The single DR β gene of the DRw8 haplotype is closely related to the DR β 3III gene encoding DRw52

In most individuals two HLA-DR genes are expressed from each chromosome. One of these genes encodes one of the classical DR specificities, while the other encodes either of the supertypic DRw52/DRw53 specificities. In addition to these genes usually one or two DR pseudogenes are present. In contrast, the DRw8 chromosomal region only contains a single DR gene. To determine the relationship of this single gene to the multiple DR genes of other DR specificities, comparisons of Southern genomic blots were carried out. In this analysis genomic clones for each individual DR chain locus were included. The DR w8 gene was indistinguishable from the DR III gene of DR3 cells (encoding DRw52), suggesting that it is closely related to the latter gene. The functional implications of this finding are discussed.     

A single amino acid deletion in the α2(I) chain of type I collagen produces osteogenesis imperfecta type III

RNase A protection analysis was used in the search for the cause of a non-lethal osteogenesis imperfecta (OI) phenotype (Sillence type III). Cleavage of the hybrid formed between a normal 2(I) sequence and RNA isolated from the patient indicated the presence of a mismatch. The position of the mismatch was determined and the corresponding area of COL1A2 was amplified using the polymerase chain reaction. Sequencing of cloned amplified DNA revealed the deletion, which was not present in either parent, of the final three bases of exon 19 in one of the patient's two COL1A2 alleles. The deletion results in the loss of amino acid 255 (a valine encoded by the last codon of exon 19) of the triple helical region of half of the 2(I) collagen chains but does not disrupt the splicing of the heterogeneous nuclear RNA (hnRNA). This provides further evidence that OI type III may result from autosomal dominant mutations rather than only from autosomal recessive mutations as had previously been believed.     

Disease-associated single amino acid mutation in the calf-1 domain of integrin α3 leads to defects in its processing and cell surface expression

Integrin α3β1, a receptor for laminins, is involved in the structural and functional organization of epithelial organs, including the lung, kidney, and skin. Recently, a missense mutation that causes substitution of Arg628 with Pro (R628P) in the calf-1 domain of human α3 was shown to be associated with disorders of the lung, kidney, and skin. Here, we found that the R628P mutation leads to aberrations in the posttranslational processing of α3. Specifically, α3 with the R628P mutation showed hardly any cleavage at the calf-2 domain, which usually occurs in the Golgi apparatus during the delivery of de novo-synthesized α3. The mutant α3 retained the ability to associate with integrin β1, but not with the tetraspanin CD151, and the bound β1 was a partially glycosylated immature form, the maturation of which also takes place in the Golgi apparatus. Furthermore, the cell surface expression of the mutant protein was markedly reduced. These results suggest that the R628P mutation leads to a deficit in the transport of α3β1 from the ER to the Golgi apparatus. When Arg628 was mutated to Gln or Glu, instead of Pro, the resulting mutants did not display aberrations in processing or CD151 binding, indicating that the presence of Pro, rather than the absence of Arg, at amino acid residue 628 of α3 is important for the abnormalities in the R628P mutant. In support of this notion, a homology modeling analysis of the calf-1 domain of α3 showed that replacement with Pro, but not with Gln or Glu, caused partial disruption of the β-sheet structures. Furthermore, the ER-associated degradation of the R628P mutant was not enhanced compared with that of the wild-type protein, suggesting that the deficits in the posttranslational processing and cell surface expression of the R628P mutant are independent of the ER-associated degradation, but arise from the defect in its export from the ER. We conclude that the calf-1 domain is required for the transport of α3 from the ER to the Golgi apparatus to maintain the integrity of epithelial tissues, and hence the impairment of the calf-1 domain by the R628P mutation leads to severe diseases of the kidneys, lungs, and skin.     

Biotransformation of oleic acid into (E)-10-hydroxy-8-octadecenoic acid and (E)-7,10-dihydroxy-8-octadecenoic acid by Pseudomonas sp. 42A2 in an immobilized system

Oleic acid was transformed into 10-hydroxy-8E-octadecenoic acid and 7,10-dihydroxy-8E-octadecenoic acid using Pseudomonas sp. 42A2 NCIMB 40045 in different immobilised supports. Celite R633 gave highest conversions in 48 h: the cellular yield (Y _p/x) g products g^–1 of cellular protein in the immobilised culture was 5 compared with the free-cell culture Y _p/x of 3.8. Conversion of oleic acid in the immobilised cell culture was 50% of the carbon source supplied.     

