SSCP detection of a Gly565Val substitution in the proα(I) collagen chain resulting in osteogenesis imperfecta type II

A patient with perinatal lethal osteogenesis imperfecta (OI) type II has been studied in order to identify the causative mutation. By analysis of the type I collagen produced by cultured fibroblasts from the patient, the defect was mapped to 1 cyanogen bromide peptide 7, a region corresponding to 271 amino acid residues of either the 1(I) or 2(I) collagen chains. Polymerase chain reaction (PCR) amplification of the corresponding region of the 1(I) mRNA followed by single-strand conformation polymorphism analysis of restriction enzyme digestions of the PCR products allowed further mapping of the mutation to a small region of COL1A1. A heterozygous transversion of G to T within the last glycine codon of exon 32 was identified by DNA sequence analysis. This resulted in the substitution of glycine-565 by a valine residue, disrupting the repeating Gly-Xaa-Yaa sequence that is obligatory for correct formation of the collagen molecule. The mutation was shown to have occurred de novo and is thought to result in the OI phenotype.     

ααααanti-3.7 type II: a new α-globin gene rearrangement suggesting that the α-globin gene duplication could be caused by intrachromosomal recombination

Summary We report here a new human -globin gene rearrangement carrying the two normal, 2 and 1, and two hybrid, 1/2, globin genes in the order 5-2-1/2-1/2-1-3. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the -thalassaemia IVS 1 nt 1 GA mutation caused thalassaemia intermedia. We also report a case of an -globin gene rearrangement in the twin of one of the -globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an -anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the -globin gene rearrangements could be of the intra-chromosomal rather than the interchromosomal type.     

Two forms of α-glucosidase from sugar-beet seeds

Two forms of -glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from sugarbeet (Beta vulgaris L.) seeds by a procedure including fractionation with ammonium sulfate and ethanol, carboxymethyl-cellulose column chromatography, and preparative disc gel electrophoresis. The two enzymes were homogeneous by polyacrylamide disc gel electrophoresis. Their molecular weights were 98,000 (I) and 60,000 (II). -Glucosidase I readily hydrolyzed maltose, isomaltose, kojibiose, maltotriose, panose, amylose, soluble starch, amylopectin and glycogen. -Glucosidase II also hydrolyzed maltose, kojibiose and maltotriose but hydrolyzed the other substrates only very weakly or not at all. -Glucosidase I hydrolyzed soluble starch at a faster rate than maltose. It produced isomaltose and panose as the main -glucosyltransfer products from maltose, whereas maltotriose was the main product of -glucosidase II. -Glucosidase I hydrolyzed amylose liberating -glucose. The neutral-sugar content was calculated to be 2.7% for -glucosidase I and 8.8% for -glucosidase II. The main neutral sugar was mannose in -glucosidase I, and glucose in -glucosidase II.     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

Localization and synthesis of an insulin-binding region on human insulin receptor

Seven regions of the subunit of human insulin receptor (HIR) were synthesized and examined for their ability to bind radioiodinated insulin. A peptide representing one of these regions (namely, residues 655–670) exhibited a specific binding activity for insulin. In quantitative radiometric titrations, the binding curves of¹²⁵I-labeled insulin to adsorbents of peptide 655–670 and of purified placental membrane were similar or superimposable. The binding of radioiodinated insulin to peptide or to membrane adsorbents was completely inhibited by unlabeled insulin, and the inhibition curves indicated that the peptide and the membrane on the adsorbents had similar affinities. Synthetic peptides that were shorter (peptide 661–670) or longer (peptide 651–670) than the region 655–670 exhibited lower insulin-binding activity. It was concluded that an insulin-binding region in the HIR subunit resides within residues 655–670. The results do not rule out the possibility that other regions of the subunit may also participate in binding of HIR to insulin, with the region described here forming a face within a larger binding site.     

