A new metabolic pathway from n-dodecane to α, ω-dodecanedioic acid in a mutant of Candida tropicalis

Summary The metabolic formation of ,-dodecanedioic acid via ,-dodecanediol from n-dodecane using a mutant S76 of Candida tropicalis was studied.It was found that resting cells of S76 produce ,-dodecanediol from n-dodecane. This intermediate was identified by different analytical methods. With n-dodecanol as substrate the quantitative changes in the concentrations of ,-dodecanediol as well as other intermediates, e.g. monoacid, -hydroxy acid and ,-dioic acid produced by resting cells of S76 for different periods of time were determined. With ,-dodecanediol as the sole carbon source, quantitative changes of -hydroxy acid and ,-dioic acid produced by S76 were also recorded.The results confirm the existence of a new metabolic pathway via ,-diol in the course of ,-dioic acid formation from n-alkane in the mutant S76 of C. tropicalis.     

Studies of α- and βL-Crystallin Complex Formation in Solution at 60°C

Studies of molecular mechanisms of chaperone-like activity of -crystallin became an active field of research over last years. However, fine interactions between -crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between - and _L-crystallins was studied during thermal denaturation of _L-crystallin at 60°C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography, and electrophoresis. A mixed solution of - and _L-crystallins at concentrations about 10 mg/ml incubated at 60°C was found to contain their soluble complexes with a mean radius of gyration 14 nm, mean molecular mass 4 MDa and maximal size over 40 nm. In pure _L-crystallin solution, no complexes were observed at 60°C. In SAXS studies, transitions in the -crystallin quaternary structure at 60°C were shown to occur and result in doubling of the molecular weight. This suggests that during the temperature-induced denaturation of _L-crystallin it binds with modified -crystallin or, alternatively, _L-crystallin complexation and -crystallin modifications are concurrent. Estimates of the -_L-crystallin complex size and relative contents of - and -_L-crystallins in the complex suggest that several -crystallin molecules are involved in complex formation.     

Structure and properties of aCephalosporium acremonium α-galactosidase

The amino acid and sugar composition of the enzyme protein, the effect of urea, sodium dodecyl sulphate and Concanavalin A on the purified -galactosidase (EC 3.2.1.22) from the moldCephalosporium acremonium has been studied. The results obtained by gas liquid chromatography indicated the presence ofN-acetylglucosamine, mannose, galactose andN-acetylneuramic acid in the molar proportions 27311. The presence of two types of Asn-linked oligosaccharide structures in the enzyme molecule is assumed. The -galactosidase liberates (1–3), (1–4) and (1–6)-linkedd-galactose units from various synthetic and natural substrates which have been tested. The effects of pH, substrate concentration and temperature on the catalytic activity of the enzyme are described. The purified -galactosidase also exhibited a lectin activity with an affinity towards glucose, and to some extent mannose.Abbreviations p-NPG p-nitrophenyl--d-galactopyranoside - 4-MUG 4-methylumbelliferyl--d-galactopyranoside - HU hemagglutinin unit - PBS phosphate buffered saline - SDS sodium dodecyl sulphate - ConA Concanavalin A - WGA wheat germ agglutinin - LCA Lens culinaris agglutinin - PHA phytohemagglutinin fromPhaseolus vulgaris     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of -l-fucosidase activity. Two natural missense mutations (G¹⁹⁷A) and (A⁸⁶⁰G) within the -l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated -l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of -l-fucosidase for the G¹⁹⁷A transition and a normal level of enzyme for A⁸⁶⁰G. We therefore conclude that the change of G¹⁹⁷A is responsible for fucosidosis in the patients while A⁸⁶⁰G is a normal polymorphic variant of -l-fucosidase.     

