Characterization of a unique IgG1 mAb CEX profile by limited Lys-C proteolysis/CEX separation coupled with mass spectrometry and structural analysis

The unique cation exchange chromatography (CEX) charge variant profile of mAb1 is characterized by a combination of mass spectrometry, limited Lys-C digestion followed by CEX separation and structural analysis. During CEX method development, mAb1 showed several unexpected phenomena, including a unique profile containing two main species (acidic 2 and main) and significant instability during stability studies of the main species. Reduced Lys-C peptide mapping identified a small difference in one of the heavy chain peptides (H4) in acidic 2 and further mass analysis identified this difference as Asn55 deamidation. However, the amount of Asn55 deamidation in acidic 2 could account for only half of the species present in this peak. Lys-C limited digest followed by CEX separated several unique peaks in the acidic peak 2 including two pre Fab peaks (LCC1 and LCC2). Whole protein mass analysis suggested that both LCC1 and LCC2 were potentially deamidated species. Subsequent peptide mapping with MS/MS determined that LCC1 contained isoAsp55 and LCC2 contained Asp55. Combining LCC1 and LCC2 CEX peak areas could account for nearly all of the species present in acidic peak 2. Subsequent detailed sequence analysis combined with molecular modeling identified Asn55 and its surrounding residues are responsible for the different CEX behavior and instability of mAb1 following forced degradation at high pH. Overall, the combinatorial approach used in this study proved to be a powerful tool to understand the unique charge variant and stability profile of a monoclonal antibody.     

Coral communities of barrier reefs of Vietnam

The composition and spatial distribution of the coral communities of the barrier reefs of Jiang Bo and of Re Island were described in detail for the first time for Vietnamese waters. Their comparability to the ribbon reefs of the Great Barrier Reef in Australia and to the barrier reefs of the Philippines and Indian Ocean was revealed by morphological parameters, species diversity and zonal distribution. Their geomorphological status, the presence of fore reef, epi-reef and back reef complexes with their specific composition of flora and fauna, and an obligatory lagoon separating the reef from fringing inshore reefs, enabled the attribution of the surveyed reefs to the barrier type of reef.     

Cohesin and recombination proteins influence the G1-to-S transition in azygotic meiosis in Schizosaccharomyces pombe

To determine whether recombination and/or sister-chromatid cohesion affect the timing of meiotic prophase events, the horsetail stage and S phase were analyzed in Schizosaccharomyces pombe strains carrying mutations in the cohesin genes rec8 or rec11, the linear element gene rec10, the pairing gene meu13, the double-strand-break formation genes rec6, rec7, rec12, rec14, rec15, and mde2, and the recombination gene dmc1. The double-mutant strains rec8 rec11 and rec8 rec12 were also assayed. Most of the single and both double mutants showed advancement of bulk DNA synthesis, start of nuclear movement (horsetail stage), and meiotic divisions by up to 2 hr. Only mde2 and dmc1 deletion strains showed wild-type timing. Contrasting behavior was observed for rec8 deletions (delayed by 1 hr) compared to a rec8 point mutation (advanced by 1 hr). An hypothesis for the role of cohesin and recombination proteins in the control of the G₁-to-S transition is proposed. Finally, differences between azygotic meiosis and two other types of fission yeast meiosis (zygotic and pat1-114 meiosis) are discussed with respect to possible control steps in meiotic G₁.     

Novel genes required for meiotic chromosome segregation are identified by a high-throughput knockout screen in fission yeast

Two rounds of chromosome segregation after only a single round of DNA replication enable the production of haploid gametes from diploid precursors during meiosis. To identify genes involved in meiotic chromosome segregation, we developed an efficient strategy to knock out genes in the fission yeast on a large scale. We used this technique to delete 180 functionally uncharacterized genes whose expression is upregulated during meiosis. Deletion of two genes, sgo1 and mde2, caused massive chromosome missegregation. sgo1 is required for retention of centromeric sister-chromatid cohesion after anaphase I. We show here that mde2 is required for formation of the double-strand breaks necessary for meiotic recombination.     

Morphology of hair pigmentation in wild red foxes,silver foxes,and their hybrids

The effects of dominant allele Ar of locus Agouti on the morphology of hair pigmentation were described in foxes. The Ar allele was shown to determine the type of melanin and its content in hair with no effect on the morphology of pigment granules and their distribution throughout a hair. Using the method of electron spin resonance (ESR), the types of melanin (eumelanin and pheomelanin) and their content in the hair of red (ArArEE) and silver (aaEE) foxes and their hybrids (AraEE) were determined. In silver foxes, only one type of melanin (eumelanin) was found. In red foxes and their hybrids (which are phenotypically similar but darker than red foxes), both types of melanin (eu- and pheomelanin) were found. The highest melanin content was detected in the coat of silver foxes. In the hybrids, the total melanin content was lower than in silver foxes, but significantly higher than in red foxes. In red foxes, the contribution of pheomelanin to the total hair melanin content was twice as large as in the hybrids.     

