Immunoglobulin heavy chain and binding protein complexes are dissociated in vivo by light chain addition

Immunoglobulin heavy chain binding protein (BiP, GRP78) associates stably with the free, nonsecreted Ig heavy chains synthesized by Abelson virus transformed pre-B cell lines. In cells synthesizing both Ig heavy and light chains, the Ig subunits assemble rapidly and are secreted. Only incompletely assembled Ig molecules can be found bound to BiP in these cells. In addition to Ig heavy chains, a number of mutant and incompletely glycosylated transport-defective proteins are stably complexed with BiP. When normal proteins are examined for combination with BiP, only a small fraction of the intracellular pool of nascent, unfolded, or unassembled proteins can be found associated. It has been difficult to determine whether these BiP-associated molecules represent assembly intermediates which will be displaced from BiP and transported from the cell, or whether these are aberrant proteins that are ultimately degraded. In order for BiP to monitor and aid in normal protein transport, its association with these proteins must be reversible and the released proteins should be transport competent. In the studies described here, transient heterokaryons were formed between a myeloma line producing BiP-associated heavy chains and a myeloma line synthesizing the complementary light chain. Introduction of light chain synthesis resulted in assembly of prelabeled heavy chains with light chains, displacement of BiP from heavy chains, and secretion of Ig into the culture supernatant. These data demonstrate that BiP association can be reversible, with concordant release of transportable proteins. Thus, BiP can be considered a component of the exocytic secretory pathway, regulating the transport of both normal and abnormal proteins.     

Skin derivatives in vertebrate ontogeny and phylogeny

The skin of vertebrates has numerous and diverse derivatives, either located within the epithelial sheet itself (glands) or extending above its surface (teeth, scales, feathers, hairs, etc.). Many of them have a modular structure and constitute structural-functional units. Ontogenetically, all skin derivatives are of ectomesodermal origin, and their morphogenesis is subject to metabolic control, heterochronies (divergence in the timing of origination and development), and regulation by means of tissue interactions and molecular signaling via similar pathways. The diversification (origination of morphological diversity) of skin derivatives within the same morphological type is explained by the development of new generations of ectomesodermal structures separated by heterochronies and regulated by changes in the gradients of molecular signaling pathways under the influence of environmental factors. Evolutionary relationships between the majority of skin derivatives are obscure, except for teeth and glands associated with sensory organs that have evolved together with these organs. Apparently, many vertebrate skin derivatives (scales, feathers, hairs, and glands) originated as novelties at nodal stages of phylogeny and subsequently evolved convergently or in parallel.     

Ca-linked phosphorylation of a light chain of vertebrate smooth-muscle myosin.

In vertebrate smooth muscle actomyosin and myofibrils a myosin light chain of molecular weight about 20,000 becomes phosphorylated at the same Ca2+ concentration as required to stimulate the actin-activated ATPase activity of myosin. Further, the degree of phosphorylation in the preparations as well as in various reconstituted actomyosins is proportional to their measured Ca2+ sensitivity. The phosphorylation process is very rapid and is essentially completed before the rise in ATPase activity. The enzyme responsible for the observed myosin phosphoylation is a specific myosin light chain kinase which is routinely co-purified with myosin. This kinase is normally present in actomyosin and its removal together with tropomyosin leads to a complete loss of the actin-activated ATPase activity. It is suggested that the Ca-dependent phosphorylation of the light chain via the light chain kinase represents the initial step in the activation of myosin that leads to contraction. Relaxation is probably effected by an as yet uncharacterised light chain phosphatase.     

Myosin light chain 2 modulates calcium-sensitive cross-bridge transitions in vertebrate skeletal muscle.

We investigated the mechanism of the Ca2+ sensitivity of cross-bridge transitions that limit the rate of force development in vertebrate skeletal muscle. The rate of force development increases with Ca2+ concentration in the physiological range. We show here that at low concentrations of Ca2+ the rate of force development increases after partial extraction of the 20-kD light chain 2 subunit of myosin, whereas reconstitution with light chain 2 fully restores native sensitivity to Ca2+ in skinned single skeletal fibers. Furthermore, elevated free Mg2+ concentration reduces Ca2+ sensitivity, an effect that is reversed by extraction of the light chain but not by disruption of thin-filament activation by partial removal of troponin C, the Ca2+ binding protein of the thin filament. Our findings indicate that the Ca2+ sensitivity of the rate of force development in vertebrate skeletal muscle is mediated in part by the light chain 2 subunit of the myosin cross-bridge.     

