The Structural Basis for the Susceptibility of Gangliosides to Enzymatic Degradation

The conformational properties of GM2, GalNac-4(Neu5Ac-3) Gal-4Glc-1Cer have been compared to those of 6-GM2, in which the linkage between the GalNAc and Gal was altered from GalNac-4Gal- to GalNac-6Gal-, and to those of GD1a, Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-3)Gal-4Glc-1Cer, and GalNAc-GD1a.Our results revealed that unlike the compact and rigid oligosaccharide head group found in GM2, where the Neu5Ac and the GalNAc residues interact, the sugar chain of 6-GM2 is in an open spatial arrangement, with the Neu5Ac no longer interacting with GalNAc, freely accessible to external interactions.The structure of GD1a can be regarded as that of GM2 with an extension of the terminal Neu5Ac-3Gal-disaccharide. The inner portion of GD1a is that of GM2 comprising the very rigid GalNAc-[Neu5Ac-]Gal trisaccharide. The terminal Neu5Ac-Gal linkage is flexible and fluctuates between two limiting conformations. In GalNAc-GD1a the outer sialic acid gains conformational rigidity due to the presence of the outer GalNAc in position 4 of galactose. This ganglioside has two core GalNAc-[Neu5Ac-]Gal trisaccharide linked in tandem.     

Pig kidney dopa decarboxylase: inactivation by iodoacetamide and sequence of the carboxyamidomethylcysteine-containing peptide

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by iodoacetamide following pseudo-first order reaction kinetics. The apparent first order rate constant for inactivation is proportional to the concentration of iodoacetamide and a second order rate constant of 37 M-1 min-1 is obtained at pH 6.8 and 25 degrees C. Cyanogen bromide fragmentation of iodo(1-14C)acetamide - modified inactivated Dopa decarboxylase followed by trypsin digestion yields a single radioactive peptide. Automated Edman degradation reveals a heptapeptide sequence which contains labeled carboxyamidomethylcysteine. This finding and the results of the incorporation of the label from ido (1-14C)acetamide into the enzyme clearly indicate that the modification of 1 mol of SH per mol of enzyme dimer is responsible for the inactivation process. The labeled peptide, which was located by means of limited proteolysis on the fragment corresponding to the COOH-terminal third of the enzyme, has been aligned with a 7 amino acid stretch of Drosophila enzyme. Although this region appears highly conserved in the Dopa decarboxylase enzymes, the cysteinyl residue is not conserved. This observation together with the spectral binding properties of the iodoacetamide inactivated enzyme argue against a functional role for the modifiable cysteine in the mechanism of action of pig kidney enzyme. It is suggested that the loss of pig kidney decarboxylase activity produced by iodoacetamide modification might be attributable to steric hindrance. This could be due to the presence of the bulky acetamidic group on a cysteine residue at, or near, the active center or in a site of strategic importance to the maintenance of the active site topography.     

Systemic Administration of NMDA and AMPA Receptor Antagonists Reverses the Neurochemical Changes Induced by Nigrostriatal Denervation in Basal Ganglia

In Parkinson's disease, nigrostriatal denervation leads to an overactivity of the subthalamic nucleus and its target areas, which is responsible of the clinical manifestations of the disease. Because the subthalamic nucleus uses glutamate as neurotransmitter and is innervated by glutamatergic fibers, pharmacological blockade of glutamate transmission might be expected to restore the cascade of neurochemical changes induced by a dopaminergic denervation within the basal ganglia. To test this hypothesis, two types of glutamate antagonists, the NMDA receptor antagonist MK-801 and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist LY293558, were administered systemically, either alone or in combination with L-DOPA, in rats with a unilateral 6-hydroxydopamine lesion of the nigrostriatal dopamine pathway. The effect of treatment was assessed neurochemically by analyzing at the cellular level the functional activity of basal ganglia output structures and the subthalamic nucleus using the expression levels of the mRNAs coding for glutamic acid decarboxylase and cytochrome oxidase, respectively, as molecular markers of neuronal activity. The present study shows that treatment with glutamate antagonists, and particularly with AMPA antagonists, alone or in combination with L-DOPA, reverses the overactivity of the subthalamic nucleus and its target areas induced by nigrostriatal denervation. These results furnish the neurochemical basis for the potential use of glutamate antagonists as therapeutic agents in Parkinson's disease.     

