Dopamine Receptor and Gα(olf) Expression in DYT1 Dystonia Mouse Models during Postnatal Development

Background

DYT1 dystonia is a heritable, early-onset generalized movement disorder caused by a GAG deletion (ΔGAG) in the DYT1 gene. Neuroimaging studies and studies using mouse models suggest that DYT1 dystonia is associated with dopamine imbalance. However, whether dopamine imbalance is key to DYT1 or other forms of dystonia continues to be debated.

Methodology/Principal Findings

We used Dyt1 knock out (Dyt1 KO), Dyt1 ΔGAG knock-in (Dyt1 KI), and transgenic mice carrying one copy of the human DYT1 wild type allele (DYT1 hWT) or human ΔGAG mutant allele (DYT1 hMT). D1R, D2R, and Gα(olf) protein expression was analyzed by western blot in the frontal cortex, caudate-putamen and ventral midbrain in young adult (postnatal day 60; P60) male mice from all four lines; and in the frontal cortex and caudate putamen in juvenile (postnatal day 14; P14) male mice from the Dyt1 KI and KO lines. Dopamine receptor and Gα(olf) protein expression were significantly decreased in multiple brain regions of Dyt1 KI and Dyt1 KO mice and not significantly altered in the DYT1 hMT or DYT1 hWT mice at P60. The only significant change at P14 was a decrease in D1R expression in the caudate-putamen of the Dyt1 KO mice.

Conclusion/Significance

We found significant decreases in key proteins in the dopaminergic system in multiple brain regions of Dyt1 KO and Dyt1 KI mouse lines at P60. Deletion of one copy of the Dyt1 gene (KO mice) produced the most pronounced effects. These data offer evidence that impaired dopamine receptor signaling may be an early and significant contributor to DYT1 dystonia pathophysiology.     

Comparison of the properties of concanavalin A and anti-α-d-glucose antibodies

Concanavalin A and anti-α-d-glucose antibodies form precipitin complexes with antigens havingα-d-glucose as terminal units. The sedimentation rates, molecular weights, gel electrophoretic mobilities, isoelectric points, and immunoglobulin type of Con A andα-Ab have been determined. The interactions of the compounds with antigens in the presence of potential inhibitors have been compared. The data show that the interaction of Con A with glucose units occurs with hydrogen bonding at hydroxyl groups at C1, 3,4, and 6 and van der Waals bonding at the pyranose ring oxygen. In theα-Ab complex with glucose units, in addition to the above bond types, a hydrogen bond at the hydroxyl at C2 occurs and this bond is essential for interaction.     

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.     

A kinetic study on the reductive opening of the diphenylmethylene acetal in methyl 2,3-O-diphenylmethylene-α-L-rhamnopyranoside

Reductive opening of the diphenylmethyl acetal in methyl 2,3-O-diphenylmethylene-α-l-rhamnopyranoside has been investigated by kinetic studies, and the results have been compared to those recently obtained by quantum chemical calculations. In contrast to the previous theoretical calculations which related only to the presumably rate limiting step of the reductive opening, the reaction system LiAlH(4), AlCl(3), and the title compound consists of at least four simultaneous reactions. Nevertheless, reasonable agreement can be found between the activation Gibbs free energy obtained from kinetic measurements and the theoretically calculated ones in spite of the experimental errors and the approximate nature of theoretical calculations.     

Microbial Baeyer–Villiger oxidation of 5α-steroids using Beauveria bassiana. A stereochemical requirement for the 11α-hydroxylation and the lactonization pathway

