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.     

Identification of an unusual [2Fe-2S]-binding motif in the CDP-6-deoxy-D-glycero-l-threo-4-hexulose-3-dehydrase from Yersinia pseudotuberculosis: implication for C-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses

CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E(1)) catalyzes the C-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. E(1) is a pyridoxamine 5'-phosphate (PMP)-dependent enzyme that also contains a [2Fe-2S] center. This iron-sulfur cluster is catalytically essential, since removal of the [2Fe-2S] center leads to inactive enzyme. To identify the [2Fe-2S] core in E(1) and to study the effect of impairing the iron-sulfur cluster on the activity of E(1), a series of E(1) cysteine mutants were constructed and their catalytic properties were characterized. Our results show that E(1) displays a cluster-binding motif (C-X(57)-C-X(1)-C-X(7)-C) that has not been observed previously for [2Fe-2S] proteins. The presence of such an unusual iron-sulfur cluster in E(1), along with the replacement of the active site lysine by a histidine residue (H220), reflects a distinct evolutionary path for this enzyme. The cysteine residues (C193, C251, C253, C261) implicated in the binding of the iron-sulfur cluster in E(1) are conserved in the sequences of its homologues. It is likely that E(1) and its homologues constitute a new subclass in the family of iron-sulfur proteins, which are distinguished not only by their cluster ligation patterns but also by the chemistry used in catalyzing a simple, albeit mechanistically challenging, reaction.     

Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection

Size exclusion chromatography (SEC) coupled with online light scattering, viscometry, refractometry, and UV-visible spectroscopy provides a very powerful tool for studying protein size, shape, and aggregation. This technique can be used to determine the molecular weight of the component peaks independent of the retention times in the SEC column and simultaneously measure the hydrodynamic radius and polydispersity of the protein. We applied this technology by coupling an Agilent Chemstation high-performance liquid chromatography system with a diode array UV-visible detector and a Viscotek 300 EZ Pro triple detector (combination of a light scattering detector, refractometer, and differential pressure viscometer) to characterize and compare the molecular properties of a number of monoclonal antibodies. Our studies reveal that different monoclonal immunoglobulin Gs (IgGs) and chimeric IgGs show slightly different retention times and therefore different molecular weights in gel filtration analysis. However, when they are analyzed by light scattering, refractometry, and viscometry, different IgGs have comparable molecular weight, molecular homogeneity (polydispersity), and size. Gel filtration coupled with UV or refractive index detection suggests that antibodies purified and formulated for preclinical and clinical development are more than 95% monomer with little or no detectable soluble aggregates. Light scattering measurements showed the presence of trace amounts of soluble aggregate in all the IgG preparations. The different IgG molecules showed different susceptibility to heat and pH. One of the murine antibodies was considerably less stable than the others at 55 degrees C. The application of this powerful technology for the characterization of monoclonal antibodies of therapeutic potential is discussed.     

Honokiol potentiates apoptosis, suppresses osteoclastogenesis, and inhibits invasion through modulation of nuclear factor-kappaB activation pathway

Recent reports have indicated that honokiol can induce apoptosis, suppress tumor growth, and inhibit angiogenesis. In this report, we found that honokiol potentiated the apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents, suppressed TNF-induced tumor cell invasion, and inhibited RANKL-induced osteoclastogenesis, all of which are known to require nuclear factor-kappaB (NF-kappaB) activation. Honokiol suppressed NF-kappaB activation induced by a variety of inflammatory stimuli, and this suppression was not cell type specific. Further studies showed that honokiol blocked TNF-induced phosphorylation, ubiquitination, and degradation of IkappaBalpha through the inhibition of activation of IkappaBalpha kinase and of Akt. This led to suppression of the phosphorylation and nuclear translocation of p65 and NF-kappaB-dependent reporter gene expression. Magnolol, a honokiol isomer, was equally active. The expression of NF-kappaB-regulated gene products involved in antiapoptosis (IAP1, IAP2, Bcl-x(L), Bcl-2, cFLIP, TRAF1, and survivin), proliferation (cyclin D1, cyclooxygenase-2, and c-myc), invasion (matrix metalloproteinase-9 and intercellular adhesion molecule-1), and angiogenesis (vascular endothelial growth factor) were also down-regulated by honokiol. Honokiol also down-regulated NF-kappaB activation in in vivo mouse dorsal skin model. Thus, overall, our results indicate that NF-kappaB and NF-kappaB-regulated gene expression inhibited by honokiol enhances apoptosis and suppresses osteoclastogenesis and invasion.     

