Ric-8A,a GEF,and a Chaperone for G Protein α-Subunits: Evidence for the Two-Faced Interface

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a prominent non-receptor GEF and a chaperone of G protein α-subunits (Gα). Recent studies shed light on the structure of Ric-8A, providing insights into the mechanisms underlying its interaction with Gα. Ric-8A is composed of a core armadillo-like domain and a flexible C-terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C-terminus appears to mediate formation of the initial Ric-8A/GαGDP intermediate, followed by the formation of a stable nucleotide-free complex. The latter event involves a large-scale dislocation of the Gα α5-helix that produces an extensive primary interface and disrupts the nucleotide-binding site of Gα. The distal portion of the C-terminal tail of Ric-8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two-site Gα interface of Ric-8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric-8A.     

Antigenic similarity between the β-subunits of the ATPases of a bacterium,a yeast and a higher plant

Antiserum raised against the β-subunit of wheat (Triticum aestivum) chloroplast ATPase cross-reacts with a 51000 protein located in the membrane fraction of Escherichia coli. The differential solubility of this polypeptide after chloroform treatment of unc⁺ and uncD409 strains indicates that this cross-reacting polypeptide is the bacterial β-subunit of ATPase. Thus a high degree of conservation of antigenic determinant sites exists between a bacterial β-subunit and the β-subunit of a monocot. This conservation also seems to extend to the β-subunit of mitochondrial ATPase of yeast (Saccharomyces cerevisiae).     

Physicochemical and functional assessments demonstrating analytical similarity between rituximab biosimilar HLX01 and the MabThera®

Development of bio-therapeutics has exhibited exponential growth in China over the past decade. However, no biosimilar drug has been approved in China (CN) due to the lack of a national biosimilar regulatory guidance. HLX01, a rituximab biosimilar developed in China under European Medicines Agency biosimilar guidelines and requirements, was the first such drug submitted for regulatory review in China, and it is expected to receive approval there as a biosimilar product. To demonstrate the analytical similarities of HLX01, CN-rituximab (sourced in China but manufactured in Europe) and EU-rituximab (sourced and manufactured in Europe), an extensive 3-way physicochemical and functional similarity assessment using a series of orthogonal and state-of-the-art techniques was conducted, following the similarity requirement guidelines recently published by China’s Center for Drug Evaluation. The results of the similarity study showed an identical protein amino acid sequence and highly similar primary structures between HLX01 and the reference product (RP) MabThera®, along with high similarities in higher order structures, potency, integrity, purity and impurity profiles, biological and immunological binding functions, as well as degradation behaviors under stress conditions. In addition, HLX01 presented slightly lower aggregates and better photostability compared with the RP. Despite slight changes in relative abundance of glycan moieties and heavy chain C-terminal lysine modification, no differences in biological activities and immunological properties were observed between the RP and HLX01. In conclusion, HLX01 is highly similar to CN- and EU-sourced RP in terms of physicochemical properties and biological activities, suggesting similar product quality, ef?cacy, and safety. The regulatory requirements interpreted and applied towards the HLX01 marketing application sets a precedent for analytical similarity assessment of biosimilar products in China.     

Demonstration of physicochemical and functional similarity between the proposed biosimilar adalimumab MSB11022 and Humira®

Biosimilars are biological products that are highly similar to existing products approved by health authorities. Demonstration of similarity starts with the comprehensive analysis of the reference product and its proposed biosimilar at the physicochemical and functional levels. Here, we report the results of a comparative analysis of a proposed biosimilar adalimumab MSB11022 and its reference product, Humira®. Three batches of MSB11022 and up to 23 batches of Humira® were analyzed by a set of state-of-the-art orthogonal methods. Primary and higher order structure analysis included N/C-terminal modifications, molecular weight of heavy and light chains, C-terminal lysine truncation, disulfide bridges, secondary and tertiary structures, and thermal stability. Purity ranged from 98.4%–98.8% for MSB11022 batches (N = 3) and from 98.4%–99.6% for Humira® batches (N = 19). Isoform analysis showed 5 isoform clusters within the pI range of 7.94–9.14 and 100% glycan site occupancy for both MSB11022 and Humira®. Functional analysis included Fab-dependent inhibition of tumor necrosis factor (TNF)-induced cytotoxicity in L929-A9 cell line and affinity to soluble and transmembrane forms of TNF, as well as Fc-dependent binding to Fcγ and neonatal Fc receptors and C1q complement proteins. All tested physicochemical and functional parameters demonstrated high similarity of MSB11022 and Humira®, with lower variability between MSB11022 and Humira® batches compared with variability within individual batches of Humira®. Based on these results, MSB11022 is anticipated to have safety and efficacy comparable to those of Humira®.     

