Characteristics of the formation of a free-amino acid brain pool in rats differing in their ethanol consumption

The concentrations of free amino acids were studied in the right and left hemispheres, cerebellum and brain stem of AA and ANA strain rats differing in their voluntary consumption of ethanol solutions. Animals of both the strains were shown to be differentiated by distribution, functional utilization and metabolism of some amino acids in the right and left hemispheres, cerebellum and brain stem.     

D-amino acids in the cell pool of bacteria

About 30 different bacterial species were tested for the possible presence of freed-amino acids in their cell pool. Gram-positive bacteria particularly the species of the genusBacillus have a fairly large pool of freely extractabled-amino acids. Varied quantities of freed-amino acids were detected inBacillus subtilis B₃,Bacillus subtilis Marburg,Bacillus licheniformis, Bacillus brevis, Bacillus stearothermophilus, Lactobacillus fermenti, Lactobacillus delbrueckii, Staphylococcus aureus andClostridium acetobutylicum. The individual components ofd-amino acids were identified in 5Bacillus species referred to above,d-alanine is the major component; the otherd-amino acids identified are aspartic acid, glutamic acid, histidine, leucines, proline, serine and tyrosine. Thed-amino acid pool size inBacillus subtilis B₃ varies with different culture conditions. The pool size is maximum when growth temperature is 30°C and it fluctuates with change in pH of the medium. The maximum quantity ofd-amino acids could be recovered when the culture was at mid log phase. O₂ supply to the medium has little effect ond-amino acid pool size. The starvation of cells leads to depletion of thed-amino acid pool which is exhausted almost completely within 4 hours by incubation in nutrient-free medium.     

Lysosomal labilization

The lysosomal compartment is the place for cellular degradation of endocytosed and autophagocytosed material and a center for normal turnover of organelles as well as most long-lived proteins. Lysosomes were long considered stable structures that broke and released their many hydrolytic enzymes only following necrotic cell death. It is now realized that lysosomes instead are quite vulnerable, although in a heterogeneous way. Their exposure to a number of events, such as oxidative stress, lysosomotropic detergents and aldhydes, as well as overexpression of the p53 protein, causes time-and-dose-dependent lysosomal rupture that is followed by apoptosis or necrosis. Partial lysosomal rupture has often been found to be an early upstream event in apoptosis, while necrosis results from fulminant lysosomal rupture. Consequently, factors influencing the stability of lysosomes, for instance their content of labile and redox-active iron, seem to be essential for the survival of cells.     

A significant pool of amino acids is adsorbed on blood cell membranes

It is well known that the amino acids in the blood are distributed between the plasma and inside the cells. This study was conducted to determine whether amino acids can be located adsorbed on blood cell membranes. The amino acid concentration in the deproteinized haemolysed blood was higher than that in the fraction of blood after removal of the blood cell membranes by centrifugation. These results showed that a pool of amino acids representing 21.1% of the whole blood cell amino acids was adsorbed on the blood cell membranes of adult Wistar rats. The non-polar amino acids showed high adsorption on the membrane, whereas out of the polar amino acid group, only the non-ionic amino acids did adsorb.Bioquimica i Biologia Molecular. Dept. de Biologia Fonamental i Ciencies de la Salut.     

Lysosomal carboxypeptidase A

Lysosomal carboxypeptidase A (cathepsin A) is synthetized in the form of preproenzyme, which undergoes to active enzyme as a result of post-translational modification. It splits off C-terminal amino acid residues from peptides and proteins and synergizes with other proteases in degradation of cellular proteins in lysosomes. Lysosomal carboxypeptidase A has an effect on peptide hormones and peptides of biological activity of tissues and body fluids as well. It forms complexes with some glycosidases that protects them against proteolytic degradation. Deficiency of this enzyme induces storage diseases. Lysosomal carboxypeptidase A as multifunctional enzyme plays an important regulatory role in organismal metabolism.     