Beauveria bassiana KCH 1065, as was recently demonstrated, is unusual amongst fungal biocatalysts in that it converts C₁₉ 3-oxo-4-ene and 3β-hydroxy-5-ene as well as 3β-hydroxy-5α-saturated steroids to 11α-hydroxy ring-D lactones. The Baeyer–Villiger monooxygenase (BVMO) of this strain is distinguished from other enzymes catalyzing BVO of steroidal ketones by the fact that it oxidizes solely substrates with 11α-hydroxyl group. The current study using a series of 5α-saturated steroids (androsterone, 3α-androstanediol and androstanedione) has highlighted that a small change of the steroid structure can result in significant differences of the metabolic fate. It was found that the 3α-stereochemistry of hydroxyl group restricted “normal” binding orientation of the substrate within 11α-hydroxylase and, as a result, androsterone and 3α-androstanediol were converted into a mixture of 7β-, 11α- and 7α-hydroxy derivatives. Hydroxylation of androstanedione occurred only at the 11α-position, indicating that the 3-oxo group limits the alternative binding orientation of the substrate within the hydroxylase. Only androstanedione and 3α-androstanediol were metabolized to hydroxylactones. The study uniquely demonstrated preference for oxidation of equatorial (11α-, 7β-) hydroxyketones by BVMO from B. bassiana. The time course experiments suggested that the activity of 17β-HSD is a factor determining the amount of produced ring-D lactones. The obtained 11α-hydroxylactones underwent further transformations (oxy-red reactions) at C-3. During conversion of androstanedione, a minor dehydrogenation pathway was observed with generation of 11α,17β-dihydroxy-5α-androst-1-en-3-one. The introduction of C1C2 double bond has been recorded in B. bassiana for the first time.     

A novel small molecule 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose mimics the antiplatelet actions of insulin

Background

We have shown that 1,2,3,4,6-penta-O-galloyl-α-D-glucopyranose (α-PGG), an orally effective hypoglycemic small molecule, binds to insulin receptors and activates insulin-mediated glucose transport. Insulin has been shown to bind to its receptors on platelets and inhibit platelet activation. In this study we tested our hypothesis that if insulin possesses anti-platelet properties then insulin mimetic small molecules should mimic antiplatelet actions of insulin.

Principal Findings

Incubation of human platelets with insulin or α-PGG induced phosphorylation of insulin receptors and IRS-1 and blocked ADP or collagen induced aggregation. Pre-treatment of platelets with α-PGG inhibited thrombin-induced release of P-selectin, secretion of ATP and aggregation. Addition of ADP or thrombin to platelets significantly decreased the basal cyclic AMP levels. Pre-incubation of platelets with α-PGG blocked ADP or thrombin induced decrease in platelet cyclic AMP levels but did not alter the basal or PGE₁ induced increase in cAMP levels. Addition of α-PGG to platelets blocked agonist induced rise in platelet cytosolic calcium and phosphorylation of Akt. Administration of α-PGG (20 mg kg⁻¹) to wild type mice blocked ex vivo platelet aggregation induced by ADP or collagen.

Conclusions

These data suggest that α-PGG inhibits platelet activation, at least in part, by inducing phosphorylation of insulin receptors leading to inhibition of agonist induced: (a) decrease in cyclic AMP; (b) rise in cytosolic calcium; and (c) phosphorylation of Akt. These findings taken together with our earlier reports that α-PGG mimics insulin signaling suggest that inhibition of platelet activation by α-PGG mimics antiplatelet actions of insulin.     

Computer modelling studies on the modes of binding of some of theα-glycosidically linked disaccharides to concanavalin A

The possible modes of binding of kojibiose, nigerose, maltose and ManPα(1 → 2)Man to concanavalin A have been investigated using computer modelling studies. While α12 linked disaccharides bind to concanavalin A in two modes,i.e. by placing the reducing as well as non-reducing sugar units in the sugar binding site, nigerose or maltose can bind only in one mode,i.e. by placing the non-reducing sugar unit in the binding site. Though, both the sugar residues in α 12 linked disaccharides can reach the binding site, the preference is high for the non-reducing unit. When the non-reducing residue, in any of these disaccharides, enters the binding site, the allowed orientations and the possible hydrogen bonds with the protein seem to be independent of the glycosidic linkage. However, the number of hydrogen bonds the outward sugar residue forms with the protein are dependent on the type of linkage. Atleast one of the hydroxyl groups adjacent to the glycosidic linkage on the outward sugar residue is involved in the formation of a hydrogen bond with the protein suggesting the presence of an extended binding site. The orientation of the reducing sugar residue in the extended binding site is dependent on the linkage. Its orientation in nigerose is flipped when compared to that found in kojibiose or maltose leading to different non-covalent interactions with the protein which affect their binding affinities.     