Evo-devo and the search for homology (“sameness”) in biological systems

Developmental biology and evolutionary studies have merged into evolutionary developmental biology (“evo-devo”). This synthesis already influenced and still continues to change the conceptual framework of structural biology. One of the cornerstones of structural biology is the concept of homology. But the search for homology (“sameness”) of biological structures depends on our favourite perspectives (axioms, paradigms). Five levels of homology (“sameness”) can be identified in the literature, although they overlap to some degree: (i) serial homology (homonomy) within modular organisms, (ii) historical homology (synapomorphy), which is taken as the only acceptable homology by many biologists, (iii) underlying homology (i.e., parallelism) in closely related taxa, (iv) deep evolutionary homology due to the “same” master genes in distantly related phyla, and (v) molecular homology exclusively at gene level. The following essay gives emphasis on the heuristic advantages of seemingly opposing perspectives in structural biology, with examples mainly from comparative plant morphology. The organization of the plant body in the majority of angiosperms led to the recognition of the classical root–shoot model. In some lineages bauplan rules were transcended during evolution and development. This resulted in morphological misfits such as the Podostemaceae, peculiar eudicots adapted to submerged river rocks. Their transformed “roots” and “shoots” fit only to a limited degree into the classical model which is based on either–or thinking. It has to be widened into a continuum model by taking over elements of fuzzy logic and fractal geometry to accommodate for lineages such as the Podostemaceae.     

Cloning of a two-component regulatory system probably involved in the regulation of chitinase inStreptomyces cœlicolor A3(2)

Using the method for the identification of promoters recognized by the sporulation specific σ factor (σ^F), we identified a positive 950 pbSau3Al DNA fragment inStreptomyces cœlicolor A3(2). High-resolution S1-nuclease mapping identified a potential promoter, P_F35, in theE. coli two-plasmid system similar to the consensus sequence ofBacillus subtilis promoters recognized by the general stress-response σ factor (σ^B). However, the putativesigF-dependent promoter, P_F35, was inactive inS. cœlicolor in the course of diffenentiation and it was located divergently in the promoter region directing expression of thechiC gene encoding chitinase. Sequence analysis of the region potentially governed by P_F35 revealed two translationally coupled genes encoding proteins similar to bacterial two-component regulatory systems, and with the highest similarity to the two-component systemchiS, chiR, regulating chitinase activity inStreptomyces thermoviolaceus. However, the genes had a divergent orientation with respect to the P_F35 promoter. Disruption of theS. cœlicolor chiR gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin. Moreover, thechiR disruption did not affect the overall chitinase activity.     

Evidence for association and linkage between atopy, airway hyper-responsiveness, and the β subunit Glu237Gly variant of the high-affinity receptor for immunoglobulin E in the French-Canadian population

Following detection of linkage between atopy and chromosome 11q13 markers, association between this disorder and variants of the beta subunit of the high-affinity receptor for immunoglobulin E (FcepsilonRI-beta, a candidate gene for asthma-related conditions co-localizing within the same region) was reported in Australian, British and Japanese populations. Investigations in several other ethnic groups failed to replicate these observations. Due to the complexity of defining intermediate phenotypes related to asthma, detection of such associations may have been hampered by clinical misclassifications. To assess whether the FcepsilonRI-beta gene was involved in atopy and/or airway hyperresponsiveness (AHR) in the French-Canadian population, we conducted a case-control study in 200 subjects using strict criteria for asthma and related conditions. The Ile181Leu and Glu237Gly FcepsilonRI-beta sequence variants were tested exploiting two amplification refractory mutation systems. No association was detected between atopy or AHR and the Ile181Leu FcepsilonRI-beta variant. However, a strong association was observed between atopy and the Glu237Gly FcepsilonRI-beta variant (odds ratio=12.25). Four large Eastern Québec families (n=106 subjects) were also recruited to perform a genetic linkage study. We observed suggestive evidence of linkage between atopy and the Glu237Gly FcepsilonRI-beta variant (Zmax=2.30). This study is the first to detect the presence of an association between atopy and the Glu237Gly FcepsilonRI-beta variant in French-Canadians. Our data suggest that a susceptibility locus for atopy is located on chromosome 11q13 in this population.     

Evidence for a direct role of α-MSH in morphological background adaptation of the skin in Sarotherodon mossambicus

Summary The skin colour of the cichlid teleost Sarotherodon mossambicus adapted rapidly to changes in background colour. The physiological adaptation was associated with morphological changes in the dermis. Differences in the dermis were found between fish adapted to a black or white background for 14 days. Number and size of the melanophores as well as the amount of pigment in the cytoplasm of the melanophores were significantly increased in fish adapted to a black background. Changes in the dermis parallelled changes in the state of activity of the two endocrine cell types in the pars intermedia of the pituitary. Both the PAS positive cells and the MSH producing cells were more active when the fish were exposed to a black rather than a white background. Fish continuously infused with -MSH, using an osmotic minipump, had more melanophore cytoplasm and pigment per dermis surface unit area than untreated fish. The activity of the MSH cells in MSH-infused fish exposed to a black background was reduced to a level comparable to the MSH cell activity of untreated fish on a white background. -MSH treated fish that were exposed to a white background had many disintegrating MSH cells. These findings point to inactivation of these cells by exogenous -MSH. The activity of the PAS positive cells was not influenced by treatment with -MSH.     