Combination Treatment with Resveratrol and Sulforaphane Induces Apoptosis in Human U251 Glioma Cells

Resveratrol is a naturally occurring polyphenolic compound highly enriched in grapes, peanuts, red wine, and a variety of food sources. Sulforaphane belongs to the family of isothiocyanates and is highly enriched in cruciferous vegetables. Our previous study showed that resveratrol, when used at high concentrations, inhibited cell proliferation, caused the cell cycle arrest and induced apoptotic cell death in glioma cells. In the current study, we tested the effect of combination treatment with resveratrol and sulforaphane, when both were used at low concentrations, on cell proliferation, migration and death in human U251 glioma cells. Our study shows that combination treatment with resveratrol and sulforaphane inhibits cell proliferation and migration, reduces cell viability, induces lactate dehydrogenase release, decreases pro-survival Akt phosphorylation and increases caspase-3 activation. The use of combination of bioactive food components, such as resveratrol and sulforaphane, may be a viable approach for the treatment of glioma.     

Population growth and development of the psocid Liposcelis rufa (Psocoptera: Liposcelididae) at constant temperatures and relative humidities

We investigated the effects of eight temperatures (22.5, 25.0, 27.5, 30.0, 32.5, 35.0, 37.5, and 40.0 degrees C) and four relative humidities (43, 55, 63, and 75%) on population growth and development of the psocid Liposcelis rufa Broadhead (Psocoptera: Liposcelididae). L. rufa did not survive at 43% RH, at all temperatures tested; at 55% RH, at the highest four temperatures; and at 63% RH and 40.0 degrees C. The greatest population growth was recorded at 35.0 degrees C and 75% RH (73-fold growth). At 40.0 degrees C, L. rufa populations declined or barely grew. L. rufa males have two to four nymphal instars, and the percentages of males with two, three, and four instars were 31, 54, and 15%, respectively. Female L. rufa have two to five instars, and the percentages of females with two, three, four, and five instars were 2, 44, 42, and 12%, respectively. The life cycle was shorter for males than females. We developed temperature-dependent developmental equations for male and female eggs, individual nymphal, combined nymphal, and combined immature stages. The ability of L. rufa to reproduce at a relative humidity of 55% and temperatures of 22.5-30.0 degrees C and at relative humidities of 63-75% and temperatures of 22.5-37.5 degrees C, in addition to being able to survive at 40.0 degrees C, suggests that this species would be expected to have a broader distribution than other Liposcelis species. These data provide a better understanding of L. rufa population dynamics and can be used to help develop effective management strategies for this psocid.     

Prophylactic efficacy of combination of DRDE-07 and its analogues with amifostine against sulphur mustard induced systemic toxicity

Sulphur mustard, [bis (2-chloroethyl)] sulphide (SM), is a bifunctional alkylating agent. SM forms sulphonium ion in the body which alkylates DNA and several other macromolecules, and induces oxidative stress. Although several antidotes have been screened for the treatment of systemic toxicity of SM in experimental animals none of them are recommended so far. In the search for more effective and less toxic antidotes, various combinations were tried against SM induced toxicity and skin lesions. SM exposed through percutaneous route was used to evaluate the prophylactic efficacy of various combinations. Low dose of DRDE-07 (S-2(2-aminoethylamino) ethyl phenyl sulphide), DRDE-30 [S-2(2-aminoethyl amino) ethyl propyl sulphide], DRDE-35 [S-2(2-aminoethyl amino) ethyl butyl sulphide] with amifostine combinations, were given orally 30 min prior to SM exposure. Significant depletion was observed in body weight, organ body weight index and hepatic GSH and GSSG content in mice after SM exposure. Pretreatment with low dose of different combinations of DRDE-07, DRDE-30 and DRDE-35 with amifostine could recover biochemical alterations and histopathological changes caused by SM exposures.     

Diversity of some insect fauna in industrial and non-industrial areas of West Bengal, India

The present work is designed to study diversity of five insect orders (viz., Hemiptera, Orthoptera, aculeate Hymenoptera, Lepidoptera and Coleoptera) in the industrial region of Haldia (India) and in non-industrial area of the same district and to evaluate the impact of industrialization on the biodiversity of those insect orders. The objective also extended to find out the possibility of existence of bioindicator, if any. Eight study sites were selected from the East part of Midnapur district covering 40 km aerial distance. Out of eight different study sites, five were distributed in and around Haldia industrial complex and three in industry-free area. During this study, a total of 120 species under 98 genera in 37 families of insects were collected. Binary data of 5 orders revealed that the species richness of Hemiptera, Orthoptera and Lepidoptera is higher in non-industrial zone in comparison to that of industrial zone. Aculeate Hymenoptera shows no particular trend whereas Coleoptera shows higher species richness in industrial areas. Results of multivariate analyses are compared with the species richness data for all the eight study sites. It is concluded that even in an apparently homogeneous ecological condition species richness may drastically change with the influence of industries. Total insect fauna decline by at least 23.33% is noticed in industrial areas. It is found that some species of lepidopteran, hemipteran and orthopteran insects are susceptible to industrial pollution and some of the members of these orders may be considered as a bioindicator group.