Enzymatic oxidation of alpha- 1-proteinase inhibitor in abnormal tissue turnover

Summary The development of pulmonary emphysema and rheumatoid arthritis is currently believed to be due to uncontrolled proteolysis by elastolytic enzymes, as a result of a deficiency in controlling proteinase inhibitors. This deficiency may arise from a number of factors, including genetically controlled improper synthesis, turnover by proteolytic enzymes not inactivated by these inhibitors, and oxidative inactivation in the case of the major plasma inhibitor, a-l-proteinase inhibitor (-1-PI).Recently, -1-PI was found to contain a methionyl residue at its reactive center which was sensitive to oxidation by either N-chlorosuccinimide or by enzymatic oxidation. Chemical oxidation revealed that two of the eight methionyl residues present in the molecule were oxidized, one of which was at the reactive center. Subsequently, it was found that the enzymatic oxidation by myeloperoxidase from neutrophil granules, in the presence of hydrogen peroxide and halide ion, could also be easily attained. Since myeloperoxidase is present in the same granules which contain proteolytic enzymes it would be expected that both types of enzymes would be released simultaneously during either phagocytosis or cell turnover.The myeloperoxidase system does not oxidize or inactivate other proteinase inhibitors or proteinases, with the exception of bovine trypsin, porcine trypsin, and human neutrophil cathepsin G, all of which are inactivated at approximately 5% of the rate for a-1-PI.Recently, it was shown that when a-1-PI was exposed to viable neutrophils in the presence of chloride ion and phorbol myristyl acetate, the ability of -1-PI to bind elastase was diminished. However, when myeloperoxidase-deficient cells or those from patients with chronic granulomatous disease were used, there was no effect on -1-PI activity. Addition of myeloperoxidase to the deficient cells or a hydrogen peroxide generating system to the other cells, however, resulted in -1-PI inactivation, confirming the involvement of myeloperoxidase and hydrogen peroxide in -1-PI inactivation.Oxidized -1-PI has been isolated from rheumatoid synovial fluid of arthritic patients and also from lung lavage fluids of smokers. Approximately four methionyl residues were found to be oxidized in either case, compared to two oxidized residues when native -1-PI was treated with either chemical or enzymatically produced oxidants. The indication is, therefore, that oxidative inactivation of -1-PI does occur in vivo. This result is also substantiated by the recent data which indicate that treatment of monkeys with the oxidizing agent, chloramine-T, resulted in their active -1-PI levels being reduced to near-zero. Furthermore, longterm treatment of dogs with chloramine-T resulted in the development of emphysematous-like lesions.The importance of oxidation to emphysema development has been shown with a rat model in which the animals were exposed to cigarette smoke. It was found that there was a 35% decrease in the elastase inhibitory capacity of the lung lavage fluids of unadapted animals but no decrease if the rats had previously been rendered tolerant to ozone. There was also a 50% decrease in active -1-PI in the lung lavage fluid of human chronic smokers tested in the same way.The myeloperoxidase system can also oxidize chemotactic peptides, including C5a, f-met-leu-phe, and f-met-leu-phe-lys. These materials lose their chemotactic activity under such conditions through oxidation of methionyl residues and it has been found that actively phagocytizing neutrophils can also inactivate the chemotactic peptides in the same manner.Although macrophages produce minimal quantities of proteinases they do produce copious amounts of oxidants, including superoxide when stimulated. This compound as well as that produced through the xanthine-xanthine oxidase generating system has been shown to inactivate -1-Pl. Furthermore, macrophages from human lung have also been found to contain a myeloperoxidase system which requires iodide ion in order to function properly. Thus, macrophages may also have the oxidizing capacity found in the neutrophils and may, therefore, play an integral role in the development of lung disease through the oxidation of -1-Pl.     

α-Thalassemia in Saudi Arabia: deletion pattern

Summary -Thalassemia exists at a high prevalence in several regions of Saudi Arabia. The restriction endonucleases Bam HI and BglII were used to investigate the molecular basis of deletion type of -thalassemia in 226 subjects from the eastern and 61 subjects from the northwestern regions of the country. The arrangements-/ and-/- were common. BglII digestion revealed the existence of rightward deletion in a majority of the cases. Leftward deletions, both homozygous and heterozygous, were also identified. Triple -gene arrangements -/ and -/- were observed at a low frequency in both regions.     

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.     

