α-Globin gene deletions associated with αAand αG Philadelphiagenes in an Algerian family that includes two Hb G homozygotes

Summary An Algerian family with a high degree of consanguinity and including two homozygotes for Hb-G Philadelphia is presented. Whether homozygotes or heterozygotes, all subjects displayed microcytosis (with various degrees of poikilocytosis) and a moderately depressed -globin chain synthesis. Hb H and Heinz bodies were absent. DNA mapping revealed the presence of 3.7 kb deletion resulting from the rightward type of recombination event between ₂ and ₁ genes on both the ^A/and the ^G chromosomes. Such data indicate that the –^A/ and –^G/ haplotypes are involved and suggest that the –^G/ haplotype, which is very rare in Algeria, has an African Black origin. In subjects with genotype (–^A/–^G/) or (–^G/–^G/), the output of the remaining genes is sufficiently high to avoid the appearance of Hb H. This situation contrasts with that reported in an Algerian patient, who had a (–^A/–^A/) genotype but who was producing Hb H (Whitelaw et al. 1980). The data collected from this family suggest that the –^A/ haplotypes are heterogeneous in Algerians.     

Orientational oscillations of the peptide groups of an α-helix

The low-energy orientational oscillations of the peptide groups of an -helix are considered and the value of the frequency is estimated to be in agreement with experiments. Approximate formulae are derived for the projection of a dipole moment on the helix axis and for the helix parameters. Within the framework of a three-chain model, the asymptotics of the soliton solution is obtained using a discrete approach.The analysis of -helix geometry exhibits two types of low-frequency oscillations of the -helix. The first one is connected with atom movements along the helix axis with the peptide groups twisting around the helix axis. Accordingly, it changes the hydrogen bond lengths between neighbouring peptide groups. In the second case, the slopes of the peptide groups to the helix axis oscillate without the helix parameters changing. Here, the energy of interactions between peptide-group dipoles is changed and, as a result, the oscillations have an optical nature. The frequency of the optical orientational oscillations is approximately 100 cm^-1.     

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.     

Replacing the 2'-oxygen with an exocyclic methylene group reverses the stabilization effects of α-L-LNA

The synthesis and hybridization properties of an α-L-LNA analog where the 2'-oxygen atom is replaced with an exocyclic methylene group is reported. Contrary to the β-D series where the exocyclic methylene group is extremely well tolerated, this group was very poorly tolerated in the α-L-series and lead to duplex destabilization. Modeling studies showed that the exocyclic methylene group results in a steric clash with the nucleobase 3' to the modified residue. Based on this structural model one can anticipate that replacing the 2'-oxygen atom of α-L-LNA with larger groups is likely to be detrimental to duplex stability. The model also provides insights into what type of 2',4'-bridges are most likely to be tolerated in α-L-LNA modified oligonucleotide duplexes.     

Hydrolysis of starches by the action of an α-amylase from Bacillus subtilis

A moderately thermophilic Bacillus subtilis strain, isolated from fresh sheep’s milk, produced extracellular thermostable α-amylase. Maximum amylase production was obtained at 40 °C in a medium containing low starch concentrations. The enzyme displayed maximal activity at 135 °C and pH 6.5 and its thermostability was enhanced in the presence of either calcium or starch. This thermostable α-amylase was used for the hydrolysis of various starches. An ammonium sulphate crude enzyme preparation as well as the cell-free supernatant efficiently degraded the starches tested. The use of the clear supernatant as enzyme source is highly advantageous mainly because it decreases the cost of the hydrolysis. Upon increase of reaction temperature to 70 °C, all substrates exhibited higher hydrolysis rates. Potato starch hydrolysis resulted in a higher yield of reducing sugars in comparison to the other starches at all temperatures tested. Soluble and rice starch took, respectively, the second and third position regarding reducing sugars liberation, while the α-amylase studied showed slightly lower affinity for corn starch and oat starch.     