α-Gustducin immunoreactivity in the airways

The G-protein subunit -gustducin is a marker of chemoreceptive cells. In the present study, we examined the immunohistochemical localization of -gustducin in rat airway epithelium both by light and electron microscopy. -Gustducin immunoreactivity was found in solitary cells that presented ultrastructural features of chemoreceptor cells, i.e. flask-shaped or pear-shaped, with an apical process with thin microvilli protruding into the lumen. The immunostaining was mainly concentrated in the apical process and along the basolateral cell surface. To investigate whether -gustducin-immunoreactive cells represented a distinct cell subset in rat airways, we performed double-label immunocytochemistry with antibodies to protein gene groduct (PGP) 9.5, a marker of neuroendocrine cells, and to phospholipase C beta2 (PLC2), a component of the bitter signalling pathway. -Gustducin-immunoreactive cells were present in a subset of PGP-9.5-immunoreactive elements, although not all -gustducin-positive cells expressed PGP 9.5 labelling. In addition, a subset of -gustducin-expressing cells colocalized PLC2. This work thus demonstrates that solitary -gustducin-immunoreactive cells exist throughout the airways and represent a specialized cell type with morphological and immunohistochemical characteristics of chemoreceptor cells.     

A route from d-galactose to the aggregation pheromone component (-)-α-multistriatin

Oxidation of 3,6-di-O-benzyl-1,2-O-isopropylidene-α-d-glucofuranose with pyridinium chlorochromate in the presence of molecular sieves, followed by conversion into the p-tolylsulfonylhydrazone, addition of methyl phenylphosphinate, and reduction with sodium borohydride, provided the key intermediate, namely, 5(R,S)-3,6-di-O-benzyl-5-deoxy-1,2-O-isopropylidene-5-C-[(methoxy)phenylphosphinyl]-α-d-xylo-hexofuranose, in 23% overall yield. Treatment of this compound with sodium dihydrobis(2-methoxyethoxy)aluminate, followed by the action of mineral acid and acetic anhydride, yielded the crystalline title compound, the structure of which was established on the basis of mass and 400-MHz, ¹H-n.m.r. spectra. A general dependence of ²J_PH values on the OPCH dihedral angles effectively served for assigning the configuration of C-1, C-5, and the ring-phosphorus atom of the present product and other such 5-C-phosphinylhexopyranoses.     

Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-α

Cytosolic phospholipase A₂-α (cPLA₂) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA₂ is dependent on a rise in cytosolic Ca²⁺ concentration, membrane association via the Ca²⁺-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA₂ via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP₂), including the role of a “pleckstrin homology (PH)-like” region of cPLA₂ (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate + the Ca²⁺ ionophore, A23187, or epidermal growth factor + A23187, expression of the PH domain of phospholipase C-δ1 (which sequesters membrane PIP₂) attenuated cPLA₂ activity. Stimulated cPLA₂ activity was also attenuated by the expression of cPLA₂ 135-366, or cPLA₂ 2-366, and expression of a PIP₂-specific 5′-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP₂, with high affinity, only cPLA₂ 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA₂s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA₂ activity can be modulated by sequestration or depletion of cellular PIP₂, although the interaction of cPLA₂ with membrane PIP₂ appears to be indirect, or of weak affinity.     

Glycoproteogenomics: A Frequent Gene Polymorphism Affects the Glycosylation Pattern of the Human Serum Fetuin/α-2-HS-Glycoprotein

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Highlights

• •Frequent genetic polymorphism affects the glycosylation pattern of fetuin.
• •Personalized in-depth proteoform profiling of fetuin purified from 20 donors.
• •Classification of serum donors into three different genotypes.
• •Septic patients show increased level of fucosylation at N-glycolation site N176.

     

Propylbenzmethylation at Val-1(α) markedly increases the tetramer stability of the PEGylated hemoglobin: A comparison with propylation at Val-1(α)

Background

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are potential pharmaceutical agents that can be used in surgery or emergency medicine. PEGylation can modulate the vasoactivity of Hb and is a widely used approach to develop HBOCs. However, PEGylation can significantly enhance the tetramer–dimer dissociation of Hb, which may perturb the structure of Hb and increase its observed adverse effect. Thus, it is necessary to increase the tetramer stability of the PEGylated Hb.

