Structure and energetic basis of overrepresented λ light chain in systemic light chain amyloidosis patients

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Association of membranous nephropathy with T-cell receptor constant β chain and immunoglobulin heavy chain switch region polymorphisms

We have investigated T-cell antigen receptor constant chain genes (Tcr C ) and immunoglobulin (Ig) heavy chain switch region genes of HLA-DR-typed patients with membranous nephropathy (MN) employing DNA restriction fragment length polymorphism (RFLP) analysis. When a Tcr C probe in conjunction with the restriction endonuclease BgI II was used, a significant increase in the frequency of a 10.0;9.2 kb heterozygous RFLP phenotype was found in MN (75.0 % versus 42.1 in controls; P=0.002). When Sst I-restricted DNA from MN patients was hybridized with a DNA probe homologous to the switch region flanking the Ig C _µ heavy chain gene (S _µ), there was a significant decrease in the frequency of the 2.1; 2.6 kb heterozygous RFLP phenotype in MN (24.0% versus 54.6% in controls; P=0.004). These results suggest that Tcr beta and Ig heavy chain loci, as well as HLA antigens, may be important in the pathogenesis of MN.     

NMR detection of side chain–side chain hydrogen bonding interactions in 13C/15N-labeled proteins

We describe the direct observation of side chain–side chain hydrogen bonding interactions in proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation between the guanidinium nitrogen ¹⁵N of arginine 71, which serves as the hydrogen donor, and the acceptor carboxylate carbon ¹³CO₂ of aspartate 100 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond ^3h J _{N CO2} coupling by employing a novel HNCO-type experiment, soft CPD-HNCO. The ^3h J _{N CO2} coupling constant appears to be even smaller than the average value of backbone ^3h J _NC couplings, consistent with more extensive local dynamics in protein side chains. The identification of trans-hydrogen bond J-couplings between protein side chains should provide useful markers for monitoring hydrogen bonding interactions that contribute to the stability of protein folds, to alignments within enzyme active sites and to recognition events at macromolecular interfaces.     

Linked genetic markers of the rabbit κ light chain are not linked to the Tcr β chain genes

In order to investigate linkage, we used serum allotypes of the two rabbit C isotypes and restriction fragment length polymorphisms (RFLPs) of the genes for V , C , and T-cell receptor C . The inheritance of these genetic markers was studied through backcross and F₂ matings. Southern analysis and hybridization of genomic DNA with a C probe detected a 5 kb Pst I fragment linked to expression of the K2bas1 allotype and the presence of the 1b ^bas gene and a 6.6 kb Pst I fragment linked to the expression of the K1b9 allotype, the presence of the 2 ^bas2 gene and lack of expression of the K2bas1 allotype. A V probe detected a 1.3 kb Eco RI fragment linked to the presence of the 1b ^bas gene and expression of the K2bas1 allotype. In contrast, the 9 or 14 kb Eco RI RFLP (C ^a or C ^b) detected with a Tcr chain probe segregated independently from C allotypes and RFLPs. It has previously been found that C and C are also unlinked in man, whereas in the mouse they are linked at a distance of 8 centimorgans.     

Identification of dog T-cell receptor β chain genes

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers D16409, D16410 and D16412     

Evolution of the rabbit immunoglobulin κ chain genes

We have analyzed the organization and the structure of rabbit chain genes encoding b allotypes in wild rabbits. The 1 gene of the b95 allotype was cloned and its structure determined. The J region is composed of five segments but only J2 appears to be functional and is identical to the J2 segment of the b4 allotype. The J region is highly conserved among the various b allotypes, whereas the constant region exon displays a high level of differences when compared with other allotypes (9%–30% of different amino acids). The b95 J region is closer to that of b4^var and the constant region to b5 allotype constant region. Alignment of nucleotide sequences revealed that the constant region exon displays segmental similarities with b4 and bas constant regions. The mosaic structure of b95 allotype gene indicates that complex allotypes of 1 genes may result from genetic exchanges of gene conversion between the different genes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide database and have been assigned the accession number M22542. Address correspondence and offprint requests to: P.-A. Cazenave.     