Reef-Building Corals of Vietnam as a Part of the Indo-Pacific Reef Ecosystem

The paper analyzes both published and unpublished results of the investigations of Vietnamese reef-building corals and reefs performed in the last decades of the twentieth century. The state of the art in the study of reef-building scleractinian corals and reefs is presented. The scleractinian fauna of Vietnam is shown to match in species diversity (366 species of 70 genera) the tropical coral fauna of the Indonesian-Malacca fertile center, from which Indo-Pacific reef-building corals originated. The whole Vietnam coast from the Gulf of Tonkin to the Gulf of Siam is a biogeographically single whole and is a part of the Indo-Polynesian Province of the Indo-Pacific Area.     

Reef-Building Corals and Reefs of Vietnam: 2. The Gulf of Tonkin

This paper deals with the history and results of the studies of reefs and coral communities of the Gulf of Tonkin based on published and unpublished materials, including the author's. The state of the art in the study of reef-building scleractinian corals and reefs of this region is reported. The peculiar nature of the reefs studied is caused by the monsoon climate in the region and river runoff waters cooled to 16–18°C, silted to 100 g/m2 per day, and freshened to 28‰ in the wintertime, i.e., conditions far from optimum for reef formation. The silting and eutrophication of the gulf waters resulted in a change in the composition and structure of the coral reef communities via the reduction or elimination of certain coral species. Instead of acroporids, typical for the majority of other reefs, reef communities of the Gulf of Tonkin are dominated by poritids and faviids, which form the framework of the reefs. These peculiarities make the reefs of the Gulf of Tonkin really unique.     

Verification of the color genotype based on morphological analysis of fur pigmentation of brown and black water voles Arvicola terrestris L.]

Qualitative and quantitative analysis of fur pigmentation in brown and black water voles (Arvicola terrestris L.) was performed. Morphology of pigment granules, their distribution along the hair layers and histology of hair bulbs were studied. Morphological data and the results of the analysis of segregation in the progeny, when brown voles were self-crossed and crossed with black ones, led to conclusion that fur colour of water voles is mediated by genes from the agouti series, precisely, black colour is determined by the extreme non-agouti allele (aeae genotype), and in brown voles which are homo- or heterozygous the colour is determined by the agouti allele (AA or Aae genotypes).     

Conformational equilibration time of unfolded protein chains and the folding speed limit

The speed with which the conformers of unfolded protein chains interconvert is a fundamental question in the study of protein folding. Kinetic evidence is presented here for the time constant for interconversion of disparate unfolded chain conformations of a small globular protein, cytochrome c, in the presence of guanidine hydrochloride denaturant. The axial binding reactions of histidine and methionine residues with the Fe(II) heme cofactor were monitored with time-resolved magnetic circular dichroism spectroscopy after photodissociation of the CO complexes of unfolded protein obtained from horse and tuna and from several histidine mutants of the horse protein. A kinetic model fitting both the reaction rate constants and spectra of the intermediates was used to obtain a quantitative estimate of the conformational diffusion time. The latter parameter was approximated as a first-order time constant for exchange between conformational subensembles presenting either a methionine or a histidine residue to the heme iron for facile binding. The mean diffusional time constant of the wild type and variants was 3 +/- 2 mus, close to the folding "speed limit". The implications of the relatively rapid conformational equilibration time observed are discussed in terms of the energy landscape and classical pathway time regimes of folding, for which the conformational diffusion time can be considered a pivot point.     

Investigation of degradation processes in IgG1 monoclonal antibodies by limited proteolysis coupled with weak cation-exchange HPLC

A new cation-exchange high-performance liquid chromatography (HPLC) method that separates fragment antigen-binding (Fab) and fragment crystallizable (Fc) domains generated by the limited proteolysis of monoclonal antibodies (mAbs) was developed. This assay has proven to be suitable for studying complex degradation processes involving various immunoglobulin G1 (IgG1) molecules. Assignment of covalent degradations to specific regions of mAbs was facilitated by using Lys-C and papain to generate Fab and Fc fragments with unique, protease-dependent elution times. In particular, this method was useful for characterizing protein variants formed in the presence of salt under accelerated storage conditions. Two isoforms that accumulated during storage were readily identified as Fab-related species prior to mass-spectrometric analysis. Both showed reduced biological activity likely resulting from modifications within or in proximity of the complementarity-determining regions (CDRs). Utility of this assay was further illustrated in the work to characterize light-induced degradations in mAb formulations. In this case, a previously unknown Fab-related species which populated upon light exposure was observed. This species was well resolved from unmodified Fab, allowing for direct and high-purity fractionation. Mass-spectrometric analysis subsequently identified a histidine-related degradation product associated with the CDR2 of the heavy chain. In addition, the method was applied to assess the structural organization of a noncovalent IgG1 dimer. A new species corresponding to a Fab–Fab complex was found, implying that interactions between Fab domains were responsible for dimerization. Overall, the data presented demonstrate the suitability of this cation-exchange HPLC method for studying a wide range of covalent and noncovalent degradations in IgG1 mAbs.