Stability of the allergenic soybean Kunitz trypsin inhibitor

The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel beta-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 degrees C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.     

C-Terminal 23 kDa polypeptide of soybean Gly m Bd 28 K is a potential allergen

Gly m Bd 28 K is a major soybean (Glycine max Merr.) glycoprotein allergen. It was originally identified as a 28 kDa polypeptide in soybean seed flour. However, the full-length protein is encoded by an open reading frame (ORF) of 473 amino acids, and contains a 23 kDa C-terminal polypeptide of as yet unknown allergenic and structural characteristics. IgE-binding (allergenic potential) of the Gly m Bd 28 K protein including the 23 kDa C-terminal portion as well as shorter fragments derived from the full-length ORF were evaluated using sera from soy-sensitive adults. All of these sera contained IgE that efficiently recognized the C-terminal region. Epitope mapping demonstrated that a dominant linear C-terminal IgE binding epitope resides between residues S256 and A270. Alanine scanning of this dominant epitope indicated that five amino acids, Y260, D261, D262, K264 and D266, contribute most towards IgE-binding. A model based on the structure of the subunit of soybean -conglycinin revealed that Gly m Bd 28 K contains two cupin domains. The dominant epitope is on the edge of the first -sheet of the C-terminal cupin domain and is present on a potentially solvent-accessible loop connecting the two cupin domains. Thus, the C-terminal 23 kDa polypeptide of Gly m Bd 28 K present in soy products is allergenic and apparently contains at least one immunodominant epitope near the edge of a cupin domain. This knowledge could be helpful in the future breeding of hypoallergenic soybeans.Abbreviations Ara h 1 Arachis hypogaea allergen 1 - Ara h 3 Arachis hypogaea allergen 3 - BCA Bicinchoninic acid - Gly m Bd 28 K Glycine max band 28 kDa allergen - Gly m Bd 30 K Glycine max band 30 kDa allergen - Gly m Bd 68 K Glycine max band 68 kDa allergen - IgE Immunoglobulin E     

Efficient high‐throughput biological process characterization: Definitive screening design with the Ambr250 bioreactor system

The burgeoning pipeline for new biologic drugs has increased the need for high‐throughput process characterization to efficiently use process development resources. Breakthroughs in highly automated and parallelized upstream process development have led to technologies such as the 250‐mL automated mini bioreactor (ambr250?) system. Furthermore, developments in modern design of experiments (DoE) have promoted the use of definitive screening design (DSD) as an efficient method to combine factor screening and characterization. Here we utilize the 24‐bioreactor ambr250? system with 10‐factor DSD to demonstrate a systematic experimental workflow to efficiently characterize an Escherichia coli (E. coli) fermentation process for recombinant protein production. The generated process model is further validated by laboratory‐scale experiments and shows how the strategy is useful for quality by design (QbD) approaches to control strategies for late‐stage characterization. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1388–1395, 2015     

Antixenosis,tolerance and genetic analysis of some rice landraces for resistance to Nilaparvata lugens (Stål.)

Twenty-six rice landraces from West Bengal, India were evaluated for antixenosis and tolerance against brown planthopper (BPH) biotype 4 at the Bidhan Chandra Krishi Viswavidyalaya (BCKV), West Bengal. High levels of resistance were observed in six landraces, namely Badshabhog, Gamra, Haldichuri, Janglijata, Kalabhat and Khara. These phenotypically resistant rice landraces including Ptb33 exhibited lowest feeding rate, fecundity, nymphal and adult preference, survival, plant dry weight loss per mg of BPH dry weight produced (PDWL), and higher functional plant loss index (FPLI), more days to wilt and unhatched eggs compared with the susceptible check Swarna. All the landraces were classified into four major clusters at 10 unit distance by the scale of similarity during genetic diversity analysis through 21 gene-linked SSR markers of BPH resistance. Some phenotypically resistant landraces were gathered under the major cluster I indicating their analogous genetic history, while some were grouped with susceptible landraces exhibiting their genetic variation. The resistant landraces can be used as potential donors in the breeding programme for the development of rice varieties with resistance to BPH.     

BJ-1108, a 6-Amino-2,4,5-Trimethylpyridin-3-ol Analog,Inhibits Serotonin-Induced Angiogenesis and Tumor Growth through PI3K/NOX Pathway

5-Hydroxytryptamine (5-HT) induces proliferation of cancer cells and vascular cells. In addition to 5-HT production by several cancer cells including gastrointestinal and breast cancer, a significant level of 5-HT is released from activated platelets in the thrombotic environment of tumors, suggesting that inhibition of 5-HT signaling may constitute a new target for antiangiogenic anticancer drug discovery. In the current study we clearly demonstrate that 5-HT-induced angiogenesis was mediated through the 5-HT₁ receptor-linked Gβγ/Src/PI3K pathway, but not through the MAPK/ERK/p38 pathway. In addition, 5-HT induced production of NADPH oxidase (NOX)-derived reactive oxygen species (ROS). In an effort to develop new molecularly targeted anticancer agents against 5-HT action in tumor growth, we demonstrate that BJ-1108, a derivative of 6-amino-2,4,5-trimethylpyridin-3-ol, significantly inhibited 5-HT-induced angiogenesis. In addition, BJ-1108 induced a significant reduction in the size and weight of excised tumors in breast cancer cell-inoculated CAM assay, showing proportionate suppression of tumor growth along with inhibition of angiogenesis. In human umbilical vein endothelial cells (HUVECs), BJ-1108 significantly suppressed 5-HT-induced ROS generation and phosphorylation of PI3K/Akt but not of Src. Unlike NOX inhibitors, BJ-1108, which showed better antioxidant activity than vitamin C, barely suppressed superoxide anion induced by mevalonate or geranylgeranyl pyrophosphate which directly activates NOX without help from other signaling molecules in HUVECs, implying that the anti-angiogenic action of BJ-1108 was not mediated through direct action on NOX activation, or free radical scavenging activity. In conclusion, BJ-1108 inhibited 5-HT-induced angiogenesis through PI3K/NOX signaling but not through Src, ERK, or p38.     

