Alpha-1-antitrypsin. Plasma survival studies in the rat of the normal and homozygote deficient forms.

Alpha-1-antitrypsin from normal individuals (Pi type MM) from those with an inherited deficiency of circulatory protein (Pi type ZZ) were labelled with 125I and plasma clearance rates measured in rats either prior to, or following treatment with neuraminidase to remove terminal sialic acid residues. In addition, these proteins and the derivatives were tested for their ability to bind to an hepatic binding protein obtained from rabbit liver membranes that has been shown to be responsible for the clearance of serum asialoglycoproteins. Finally, the two native forms of alpha-1-antitrypsin were treated with galactose oxidase followed by reduction with tritiated potassium borohydride and then analyzed for tritium incorporation in the neutral sugar fraction. The results indicate: (a) clearance from plasma for both forms of alpha-1-antitrypsin is dramatically enhanced upon the loss of terminal sialic acid residues to the liver membrane protein; (b) Z protein does not exhibit terminal galactosyl residues; (c) the low level of Z protein in plasma cannot be accounted for by a faster rate of clearance relative to M protein. The relevance of these findings to the alpha-1-antitrypsin deficiency state are discussed.     

Message amplification phenotyping of an inherited delta-aminolevulinate dehydratase deficiency in a family with acute hepatic porphyria

The molecular basis of the enzymatic defect responsible for acute hepatic porphyria due to delta-aminolevulinate dehydratase (ALAD) deficiency was investigated in a family including a proband with the acute disease. In order to delineate the mutation in the proband, cDNA for deficient ALAD was synthesized from the proband's cells. The ALAD phenotype was studied by message amplification phenotyping with total RNA extracted from lymphoblastoid cells of the proband and his family members. Two independent mutant alleles of ALAD were identified in the proband's cells. One mutant allele was shown to result in an amino acid substitution at residue 274 (Ala274----Thr). Message amplification phenotyping studies have also permitted us to define the ALAD phenotype of each subject in the family. This is the first mutation to be recognized in the human ALAD gene.     

Structure of glycerol dehydratase reactivase: a new type of molecular chaperone

The function of glycerol dehydratase (GDH) reactivase is to remove damaged coenzyme B(12) from GDH that has suffered mechanism-based inactivation. The structure of GDH reactivase from Klebsiella pneumoniae was determined at 2.4 A resolution by the single isomorphous replacement with anomalous signal (SIR/AS) method. Each tetramer contains two elongated 63 kDa alpha subunits and two globular 14 kDa beta subunits. The alpha subunit contains structural features resembling both GroEL and Hsp70 groups of chaperones, and it appears chaperone like in its interactions with ATP. The fold of the beta subunit resembles that of the beta subunit of glycerol dehydratase, except that it lacks some coenzyme B(12) binding elements. A hypothesis for the reactivation mechanism of reactivase is proposed based on these structural features.     

Female hemophilia. Apropos of a homozygote woman

About one homozygote female for haemophilia, authors report circumstances where we can see an haemophilic female: lyonisation, chromosomal abnormalities of X chromosome, and homozygosis. They report the cases of literature.     

Import of host delta-aminolevulinate dehydratase into the malarial parasite: identification of a new drug target

The parasite Plasmodium berghei imports the enzyme delta-aminolevulinate dehydratase (ALAD), and perhaps the subsequent enzymes of the pathway from the host red blood cell to sustain heme synthesis. Here we have studied the mechanism of this import. A 65-kDa protein on the P. berghei membrane specifically bound to mouse red blood cell ALAD, and a 93-amino-acid fragment (ALAD-DeltaNC) of the host erythrocyte ALAD was able to compete with the full-length enzyme for binding to the P. berghei membrane. ALAD-DeltaNC was taken up by the infected red blood cell when added to a culture of P. falciparum and this led to a substantial decrease in ALAD protein and enzyme activity and, subsequently, heme synthesis in the parasite, resulting in its death.     

Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides.

The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a alpha2beta2gamma2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 micro m. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 degrees C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway.     

Use of metabolic and molecular methods for the identification of a Bacillus strain isolated from paper affected by foxing

Foxing of paper is a deterioration phenomenon occurring in the form of brown-yellowish spots, the abiotic and/or biotic causes of which are not yet completely understood. Nevertheless, microbiological infection has been recognized that may contribute to paper damage and therefore it becomes important to know the taxonomic position and the degradative activity of the potential infectious biological agents which mostly are fungi, but also bacteria and yeasts. A cellulolytic bacterial strain isolated from a foxed paper sample exhibited morphological and physiological characteristics of the Bacillus genus. To study its taxonomic position, different identification methods were used: the Biolog system, the direct amplified polymorphic DNA-polymerase chain reaction analysis (DAPD-PCR) and the partial sequencing of the 16S rDNA gene. Biolog system and partial sequencing of 16S rDNA gene assigned the strain to the Paenibacillus polymyxa species. DAPD-PCR analysis indicated a high similarity with Bacillus circulans, by comparing the isolated strain with some closely related Bacillus species.     

