Quantitative analysis of glycation and its impact on antigen binding

Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1. Heavy chain Lys100, which is located in the complementary-determining region of mAb1, had the highest levels of glycation in both stressed and unstressed samples, and glycation of this residue was likely responsible for the loss of antigen binding based on hydrogen/deuterium exchange mass spectrometry analysis. Peptide mapping and intact liquid chromatography-mass spectrometry (LC-MS) can both be used to monitor the glycation levels. Peptide mapping provides site specific glycation results, while intact LC-MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing. Capillary isoelectric focusing (cIEF) can also be used to monitor glycation because glycation induces an acidic shift in the cIEF profile. As expected, total glycation measured by intact LC-MS correlated very well with the percentage of total acidic peaks or main peak measured by cIEF. In summary, we demonstrated that glycation can affect the function of a representative IgG1 mAb. The analytical characterization, as described here, should be generally applicable for other therapeutic mAbs.     

Serological Indicators of Helicobacter pylori Infection in Adult Dyspeptic Patients and Healthy Blood Donors

The levels of IgM, IgG and IgA antibodies reacting with two Helicobacter pylori antigens (glycine acid extract (GE) and a recombinant CagA protein) were determined in the sera from adult dyspeptic patients, positive (H.p.⁽⁺⁾) or negative (H.p.^(-)) for H. pylori urease/culture, and from healthy blood donors. All sera were also examined against GE by Western blot (immunoblot) technique. Similar levels of anti-GE IgG were detected in the sera from all H.p.⁽⁺⁾ and almost all H.p.^(-) patients and from over 40% of the healthy volunteers. In contrast, higher levels of anti-GE IgA were found in the sera from patients than that from healthy subjects, although such antibodies were not detected in the sera from 30% of the H.p.⁽⁺⁾ patients. In general, our results suggest that a combination of ELISA and immunoblot may be more sensitive in the detection of H. pylori infection in dyspeptic patients than the examination of biopsy specimens by culturing or histology.     

Mechanism of the interactions of aliphatic alcohols with bovine serum albumin in the ternary systems—1H n.m.r. study: 1. Aqueous solutions BSA-alcohols-urea

The molecular mechanism of the interaction of aliphatic alcohols (A) with bovine serum albumin (BSA) protein was studied in aqueous solutions at increasing concentrations (0–8 m) of urea (U). ¹H n.m.r. spectra of alcohols were monitored in D₂O in the control binary systems (A—U) and (A—BSA), and in the ternary systems (A—U—BSA) at pH 7.0. Marked and selective broadening of the n.m.r. lines of alcohols in the system (A—BSA) was reduced upon addition of urea, indicating that alcohols are poorly bound by urea-denaturated BSA. The reduction in the ability to associate with BSA depends on chain position of the alcohol molecule and is much higher for α-methylenes (next to ?OH) than for other proton groups. Besides this reduction seems to be a two-step phenomenon dependent upon urea concentration. The results obtained can be explained by competition in formation by the peptide linkages of a protein of the hydrogen bonds with ?OH group of alcohols or fragments of urea molecules.     

Ultrastructural and molecular characterization of diversity among small araphid diatoms all lacking rimoportulae. I. Five new genera,eight new species

Pennate diatoms are important contributors to primary production in freshwater and marine habitats. But the extent of their diversity, ecology, and evolution is still largely unknown. This is particularly evident among the clades of pennate diatoms without raphe slits, whose diversity is likely underestimated due to their small size and features that can be difficult to discern under light microscopy. In this study, we described five new araphid genera with eight new species based on morphological observations (light and electron microscopy) and molecular data (nuclear‐encoded small subunit ribosomal RNA and chloroplast‐encoded rbcL and psbC): Serratifera varisterna, Hendeyella rhombica, H. dimeregrammopsis, H. lineata, Psammotaenia lanceolata, Castoridens striata, C. hyalina, and Cratericulifera shandongensis. We also transferred Dimeregramma dubium to Hendeyella dubia. Phylogenetic analysis of the molecular data revealed that all the newly established taxa fell into a monophyletic group, with Fragilariforma virescens located at the base. The group was composed by two subclades: one comprising Castoridens, Cratericulifera, and Plagiostriata, and the larger including also the rest of the new genera plus some of the smallest known diatoms, such as Nanofrustulum, Opephora, Pseudostaurosira, Staurosirella, and Staurosira with a high level of support. This study enhances the general knowledge on the phylogeny and biodiversity of a group of small araphid diatoms that have been generally poorly described both by electron microscopy and DNA sequence data.     

Changes in fatty acid composition of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> KB2 during co-metabolic degradation of monochlorophenols

The changes in the cellular fatty acid composition of Stenotrophomonas maltophilia KB2 during co-metabolic degradation of monochlorophenols in the presence of phenol as well as its adaptive mechanisms to these compounds were studied. It was found that bacteria were capable of degrading 4-chlorophenol (4-CP) completely in the presence of phenol, while 2-chlorophenol (2-CP) and 3-chlorophenol (3-CP) they degraded partially. The analysis of the fatty acid profiles indicated that adaptive mechanisms of bacteria depended on earlier exposure to phenol, which isomer they degraded, and on incubation time. In bacteria unexposed to phenol the permeability and structure of their membranes could be modified through the increase of hydroxylated and cyclopropane fatty acids, and straight-chain and hydroxylated fatty acids under 2-CP, 3-CP and 4-CP exposure, respectively. In the exposed cells, regardless of the isomer they degraded, the most important changes were connected with the increase of the contribution of branched fatty acid on day 4 and the content of hydroxylated fatty acids on day 7. The changes, particularly in the proportion of branched fatty acids, could be a good indicator for assessing the progress of the degradation of monochlorophenols by S. maltophilia KB2. In comparison, in phenol-degrading cells the increase of cyclopropane and straight-chain fatty acid content was established. These findings indicated the degradative potential of the tested strain towards the co-metabolic degradation of persistent chlorophenols, and extended the current knowledge about the adaptive mechanisms of these bacteria to such chemicals.     

Toxicological evaluation of multi-class pesticide residues in vegetables and associated human health risk study for adults and children

This article describes, for the first time in 10 years, the toxicological evaluation of pesticide residues in highly consumed vegetables (35 commodities) and health risk studies. Among 1075 vegetable samples consisting mainly of tuber, root, fruiting, and Brassica, 20% of samples contained 38 pesticides in the range of 0.005–18.7 mg/kg and 1% > maximum residue limits (MRLs). Organophosphates and chloronitrile were most frequently found (20%) in the pesticide class. The highest concentration of 18.7 mg/kg was noted for dichlofluanid. Fruiting vegetables (8%), especially tomatoes (5%), were the most contaminated, while multi-residues were determined in 5% of the samples. Risk assessment was performed by analytical results and consumption on the 97.5 percentile expressed as hazard index (HI) and hazard quotient (HQ). The highest chronic HI was calculated for diazinon in lettuce (32% Acceptable Daily Intake (ADI) adults and 36% children) and for forbidden dieldrin in carrots (26%, 62% ADI). The highest acute HI was estimated for dichlofluanid in lettuce (69% Acute Reference Dose (ARfD)) for adults, whereas for children it was above the acceptance values of ARfD (168%). Organophosphate insecticides with common mode of action showed the greatest HQ (108% ADI). Vegetables may not be a serious problem for consumers, but investigations on pesticide residues are necessary to ensure food safety and the protection of human health, especially toward children who are more highly exposed to pesticides than adults.