Glycomodification and characterization of anti-colorectal cancer immunotherapeutic monoclonal antibodies in transgenic tobacco

Anti-colorectal cancer mAb CO17-1A (IgG_2a) recognizes the antigen GA733, which is highly expressed on the surface membrane of human colorectal carcinoma cells. In this study, a transgenic tobacco system for the production of mAb CO17-1A was developed. The mAb construct included a KDEL sequence, an endoplasmic reticulum (ER) retention signal attached to the C-terminus of the heavy chain, to target accumulation of mAb into ER. An immunoblot showed significantly enhanced levels of expression of the plant-derived mAbK (mAb^PK) CO17-1A compared to mAb^P CO17-1A mAb without the KDEL sequence. An ELISA assay using human colorectal carcinoma cells confirmed that expression of mAb^PK was also significantly higher than that of mAb^P. Glycosylation analysis revealed that mAb^P had plant-specific glycans; whereas, mAb^PK primarily had oligomannose glycans. FACS showed that the Fc domains of both mAb^PK and mammalian-derived mAb (mAb^M) had similar binding activity to the FcγRI receptor (CD64). However, the Fc domains of the mAb^P had slightly lower binding activity to the Fc_γRI receptor than both mAb^PK and mAb^M. The antibody-dependent cell cytotoxicity of mAb^PK, against human colorectal cancer cells, was as efficient as mAb^M; whereas mAb^P was very low. These results suggest that KDEL localized and accumulated mAb^P in the ER and eventually enhanced the expression of mAb^P with oligomannose glycan and similar anti-cancer biological activity to the parental mAb^M.     

Selective Inhibition of Phosphoinositide 3-Kinase p110α Preserves Lymphocyte Function

Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors.     

Measurement of water fluxes and potentials in a single root-soil system

Summary The construction and operation of a novel tensiometer-potometer system capable of measuring the xylem water potential and flux of water into the root is described. The validity of its measurements has been illustrated and it was shown that a unique linear relationship exists between the resistance to water flow and the water status of the root tissues.     

Quantitative relationship between the protein secondary structure in cardiac sarcolemma and the activity of the membrane-bound Ca2+-ATPase

In the absence of ATP, increasing concentrations of calcium within a range between 0.1--8.0 mmol . 1(-1) gradually lowered the alpha-helix content of proteins in rat heart sarcolemma requiring no energy supply. In the presence of ATP, similar concentrations of calcium stepwise activated the sarcolemmal low-affinity Ca2+-ATPase. A mathematical analysis of the data obtained revealed a quantitative relationship between calcium-induced stimulation of the Ca2+-ATPase activity and a diminution of the alpha-helix contents of membrane proteins in cardiac sarcolemma. The cooperation between changes in protein conformation and energy consumption in relation to the supposed role of low-affinity Ca2+-ATPase in gating the calcium channel are discussed.     

Age-dependent changes in the distribution and concentration of human lens cholesterol and phospholipids

Analyses of total lipid in individual lenses 1.8-63 years of age indicate that both the cholesterol and the phospholipid concentrations have reached a high level of 10 and 14 micrograms/mg lens dry weight, respectively, after the first ten years of growth. Thereafter, the rate of phospholipid accumulation was greatly reduced to a value of 0.05 microgram/mg per year while that of cholesterol reduced to 0.19. Analyses of the distribution of lipid in successive lens fiber layers indicate that both the cholesterol and phospholipid levels increase in the entire lens between the age of 1.8 and 9 years. Older lenses showed a continuous increase in the accumulation of cholesterol in the deep cortical fibers, while little or no increase in phospholipid concentration was observed. These results indicate that the accumulation of lipids is greater than that of lens dry mass (protein) during the first decade of lens growth. Since more than 90% of lenticular lipids are associated with fiber cell membranes, these data suggest a gradual change in the differentiation of the newly formed secondary fibers from the epithelium during this period. Analyses of the phospholipid composition of the successive fiber fractions indicate that the major phospholipids of phosphatidyl ethanolamine (PE), phosphatidylserine (PS) and sphingomyelin maintained a uniform distribution in the 1.8- and 5-year-old lenses. While no change was observed with the cortical fibers, older lenses showed a gradual loss of PE and PS in the nuclear fiber up to 63 years of age. By the late teen years, nuclear PS can no longer be detected, while high levels of PE are maintained in lens nucleus. The disappearance of nuclear PE begins in the teen years and is completed by the age of 40. The decrease in PE and PS resulted in a continuous increase in the cholesterol/phospholipid ratio, a measure of membrane rigidity in the nuclear fiber in lenses 20 years of age and older. This decrease is also responsible for the exceedingly high rigidity of the nuclear fibers of lenses 60 years of age and older. Possible lamellar cholesterol organization in the lens fiber membrane is discussed.     

Antimicrobial effect and membrane-active mechanism of Urechistachykinins, neuropeptides derived from Urechis unicinctus

We investigated the antimicrobial effects of Urechistachykinins I and II (UI and UII) and their modes of action. UI and UII showed antimicrobial activities without a hemolytic effect. To investigate the mechanism(s) of UI and UII, cellular localization was examined. Confocal microscopy results showed that peptides were located in the cell envelope. To elucidate the physical changes of membrane induced by UI and UII in Candida albicans, flow cytometry analyses were performed by using bis-(1,3-dibutylbarbituric acid) trimethine oxonol, and changes in membrane dynamics were assessed using 1,6-diphenyl-1,3,5-hexatriene. The results suggest that UI and UII may exert their antimicrobial effect by disrupting the cell membranes.