Lysosomal ROS formation

Ubiquinone is inhomogenously distributed in subcellular biomembranes. Apart from mitochondria, where ubiquinone has bioenergetic and pathophysiological functions, unusually high levels of ubiquinone have also been reported in Golgi vesicles and lysosomes. In lysosomes, the interior differs from other organelles in its low pH value which is important to ensure optimal activity of hydrolytic enzymes. Since redox-cycling of ubiquinone is associated with the acceptance and release of protons, we assumed that ubiquinone is part of a redox chain contributing to unilateral proton distribution. A similar function of ubiquinone was earlier suggested by Crane to operate in Golgi vesicles. Support for the involvement of ubiquinone in a presumed couple of redox carriers came from our observation that almost 70% of total lysosomal ubiquinone was in the divalently reduced state. Further reduction was seen in the presence of external NADH. Analysis of the components involved in the transfer of reducing equivalents from cytosolic NADH to ubiquinone revealed the existence of an FAD-containing NADH dehydrogenase. The latter was found to reduce ubiquinone by means of a b-type cytochrome. Proton translocation into the interior was linked to the activity of the novel lysosomal redox chain. Oxygen was found to be the terminal electron acceptor, thereby also regulating acidification of the lysosomal matrix. In contrast to mitochondrial respiration, oxygen was only trivalently reduced giving rise to the release of HO radicals. The role of this novel proton-pumping redox chain and the significance of the associated ROS formation has to be elucidated.     

Lysosomal involvement in apoptosis

Abstract

The involvement of reactive oxygen species (ROS) in the induction of DNA damage to Escherichia coli cells caused by UVC (254 nm) irradiation was studied. We verified the expression of the soxS gene induced by UVC (254 nm) and its inhibition by sodium azide, a singlet oxygen (¹O₂) scavenger. Additional results showed that a water-soluble carotenoid (norbixin) protects against the lethal effects of UVC. These results suggest that UVC radiation can also cause ROS-mediated lethality.     

Lysosomal glycosidases and glycoproteinoses

The development of chemicall, physical and enzymatic methods lead to the determination of numerous structures of glycoprotein glycans and allowed to classify them into "structural families". On the basis of this knowledge, it has been possible, 1) to demonstrate that the oligosaccharides and glycoasparagines accumulating in tissues and urines of patients with diseases characterized by a lack in lysosomal glycosidases originate from glycoprotein glycans incompletely catabolized; 2) to propose a scheme for the normal and pathological catabolism of glycoproteins and 3) to elucidate the problem of the origin of lysosomal glycosidases. These latter are internalized into the lysosomes either through a mechanism of secretion-reuptake, or by following an intracellular traffic, or via the cell plasma membrane. In al cases, membrane receptors intervene which specifically recognize phosphorylated oligomannosidic structures carried by the acidic hydrolases.     

Lysosomal dysfunction in neurodegeneration

Neuronal homeostasis and survival critically depend on an efficient autophagy-lysosomal degradation pathway, especially since neurons cannot reduce the concentration of misfolded proteins and damaged organelles by cell division. While increasing evidence implicates lysosomal dysfunction in the pathogenesis of neurodegenerative disorders, the molecular underpinnings of the role of lysosomes in neurodegeneration remain largely unknown. To this end, studies of neurodegenerative disorders caused by mutations in lysosomal proteins offer an opportunity to elucidate such mechanisms and potentially identify specific therapeutic targets. One of these disorders is Kufor-Rakeb syndrome, caused by mutations in the lysosomal protein ATP13A2/PARK9 and characterized by early-onset Parkinsonism, pyramidal degeneration and dementia. We found that loss of ATP13A2 function results in impaired lysosomal function and, consequently, accumulation of SNCA/α-synuclein and neurotoxicity. Our results suggest that targeting of ATP13A2 to lysosomes to enhance lysosomal function may result in neuroprotection in Kufor-Rakeb syndrome. From a broader perspective, these findings, together with other recent studies of lysosomal dysfunction in neurodegeneration, suggest that strategies to upregulate lysosomal function in neurons represent a promising therapeutic approach for neurodegenerative disorders.     