Primary structure of hemoglobin α-chain ofColumba livia (gray wild pigeon)

Primary structure of hemoglobin of -chain ofColumba livia is presented. The separation of -chain was obtained from globin by ion-exchange chromatography (CMC-52) and reversed-phase HPLC (RP-2 column). Amino acid sequence of intact as well as tryptic digested chain was determined on gas-phase sequencer. Structure is aligned homologously with 21 other species. Among different exchanges, positions 24 (TyrLeu), 26 (AlaGly), 32 (MetLeu), 64 (AspGlu), 113 (LeuPhe), and 129 (LeuVal) are unique to pigeon hemoglobin. The various exchanges in -chain are discussed with reference to evolution and phylogeny. The results show that the order Columbiformes is evolutionarily closer to the order Anseriformes. Since the pigeon is homogeneous, having HbA (^A-chain) and lacks ^D-chain, its phylogenetic placement could be established among birds having single hemoglobin components.     

Physicochemical characterization of alpha-crystallins from bovine lenses: hydrodynamic and conformational properties

A detailed investigation of hydrodynamic and conformational behavior has been made of the HM-crystallin and -crystallins of bovine lens. Results from this study indicated that HM (high-molecular-weight -crystallin) and (low-molecular-weight -crystallin) possess considerable size and charge heterogeneities in their native structures and subunit polypeptides, respectively. Sedimentation velocity showed a heterogeneous polydisperse system of HM with an average sedimentation coefficient of about 50 S and a more homogeneous system of -crystallin of 20 S. Viscosity and circular dichroism studies pointed to a compact and globular shape of dominant -sheet conformation for -crystallin, yet a highly asymmetrical and aggregated form for HM. The conformational stability of -crystallin was investigated in the presence of various denaturants. The evidence presented shows that hydrogen bonding is the main force in maintaining the quaternary structure of compact native -crystallin. Conformational flexibility of -crystallin demonstrated in the equilibrium unfolding study indicated a multistep transition that made the extraction of thermodynamic data from the heat denaturation study difficult. Temperature perturbation on -crystallin suggested the possible involvement of hydrophobic interaction in the aggregation process, leading to the formation of HM from -crystallin. The comparison of conformational properties between HM and -crystallin strongly indicated that HM is a denatured form of -crystallin.     

Isolation and partial sequence of goat spleen prothymosin α

Goat prothymosin , a highly acidic polypeptide of pl 3.5, 109 amino acid residues, has been isolated from lymphoid and non-lymphoid tissues of young female goats. Unlike rat, murine and porcine prothymosins , goat prothymosin appears at a higher concentration in the spleen compared with the thymus. The sequence of segments of the polypeptide involving known mutations has been determined, by automatic sequencing of its tryptic peptide fragments. The acidic amino acid-rich segment in the middle of the molecule, including residues 49–83, has not been sequenced. Goat prothymosin closely resembles bovine prothymosin , with only one substitution, proline for alanine at position 85. It also resembles human prothymosin , with only three substitutions. It differs more significantly from rat and murine prothymosins , by two deletions and three substitutions. The results show the highly conserved nature of the molecule, with substitutions at given positions only.Abbreviations ProT Prothymosin - T₁ Thymosin ₁ - MLR Mixed Lymphocyte Response - HPLC High Performance Liquid Chromatography - RIA Radioimmunoassay - B Aspartic acid or Asparagine - Z Glutamic acid or Glutamine     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

αααanti-4.2 Haplotype and heterozygous β° thalassemia in a Sicilian family

Summary The presence of the ^anti-4.2 haplotype and heterozygous ° thalassemia in a Sicilian family is described. These findings confirm the presence in Italy of a leftward deletion (–^4.2) and indicate that this may not be rare. Furthermore, although the thalassemia determinant in this family has a severe expression, the interaction with the triplicated gene does not necessarily express itself as thalassemia intermedia.     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

Phylogenetic Analysis of Vertebrate Fibrillar Collagen Locates the Position of Zebrafish α3(I) and Suggests an Evolutionary Link Between Collagen α Chains and Hox Clusters

Type I collagen in tetrapods is usually a heterotrimeric molecule composed of two 1 and one 2 chains. In some teleosts, a third chain has been identified by chromatography, suggesting that type I collagen should also exist as an 1(I)2(I)3(I) heterotrimer. We prepared, from zebrafish, three distinct cDNAs identified to be those of the collagen 1(I), 2(I), and 3(I) chains. In this study on the evolution of fibrillar collagen chains and their relationships, an exhaustive phylogenetic analysis, using vertebrate fibrillar collagen sequences, showed that each chain constitutes a monophyletic cluster. Results obtained with the newly isolated sequences of the zebrafish showed that the 3(I) chain is phylogenetically close to the 1(I) chain and support the hypothesis that the 3(I) chain arose from a duplication of the 1(I) gene. The duplication might occur during the duplication of the actinopterygian genome, soon after the divergence of actinopterygians and sarcopterygians, a hypothesis supported by the demonstration of a syntenic evolution between a set of fibrillar collagen genes and Hox clusters in mammals. An evolutionary scenario is proposed in which phylogenetic relationships of the chains of fibrillar collagens of vertebrates could be related to Hox cluster history. Present address (Laure Bonnaud): Institut Jacques Monod, Tour 43, CNRS, Universités Paris 6 et 7, Equipe Evolution du Développement des Nématodes, 2 place Jussieu, 75005 Paris, France     