Toward a structural understanding of the dehydratase mechanism.

dTDP-D-glucose 4,6-dehydratase (RmlB) was first identified in the L-rhamnose biosynthetic pathway, where it catalyzes the conversion of dTDP-D-glucose into dTDP-4-keto-6-deoxy-D-glucose. The structures of RmlB from Salmonella enterica serovar Typhimurium in complex with substrate deoxythymidine 5'-diphospho-D-glucose (dTDP-D-glucose) and deoxythymidine 5'-diphosphate (dTDP), and RmlB from Streptococcus suis serotype 2 in complex with dTDP-D-glucose, dTDP, and deoxythymidine 5'-diphospho-D-pyrano-xylose (dTDP-xylose) have all been solved at resolutions between 1.8 A and 2.4 A. The structures show that the active sites are highly conserved. Importantly, the structures show that the active site tyrosine functions directly as the active site base, and an aspartic and glutamic acid pairing accomplishes the dehydration step of the enzyme mechanism. We conclude that the substrate is required to move within the active site to complete the catalytic cycle and that this movement is driven by the elimination of water. The results provide insight into members of the SDR superfamily.     

Genetic deficiency of purine nucleoside phosphorylase in the mouse. Characterization of partially and severely enzyme deficient mutants

Two independent mutations of purine nucleoside phosphorylase were identified in the first-generation progeny of male mice that had been treated with the mutagen N-ethylnitrosourea and mated to untreated females. The common allele in inbred strains is Np-1a and the mutants are assigned the gene symbols Np-1e and Np-1f. Heterozygotes had approximately half normal purine nucleoside phosphorylase activity in erythrocytes and activity of homozygotes was 17 and 5% of NP-1A for NP-1E and NP-1F, respectively. The following properties are consistent with both Np-1e and Np-1f being point mutations: the expression of residual but markedly reduced activity with normal Michaelis constants for inosine and phosphate, altered isoelectric points, and increased thermal lability. The reduction in erythrocyte activity was also evident in other tissues. A metabolic consequence of the mutations was increased purine nucleoside excretion. Inosine and guanosine, total 150 +/- 84 microM, and inosine, deoxyinosine, guanosine, and deoxyguanosine, total 1490 +/- 190 microM, were present in urine of Np-1e/Np-1e and Np-1f/Np-1f mice, respectively, but not in normal urine, less than 10 microM.     

A molecular study of congenital erythropoietic porphyria in cattle

Previous studies have shown that congenital erythropoietic porphyria (CEP) in cattle is caused by an inherited deficiency of the enzyme uroporphyrinogen III synthase (UROS) encoded by the UROS gene. In this study, we have established the pedigree of an extended Holstein family in which the disease is segregating in a manner consistent with autosomal recessive inheritance. Biochemical analyses demonstrated accumulation of uroporphyrin, thus confirming that it is indeed insufficient activity of UROS which is the cause of the disease. We have therefore sequenced all nine exons of UROS in affected and non-affected individuals without detecting any potential causative mutations. However, a single nucleotide polymorphism (SNP) located within the spliceosome attachment region in intron 8 of UROS is shown to segregate with the disease allele. Our study supports the hypothesis that CEP in cattle is caused by a mutation affecting UROS; however, additional functional studies are needed to identify the causative mutation.     

Evolution of biosynthetic pathways: a common ancestor for threonine synthase, threonine dehydratase and D-serine dehydratase.

The Bacillus subtilis genes encoding threonine synthase (thrC) and homoserine kinase (thrB) have been cloned via complementation of Escherichia coli thr mutants. Determination of their nucleotide sequences indicates that the thrC stop codon overlaps the thrB start codon; this genetic organization suggests that the two genes belong to the same operon, as in E. coli. However, the gene order is thrC-thrB in B. subtilis whereas it is thrB-thrC in the thr operon of E. coli. This inversion of the thrC and thrB genes between E. coli and B. subtilis is indicative of a possible independent construction of the thr operon in these two organisms. In other respects, comparison of the predicted amino acid sequences of the B. subtilis and E. coli threonine synthases with that of Saccharomyces cerevisiae threonine dehydratase and that of E. coli D-serine dehydratase revealed extensive homologies between these pyridoxal phosphate-dependent enzymes. This sequence homology, which correlates with similarities in the catalytic mechanisms of these enzymes, indicates that these proteins, catalyzing different reactions in different metabolic pathways, may have evolved from a common ancestor.     