A new heterobinuclear FeIIICuII complex with a single terminal FeIII–O(phenolate) bond. Relevance to purple acid phosphatases and nucleases

A novel heterobinuclear mixed valence complex [Fe^IIICu^II(BPBPMP)(OAc)₂]ClO₄, 1, with the unsymmetrical N₅O₂ donor ligand 2-bis[{(2-pyridylmethyl)aminomethyl}-6-{(2-hydroxybenzyl)(2-pyridylmethyl)}aminomethyl]-4-methylphenol (H₂BPBPMP) has been synthesized and characterized. A combination of data from mass spectrometry, potentiometric titrations, X-ray absorption and electron paramagnetic resonance spectroscopy, as well as kinetics measurements indicates that in ethanol/water solutions an [Fe^III–()OH–Cu^IIOH₂]⁺ species is generated which is the likely catalyst for 2,4-bis(dinitrophenyl)phosphate and DNA hydrolysis. Insofar as the data are consistent with the presence of an Fe^III-bound hydroxide acting as a nucleophile during catalysis, 1 presents a suitable mimic for the hydrolytic enzyme purple acid phosphatase. Notably, 1 is significantly more reactive than its isostructural homologues with different metal composition (Fe^IIIM^II, where M^II is Zn^II, Mn^II, Ni^II, or Fe^II). Of particular interest is the observation that cleavage of double-stranded plasmid DNA occurs even at very low concentrations of 1 (2.5 M), under physiological conditions (optimum pH of 7.0), with a rate enhancement of 2.7×10⁷ over the uncatalyzed reaction. Thus, 1 is one of the most effective model complexes to date, mimicking the function of nucleases.Electronic Supplementary Material Supplementary material is available for this article at .     

A single dose of the DENV-1 candidate vaccine rDEN1Δ30 is strongly immunogenic and induces resistance to a second dose in a randomized trial

Dengue is an emerging infectious disease that has become the most important arboviral infection worldwide. There are four serotypes of dengue virus, DENV-1, DENV-2, DENV-3, and DENV-4, each capable of causing the full spectrum of disease. rDEN1Δ30 is a live attenuated investigational vaccine for the prevention of DENV-1 illness and is also a component of an investigational tetravalent DENV vaccine currently in Phase I evaluation. A single subcutaneous dose of rDEN1Δ30 was previously shown to be safe and immunogenic in healthy adults. In the current randomized placebo-controlled trial, 60 healthy flavivirus-naive adults were randomized to receive 2 doses of rDEN1Δ30 (N = 50) or placebo (N = 10), either on study days 0 and 120 (cohort 1) or 0 and 180 (cohort 2). We sought to evaluate the safety and immunogenicity of this candidate vaccine in 50 additional vaccinees and to test whether the humoral immune response could be boosted by a second dose administered 4 or 6 months after the first dose. The first dose of vaccine was well tolerated, infected 47/50 vaccinees and induced seroconversion in 46/50 vaccinees. Irrespective of dosing interval, the second dose of vaccine was also well tolerated but did not induce any detectable viremia or ≥4-fold rise in serum neutralizing antibody titer.Only five subjects had an anamnestic antibody response detectable by ELISA following a second dose of vaccine, demonstrating that the vaccine induced sterilizing humoral immunity in most vaccinees for at least six months following primary vaccination.The promising safety and immunogenicity profile of this vaccine confirms its suitability for inclusion in a tetravalent dengue vaccine.     

Mycobacterium tuberculosis chaperonin 10 is secreted in the macrophage phagosome: is secretion due to dissociation and adoption of a partially helical structure at the membrane?

