Partial mispairing and crossing-over between β0 and δ genes as the origin of the δ β0 thalassemia gene

Summary Two double heterozygous ⁰/⁰ thalassemic sibs of Mexican descent were studied. The father had a ⁰/⁰ genotype, while the mother, one sib and several maternal relatives were ⁰/⁰ heterozygotes. Parental consanguinity and an apparently low frequency of thalassemia among Mexicans suggested a possible common origin of both ⁰ and ⁰ genes. A hypothesis to explain such a possibility is proposed on the basis of a partial mispairing between ⁰ and genes followed by a crossing-over which would results in a ⁰ recombinant gene. This hypothesis could also be extended to explain the 22 gluala, 22 alaglu and 116 arghis Hb variants as recombinants from double crossing-over between and mispaired genes for which the name interstitial-Lepore is proposed.     

Gaussian-based Alignment of Protein Structures: Deriving a Consensus Superposition when Alternative Solutions Exist

The use of a Gaussian-based representation of protein structures for evaluating protein-structure similarities and deriving three-dimensional superpositions is presented. The approach, as implemented in the program GAPS, is applied to three pairs of proteins with different topological characteristics (rich -helix, mixed -helix/-strand, and rich -strand), low sequence identities (10–30%), and recognized difficulties to define a unique optimum alignment.Validation of the GAPS superpositions is done by comparison with superpositions obtained by the TOP, GA_FIT, and ALIGN programs and those directly extracted from the FSSP database. Results suggest that a Gaussian-based methodology offers an objective means to, depending on the Gaussian-based representation, derive a consensus three-dimensional superposition when alternative superposition solutions exist.     

Synthesis of Galβ1-4GlcNAcβ- and Galβ1-3GalNAcα-O-L-serine Derivatives employing glycosidases

A new approach for the highly specific preparation of L-serine conjugates of lactosamine and Gal1-3GalNAc is described. Thus, the L-serine derivative of lactosamine Gal1-4GlcNAc-O-(N-Z)-Ser-OEt, was obtained from lactose, employing GlcNAc-O-(N-Z)-Ser-OEt as acceptor and a yeast -galactosidase as catalyst Galp 1-3GalNAc-O-(N-Alloc)-Ser-OMe was obtained from lactose, employing GalNAc-O-(N-Alloc)-Ser-OMe as acceptor and -galactosidase from bovine testes as catalyst.     

No linkage to the 3β-HSD gene cluster in a kindred affected with 3β-hydroxy-Δ5-C27steroid dehydrogenase deficiency and early onset hepatic failure

We studied the segregation of the genes for 3-hydroxy-C_19/21-steroid dehydrogenase types I and II (3-HSD I and II) in a consanguineous family affected with 3-hydroxy-⁵-C₂₇steroid dehydrogenase (3-OH-C₂₇-SD) deficiency. The results show that the C₂₇ and C_19/21 steroid dehydrogenase activities are encoded by distinct genes that are not in genetic linkage. Further kindreds would assist in screening for linkage of 3-OH-C₂₇-SD to other members of the 3-hydroxysteroid dehydrogenase gene family.     

Production of steroid-ring-B-seco acid from β-sitosterol and influence of surfactants on sterol uptake in Nocardia sp. M 29–40

Summary The effect of several parameters (temperature; pH; main carbon source; time and amount of -sitosterol addition; Tween 20, 40, 60, 80; Span 20; pluronic F 68, L 64) on the conversion of -sitosterol to 3-(5-hydroxy-7a-methyl-1-oxo-3a-H-hexahydroindan-4-yl) propionic acid (I) by Nocardia sp. M. 29–40 was investigated. A maximal theoretical yield of 65 mol% I (with respect to substrate added) could be achieved during cultivation at pH 8.0 in presence of 6 g/l Tween 40 or Tween 60. Tween 40 and Tween 60 stimulate -sitosterol cooxidation not by improving the substrate suspension but by providing a fatty acid component as precursor for biosynthesis of surface active cell wall lipids.In memory of Professor David Perlmann     

Curdlan and other bacterial (1→3)-β-d-glucans

Three structural classes of (13)--d-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. Linear (13)--glucans and side-chain-branched (13,12)--glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage. Cyclic (13,16)--glucans are predominantly periplasmic, serving in osmotic adaptation. Curdlan, the linear (13)--glucan from Agrobacterium, has unique rheological and thermal gelling properties, with applications in the food industry and other sectors. This review includes information on the structure, properties and molecular genetics of the bacterial (13)--glucans, together with an overview of the physiology and biotechnology of curdlan production and applications of this biopolymer and its derivatives.     