Partial structure of the human α2(IV) collagen chain and chromosomal localization of the gene (COL4A2)

Summary We have isolated a 2.1-kb cDNA clone from a human placental library encoding part of the 2 chain of collagen IV, a major structural protein of basement membranes. The DNA sequence encodes 446 amino acids in the triplehelical domain plus the 227 amino acids of the carboxy-terminal globular domain. The latter structure is composed of two homologous subdomains and is highly conserved between the 1 and 2 chains. The triple-helical domain contained seven interruptions of the Gly-X-Y repeat and these interruptions were in general larger than their counterparts in the 1 chain. DNA from human rodent hybrid cell lines was analyzed under conditions in which there was no cross-hybridization of the 2(IV) cDNA probe with the gene for the 1(IV) collagen chain. An Eco RI fragment characteristic of the 2 chain had a concordance of 0.97 with chromosome 13. This result was confirmed and extended with in situ localization of the gene at 13q34. Since the 1(IV) gene has previously been localized to 13q34, the two type IV collagen genes reside in the same chromosome region (13q34), possibly in a gene cluster. The presence of the genes for type IV collagen chains on chromosome 13 excludes a primary role for these genes in adult polycystic kidney disease and X-linked forms of hereditary nephritis.     

Structures of the N-linked sugar chains in the PAS-6 glycoprotein from the bovine milk fat globule membrane

The structures of the N-linked sugar chains in the PAS-6 glycoprotein (PAS-6) from the bovine milk fat globule membrane were determined. The sugar chains were liberated from PAS-6 by hydrazinolysis, and the pyridylaminated sugar chains were separated into a neutral (6N) and two acidic chains (6M and 6D), the acidic sugar chains then being converted to neutral sugar chains (6MN and 6DN). 6N was separated into two neutral fractions (6N13 and 6N5.5), while 6MN and 6DN each gave a single fraction (6MN13 and 6DN13). The structure of 6N5.5, which was the major sugar chain in PAS-6, is proposed to be Man16 (Man13) Man14GlcNAc14GlcNAc-PA; 6N13, 6MN13 and 6DN13 are proposed to be Gal13Gal14GlcNAc12Man16 (Gal13Gal14GlcNAc12Man13) Man14GlcNAc14 (Fuc16)GlcNAc-PA;6M and 6D had 1 or 2 additional NeuAc residues at the non-reducing ends of 6MN13 and 6DN13, respectively. © 1998 Rapid Science Ltd     

Long-range physical mapping of the alpha-amylase-1 (alpha-Amy-1) loci on homoeologous group 6 chromosomes of wheat

Summary Long-range physical maps of the small multigene family of the malt -amylase genes (-Amy-1) located on the long arms of wheat chromosomes 6A (the -Amy-A1 locus) and 6B (-Amy-B1) were generated by pulsed-field gel electrophoresis analysis. By using three methylation-sensitive rare-cutter restriction endonucleases, NotI, NruI and MluI, and an -Amy-1 cDNA probe and four gene-specific genomic probes from the -Amy-B1 locus, the size of the -Amy-B1 locus was estimated to be about 700 kb and of the -Amy-B1 locus to be about approximately 4300 kb. These two maps indicate clustering of GC-rich and C-methylation-sensitive restriction enzyme recognition sites. At least five regions reminiscent of CpG islands are apparent in -Amy-B1, and three in -Amy-A1. Correlation between recombination frequency and physical distance within the -Amy-B1 locus suggests that 1 cM approximates to 1 Mb in physical distance.     

Is haemoglobin Gα Philadelphia linked to α-thalassaemia?