To confirm that Mycobacterium tuberculosis chaperonin 10 (Cpn10) is secreted outside the live bacillus, infected macrophages were examined by electron microscopy. This revealed that the mycobacterial protein accumulates both in the wall of the bacterium and in the matrix of the phagosomes in which ingested mycobacteria survive within infected macrophages. To understand the structural implications underlying this secretion, a structural study of M. tuberculosis Cpn10 was performed under conditions that are generally believed to mimic the membrane environment. It was found that in buffer-organic solvent mixtures, the mycobacterial protein forms two main species, namely, a partially helical monomer that prevails in dilute solutions at room temperature and a dimer that folds into a beta-sheet-dominated structure and prevails in either concentrated protein solutions at room temperature or in dilute solutions at low temperature. A partially helical monomer was also found and was completely associated with negatively charged detergents in a micelle-bound state. Remarkably, zwitterionic lipids had no effect on the protein structure. By using N- and C-truncated forms of the protein, the C- and N-terminal sequences were identified as possessing an amphiphilic helical character and as selectively associating with acidic detergent micelles. When the study was extended to other chaperonins, it was found that human Cpn10 is also monomeric and partially helical in dilute organic solvent-buffer mixtures. In contrast, Escherichia coli Cpn10 is mostly dimeric and predominately beta-sheet in both dilute and concentrated solutions. Interestingly, human Cpn10 also crosses biological membranes, whereas the E. coli homologue is strictly cytosolic. These results suggest that dissociation to partially helical monomers and interaction with acidic lipids may be two important steps in the mechanism of secretion of M. tuberculosis Cpn10 to the external environment.     

Structural and functional roles of small group-conserved amino acids present on helix-H7 in the β(2)-adrenergic receptor

Sequence analysis of the class A G protein-coupled receptors (GPCRs) reveals that most of the highly conserved sites are located in the transmembrane helices. A second level of conservation exists involving those residues that are conserved as a group characterized by small and/or weakly polar side chains (Ala, Gly, Ser, Cys, Thr). These positions can have group conservation levels of up to 99% across the class A GPCRs and have been implicated in mediating helix-helix interactions in membrane proteins. We have previously shown that mutation of group-conserved residues present on transmembrane helices H2-H4 in the β(2)-adrenergic receptor (β(2)-AR) can influence both receptor expression and function. We now target the group-conserved sites, Gly315(7.42) and Ser319(7.46), on H7 for structure-function analysis. Replacing Ser319(7.46) with smaller amino acids (Ala or Gly) did not influence the ability of the mutant receptors to bind to the antagonist dihydroalprenolol (DHA) but resulted in ~15-20% agonist-independent activity. Replacement of Ser319(7.46) with the larger amino acid leucine lowered the expression of the S319L mutant and its ability to bind DHA. Both the G315A and G315S mutants also exhibited agonist-independent signaling, while the G315L mutant did not show specific binding to DHA. These data indicate that Gly315(7.42) and Ser319(7.46) are stabilizing β(2)-AR in an inactive conformation. We discuss our results in the context of van der Waals interactions of Gly315(7.42) with Trp286(6.48) and hydrogen bonding interactions of Ser319(7.46) with amino acids on H1-H2-H7 and with structural water.     

γ-Aminobutyric acid (GABA) and other amino acids in tissues of the tick,Amblyomma hebraeum (Acari: Ixodidae) throughout the feeding and reproductive periods

We assayed a variety of tick (Amblyomma hebraeum Koch; Acari, Ixodidae) tissues for a number of amino acids throughout the feeding and early reproductive periods. Our HPLC assay could detect as little as 2–5 pmol per sample of the following: GABA, glycine, serine, glutamine, alanine, taurine, glutamate and aspartate. All of these amino acids could be detected in the salivary gland, synganglion (=total CNS in acarines), haemolymph, Gené's organ, seminal receptacle and ovary. GABA reached high levels in the salivary gland of freshly engorged ticks (685 nmol g^-1) and in the synganglion it exceeded 1000 nmol g^-1 throughout most of the feeding cycle and the first week post-engorgement. GABA also reached a peak titre in the haemolymph of 40 nmol ml^-1. Taurine levels peaked at 1065 nmol g^-1 in the salivary gland from large partially fed ticks. Glutamate and aspartate were likewise found in the salivary gland and synganglion at high concentrations. For most of the amino acids there is insufficient information to correlate these titres (and fluctuations of titres) to neuromodulatory functions. It is possible, however, that the high GABA titre in the salivary gland of engorged ticks is correlated with an augmented level of fluid secretion.Deceased: Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9.     