Immunoglobulin kappa light chain and its amyloidogenic mutants: a molecular dynamics study

AL amyloidosis and LCDD are pathological conditions caused by extracellural deposition of monoclonal Ig light chain variable domains. In the former case, deposits have a form of amyloid fibrils, in the latter, amorphous aggregates. 1REI kappa light chain variable domain and its two point mutants, R61N and D82I, were chosen for the analysis in this work. Wild 1REI does not create deposits in vitro, while R61N aggregates as amyloid fibrils and D82I creates amorphous aggregates. Both mutated residues create a conserved salt bridge; thus, substitution of any of them should decrease V(L) domain stability. For these three proteins, 5 ns MD simulations were conducted in temperatures of 300 K and 400 K, with protonated and unprotonated acidic residues, mimicking acidic and neutral experimental pH conditions (3 sets: N300, N400, and A400). The analysis of trajectories focused on characterization of changes in conformational behavior and stability of Ig kappa light chain variable domain caused by single aminoacid substitutions that were experimentally proved to enhance aggregation propensity, both in the form of amyloid and amorphous aggregates. Residue D82 turns out to be involved not only in R61-D82 but also in K45-D82 interaction, which was not observed in the X-ray structure, but frequently populated simulations of 1REI. The substitution D82I excludes both interactions, resulting in substantial destabilization (i.e., easier aggregation). Examination of behavior of edge regions of V(L) beta-sandwich reveals significant alterations in D82I mutant compared to wild 1REI, while relatively small changes occur in R61N. This suggests that mild and slow destabilization is the reason of the conversion of V(L) to partially folded amyloidogenic intermediate structure.     

Conodont anatomy, chordate phylogeny and vertebrate classification

Interpretations of conodont anatomy and affinity continue to generate controversy. Fossilized soft-tissue evidence indicates that conodonts possessed eyes, extrinsic eye muscles, a notochord, myomeres, a differentiated tail with fin radiais, possible otic capsules and possible branchial structures. Indirect evidence suggests a differentiated brain and cartilaginous head skeleton. The multi-component phosphatic tissue complexes of the conodont feeding apparatus cannot be compared to the amorphous apatite of extant agnathan otoliths. By limiting cladistic analysis to a restricted selection of these characters the hypothesis that conodonts are a sister group of the clade comprising extant hagfish, lampreys and gnathostomes can be supported. However, exhaustive analysis of a more complete character-set strongly supports the hypothesis that conodonts are more derived than hagfish. From a taxonomic perspective, these two hypotheses have no effect on how conodonts should be classified. Whether they are a stem group (the former hypothesis) or part of the crown group (the latter), conodonts are clearly part of the total group Vertebrata (=Craniata).     

Immunoglobulin lambda light chain evolution: Igl and Igl-like sequences form three major groups

The nucleotide sequences, and the derived protein sequences, of immunoglobulin (Ig) Igl, Igl-likeVpreB genes and the protein sequences ofIgl-C regions were aligned and compared. A classification of the Igl and Igl-likeVpreB sequences into three categories, designated groups I, II, and III, is proposed. Group I contains the human and mouse Igl-likeVpreB genes. Group II containsIgl-V genes of the rabbit and the recently described mouseIgl-Vx gene. Group III includes theIgl-V genes, encoding all other knownIgl-V region protein sequences, of mouse, rat, human, pig, sheep, and chicken. An evolutionary analysis of the three groups is presented, and suggests that the group III genes are evolving at a faster rate than those of the other groups and that within this group a further subdivision is possible; the V-encoding genes of mouse, rat, and one human subgroup evolve faster than other group III genes. It is suggested that all mammalian species containIgl-V genes of each group. A similar comparison between the protein sequences encoded by the knownIgl-C genes indicates that the duplication of theIgl-J-C gene pairs occured independently in each species, after mammalian speciation, and that theIgI-V-(J-C)(J-C) gene clusters of the mouse may not have their homologues in other species.     