Molecular heterogeneity of acute intermittent porphyria: identification of four additional mutations resulting in the CRIM-negative subtype of the disease.

Four mutations of the porphobilinogen (PBG) deaminase gene that result in cross-reacting immunological material (CRIM)-negative forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA from patients and by cloning of the amplified products in a bacterial expression vector. One mutation is a single base deletion which causes a frameshift and which is expected to result in the synthesis of a truncated protein. Two other mutations consist of single base substitutions and lead to amino acid changes. The fourth mutation is a single base substitution producing an aberrant splicing and resulting in an mRNA which would encode a protein missing three amino acids. DNAs from 16 unrelated CRIM-negative AIP patients were screened for the presence of these four mutations, by hybridization with oligonucleotides specific for each of the mutations, but none of the four mutations was identified in additional patients. The results indicate that mutations responsible for CRIM-negative AIP are highly heterogenous.     

Mechanism of reactivation of coenzyme B12-dependent diol dehydratase by a molecular chaperone-like reactivating factor.

The mechanism of reactivation of diol dehydratase by its reactivating factor was investigated in vitro by using enzyme. cyanocobalamin complex as a model for inactivated holoenzyme. The factor mediated the exchange of the enzyme-bound, adenine-lacking cobalamins for free, adenine-containing cobalamins through intermediate formation of apoenzyme. The factor showed extremely low but distinct ATP-hydrolyzing activity. It formed a tight complex with apoenzyme in the presence of ADP but not at all in the presence of ATP. Incubation of the enzyme.cyanocobalamin complex with the reactivating factor in the presence of ADP brought about release of the enzyme-bound cobalamin, leaving the tight apoenzyme-reactivating factor complex. Although the resulting complex was inactive even in the presence of added adenosylcobalamin, it dissociated by incubation with ATP, forming the apoenzyme, which was reconstitutable into active holoenzyme with added coenzyme. Thus, it was established that the reactivation of the inactivated holoenzyme by the factor in the presence of ATP and Mg2+ takes place in two steps: ADP-dependent cobalamin release and ATP-dependent dissociation of the apoenzyme.factor complex. ATP plays dual roles as a precursor of ADP in the first step and as an effector to change the factor into the low-affinity form for diol dehydratase. The enzyme-bound adenosylcobalamin was also susceptible to exchange with free adeninylpentylcobalamin, although to a much lesser degree. The mechanism for discrimination of adenine-containing cobalamins from adenine-lacking cobalamins was explained in terms of formation equilibrium constants of the cobalamin.enzyme.reactivating factor ternary complexes. We propose that the reactivating factor is a new type of molecular chaperone that participates in reactivation of the inactivated enzymes.     

Species identification in cell culture: a two-pronged molecular approach

Species identification of cell lines and detection of cross-contamination are crucial for scientific research accuracy and reproducibility. Whereas short tandem repeat profiling offers a solution for a limited number of species, primarily human and mouse, the standard method for species identification of cell lines is enzyme polymorphism. Isoezymology, however, has its own drawbacks; it is cumbersome and the data interpretation is often difficult. Furthermore, the detection sensitivity for cross-contamination is low; it requires large amounts of the contaminant present and cross-contamination within closely related species may go undetected. In this paper, we describe a two-pronged molecular approach that addresses these issues by targeting the mitochondrial genome. First, we developed a multiplex PCR-based assay to rapidly identify the most common cell culture species and quickly detect cross-contaminations among these species. Second, for speciation and identification of a wider variety of cell lines, we amplified and sequenced a 648-bp region, often described as the “barcode region” by using a universal primer mix targeted at conserved sequences of the cytochrome C oxidase I gene (COI). This method was challenged with a panel of 67 cell lines from 45 diverse species. Implementation of these assays will accurately determine the species of cell lines and will reduce the problems of misidentification and cross-contamination that plague research efforts.     

Peripheral olfactory projections are differentially affected in mice deficient in a cyclic nucleotide-gated channel subunit

Axons of olfactory sensory neurons expressing a given odorant receptor converge to a few glomeruli in the olfactory bulb. We have generated mice with unresponsive olfactory sensory neurons by targeted mutagenesis of a cyclic nucleotide-gated channel subunit gene, OCNC1. When these anosmic mice were crossed with mice in which neurons expressing a given odorant receptor can be visualized by coexpression of an axonal marker, the pattern of convergence was affected for one but not another receptor. In a novel paradigm, termed monoallelic deprivation, axons from channel positive or negative neurons that express the same odorant receptor segregate into distinct glomeruli within the same bulb. Thus, the peripheral olfactory projections are in part influenced by mechanisms that depend on neuronal activity.