Why is a specific amino acid sequence of F glycoprotein required for the membrane fusion reaction between envelope of HVJ (Sendai virus) and target cell membranes?

Since homology among the fusogenic sequences of the F glycoprotein of paramyxoviruses is high compared with other hydrophobic sequences such as transmembrane sequences and signal peptides, specific binding of this segment to some membrane component was suspected. Thus, temperature-dependent binding of cholesterol to F protein was found. A virus inhibitory peptide (Z-D-Phe-L-Phe-Gly) was found to bind to virus particles also temperature dependently. These dependency are very similar to that of the fusion reaction. Binding of cholesterol and the peptide to the fusogenic sequence of F protein was suggested based on construction of molecular models. Importance of these bindings for understanding the fusion mechanism is discussed.     

Sheep haemoglobin i or β b13(a10)gly → ser: an example of a cpg mutation in vertebrates. Characterization using fab-mass spectrometry and amino acid sequencing

• 1.1. The amino acid substitution which characterizes the haemoglobin I variant from sheep has been ascertained using a combination of Fast Atom Bombardment mass spectrometry and protein sequencing.
• 2.2. A Ser for Gly substitution at position 13 (10 of the A helix) was found in a polypeptide with the overall sequence of the β^B globin.
• 3.3. On the basis of the nucleotide sequence of the β^B-globin gene, a C to T transition occurring on a CpG doublet is considered to be responsible for the amino acid substitution.
• 4.4. This represents the first observation of a variant sheep Hb due to a mutation which is rather common in the human genome.
• 5.5. Amongst ruminants, serine is normally present at position 13 of goat and sheep ε¹¹ and γ chains and of bovine γ chain which had an independent and more ancient evolutionary origin than the β chains.

     

Cloning of the lanosterol 14-α-demethylase ( ERG11 ) gene in Trichosporon asahii: a possible association between G453R amino acid substitution and azole resistance in T. asahii

Lanosterol 14-α-demethylase ( Erg11 protein; Erg11p ), encoded by the ERG11 gene, is the primary target of azoles. Recently, a change in affinity of this enzyme for azoles has been reported as a resistance mechanism in several fungal species. Trichosporon asahii ( T.?asahii) is susceptible to fluconazole (FLC). This report identified the ERG11 gene of T.?asahii (NCBI accession; HQ176415). The Erg11p of T.?asahii, presumed from the DNA sequence, was closely related to the Erg11p of Cryptococcus neoformans. Furthermore, a FLC-susceptible strain was cultured in medium containing FLC at concentrations from 4.0 to 16?μg?mL(-1) in order to analyze the development of FLC resistance in T.?asahii. The degree of resistance was related to the FLC concentration of the growth medium. One highly resistant strain that was cultured in the medium containing 16?μg?mL(-1) FLC contained 1 point mutation (G1357C) that caused a single amino acid substitution at G453R. This amino acid is highly conserved among major fungal pathogens, and it is in a region very close to the heme-binding domain, which is characteristic of the cytochrome P450 superfamily, the primary target for the azole class of antifungal agents. This amino acid substitution may have caused the high resistance to azoles in T.?asahii.     

α1,6-Fucosyltransferase (Fut8) is implicated in vulnerability to elastase-induced emphysema in mice and a possible non-invasive predictive marker for disease progression and exacerbations in chronic obstructive pulmonary disease (COPD)

Fut8 (α1,6-Fucosyltransferase) heterozygous knock-out (Fut8(+/-)) mice had an increased influx of inflammatory cells into the lungs, and this was associated with an up-regulation of matrix metalloproteinases, MMP-2 and MMP-9, after treatment with porcine pancreatic elastase (PPE), exhibiting an emphysema-prone phenotype as compared with wild type mice (Fut8(+/+)). The present data as well as our previous data on cigarette-smoke-induced emphysema [8] led us to hypothesize that reduced Fut8 levels leads to COPD with increased inflammatory response in humans and is associated with disease progression. To test this hypothesis, symptomatic current or ex-smokers with stable COPD or at risk outpatients were recruited. We investigated the association between serum Fut8 activity and disease severity, including the extent of emphysema (percentage of low-attenuation area; LAA%), airflow limitation, and the annual rate of decline in forced expiratory volume in 1 s (FEV(1)). Association with the exacerbation of COPD was also evaluated over a 3-year period. Serum Fut8 and MMP-9 activity were measured. Fut8 activity significantly increased with age among the at risk patients. In the case of COPD patients, however, the association was not clearly observed. A faster annual decline of FEV(1) was significantly associated with lower Fut8 activity. Patients with lower Fut8 activity experienced exacerbations more frequently. These data suggest that reduced Fut8 activity is associated with the progression of COPD and serum Fut8 activity is a non-invasive predictive biomarker candidate for progression and exacerbation of COPD.     