Methods

Propylbenzmethylation at Val-1(α) of HbA was carried out to stabilize the Hb tetramer. The propylbenzmethylated Hb at Val-1(α) (PrB-Hb) was used as the starting material for site-specific PEGylation at Cys-93(β) of Hb using maleimide PEG. Structural and functional properties, autoxidation rate and thermal stability of the resultant product (PEG-PrB-Hb) were measured.

Results

Propylbenzmethylation at Val-1(α) led to 25-fold and 24-fold decreases in the tetramer–dimer dissociation constant of HbA and PEG-Hb, respectively. The increased tetramer stability is due to the enhanced hydrophobicity of the area around Val-1(α) and the increased polar interaction of Hb upon propylbenzmethylation. Thus, the structural and functional properties of PEG-Hb were improved, and its autoxidation rate and thermal denaturation were decreased.

Conclusion

Propylbenzmethylation at Val-1(α) showed higher ability than propylation at Val-1(α) to improve the structural and functional properties and decrease the side effect of PEG-Hb.

General significance

Our study can facilitate the biotechnological development of stable PEGylated Hb as more advanced HBOC. Our study is also expected to improve the stability of the tetrameric or dimeric proteins (e.g., uric oxidase) by propylbenzmethylation at their N-terminus.     

Metabolism of 17α-methyl-5α-dihydrotestosterone in the rabbit

17α-Methyl-5α-dihydrotestosterone and the reduced metabolites, 17α-methyl-5α-androstane-3α, 17β-diol and -3β, 17β-diol together with two hydroxylated metabolites, 17α-methyl-5α-androstane-3β, 15α, 17β-triol and 17α-methyl-5α-androstane-3α, 6α, 17β-triol were isolated and identified in the urine of rabbits orally dosed with 17α-methyl-5α-dihydrotestosterone. Formation of the C-6 hydroxylated derivative demonstrates that the 4,6-enolization of a 4-en-3-one is not a necessary requirement for hydroxylation at C-6 of the androstane nucleus in the rabbit. No evidence was obtained for the presence of 17α-methyl/17β-hydroxyl epimerization.     

5-α-androst-16en-3α-on: A male pheromone? A brief report

Olfactory cues play a prominent, yet underestimated role in shaping emotional attitudes towards conspecifics. Among humans olfactory cues can have effects on behavior. In a rating study (n = 290) females rated the smell of androstenone. The emotional reaction to androstenone changed during the menstrual cycle. Females rated the main component of male body odor unattractive. This changed to a neutral emotional response at the conceptive optimum around ovulation. The finding has direct consequences for hypotheses concerning the evolutionary loss of estrus. It is suggested that the cyclic-dependent emotional rating of androstenone may facilitate active female choice of sex partners and may be a proximate cue for female mate-choice.     

A 1-step microplate method for assessing the substrate range of l-α-amino acid aminotransferase

Aminotransferase enzymes catalyse the reversible substitution of a keto group for an amino group. While this reaction is highly stereoselective with respect to the amino group, each enzyme can usually catalyse the turnover of a number of different substrates. As the substrate range cannot be inferred from the sequence, it remains an early bottleneck when selecting an enzyme for a biocatalysis application. We have developed a simple first round characterisation method applicable to the broad range of aminotransferases that accept l-glutamate, the central junction of cellular transamination, as one of the amino donors. The assay is based on l-glutamate detection by its highly specific dehydrogenase enzyme in a coupled assay, ending in the reduction of the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-tetrazolium-5-carboxanilide (XTT). While products of most tetrazolium salts are water-insoluble, XTT is reduced to a water soluble colored formazan, allowing direct spectrophotometric detection. The reaction is carried out in microplate format using a single endpoint measurement and is thus suitable for automation.The setup was tested with 7 aminotransferase enzymes: Escherichia coli branched chain amino acid aminotransferase, Pseudomonas aeruginosa and Klebsiella pneumoniae aromatic amino acid AT, Bacillus subtilis histidinol-phosphate AT, and Thermus aquaticus aspartate, serine and histidinol-phosphate AT. In addition to 17 of the 20 proteinogenic amino acids, 32 alternative substrates were tested.