Characterization of an exo-acting intracellular α-amylase from the hyperthermophilic bacterium Thermotoga neapolitana

We cloned and expressed the gene for an intracellular α-amylase, designated AmyB, from the hyperthermophilic bacterium Thermotoga neapolitana in Escherichia coli. The putative intracellular amylolytic enzyme contained four regions that are highly conserved among glycoside hydrolase family (GH) 13 α-amylases. AmyB exhibited maximum activity at pH 6.5 and 75°C, and its thermostability was slightly enhanced by Ca²⁺. However, Ca²⁺ was not required for the activity of AmyB as EDTA had no effect on enzyme activity. AmyB hydrolyzed the typical substrates for α-amylase, including soluble starch, amylose, amylopectin, and glycogen, to liberate maltose and minor amount of glucose. The hydrolytic pattern of AmyB is most similar to those of maltogenic amylases (EC 3.2.1.133) among GH 13 α-amylases; however, it can be distinguished by its inability to hydrolyze pullulan and β-cyclodextrin. AmyB enzymatic activity was negligible when acarbose, a maltotetraose analog in which a maltose residue at the nonreducing end was replaced by acarviosine, was present, indicating that AmyB cleaves maltose units from the nonreducing end of maltooligosaccharides. These results indicate that AmyB is a new type exo-acting intracellular α-amylase possessing distinct characteristics that distinguish it from typical α-amylase and cyclodextrin-/pullulan-hydrolyzing enzymes.     

A Fos-Jun element in the first intron of an α2u-globulin gene

The hepatic expression of the _2u gene family is controlled by a variety of hormones including steroids, growth hormone and insulin. The mechanisms by which these hormones affect -globulin expression are only partially understood. Recently we isolated and characterized clone RAP 01, an _2u-globulin gene expressed in the liver. In preliminary experiments we noted that partial hepatectomy, a procedure which results in a sharp rise in the level of the oncoproteins c-Fos and c-Jun, also causes a transient induction of the messenger RNA corresponding to clone RAP 01. Using the DNAseI footprinting technique we were able to show that this clone contains a TPA (phorbol 12-myristate 13-acetate)-responsive element (TRE) in its first intron. This element (denoted as element X) is identical to the consensus AP-1 binding site (TGACTCAG) and is protected by rat liver nuclear extracts as well as by purified c-Jun. Gel retardation experiments show that an oligonucleotide containing the TRE consensus sequence competes for binding of liver nuclear proteins to element X and that antibodies directed against the M₂ peptide of the mouse Fos protein or the PEP-2 peptide of Jun prevent the formation of specific complexes with the same element. Moreover, element X functions as a TRE in transfected BWTG₃ hepatoma cells treated with TPA. Co-transfection withfos andjun expression vectors mimics the effects of TPA suggesting that AP-1 is in fact the mediator of the observed response. It is concluded that the first intron of RAP 01 contains a functional Fos-Jun element.     

Illuminating the Off-Pathway Nature of the Molten Globule Folding Intermediate of an α-β Parallel Protein

Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin’s molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an α-β parallel protein.     

Use of an α-d-glucosidase for the specific cytochemical identification of lateral α-d-xylosyl end groups in plant xyloglucans

Summary α-Linked d-xylosyl side chains represent the typical feature common to all xyloglucans not shared by other cell wall polysaccharides. Since no easily available α-d-xyloxidase is known, advantage was taken of the conformational and configurational homologies between α-d-xylopyranose and α-d-glucopyranose to make an α-d-glucosidase-gold complex which was able to recognize α-d-xylosyl terminal residues of xyloglucans. This marker was used together with α-l-fucosidase gold complex for the double labeling on two different structural features of the same macromolecule in plant primary cell wall.     

Collisionless current generation in the center of the tokamak plasma by an isotropic source of α-particles

The density of the noninductive current generated due to collisionless motion of α-particles in the tokamak magnetic field is calculated. The analysis is based on fully three-dimensional calculations of charged particle trajectories without simplifying assumptions typical for drift and neoclassical approaches. The current is calculated over the entire cross section of the plasma column, including the magnetic axis. It is shown that the current density is not a function of a magnetic surface and is strongly polarized over the poloidal angle. The current density distribution in the tokamak poloidal cross section is obtained, and the current density as a function of the safety factor, the tokamak aspect ratio, and the ratio of the particle Larmor radius on the axis to the tokamak minor radius is determined. It is shown that, when the source of α-particles is spatially nonuniform, the current density in the center of the tokamak is nonzero due to asymmetry of the phase-space boundary between trapped and passing particles. The current density scaling in the tokamak center differs from the known approximations for the bootstrap current and is sensitive to the spatial distribution of α-particles.     

