The use of monoclonal antibodies (mAbs) for the manufacture of innovator and biosimilar biotherapeutics has increased tremendously in recent years. From a structural perspective, mAbs have high ...disulfide bond content, and the correct disulfide connectivity is required for proper folding and to maintain their biological activity. Therefore, disulfide linkage mapping is an important component of mAb characterization for ensuring drug safety and efficacy. The native disulfide linkage patterns of all four subclasses of IgG antibodies have been well established since the late 1960s. Among these IgG subtypes, disulfide mediated isoforms have been identified for IgG2 and IgG4, and to a lesser extent in IgG1, which is the most studied IgG subclass. However, no studies have been carried out so far to investigate whether different IgG3 isoforms exist due to alternative disulfide connectivity. In an effort to investigate the presence of disulfide-mediated isoforms in IgG3, we employed a bottom-up mass spectrometry approach to accurately determine the disulfide bond linkages in endogenous human IgG3 monoclonal antibody, and our results show that no such alternative disulfide bonds exist. While many antibody-based drugs are developed around IgG1, IgG3 represents a new, and in some cases, more desirable drug candidate. Our data represent the first demonstration that alternative disulfide bond arrangements are not present in endogenous IgG3; and therefore, they should not be present in recombinant forms used as antibody-based therapeutics.
The use of monoclonal antibodies (mAbs) for the manufacture of innovator and biosimilar biotherapeutics has increased tremendously in recent years.
Excesses of digestive and respiratory cancers have been reported previously in association with exposure to machining fluids, agents in widespread use as coolants and lubricants in machining ...operations. Previous studies have had limited power to distinguish the effects of the different types of machining fluids in use. In a cohort of over 30 000 workers employed at two automotive plants in Michigan, mortality patterns were studied in relation to exposure to each of the three major fluid types — straight oils, soluble oils, and synthetic fluids. Standardized mortality ratios were estimated for subgroups of the cohort ever exposed to each of the three fluid types, and Poisson regression analyses were used to assess trends in risk with duration of exposure. The data suggest modest positive associations between exposure to straight oils and rectal, laryngeal, and prostatic cancer and a negative association between soluble and synthetic fluid exposure and lung cancer.
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The enzymatic conversion of glucose into ATP, GTP, UTP, and CTP with several different isotopic labeling patterns is described. Enzymes of the pentose phosphate pathway and enzyme-catalyzed hydrogen ...exchange were used to convert three types of isotopically labeled glucose into 1‘,2‘,3‘,4‘,5‘,5‘-2H6NTPs (1−4), 3‘,4‘,5‘,5‘-2H4UTP (5), 1‘,2‘,3‘,4‘,5‘-13C5NTPs (6−9), and 3‘,4‘,5‘,5‘-2H4-1‘,2‘,3‘,4‘,5‘-13C5NTPs (10−13), which were then used to synthesize a 30 nucleotide HIV TAR RNA. Representative NOESY and HSQC spectra were acquired to demonstrate the utility of the new labeling patterns. The spectral editing afforded by 2H and 13C labeling dramatically simplifies the crowded NOESY and HSQC spectra of RNA molecules. The synthetic methods described here will permit the preparation of several specifically deuterated and/or 13C-labeled forms of RNA which should be useful in NMR structural studies of large RNAs.
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IJS, KILJ, NUK, PNG, UL, UM
Machining fluids are widely used in a variety of common industrial metalworking operations to lubricate and cool both the tool and the working surfaces. Previous studies have suggested elevated ...respiratory, digestive, and skin cancers in exposed populations. This cohort study was initiated to assess whether long-term exposure to machining fluids in the course of machining, grinding, and other cutting operations is associated with excess cancer mortality. The cohort includes more than 45,000 automobile production workers from 3 plants, almost 1 million years of follow-up, over 10,000 deaths, and an extensive exposure assessment component. Standardized mortality ratios (SMRs) have been estimated for each of the 3 plants, using both U.S. as well as local populations as reference. Relative risks of 1.2-3.1 have been observed for several specific respiratory and digestive cancers of a priori interest, including cancer of the stomach, large intestine, pancreas, lung, and larynx. In addition, elevated risks for leukemia and asthma were noted. Future exposure-response analyses will provide the opportunity to identify relatively modest excesses in cause-specific mortality risk associated with exposure to specific types (straight, soluble, or synthetic), additives, or components of machining fluids.
Functional single‐stranded oncogenic mRNAs such as the breakpoints of the PAX3 – FKHR and Bcr – Abl oncogenes bind selectively and with high affinity to naturally occurring, as well as synthetic, ...aminoglycosides. The latter, neamine‐derived compounds were designed to mimic their natural counterparts in recognizing RNA (the proposed mode of binding to a phosphodiester is shown) and were obtained by parallel synthesis.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Contemporary approaches to the synthesis of chemically modified proteins not only require efficient means to control conjugation and the specific site of attachment of the conjugated moiety, but also ...the effective use of recent developments in microarray technologies and nanobiotechnology. In Bioconjugation Protocols: Strategies and Methods, expert laboratorians update the classic methods and introduce valuable new approaches that go beyond basic conjugation techniques to include elements from advanced organic synthesis, molecular biology, and materials science. These readily reproducible methods cover the preparation of protein conjugates using covalent and noncovalent conjugation, the synthesis of nucleic acid conjugates using a variety of labeling techniques, and approaches to semisynthetic conjugates of proteins. Additional chapters address the biofunctionalization of inorganic surfaces, including the on-chip synthesis of peptide nucleic acids to generate microarrays for the high-throughput analysis of RNA and DNA, gold nanoparticles, and carbon nanotube probes for atomic force microscopy. All the protocols follow the successful Methods in Molecular Biology™ series format, each one offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and highly practical, Bioconjugation Protocols: Strategies and Methods offers both novice and experienced researchers access to the broad array of techniques needed to master the semisynthesis of functional biomolecular reagents for a wide variety of applications-ranging from novel biomedical diagnostics, to powerful new therapeutics, to advanced biomaterials.
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FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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