C-peptide inhibitors of Ebola virus glycoprotein-mediated cell entry: Effects of conjugation to cholesterol and side chain–side chain crosslinking

We previously described potent inhibition of Ebola virus entry by a ‘C-peptide’ based on the GP2 C-heptad repeat region (CHR) targeted to endosomes (‘Tat-Ebo’). Here, we report the synthesis and evaluation of C-peptides conjugated to cholesterol, and Tat-Ebo analogs containing covalent side chain–side chain crosslinks to promote α-helical conformation. We found that the cholesterol-conjugated C-peptides were potent inhibitors of Ebola virus glycoprotein (GP)-mediated cell entry (～10³-fold reduction in infection at 40 μM). However, this mechanism of inhibition is somewhat non-specific because the cholesterol-conjugated peptides also inhibited cell entry mediated by vesicular stomatitis virus glycoprotein G. One side chain–side chain crosslinked peptide had moderately higher activity than the parent compound Tat-Ebo. Circular dichroism revealed that the cholesterol-conjugated peptides unexpectedly formed a strong α-helical conformation that was independent of concentration. Side chain–side chain crosslinking enhanced α-helical stability of the Tat-Ebo variants, but only at neutral pH. These result provide insight into mechanisms of C-peptide inhibiton of Ebola virus GP-mediated cell entry.     

Are crustaceans linked to the ciguatera food chain?

Synopsis Adult brine shrimp, Artemia spp., were used as an experimental organism to elucidate the role that crustacea may play in the transference of ciguatera toxins. Some ciguatera-implicated dinoflagellates were highly toxic to brine shrimp that had consumed them. Four clones of Gambierdiscus toxicus were fed in four trials at rates ranging from 2 to 480 cells per adult brine shrimp; the 24 h LD₅₀ for the four clones were 2.8, 33.4, 41.1, and 104.5 cells per brine shrimp. Dinoflagellates Prorocentrum concavum and P. lima were also fed to adult brine shrimp, but minimal mortalities occurred at cell concentrations ranging up to 1000 cells per test animal. Tilapine cichlid (Oreochromis niloticus ×O. mossambicus) young fed brine shrimp containing G. toxicus cells displayed behavioral abnormalities ranging from spiral swimming to loss of equilibrium. The present data suggest that toxins accumulated by dinoflagellate-consuming crustaceans could produce toxicity in zooplanktivorous fish species, or to detritivores in cases where dinoflagellate consumption resulted in crustacean mortalities. Field studies of the ciguatera food chain should be expanded to include examination of crustacean diets to more fully define their role in toxin transfer.     

Phylogeny of immunoglobulin heavy chain isotypes: structure of the constant region of Ambystoma mexicanum υ chain deduced from cDNA sequence

An RNA polymerase chain reaction strategy was used to amplify and clone a cDNA segment encoding for the complete constant part of the axolotl IgY heavy (C) chain. C is 433 amino acids long and organized into four domains (C1–C4); each has the typical internal disulfide bond and invariant tryptophane residues. Axolotl C is most closely related to Xenopus C (40% identical amino acid residues) and C1 shares 46.4% amino acid residues among these species. The presence of additional cysteines in C1 and C2 domains is consistent with an additional intra-domain S-S bond similar to that suggested for Xenopus C and C, and for the avian C and the human C. C4 ends with the Gly-Lys dipeptide characteristic of secreted mammalian C3, human C4, and avian and anuran C4, and contains the consensus [G/GT(AA)] nucleotide splice signal sequence for joining C4 to the transmembrane region. These results are consistent with the hypothesis of an ancestral structural relationship between amphibian, avian chains, and mammalian chains. However, these molecules have different biological properties: axolotl IgY is secretory Ig, anuran and avian IgY behave like mammalian IgG, and mammalian IgE is implicated in anaphylactic reactions.The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number X69492. Correspondence to: J. S. Fellah.     