A comparison of the alkali light chain subunits of vertebrate skeletal muscle myosin in free and heavy chain associated states

The conformations of the alkali light-chain subunits A1 and A2 of vertebrate fast-twitch muscle myosin have been compared for these chains both in their free state and their heavy-chain-associated states by examining the fluorescence parameters of the extrinsic probe 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid attached covalently to the two light chains. The effect of temperature, salt concentration, and ligands such as Mg²⁺ ions, MgADP, and MgATP has also been investigated. In spite of the extensive sequence homology between the two light chains the data indicate that in their free states the fluorophore in the A2 chain resides in a considerably higher hydrophobic environment. It was also found that the presence of the bulky fluorophore on these light chains does not adversely affect their ability to hybridize with Subfragment 1 heavy chains to form ATPase active hybrids. This association to the heavy chains is accompanied by significant changes in the quantum yields of the 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid label indicating that conformational changes do occur during this transition. Mg²⁺ ions were found to cause either an enhancement or a decrease in fluorescence intensity depending on whether the alkali light chains were free or combined to the heavy chains, respectively. Fluorescence perturbation by nucleotide was only observed for the heavy-chain-associated state.     

Amphibian alcohol dehydrogenase, the major frog liver enzyme. Relationships to other forms and assessment of an early gene duplication separating vertebrate class I and class III alcohol dehydrogenases

Submammalian alcohol dehydrogenase structures can be used to evaluate the origins and functions of the different types of the mammalian enzyme. Two avian forms were recently reported, and we now define the major amphibian alcohol dehydrogenase. The enzyme from the liver of the Green frog Rana perezi was purified, carboxymethylated, and submitted to amino acid sequence determination by peptide analysis of six different digests. The protein has a 375-residue subunit and is a class I alcohol dehydrogenase, bridging the gap toward the original separation of the classes that are observable in the human alcohol dehydrogenase system. In relation to the human class I enzyme, the amphibian protein has residue identities exactly halfway (68%) between those for the corresponding avian enzyme (74%) and the human class III enzyme (62%), suggesting an origin of the alcohol dehydrogenase classes very early in or close to the evolution of the vertebrate line. This conclusion suggests that these enzyme classes are more universal among animals than previously realized and constitutes the first real assessment of the origin of the duplications leading to the alcohol dehydrogenase classes. Functionally, the amphibian enzyme exhibits properties typical for class I but has an unusually low Km for ethanol (0.09 mM) and Ki for pyrazole (0.15 microM) at pH 10.0. This correlates with a strictly hydrophobic substrate pocket and one amino acid difference toward the human class I enzyme at the inner part of the pocket. Coenzyme binding is highly similar, while subunit-interacting residues, as in other alcohol dehydrogenases, exhibit several differences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heavy chain variable region gene families evolved early in phylogeny. Ig complexity in fish

The V regions of channel catfish H chain cDNA clones have been analyzed. Based upon sequence relationships and hybridization analyses, five different groups of VH genes are identified whose definition is consistent with that of five different VH families. Genomic Southern blots indicate that as many as 100 different germ-line VH genes are likely represented by these families. The sequence diversity between identified members of these different families is similar in magnitude to the divergence represented between members of different human or mouse VH families. The FR regions are the most conserved regions when members of different catfish VH families are compared; specific amino acid positions appear to be highly conserved in phylogeny. Equally important is that diversity is represented in complementarity-determining regions CDR1 and CDR2 in members of the different families as well as in members of the same VH family. These results suggest that an extensive repertoire of VH genes can contribute to antibody diversity in this lower vertebrate. Sequence comparisons indicate that one of the catfish VH families shares considerable structural similarity to several higher vertebrate VH gene families--a relationship which suggests that this VH family may be ancestral to some VH gene families of higher vertebrates. Characteristic of the genomic organization of higher vertebrate H chains, catfish appear to have different VH families wherein a VH gene likely undergoes functional recombination with putative DH gene segments and one of apparently several different JH segments. The recombined V region is expressed with the same C region gene. These combined results suggest that bony fishes are the earliest known phylogenetic representatives to have evolved extensive V region gene families.     

Antibody elbow angles are influenced by their light chain class

We have examined the elbow angles for 365 different Fab fragments, and observe that Fabs with lambda light chains have adopted a wider range of elbow angles than their kappa chain counterparts, and that the lambda light chain Fabs are frequently found with very large (>195 degrees ) elbow angles. This apparent hyperflexibility of lambda chain Fabs may be due to an insertion in their switch region, which is one residue longer than in kappa chains, with glycine occurring most frequently at the insertion position. A new, web-based computer program that was used to calculate the Fab elbow angles is described.     