Study of beta-amyloid peptide (Abeta40) insertion into phospholipid membranes using monolayer technique

-Amyloid peptide (A), a normal constituent of neuronal and non-neuronal cells, has been proved to be the major component of extracellular plaque of Alzheimer's disease (AD). The interaction of A with lipid membranes may be essential for its neurotoxicity. Our previous study revealed that membrane insertion may provide a possible pathway by which A prevents itself from aggregation and fibril formation. In the present work we studied the membrane insertion of A and the factors that affect its insertion ability using a monolayer approach. The results show that A is surface active and can insert into lipid monolayers. When a high level of cholesterol is present, A40 can insert into the phospholipid mixtures simulating physiological membrane composition. Acidic pH enhances A insertion, while the effect of ionic strength is rather complex. A insertion ability may be ultimately relative to cholesterol-rich domains in the monolayers, which indicates strong interaction between A and cholesterol.     

Steroid Polyols from the Far Eastern Starfish Henricia sanguinolenta and H. leviuscula leviuscula

Two new asterosaponins, (20R)-3-O--D-(2-O-methylxylopyranosyl)-24-propylcholest-4-ene-3,6,8,15,16,29-hexaol (sanguinoside A) and (20R,24S)-3-O--D-(2,3,4-tri-O-methylxylopyranosyl)-5-cholestane-3,4,6,8,15,24-hexaol (sanguinoside B), were isolated from two species of Pacific Far Eastern Starfish Henricia sanguinolenta and H. leviuscula leviuscula, collected in the Sea of Okhotsk. Both glycosides contain aglycones with pentahydroxysteroid nuclei of similar structures, which are substituted at the 3-hydroxy group with differently methylated -D-xylosyl residues. Sanguinoside A has an unusual structure of its aglycone side chain, whereas sanguinoside B has a unique permethylated carbohydrate chain. In addition, laevisculoside G, a known glycoside, was identified in the H. leviuscula starfish. The structures of the isolated glycosides were established by interpreting their spectral data and by comparing their spectral characteristics with those of known compounds.     

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of -helix, -sheet, and unordered form in -lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of -lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% -helix and 41% -sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased -sheet up to 48% but did not affect the -helical proportion. The -helical proportions of nonreduced -lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the -helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The -sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

Synthesis of Gal-β-(1→4)-GlcNAc-β-(1→6)-[Gal-β- (1→3)]-GalNAc-α-OBn oligosaccharides bearing O-methyl or O-sulfo groups at C-3 of the Gal residue: specific acceptors for Gal: 3-O-sulfotransferases

Our recent studies have revealed the existence of two distinct Gal: 3-O-sulfotransferases capable of acting on the C-3 position of galactose in a Core 2 branched structure, e.g., Gal14GlcNAc16(Gal13)GalNac1OBenzyl as acceptor to give 3-O-sulfoGal14GlcNAc13(Gal13)GalNAc1OB 20 and Gal14GlcNAc16(3-O-sulfoGal13)GalNAc1OB 23. We herein report the synthesis of these two compounds and also that of other modified analogs that are highly specific acceptors for the two sulfotransferases. Appropriately protected 1-thio-glycosides 7, 8, and 10 were employed as glycosyl donors for the synthesis of our target compounds.     

Partitioning and separation of α-lactalbumin and β-lactoglobulin in polyethylene glycol/ammonium sulphate aqueous two-phase systems

The partition behaviour of -lactalbumin (la) and -lactoglobulin (lg) on PEG/(NH₄)₂SO₄ system was studied. For purified proteins, a partition coefficient of 12.8 for la and 0.34 for lg, with mass recovery yields of 96.7% for la in the upper phase and 83.8% for lg in the lower phase was obtained, in 18% (w/w) PEG 900/14% (w/w) (NH₄)₂SO₄ system, at pH 7. PEG/(NH₄)₂SO₄ system was an economical alternative for the recovery and separation of the two proteins in cheese whey, allowing a 50% reduction in costs. An efficient and inexpensive separation of both proteins in cheese whey could be achieved, by using 16% (w/w) PEG 900/15% (w/w) (NH₄)₂SO₄, at pH 7.5.     