Side chain SAR of bicyclic β-lactamase inhibitors (BLIs). 2. N-Alkylated and open chain analogs of MK-8712

The bridged monobactam β-lactamase inhibitor MK-8712 (1) effectively inhibits class C β-lactamases. Side chain N-alkylated and ring-opened analogs of 1 were prepared and evaluated for combination with imipenem to overcome class C β-lactamase mediated resistance. Although some analogs were more potent inhibitors of AmpC, none exhibited better synergy with imipenem than 1.     

Is outer arm dynein intermediate chain 1 multifunctional?

The outer arm dynein of sea urchin sperm axoneme contains three intermediate chains (IC1, IC2, and IC3; M(r) 128,000, 98,000, and 74,000, respectively). IC2 and IC3 are members of the WD family; the WD motif is responsible for a protein-protein interaction. We describe here the molecular cloning of IC1. IC1 has a unique primary structure, the N-terminal part is homologous to the sequence of thioredoxin, the middle part consists of three repetitive sequences homologous to the sequence of nucleoside diphosphate kinase, and the C-terminal part contains a high proportion of negatively charged glutamic acid residues. Thus, IC1 is a novel dynein intermediate chain distinct from IC2 and IC3 and may be a multifunctional protein. The thioredoxin-related part of IC1 is more closely related to those of two redox-active Chlamydomonas light chains than thioredoxin. Antibodies were prepared against the N-terminal and middle domains of IC1 expressed as His-tagged proteins in bacteria. These antibodies cross-reacted with some dynein polypeptides (potential homologues of IC1) from distantly related species. We propose here that the three intermediate chains are the basic core units of sperm outer arm dynein because of their ubiquitous existence. The recombinant thioredoxin-related part of IC1 and outer arm dyneins from sea urchin and distantly related species were specifically bound to and eluted from a phenylarsine oxide affinity column with 2-mercaptoethanol, indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction.     

Ia antigens contain two distinct forms of β chain

Two new forms of chain, ₁ and ₂, in I-A immunoprecipitates are described, which differ in their migration values in SDS-PAGE under nonreducing conditions, but which migrate identically in a reduced form. This behavior is very likely due to a different arrangement of intramolecular disulfide bonds which may influence mobility in SDS-PAGE. Peptide map analysis confirmed that ₁ and ₂, possess identical primary polypeptide structures. These two forms of chain are also expressed on the cell surface and it is suggested that both associate with chains. The structural differences in these complexes may lead to an increase in heterogeneity of la antigens which could be of importance for T-cell recognition.     

Can sucrose affect polymerase chain reaction product formation?

Sucrose or trehalose at 0.3 M stabilised double-stranded DNA in PCR. Furthermore, the DNA polymerase from Thermus aquaticus was stabilised towards thermo-denaturation at 98°C by sucrose or trehalose. The effect was limited to these sugars and was shown not to be general for carbohydrates.     

Separation and characterization of the A chain and B chain in β1-bungarotoxin fromBungarus multicinctus (Taiwan banded krait) venom

The interchain disulfide bond between A chain and B chain of β₁-bungarotoxin (β₁-Bgt) was selectively cleaved by dithiothreitol, and the A and B chains were separated by HPLC. The separated A and B chains did not show detectable enzymatic activity and lethal toxicity, but exhibited an immunoreactivity with anti-β₁-Bgt antibody. Analytical isoelectrofocusing revealed that the A chain is a neutral subunit with pI=7.4, and the B chain is a basic one with pI=9.6. The A chain exhibited a Ca²⁺-binding ability as revealed by fluorescence measurement. Moreover, fluorescence studies showed that the intact interchain disulfide bond is essential for maintaining the hydrophobic character of substrate binding site in β₁-Bgt and stabilizing the architectural environment of Trp-19 in the A chain. However, combination of the A chain and B chain failed to restore the biological activities and physicochemical properties which the intact β₁-Bgt possessed. These, together with our previous result that the Trp-19 of the A chain is involved in substrate binding, suggest that the integrity of the interchain disulfide bond favors the maintenance of the active conformation of β₁-Bgt.