Analysis of the DR β chains from two DRw6 cell lines (WT46 and WT52): Recombination in vivo may have generated new haplotypes

The HLA-DRw6 haplotype of the class II major histocompatibility complex (MHC) antigens exhibits unusual complexity and cannot be uniquely typed serologically. The DR chains expressed by consanguineous homozygous DRw6 typing cells WT46 and WT52 were biochemically analyzed using three monoclonal antibodies (mAb) that recognize denatured DR chains. The results of isoelectric focusing and N-terminal sequencing demonstrate that each DRw6 B-cell line expresses two DR chains. Evidence of an exchange of mAb epitopes involving the two DR chains of one of these cell lines was obtained and may be explained by a recombinational mechanism involving reciprocal exchange of genetic segments of the DR chains, one of which may encode the putative DRw6 chain and the other the chain carrying the MT2 allotypic determinant. Since a recombinational hot spot has been shown to occur uniquely in the mouse MHC within the E gene, the occurrence of a recombination within the human homolog, DR(MT2) , could reflect some specific feature of this MHC region. Comparison of the DR chains of the WT46 and WT52 cell lines with those of a third DRw6 cell line, LB, suggests that two alleles of MT2 occur.Abbreviations used in this paper IEF isoelectric focusing - mAb monoclonal antibody - MHC major histocompatibility complex - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Information contained in the amino acid sequence of theα1(I)-chain of collagen and its consequences upon the formation of the triple helix,of fibrils and crosslinks

The molecule of type I collagen from skin consists of two alpha1(I)-chains and one alpha2-chain. The sequence of the entire alpha1-chain comprising 1052 residues is summarily presented and discussed. Apart from the 279 residues of alpha1(I)-CB8 whose sequence has been established for rat skin collagen, all sequences have been determined for calf skin collagen. In order to facilitate sequence analysis, the alpha1-chain was cleaved into defined fragments by cyanogen bromide or hydroxylamine or limited collagenase digestion. Most of the sequence was established by automated stepwise Edman degradation. The alpha1-chain contains two basically different types of sequences: the triple helical region of 1011 amino acid residues in which every third position is occupied by glycine and the N- and C-terminal regions not displaying this type of regularity. Both of these non-triple helical regions carry oxidizable lysine or hydroxylysine residues as functional sites for the intermolecular crosslink formation. Implications of the amino acid sequence for the stability of the triple helix and the fibril as well as for formation of crosslinks are discussed. Evaluation of the sequence in connection with electron microscopical investigations yielded the parameters of the axial arrangement of the molecules within the fibrils. Axial stagger of the molecules by a distance D = 670 angstrom = 233 amino acid residues results in maximal interaction of polar sequence regions of adjacent molecules and similarly of regions of hydrophobic residues. Ordered aggregation of molecules into fibrils is, therefore, regulated by electrostatic and electrophobic forces. Possible loci of intermolecular crosslinks between the alpha1-chains of adjacent molecules may be deduced from the dimensions of the axial aggregation of molecules.     

Genetic polymorphism of lactate dehydrogenase isoenzymes in the carp (Cyprinus carpio) apparently due to a “null allele”

As a consequence of polyploidization, the carp is endowed with three genetic loci coding for LDH in most tissues (and additional ones in liver). However, each such duplicated gene may not be functionally essential for the organism and could be eliminated. In a population survey, an electrophoretic polymorphism was detected which may best be interpreted by the assumption of a null allele, which is apparently not subject to selective pressure. Thus this originally duplicated gene would have diverged in the course of evolution without the origin of differences in function, so that one or the other of the genes is dispensable.