Prediction of the Secondary Structure Contents of Globular Proteins Based on Three Structural Classes

The prediction of the secondary structural contents (those of -helix and -strand) of a globular protein is of great use in the prediction of protein structure. In this paper, a new prediction algorithm has been proposed based on Chou's database [Chou (1995), Proteins 21, 319–344]. The new algorithm is an improved multiple linear regression method, taking into account the nonlinear and coupling terms of the frequencies of different amino acids and the length of the protein. The prediction is also based on the structural classes of proteins, but instead of four classes, only three classes are considered, the class, class, and the mixed + and / class or simply the class. Thus the ambiguity that usually occurs between + proteins and / proteins is eliminated. A resubstitution examination for the algorithm shows that the average absolute errors are 0.040 and 0.035 for the prediction of -helix content and -strand content, respectively. An examination of cross-validation, the jackknife analysis, shows that the average absolute errors are 0.051 and 0.045 for the prediction of -helix content and -strand content, respectively. Both examinations indicate the self-consistency and the extrapolating effectiveness of the new algorithm. Compared with other methods, ours has the merits of simplicity and convenience for use, as well as high prediction accuracy. By incorporating the prediction of the structural classes, the only input of our method is the amino acid composition and the length of the protein to be predicted.     

Variable Deposition of Amyloid β-Protein (Aβ) with the Carboxy-Terminus that Ends at Residue Valine40 (Aβ40) in the Cerebral Cortex of Patients with Alzheimer's Disease: A Double-Labeling Immunohistochemical Study with Antibodies Specific for Aβ40 and the Aβ that Ends at Residues Alanine42/Threonine43 (Aβ42)

Amyloid -protein (A) deposits in the cerebral cortices of patients with Alzheimer's disease (AD) were investigated immunohistochemically to determine their carboxy terminal sequences. Antibodies specific for A terminating at residue valine₄₀ (A40) and at residues alanine₄₂/threonine₄₃ (A42) were used. Virtually all parenchymal A deposits were positive for A42. Many of these deposits were also partially or completely labeled for A40. The degree of A40 labeling varied from area to area within a given brain and from AD case to AD case. In contrast to parenchymal deposits, A40 labeled essentially all the vascular deposits which constitute amyloid angiopathy (AA), with A42 occurring variably in some of these deposits. Occasional AA was found, however, in which A42 predominated or was exclusively deposited. Such a diversity of A species, both in brain parenchyma and in AA, suggests that multiple C-terminal processing mechanisms occur in the cell types responsible for these deposits.     

Variant forms ofα-2-l-fucosyltransferase in human submaxillary glands from blood group ABH “secretor” and “non-secretor” individuals

The properties of the -2-l-fucosyltransferases in submaxillary gland preparations from blood group ABH secrefors and non-secretors were compared. The level of activity in the non-secretor gland homogenates amounted to about 5% only of that found in the secretor gland preparations. The enzymes from the two sources differed in solubility properties, charge and affinities for donor and acceptor substrates. The enzyme from secretor glands showed a preference for acceptors with Type 1 [d-galactosyl(1–3)-N-acetyl-d-glucosamine] structures whereas the enzyme from non-secretor glands had a preference for Type 2 [d-galactosyl(1–4)-N-acetyl-d-glucosamine] structures.These results demonstrate that expression of the secretor gene (Se) is associated with a molecular form of the -2-l-fucosyltransferase that is different from the species present in the same tissue when theSe gene is not expressed.     

