1 is bound by a pair of 40-bp imperfect inverted repeats while that of IS1489v2 is flanked only by the left inverted repeat. The tnpA gene codes for a putative 53-kDa polypeptide of 456 amino acids bearing similarity to transposases encoded on IS elements of P. alcaligenes, P. aeruginosa, P. stutzeri, and Serratia marcescens. The basic nature of the putative TnpA protein with a deduced pI of 8.93 is typical of IS-encoded transposases. Similar to other IS elements, an outward facing promoter was detected at the right end of IS1489v1. Experiments involving the suicide vector, pKNG101, failed to show transposition of Tn5542.     

Identification of functional elements of the GDP-fucose transporter SLC35C1 using a novel Chinese hamster ovary mutant

The GDP-fucose transporter SLC35C1 critically regulates the fucosylation of glycans. Elucidation of its structure-function relationships remains a challenge due to the lack of an appropriate mutant cell line. Here we report a novel Chinese hamster ovary (CHO) mutant, CHO-gmt5, generated by the zinc-finger nuclease technology, in which the Slc35c1 gene was knocked out from a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter (CST) gene (Slc35a1). Consequently, CHO-gmt5 harbors double genetic defects in Slc35a1 and Slc35c1 and produces N-glycans deficient in both sialic acid and fucose. The structure-function relationships of SLC35C1 were studied using CHO-gmt5 cells. In contrast to the CST and UDP-galactose transporter, the C-terminal tail of SLC35C1 is not required for its Golgi localization but is essential for generating glycans that are recognized by a fucose-binding lectin, Aleuria aurantia lectin (AAL), suggesting an important role in the transport activity of SLC35C1. Furthermore, we found that this impact can be independently contributed by a cluster of three lysine residues and a Glu-Met (EM) sequence within the C terminus. We also showed that the conserved glycine residues at positions 180 and 277 of SLC35C1 have significant impacts on AAL binding to CHO-gmt5 cells, suggesting that these conserved glycine residues are required for the transport activity of Slc35 proteins. The absence of sialic acid and fucose on Fc N-glycan has been independently shown to enhance the antibody-dependent cellular cytotoxicity (ADCC) effect. By combining these features into one cell line, we postulate that CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC effect.