Anion inhibition studies of an α-carbonic anhydrase from the living fossil Astrosclera willeyana

An α-carbonic anhydrase (CA, EC 4.2.1.1) isolated from the living fossil sponge Astrosclera willeyana, Astrosclerin, was investigated for its inhibition profile with simple inorganic anions, complex anions and other small molecules known to interact with these zinc enzymes. Astrosclerin is a catalytically highly efficient enzyme, and is inhibited in the low micromolar range by sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, and in the submillimolar range by a variety of anions including fluoride, chloride, cyanate, thiocyanate, cyanide, hydrogen sulfide, bisulfate, stannate, perosmate, divanadate, perrhenate, perruthenate, selenocyanide, trithiocarbonate, diethyldithiocarbamate and iminodisulfonate. Less efficient Astrosclerin inhibitors were sulfate, bromide, iodide, azide, bicarbonate, carbonate, tetraborate and perchlorate (K(I)s of 5.11-30.6mM) whereas tetrafluoroborate was not at all inhibitory. Because Astrosclerin is involved in calcification processes in vivo, its anion inhibition profile may be important for future studies designed to shed light on the physiologic functions of α-CAs in marine organisms.     

Biochemical characterization of an extracellular polyextremophilic α-amylase from the halophilic archaeon Halorubrum xinjiangense

An extracellular haloalkaliphilic thermostable α-amylase producing archaeon was isolated from the saltwater Lake Urmia and identified as Halorubrum xinjiangense on the basis of morphological, biochemical, and molecular properties. The enzyme was purified to an electrophoretically homogenous state by 80 % cold ethanol precipitation, followed by affinity chromatography. The concentrated pure amylase was eluted as a single peak on fast protein liquid chromatography. The molecular mass of the purified enzyme was about 60 kDa, with a pI value of 4.5. Maximum amylase activity was at 4 M NaCl or 4.5 M KCl, 70 °C, and pH 8.5. The K _m and V _max of the enzyme were determined as 3.8 mg ml^?1 and 12.4 U mg^?1, respectively. The pure amylase was stable in the presence of SDS, detergents, and organic solvents. In addition, the enzyme (20 U) hydrolyzed 69 % of the wheat starch after a 2-h incubation at 70 °C in an aqueous/hexadecane two-phase system.     

Model studies of the α-peroxidase system: Formation of an electronically excited product

Horseradish peroxidase—as an oxidase—converts propanaldehyde to acetaldehyde and formic acid. To some extent the enzyme also acts upon linear acids, thus mimicking even better the α-peroxidase activity of higher plants. In these reactions an electronically excited species—presumably the aldehyde—is generated, as revealed by sensitized emission. The species is long-lived; in accord with its triplet nature heavy substituents are required in the acceptor for efficient sensitization. Energy transfer occurs noncollisionally and does not appear to proceed by a long-range Förster-type T-S mechanism. A long-range triplet-triplet exciton transfer to an upper triplet state of the acceptor is proposed; then ISC occurs to the fluorescent state of the acceptor. Biological compounds which might originate from excited aldehydes are pointed out.     

Fractalkine Signaling Regulates the Inflammatory Response in an α-Synuclein Model of Parkinson Disease

Background

Parkinson disease (PD) is a progressive neurodegenerative disorder characterized by loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and widespread aggregates of the protein alpha-synuclein (α-syn). Increasing evidence points to inflammation as a chief mediator; however, the role of α-syn in triggering and sustaining inflammation remains unclear. In models of Alzheimer’s disease (AD), multiple sclerosis (MS) and neurotoxin models of PD, the chemokine CX3CL1 (fractalkine) and its receptor (CX3CR1) have important roles in modulating neuroinflammation.