Genetic heterogeneity of propionic acidemia: Analysis of 15 Japanese patients

Summary Propionic acidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl CoA carboxylase (PCC) activity. We have analyzed the molecular heterogeneity of Japanese propionic acidemia patients using anti-human PCC antiserum and cDNA clones coding for the two protein subunits ( and ) of the enzyme. The steady state levels of both and subunits of PCC from 15 Japanese patients were determined by Western blot. Three patients had neither nor subunits, and the amounts of both and subunits were low in 3 other patients. According to our previous data, we classified these 6 patients as having subunit deficiency. In the remaining 8 patients, subunits were normal, but the subunits were aberrant. Two patients had low levels of normal-sized subunits and 6 had subunits smaller than normal in size and greatly reduced in quantity. These 8 patients were assigned to the subunit deficiency category. One patient had apparently normal and subunits. We could not determine this patient's primary defect. These data reveal the genetic heterogeneity of molecular defects causing propionic acidemia in the Japanese. Southern blot analysis did not reveal any gross alteration in gene structure when DNA was digested withHindIII,EcoRI andTaqI. However, DNA from 3 -subunit-deficient patients, when digested withMspI and probed with PCC cDNA, revealed a unique 2.7-kb band not observed in blots of DNA from any other patient or 15 normal controls. We conclude that this alteredMspI restriction map is the result of a mutation in the subunit gene of these patients.     

Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.)

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-¹⁴C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg²⁺: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg²⁺, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca²⁺ dependent and this dependence could be abolished by proteinase treatment. The K _m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.     

Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled withp-aminobenzoic acid ethyl ester (ABEE) afterN-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz¹H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6%, Glc1-3Glc1-3Man₉GlcNAc₂; 35.6%, Glc1-3Man_7–9GlcNAc₂). oligomannose type (15.0%, with the structure Man_5–9GlcNAc₂) and biantennary complex type with core structures of-Man1-3(-Man1-6)Man1-4GlcNAc1-4GlcNAc (9.9%),-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4GlcNAc (25.1%) and-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc₁Man_7–9GlcNAc₂) have been located previously in hen IgY.Abbreviations IgG, IgM, IgA, IgY immunoglobulin G, M, A and Y, respectively - ABEE p-aminobenzoic acid ethyl ester     

Differential sensitivity of astrocyte primary cultures and derived spontaneous transformed cell lines to 70-hydroxycholesterol: effect on plasma membrane lipid composition and fluidity,and on cell surface protein expression

Summary The cytotoxicity of 7-hydroxycholesterol (7-OHC) was investigated on rat astrocyte primary cultures and spontaneously transformed cell lines derived from them. Confluent astrocyte primary cultures (normal cells) were unaffected by 20 µM 7(3-OHC over a period of 72 h whereas 30 µM markedly affected the viability of the transformed cells within the first 72 h. Both cell types incorporated 18% of the total amount of 7-OHC added to the cultures at concentrations of 20 µM or 30 µM. Cellular fractionation after incubation with 20 µM or 30 µM 7-OHC indicated that the plasma membrane incorporated 2 or 6 fold more 7-OHC than the intracellular one's respectively. Plasma membrane cholesterol (CH) and phospholipid (PL) analysis showed that 20 µM 7-OHC did not affect CH/PL in normal cells; in contrast, plasma membranes of transformed cells displayed a significant CH/PL decrease, which was more pronounced with 30 µM 7-OHC treatment. Fluorescence anisotropy measurements indicated that 20 µM 7-OHC slightly fluidified the plasma membrane of normal cells whereas it has not effect on that of the transformed cells one; however, an increase in plasma membrane fluidity was observed when the transformed cells were treated with 30 µM 7-OHC. Lactoperoxidase catalyzed radioiodination of cell surface proteins and subsequent autoradioelectrophoretic analysis demonstrated that the labelled protein pattern was unchanged when both cell types were incubated with 30 µM 7-OHC.     

The structure of the incompatibility factors of Schizophyllum commune: Constitution of the three classes of B factors

Summary The B factors of Schizophyllum commune are of 3 classes: The high recombining class I has 7 alleles and 7 alleles; the low recombining classes are class II with 7 allels and probably 2 alleles and class III with probably 2 (or also 2) alleles and 7 allels. A fourth hypothetical class (-) was not found and either does not exist or is indistinguishable from class III by the tests employed. The and alleles differ from and by either (a) mutations affecting both mating specificity and recombination frequency, or (b) deletions involving most of the B region.The research was supported by a grant from the Atomic Energy Commission of the U.S. No. (30-1)-3875 and was performed at the Biological Laboratories, Harvard University, Cambridge, Mass., U.S.A.