Summary The question, Is Hb G Philadelphia linked to -thalassaemia? was first posed because the abnormal haemoglobin is found in heterozygotes at a concentration greater than 25%, the proportion predicted from a 4 -chain gene model. Globin chain biosynthesis was studied in a West Indian family in which one parent had ⁺ thalassaemia and the other was heterozygous for the G Philadelphia chain gene. The former had a globin chain production ratio / well above 1, while the latter had a ratio significantly less than 1. One child of the marriage had inherited the ⁺ thallassaemia from one parent and the G Philadelphia chain gene from the other and showed the typical picture of /-thalassaemia (/ ratio slightly above normal). It is explained in the discussion that the evidence favours a close linkage of 2 -chain genes.     

Selective down-regulation of the Cqα/G11α G-protein family in tumour necrosis factor-α induced cell death

Investigations into the regulation of heterotrimeric GTP-binding protein a-subunits in models of tumour necrosis factor- (TNF)-induced cell death, revealed the selective down-regulation of the G_q/G₁₁ family of G-proteins. The human HeLa and murine L929 cells treated with recombinant human TNF for up to 24 h displayed down-regulated G_q/G₁₁ family protein levels, but not G_s, G_i and G_o protein levels as determined by Western analyses. This effect of TNF was observed in a concentration - and time-dependent manner, consistent with the profiles of TNF-induced cell death observed. Moreover, the functioning of G_q/G₁₁ family proteins were found to be impaired in TNF-treated cells, as measured by agonist-induced [Ca²⁺],_i release. In contrast, G_s activity was unaltered by TNF-treatment, determined by measurement of agonist-induced intracellular cyclic AMP generation. These findings in TNF-induced cytotoxic models, indicate a novel 'cross-talk' mechanism by which TNF alters Ca²⁺-signalling mechanisms, which may contribute towards the apoptotic and necrotic cell death.     

Glycosylated human interleukin-1α, neoglyco IL-1α, coupled with N-acetylneuraminic acid exhibits selective activities in vivo and altered tissue distribution

In order to study the effect of glycosylation on its biological activities and to develop IL-1 with less deleterious effects, N-acetylneuraminic acid (NeuAc) with C9 spacer was chemically coupled to human recombinant IL-1. NeuAc-coupled IL-1 (NeuAc-IL-1) exhibited reduced activities in vitro and receptor-binding affinities by about ten times compared to IL-1. In this study, we examined a variety of IL-1 activities in vivo. NeuAc-IL-1 exhibited a marked reduction in the activity to up-regulate serum IL-6, moderate reduction in the activities to up-regulate serum amyloid A and NOx. However, it exhibited comparable activities as IL-1 to down-regulate serum glucose and to improve the recovery of peripheral white blood cells from myelosuppression in 5-fluorouracil-treated mice. In addition, tissue level of NeuAc-IL-1 was high compared to IL-1. These results indicate that coupling with NeuAc enabled us to develop neo-IL-1 with selective activities in vivo and enhanced tissue level.     

Primary structure of the marmoset (Saguinus fuscicollis) hemoglobin. I. Use of tryptic maleylated peptides in the solubilization and sequence elucidation of the α- and β-chains

The primary structure of adult marmoset hemoglobin has been determined. The - and -chains of HbA were separated on a CM23 column in 8 M urea using a sodium phosphate gradient. Tryptic digests of the - and -chains were fractionated on a Dowex 50W-X2 column using a pH and pyridine acetate gradient. Large peptide fragments were obtained by the cyanogen bromide cleavage of the - and -chains, as well as by tryptic digestion of the maleylated - and -chains. The sequence was derived from the amino acid compositions and sequences of the individual tryptic peptide, automated sequence determination of intact - and -chains, as well as automated sequence determination of cyanogen bromide fragments and tryptic maleylated peptides derived from the - and -chains. The complete structure of marmoset adult hemoglobin is closely homologous to that of other primate hemoglobins. The sequence of the marmoset -chain differs from the -chain of human HbA at positions 8, 19, 23, 68, and 116. The -chain from marmoset HbA differs from the -chain of human HbA at positions 5, 13, 21, 50, 87, and 125.This work was supported in part by funds from a Physicians' Medical Education and Research Foundation Grant of the University of Tennessee Memorial Research Hospital and by NIH General Research Support Grant FR-5541 to the institution.