Insulin substrates 1 and 2 are corequired for activation of atypical protein kinase C and Cbl-dependent phosphatidylinositol 3-kinase during insulin action in immortalized brown adipocytes

Phosphatidylinositol 3-kinase (PI3K)-dependent activation of atypical protein kinase C (aPKC) is required for insulin-stimulated glucose transport. Although insulin receptor substrate-1 (IRS-1) and IRS-2, among other factors, activate PI3K, there is little information on the relative roles of IRS-1and IRS-2 during aPKC activation by insulin action in specific cell types. Presently, we have used immortalized brown adipocytes in which either IRS-1 or IRS-2 has been knocked out by recombinant methods to examine IRS-1 and IRS-2 requirements for activation of aPKC. We have also used these adipocytes to see if IRS-1 and IRS-2 are required for activation of Cbl, which is required for insulin-stimulated glucose transport and has been found to function upstream of both PI3K/aPKC and Crk during thiazolidinedione action in 3T3/L1 adipocytes [Miura et al. (2003) Biochemistry 42, 14335]. In brown adipocytes in which either IRS-1 or IRS-2 was knocked out, insulin-induced increases in aPKC activity and glucose transport were markedly diminished. These effects of insulin on aPKC and glucose transport were fully restored by retroviral-mediated expression of IRS-1 or IRS-2 in their respective knockout cells. Knockout of IRS-1 or IRS-2 also inhibited insulin-induced increases in Cbl binding to the p85 subunit of PI3K, which, along with IRS-1/2, may be required for activation of PI3K, aPKC, and glucose transport during insulin action in 3T3/L1 adipocytes. These findings provide evidence that directly links both IRS-1 and IRS-2 to aPKC activation in immortalized brown adipocytes, and further suggest that IRS-1 and IRS-2 are required for the activation of Cbl/PI3K during insulin action in these cells.     

Slow ligand binding kinetics dominate ferrous hexacoordinate hemoglobin reactivities and reveal differences between plants and other species

Hexacoordinate hemoglobins are found in many living organisms ranging from prokaryotes to plants and animals. They are named "hexacoordinate" because of reversible coordination of the heme iron by a histidine side chain located in the heme pocket. This endogenous coordination competes with exogenous ligand binding and causes multiphasic relaxation time courses following rapid mixing or flash photolysis experiments. Previous rapid mixing studies have assumed a steady-state relationship between hexacoordination and exogenous ligand binding that does not correlate with observed time courses for binding. Here, we demonstrate that this assumption is not valid for some hexacoordinate hemoglobins, and that multiphasic time courses are due to an appreciable fraction of pentacoordinate heme resulting from relatively small equilibrium constants for hexacoordination (K(H)). CO binding reactions initiated by rapid mixing are measured for four plant hexacoordinate hemoglobins, human neuroglobin and cytoglobin, and Synechocystis hemoglobin. The plant proteins, while showing a surprising degree of variability, differ from the others in having much lower values of K(H). Neuroglobin and cytoglobin display dramatic biphasic time courses for CO binding that have not been observed using other techniques. Finally, an independent spectroscopic quantification of K(H) is presented that complements rapid mixing for the investigation of hexacoordination. These results demonstrate that hexacoordination could play a much larger role in regulating affinity constants for ligand binding in human neuroglobin and cytoglobin than in the plant hexacoordinate hemoglobins.     

Structure-based design of selective phosphodiesterase 4B inhibitors based on ginger phenolic compounds

Phosphodiesterase 4 (PDE4) has been established as a drug target for inflammatory diseases of respiratory tract like asthma and chronic obstructive pulmonary disease. The selective inhibitors of PDE4B, a subtype of PDE4, are devoid of adverse effects like nausea and vomiting commonly associated with non-selective PDE4B inhibitors. This makes the development of PDE4B subtype selective inhibitors a desirable research goal. Thus, in the present study, molecular docking, molecular dynamic simulations and binding free energy were performed to explore potential selective PDE4B inhibitors based on ginger phenolic compounds. The results of docking studies indicate that some of the ginger phenolic compounds demonstrate higher selective PDE4B inhibition than existing selective PDE4B inhibitors. Additionally, 6-gingerol showed the highest PDE4B inhibitory activity as well as selectivity. The comparison of binding mode of PDE4B/6-gingerol and PDE4D/6-gingerol complexes revealed that 6-gingerol formed additional hydrogen bond and hydrophobic interactions with active site and control region 3 (CR3) residues in PDE4B, which were primarily responsible for its PDE4B selectivity. The results of binding free energy demonstrated that electrostatic energy is the primary factor in elucidating the mechanism of PDE4B inhibition by 6-gingerol. Dynamic cross-correlation studies also supported the results of docking and molecular dynamics simulation. Finally, a small library of molecules were designed based on the identified structural features, majority of designed molecules showed higher PDE4B selectivity than 6-gingerol. These results provide important structural features for designing new selective PDE4B inhibitors as anti-inflammatory drugs and promising candidates for synthesis and pre-clinical pharmacological investigations.