Carbohydrate antigen expression in murine embryonic stem cells and embryos. II. Sialylated antigens and glycolipid analysis

Summary A mouse embryonic stem (ES) cell line E14 and early mouse embryos were stained with a panel of 15 monoclonal antibodies recognizing sialylated or potentially sialylated carbohydrate determinants, Sialyl Le-x and sialyl Le-a were detected on the pre-implantation embryo from the 8-cell stage, and sialyl Le-a weakly on undifferentiated ES cells. Changes in cell surface carbohydrates occurred after induction of ES cell differentiation with retinoic acid (RA) and dibutyryl cAMP. Qualitative analysis of the neutral glycolipids of untreated and RA-treated ES cells using high-performance thin-layer chromatography (HPTLC) revealed few differences between the two types of culture. The major gangliosides in both cultures were indicative of an active a ganglioside synthesis pathway. GD₃, a precursor of the b synthesis pathway, previously reported to be characteristic of embryonal carcinoma (EC) cells, was absent. RA-induced differentiation caused a shift in the spectrum to more complex gangliosides. Application of fast atom bombardment mass spectrometry (FAB-MS) to permethylated derivatives of individual bands permitted partial characterization of an unusual sialylated glycolipid and a rare ganglioside with the suggested structure of GalNAc-GD1a.Abbreviations NeuAc N-acetylneuraminic acid - Cer ceramide - CMH monohexosylceramide - CDH lactosylceramide (Gal1-4Glc1-Cer) - CTH ceramide trihexoside (Gal1-4Gal1-4Glc1-Cer) - globoside (GalNAc1-3 Gal1-4Gal1-4Glc1-Cer) - Forssman antigen (GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-Cer) - GM₃ (NeuAc2-3Gal1-4Glc1-Cer) - GD₃ (NeuAc2-8NeuAc2-3Gal1-4Glc1-Cer) - GM1 (Gal1-3GalNAc1-4[NeuAca2-3]Gal1-4Glc1-Cer) - GD1a (NeuAc2-3Gal1-3GalNAc1-4[NeuAc2-3]Gal1-4Glc1-Cer) - GT1b (Neu5Ac2-3Gal1-3GalNAc1-4[Neu5Ac2-8Neu5Ac2-3]Gal1-4Glc1-Cer) The glycolipids are named according to Svennerholm (1963) and the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (1978).     

Purification and partial characterisation of and α-tocopherol-binding protein from rabbit heart cytosol

An -tocopherol-binding protein has been isolated and purified from rabbit heart cytosol. The purified protein had an apparent molecular mass of 14,200, as derived from SDS-PAGE. The content of the protein in rabbit heart was around 11.8 g per g of tissue. The binding of -tocopherol to the purified protein was rapid, reversible, and saturable. Neither nor tocopherol could displace the bound -tocopherol from the protein, suggesting a high specificity for -tocopherol. -Tocopherol-binding protein did not bind oleate. Transfer of -tocopherol from liposomes to mitochodria was stimulated 8-fold in the presence of the binding protein, suggesting that this protein may be involved in the intracellular transport of -tocopherol in the heart.     

Na-butyrate increases the production and α2,6-sialylation of recombinant interferon-γ expressed by α2,6- sialyltransferase engineered CHO cells

A non-human like glycosylation pattern in human recombinant glycoproteins expressed by animal cells may compromise their use as therapeutic drugs. In order to correct the CHO glycosylation machinery, a CHO cell line producing recombinant human interferon- (IFN) was transformed to replace the endogenous pseudogene with a functional copy of the enzyme 2,6-sialyltransferase (2,6-ST). Both the parental and the modified CHO cell line were propagated in serum-free batch culture with or without 1 mM sodium butyrate. Although Na-butyrate inhibited cell growth, IFN concentration was increased twofold. The IFN sialylation status was determined using linkage specific sialidases and HPLC. Under non- induced conditions, IFN expressed by 2,6-engineered cells contained 68% of the total sialic acids in the 2,6- conformation and the overall molar ratio of sialic acids to IFN was 2.3. Sodium butyrate addition increased twofold the molar ratio of total sialic acids to IFN and 82% of total sialic acids on IFN were in the 2,6-conformation. In contrast, no effect of the sodium butyrate was noticed on the sialylation of the IFN secreted by the 2,6-ST deficient parental cell line. This study deals for the first time with the effect of Na-butyrate on CHO cells engineered to produce human like sialylation.     