Methods

To examine the role of fractalkine signaling in α-syn-induced-neuroinflammation and neurodegeneration, we used an in vivo mouse model in which human α-syn is overexpressed by an adeno associated viral vector serotype 2 (AAV2) and in vitro phagocytosis and protein internalization assays with primary microglia treated with aggregated α-syn.

Results

We observed that loss of CX3CR1 expression led to a reduced inflammatory response, with reduced IgG deposition and expression of MHCII 4 weeks post-transduction. Six months post transduction, AAV2 mediated overexpression of α-syn leads to loss of dopaminergic neurons, and this loss was not exacerbated in animals with deletion of CX3CR1. To determine the mechanism by which CX3CR1affects inflammatory responses in α-syn-induced inflammation, phagocytosis was assessed using a fluorescent microsphere assay as well as by microglial uptake of aggregated α-syn. CX3CR1-/- microglia showed reduced uptake of fluorescent beads and aggregated α-syn.

Conclusion

Our results suggest that one mechanism by which CX3CR1-/- attenuates inflammation is at the level of phagocytosis of aggregated α-syn by microglia. These data implicate fractalkine signaling as a potential therapeutic target for regulating inflammatory response in α-syn models PD.     

Isolation and Growth Characteristics of an EDTA-degrading Member of the α-subclass of Proteobacteria

A Gram-negative, ethylenediaminetetraacetic acid (EDTA)-degrading bacterium (deposited at the German Culture Collection as strain DSM 9103) utilising EDTA as the only source of carbon, energy and nitrogen was isolated from a mixed EDTA-degrading population that was originally enriched in a column system from a mixture of activated sludge and soil. Chemotaxonomic analysis of quinones, polar lipids and fatty acids allowed allocation of the isolate to the alpha-subclass of Proteobacteria. 16S rDNA sequencing and phylogenetic analysis revealed highest similarity to the Mesorhizobium genus followed by the Aminobacter genus. However, the EDTA-degrading strain apparently forms a new branch within the Phyllobacteriaceae/Mesorhizobia family. Growth of the strain was rather slow not only on EDTA (micro(max) = 0.05 h(-1)) but also on other substrates. Classical substrate utilisation testing in batch culture suggested a quite restricted carbon source spectrum with only lactate, glutamate, and complexing agents chemically related to EDTA (nitrilotriacetate, iminodiacetate and ethylenediaminedisuccinate) supporting growth. However, when EDTA-limited continuous cultures of strain DSM 9103 were pulsed with fumarate, succinate, glucose or acetate, these substrates were assimilated immediately. Apparently, the strain can use a broader spectrum than indicated by traditional substrate testing techniques. The EDTA species CaEDTA and MgEDTA served as growth substrates of the strain because in the mineral medium employed EDTA was predicted to be mainly present in the form of these two complexes. The bacterium was not able to degrade Fe3+-complexed EDTA.     

Functionalization of fullerene via the Bingel reaction with α-chlorocarbanions: an ONIOM approach

Two-layer ONIOM method at the ONIOM(B3LYP/6-31G(d):PM6) level of theory was applied to study the cycloaddition reaction of α-chlorocarbanions (CR₂Cl^─, where R is H, Cl, CH₃ CN, and NO₂) and fullerene. The results show that the reaction pathways depend on the electron withdrawing functional groups or the electron donating functional groups contained in the α-chlorocarbanions. The energy profile analysis reveals that functionalization of fullerene by CCl₃ ^─, C(CH₃)₂Cl^─, and CH₂Cl^─ is more favorable than by C(CN)₂Cl^─ and C(NO₂)₂Cl^─ in terms of the thermodynamic point of view.     

Adrenergic innervation of the mouse heart revealed at an early stage using α-methylnoradrenaline

Summary The development of noradrenergic innervation was studied in the mouse heart using fluorescence histochemistry. Following incubation of hearts with -methylnoradrenaline fluorescent nerve fibres were seen as early as 13 days in utero. It is suggested that the neuronal uptake mechanism for noradrenaline is functional at an early stage.