Duplicated α-Chain Genes in Hopkins-2 Haemoglobin of Man and Evidence for Unequal Crossing Over between Them

SYNTHESIS of the α and β-chains for haemoglobin is dictated by independent genetic loci. The first evidence for this notion came from Smith and Torbert's observation that inheritance of haemoglobin Hopkins-2 (Ho-2) was independent from that of haemoglobin S¹ and that Ho-2 was an α-chain variant². We wish to report the amino-acid replacements involved. These structural changes establish the presence of at least two α-chain genes in man. Some physical and physiological properties of the abnormal haemoglobin and the clinical status of carriers, have been reported in another article³.     

Evidence for the Interaction between (−)-Epigallocatechin Gallate and Human Plasma Proteins Fibronectin,Fibrinogen, and Histidine-rich Glycoprotein

Human plasma proteins were subjected to affinity chromatography with (–)-epigallocatechin gallate (EGCg)-agarose, and the bound proteins were examined by sodium dodecylsulfate–polyacrylamide gel electrophoresis. A molecular weight evaluation of the protein bands suggested the presence of three proteins, fibronectin, fibrinogen, and a 75-kDa protein. When human serum was used, the 75-kDa protein dominated the bound fraction. The determination of the partial amino acid sequence of a peptide derived by endopeptidase digestion of this fraction suggested the 75-kDa protein to be histidine-rich glycoprotein (HRG). The presence of these proteins in the bound fraction was confirmed by the immunoblotting method. Affinity chromatography of the individual proteins indicated that fibrinogen and HRG had direct affinity for EGCg. Dot binding assays demonstrated the interaction of EGCg with these proteins. The method also showed that only gallate-containing catechins were bound by these proteins. These data suggest that when EGCg is absorbed in the body through the digestive system, it may interact with these proteins in blood plasma.     

Mutants of β-strand β3 and the loop B in the interface between α7 subunits of a homomeric acetylcholine receptor show functional and pharmacological alterations

Activation of nicotinic acetylcholine receptors (nAChR) requires a global conformational change involving a number of domains of the protein. Structural data from Torpedo nAChR suggest that adjacent subunits might be functionally coupled at the interface between the β-strand β3 and the loop B through a salt bridge between α1Asp152 and γArg78. We have checked this hypothesis in homomeric α7 nAChRs by mutating residues at these (Gly152 and Arg79) and neighboring locations and analyzing the results obtained after expression of single and double mutants in Xenopus oocytes. We found that Arg79 mutants showed a decreased gating function when challenged with different agonists, being the reduction more important for dimethylphenylpiperazinium. EC(50) values in these mutants were also increased up to 30-fold. In contrast, mutating Gly152 only showed significant higher EC(50) values for ACh. However, all Gly153 mutants presented increased gating function and lower EC(50) values with no significant differences among them. When analyzing several mutant cycles it is concluded that Arg79 is functionally coupled to Gly152, but neither to Gly153 nor to Asp157. These data suggest an involvement of the minus side of homomeric α7 nAChRs in their gating function, reinforcing the significance of complementary subunits in the gating of neuronal nAChRs.     

Interactions between different predator–prey states: a method for the derivation of the functional and numerical response

Journal of Mathematical Biology - In this paper we introduce a formal method for the derivation of a predator’s functional response from a system of fast state transitions of the prey or...     

Evidence for cospeciation events in the host–symbiont system involving crinoids (Echinodermata) and their obligate associates,the myzostomids (Myzostomida,Annelida)

Although molecular-based phylogenetic studies of hosts and their associates are increasingly common in the literature, no study to date has examined the hypothesis of coevolutionary process between hosts and commensals in the marine environment. The present work investigates the phylogenetic relationships among 16 species of obligate symbiont marine worms (Myzostomida) and their echinoderm hosts (Crinoidea) in order to estimate the phylogenetic congruence existing between the two lineages. The combination of a high species diversity in myzostomids, their host specificity, their wide variety of lifestyles and body shapes, and millions years of association, raises many questions about the underlying mechanisms triggering their diversification. The phylogenetic relationships, inferred using a three-genes dataset (18S rDNA, 16S rDNA, and COI) and two-genes dataset (18S rDNA, and COI) for the myzostomids and crinoids, respectively, were congruent with the literature. The overall congruence between the two phylogenies was statistically significant according to topology-based, distance-based, and data-based approaches: a significant pattern of cophylogeny was found, though not perfect probably resulting from occasional host switches, duplications or extinction events. A minimum of 8 cospeciation events was estimated, which is significantly higher than it would have been expected due to chance alone.     