Going nuclear: gene family evolution and vertebrate phylogeny reconciled

Gene duplications have been common throughout vertebrate evolution, introducing paralogy and so complicating phylogenetic inference from nuclear genes. Reconciled trees are one method capable of dealing with paralogy, using the relationship between a gene phylogeny and the phylogeny of the organisms containing those genes to identify gene duplication events. This allows us to infer phylogenies from gene families containing both orthologous and paralogous copies. Vertebrate phylogeny is well understood from morphological and palaeontological data, but studies using mitochondrial sequence data have failed to reproduce this classical view. Reconciled tree analysis of a database of 118 vertebrate gene families supports a largely classical vertebrate phylogeny.     

Glutamate-operated channels: developmentally early and mature forms arise by alternative splicing.

The expression of two alternative splice variants, Flip and Flop, in mRNAs encoding the four AMPA-selective glutamate receptors (GluR-A, -B, -C, and -D) was studied in the developing brain by in situ hybridization. These receptors are expressed prominently before birth, and patterns of distribution for Flip versions remain largely invariant during postnatal brain development. In contrast, the Flop versions are expressed at low levels prior to postnatal day 8. Around this time, the expression of Flop mRNAs increases throughout the brain, reaching adult levels by postnatal day 14. Thus, receptors carrying the Flop module appear to participate in mature receptor forms.     

中国脊椎动物2020年新增物种

为了及时掌握中国脊椎动物新增物种情况, 本文系统检索和整理了2020年发表的分类学文献, 汇总结果表明: 2020年中国脊椎动物共新增109种, 其中新物种100种, 国家级新记录种9种。包括鱼类新种24种、两栖类新种41种和新记录4种、爬行类新种30种和新记录4种、鸟类新种1种、哺乳类新种4种和新记录1种。上述新增物种中有92种描述报道时应用了分子遗传学证据, 占新增物种数量的84.4%。在新增脊椎动物物种中, 两栖类集中于无尾目、爬行类全部属于有鳞目, 分别有43种和34种, 其累计占比超新增物种总数的70%。在云南、西藏、湖南、贵州、四川等省区发现新物种数量较多, 均有10种及以上, 累计占比超新增物种总数的60%。绝大部分物种为中国学者发表, 绝大多数论文发表于英文期刊。总结数据提示今后需持续加强我国低等脊椎动物多样性的调查研究, 重视运用分子系统学技术进行物种识别。     

中国脊椎动物2021年度新增物种报告

为了及时掌握脊椎动物在中国的新增情况, 本文汇总了2021年发表的脊椎动物新物种及新记录种的基本信息。结果表明, 2021年中国新增脊椎动物95种, 包括新种80种, 新记录15种。其中鱼类新种15种、两栖类新种28种、爬行类新种31种和新记录10种、鸟类新种1种和新记录3种、哺乳类新种5种和新记录2种。在新增物种中, 冷血脊椎动物占绝大多数(占总数的88%), 提示这些类群可能仍是以后探索的重点; 两栖类新增物种集中于无尾目、爬行类集中于有鳞目, 分别为27种和40种, 各占其新增物种总数的96%和98%; 新增哺乳类动物全部为小型兽类。本次新增物种的分布涉及30个省区, 其中云南33种、四川11种、广西10种、西藏和广东均为7种、台湾6种, 累计约占新增物种总数的70.5%; 其余省区新增物种在5种或以下。有84个物种(占总数的88%)发表时应用了分子系统学研究, 提示这一技术手段是分类工作的重要支撑。在新发现的95个物种中, 绝大部分物种为中国学者发表; 除3种鸟类新记录种外, 其余的新种和新记录均正式发表于英文期刊, 其中在中国出版的3种期刊发表了21个新种和2个新记录种。本文工作可为中国脊椎动物的分类和保护等相关工作提供基础信息。     

Structure and polymorphism of the HLA class II SB light chain genes

The HLA Class II region contains at least three groups of loci, DR, DC and SB, which play an important role in the immune response. The antigens encoded at these loci are heterodimers composed of an alpha and a beta chain. The sequence of a complete Class II beta cDNA clone whose sequence agrees closely with the limited N-terminal protein sequence available for the SB beta chain is reported. In addition the structure and coding sequence of genomic SB beta clones of two different SB haplotypes has been obtained and allows definition of some polymorphic regions. The SB beta gene appears to undergo alternate splicing at its 3' end, resulting in expression of two different intracytoplasmic regions. Partial sequencing of a second non-allelic SB beta-like gene, SX beta, indicates that it is a pseudogene.