C3a and C3b activation products of the third component of complement (C3) are critical for normal liver recovery after toxic injury

Although the complement system has been implicated in liver regeneration after toxic injury and partial hepatectomy, the mechanism or mechanisms through which it participates in these processes remains ill-defined. In this study, we demonstrate that complement activation products (C3a, C3b/iC3b) are generated in the serum of experimental mice after CCl(4) injection and that complement activation is required for normal liver regeneration. Decomplementation by cobra venom factor resulted in impaired entry of hepatocytes into S phase of the cell cycle. In addition, livers from C3-deficient (C3(-/-)) mice showed similarly impaired proliferation of hepatocytes, along with delayed kinetics of both hepatocyte hyperplasia and removal of injured liver parenchyma. Restoration of hepatocyte proliferative capabilities of C3(-/-) mice through C3a reconstitution, as well as the impaired regeneration of C3a receptor-deficient mice, demonstrated that C3a promotes liver cell proliferation via the C3a receptor. These findings, together with data showing two waves of complement activation, indicate that C3 activation is a pivotal mechanism for liver regeneration after CCl(4) injury, which fulfills multiple roles; C3a generated early after toxin injection is relevant during the priming of hepatocytes, whereas C3 activation at later times after CCl(4) treatment contributes to the clearance of injured tissue.     

Transgenic expression of Cre recombinase from the tyrosine hydroxylase locus

Catecholaminergic neurons are affected in several neurological and psychiatric diseases. Tyrosine hydroxylase (TH) is the first, rate-limiting enzyme in catecholamine synthesis. We report a knockin mouse expressing Cre-recombinase from the 3'-untranslated region of the endogenous Th gene by means of an internal ribosomal entry sequence (IRES). The resulting Cre expression matches the normal pattern of TH expression, while the pattern and level of TH are not altered in the knockin mouse. Crossings with two different LacZ reporter mice demonstrated Cre-mediated genomic recombination in TH expressing tissues. In addition, LacZ was found in some unexpected cell populations (including oocytes), indicating recombination due to transient developmental TH expression. Our novel knockin mouse can be used for generation of tissue-specific or general knockouts (depending on scheme of crossing) in mice carrying genes flanked by loxP sites. This knockin mouse can also be used for tracing cell lineages expressing TH during development.     

Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).     

Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.     

Tubulin must be acetylated in order to form a complex with membrane Na+,K+-ATPase and to inhibit its enzyme activity

In cells of neural and non-neural origin, tubulin forms a complex with plasma membrane Na⁺,K⁺-ATPase, resulting in inhibition of the enzyme activity. When cells are treated with 1 mM L-glutamate, the complex is dissociated and enzyme activity is restored. Now, we found that in CAD cells, ATPase is not activated by L-glutamate and tubulin/ATPase complex is not present in membranes. By investigating the causes for this characteristic, we found that tubulin must be acetylated in order to associate with ATPase and to inhibit its catalytic activity. In CAD cells, the acetylated tubulin isotype is absent. Treatment of CAD cells with deacetylase inhibitors (trichostatin A or tubacin) caused appearance of acetylated tubulin, formation of tubulin/ATPase complex, and reduction of membrane ATPase activity. In these treated cells, addition of 1 mM L-glutamate dissociated the complex and restored the enzyme activity. Cytosolic tubulin from trichostatin A-treated but not from non-treated cells inhibited ATPase activity. These findings indicate that the acetylated isotype of tubulin is required for interaction with membrane Na⁺,K⁺-ATPase and consequent inhibition of enzyme activity.