α-Globin gene deletion causes α-thalassemia syndromes in two German families

Summary Restriction endonuclease mapping of chromosomal DNA has been used to determine whether the -globin gene deletion or non-deletion form of -thalassemia is the underlying molecular defect in individuals of two unrelated German families with -thalassemia syndromes. The obtained DNA pattern in all cases indicated loss of -globin genes resulting in-/,--/, and--/- genotypes in thalassemia-2, -thalassemia-1, and Hb H individuals respectively. The chromosomes showing loss of one -globin gene in -thalassemia-2 and Hb H disease were characterized by the so-called rightward deletion form exhibiting loss of a 3.7 kb DNA fragment in the -gene cluster.     

Primary structure of alpha-globin chains from river buffalo (Bubalus bubalis L.) hemoglobins

Primary structure analysis of the four river buffalo -globin chains showed that haplotypes A and B differ from each other by a substitution at codon 64 that may encode Ala or Asn. The A haplotype encodes two -globin chains, ^I1 and ^II3, which differ at positions 129 and 131: ^I1 has 64 Ala, 129 Phe, 131 Asn; ^II3 has 64 Ala, 129 Leu, 131 Ser. The B haplotype encodes two -globin chains, ^I2 and ^II4, which differ at positions 10 and 11: ^I2 has 10 Ile, 11 Gln, 64 Asn; ^II4 has 10 Val, 11 Lys, 64 Asn. Apart from the Ala/Asn polymorphism at position 64, amino acid substitutions in allelic and nonallelic -globin chains seem to have arisen by single point mutations. Detection of electrophoretically silent mutations due to neutral amino acid substitutions and their influence on the isoelectric point are discussed. Furthermore, primary structures of river buffalo -globin chains are compared to other species of the Bovidae family to suggest evolutionary events that have characterized the amino acid substitutions of river buffalo hemoglobin.     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

Detection of a new hybrid α2 globin gene among American Blacks

Summary We have examined the globin gene complex for 49 individuals with -thalassemia-2 (–^3.7). Crossovers resulting in -thalassemia-2 (type I) were observed in all 57 chromosomes with the –^3.7 defect. Except for one -thalassemia-2 chromosome, all were linked to the absence of an Rsa I restriction site located 0.7 kb 5 to the 2-globin gene; this polymorphic site was observed for 10 of 38 non--thalassemia chromosomes from Black Americans. In four Black families with a heterozygous -thalassemia-2 [–^3.7 (I)], an Apa I restriction site has been identified in the IVS-2 of the 2 gene of the normal chromosome (labeled the ^*2 gene). The ^*2 gene of one Black subject was cloned and a segment located 5 to the Cap site as well as the IVS-2, exon 3, and a 3 segment were sequenced. The data show that the ^*2 gene is an 2 gene except for a segment between nucleotides (nts) 580–81 and nt 509 (Cap site=nt 1), and perhaps as far upstream as nt-634, which has an 1 sequence. This ^*2 hybrid gene probably originated through a double crossover; the structural identity of its IVS-2 with that of the 1 gene adequately explains the presence of the Apa I restriction site.     

Quantitative analysis of type IV collagen alpha chains in the basement membrane of human urogenital epithelium

Type IV collagen is a major component of the basement membrane (BM), which consists of six genetically distinct (IV) chains. In this study the expression of these six (IV) chains was demonstrated immunohistochemically. In addition, the 2(IV) and 5(IV) chains were analysed quantitatively by confocal laser scanning microscopy in human urogenital epithelial BM. The 1/2(IV) and 5/6(IV) chains were immunoreactive in the epithelial BM, whereas, 3/4(IV) chains were not. The quantitative analysis revealed that the amount of 2(IV) and 5(IV) chains differed in each urogenital epithelial BM. The content of 5(IV) chains in the epithelial BM of the bladder was differentially high, and that of the foreskin was differentially low. It is concluded that the elasticity of epithelial BM of the bladder may be structurally related to the high content of 5/6(IV) chains.     

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.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.