The Genetics of α-Hydroxyacid Oxidase and Alcohol Dehydrogenase in the Mouse: Evidence for Multiple Gene Loci and Linkage between Hao-2 and Adh-3

Electrophoretic polymorphisms for stomach alcohol dehydrogenase (ADH-C2) and kidney L-alpha-hydroxyacid oxidase (HAOX-B4) have been identified in an Asian subspecies of mouse, Musmusculus castaneous. These variants are inherited in a normal Mendelian fashion with two alleles in each case showing codominant expression. The structural gene loci for those enzymes (Adh-3 and Hao-2, respectively) are apparently linked (17.6% recombinants) in this organism, whereas the multiple gene loci for HAOX, Hao-1 (encoding the A4 liver isozyme) and Hao-2, exhibited independent segregation and are unlinked (50% recombinants). Evidence is presented for 3 ADH loci: ADH-1, encoding liver ADH-A2 which exhibits high activity with ethanol (SELANDER, HUNT and YANG 1969; ADH-2, liver and stomach ADH-B2 using 2-hexene-1-ol as substrate; and Adh-3, stomach ADH-C2 using both benzyl alcohol and 2-hexene-1-ol as substrate.     

Linkage between vitamin D-binding protein and α-fetoprotein in the mouse

The albumin gene family consists of four evolutionarily related genes that code for serum transport proteins. In rodents, the genes for albumin, |ga-fetoprotein, and |gaALB are physically linked within 100 kilobases of DNA. The fourth gene, Gc, encoding vitamin D-binding protein or group-specific component, maps to the same chromosome as the other family members, but linkage has not been established. This report describes the genetic and physical mapping of Gc in mouse and establishes that, although Gc is genetically linked to the other genes, its physical distance from them extends beyond the resolution range of yeast artificial chromosome cloning and pulsed-field gel electrophoresis. Received: 18 July 1995 / Accepted: 9 September 1995     

Formation of the functional organization of the cerebral cortex at rest in young schoolchildren varying in the maturity of cerebral regulatory systems: II. Analysis of EEG α-rhythm coherence

Coherence (COH) of rhythmic components of the EEG α rhythm at rest was analyzed to demonstrate that the maturation of deep regulatory systems (RSs) of the brain at different levels substantially affected the functional organization of the cerebral cortex and the time course of the formation of intercentral connections in young schoolchildren. The specific effect of fronto-thalamic regulatory system immaturity (FRSI) was a decrease in the α-rhythm COH predominantly in neighboring left-hemispheric derivations of foci in the anterior temporal area. A deficit of nonspecific activation had the strongest effect on the α-rhythm integration of right-hemispheric areas, although this effect was less distinct than that of FRSI and remained only as a tendency by the age of nine to ten years. Children with normal and functionally immature cerebral RSs differed from each other with respect to age-related changes in the corticocortical connections, especially in the left hemisphere. In the norm, intense growth of functional connections in the left hemisphere ceased in the period between seven to eight and nine to ten years of age; in contrast, children with RS immaturity exhibited a trend towards an increasingly greater amount of these connections in both hemispheres, which, apparently, corresponded to an earlier stage of ontogeny.     

Potential implications of the glycosylation patterns in collagen α1(I) and α2(I) chains for fibril assembly and growth

O-Glycosylation of hydroxylysine (Hyl) in collagen occurs at an early stage of biosynthesis before the triple-helix has formed. This simple post-translational modification (PTM) of lysine by either a galactosyl or glucosylgalactosyl moiety is highly conserved in collagens and depends on the species, type of tissue and the collagen amino acid sequence. The structural/functional reason why only specific lysines are modified is poorly understood, and has led to increased efforts to map the sites of PTMs on collagen sequences from different species and to ascertain their potential role in vivo. To investigate this, we purified collagen type I (Col1) from the skins of four animals, then used mass spectrometry and proteomic techniques to identify lysines that were oxidised, galactosylated, glucosylgalactosylated, or glycated in its mature sequence. We found 18 out of the 38 lysines in collagen type Iα1, (Col1A1) and 7 of the 30 lysines in collagen type Iα2 (Col1A2) were glycosylated. Six of these modifications had not been reported before, and included a lysine involved in crosslinking collagen molecules. A Fourier transform analysis of the positions of the glycosylated hydroxylysines showed they display a regular axial distribution with the same d-period observed in collagen fibrils. The significance of this finding in terms of the assembly of collagen molecules into fibrils and of potential restrictions on the growth of the collagen fibrils is discussed.