Lysosomal Lipid Storage Diseases

Lysosomal lipid storage diseases, or lipidoses, are inherited metabolic disorders in which typically lipids accumulate in cells and tissues. Complex lipids, such as glycosphingolipids, are constitutively degraded within the endolysosomal system by soluble hydrolytic enzymes with the help of lipid binding proteins in a sequential manner. Because of a functionally impaired hydrolase or auxiliary protein, their lipid substrates cannot be degraded, accumulate in the lysosome, and slowly spread to other intracellular membranes. In Niemann-Pick type C disease, cholesterol transport is impaired and unesterified cholesterol accumulates in the late endosome. In most lysosomal lipid storage diseases, the accumulation of one or few lipids leads to the coprecipitation of other hydrophobic substances in the endolysosomal system, such as lipids and proteins, causing a “traffic jam.” This can impair lysosomal function, such as delivery of nutrients through the endolysosomal system, leading to a state of cellular starvation. Therapeutic approaches are currently restricted to mild forms of diseases with significant residual catabolic activities and without brain involvement.Lysosomal lipid storage diseases are a group of inherited catabolic disorders in which typically large amounts of complex lipids accumulate in cells and tissues. Macromolecules such as complex lipids and oligosaccharides are constitutively degraded in the acidic compartments of the cell, the endosomes, and lysosomes, into their building blocks. The resulting catabolites are exported to the cytosol and reused in cellular metabolism. When lysosomal function is impaired because of a defect in a catabolic step, degradation cannot proceed normally and undegraded compounds accumulate. Lysosomal lipid storage diseases comprise mainly the sphingolipidoses, Niemann-Pick type C disease (NPC), and Wolman disease, including the less severe form of this disease, called cholesteryl ester storage. NPC is a complex lipid storage disease mainly characterized by the accumulation of unesterified cholesterol in the late endosomal/lysosomal compartment (Bi and Liao 2010). The sphingolipidoses are caused by defects in genes encoding proteins involved in the lysosomal degradation of sphingolipids (Kolter and Sandhoff 2006). First reports on these diseases were given more than a century ago. Already in 1881, Warren Tay described the clinical symptoms of a disease, which is today called Tay-Sachs disease (Tay 1881). After Christian de Duve discovered the lysosome in 1955 (de Duve 2005), Henri-Géry Hers established the first correlation between an enzyme deficiency and a lysosomal storage disorder (Pompe’s disease) in 1963 (Hers 1963). In the following decades, the enzymes and cofactors deficient in the sphingolipidoses have been identified. Though lysosomal lipid storage diseases have been known for a long time, treatment is only available for a few mild forms of the diseases, such as the adult forms of Gaucher disease (Barton et al. 1991). For several lysosomal storage diseases, therapies like enzyme replacement or bone marrow transplantation are in the clinical trial stage (Platt and Lachmann 2009). For a long time, lysosomal diseases have been considered a problem of superabundance (storage) in which the storage material can slowly spread to other cellular membranes, impairing their function. More recently, it came into focus that massive storage prevents lysosomal functions such as nutrition delivery through the endolysosomal system, leading to a state of cellular starvation. In mouse models of both GM1 and GM2 gangliosidoses iron is progressively depleted in brain tissue. Administration of iron prolonged survival in the diseased mice by up to 38% (Jeyakumar et al. 2009).     

A physical-chemical model for cellular uptake of fatty acids: prediction of intracellular pool sizes

If the uptake of fatty acids by liver is a physical, not a biological, process, then the size and location of the intrahepatic pool of fatty acids can be predicted from uptake rates and thermodynamic data. The purpose of the experiments in this paper was to test the accuracy of this idea. Rat livers were perfused with palmitate bound to albumin, and the total amounts of palmitate removed from the perfusate were measured at 3-s intervals. The intrahepatic pools of palmitate calculated from these data were 13.8 and 23.0 nmol/g of liver at ratios of palmitate/albumin (mol/mol) (afferent side) of 2/1 and 4/1, respectively, in the steady state. The intrahepatic pools of palmitate calculated from the distributions of palmitate between membranes, H2O, albumin, and fatty acid binding protein and the measured first-order rate constants for acyl-CoA ligases in mitochondria and microsomes were 12.1 and 34.6 nmol/g for perfusate ratios of palmitate/albumin of 2/1 and 4/1, in the steady state. Intrahepatic pools of palmitate measured after establishment of a steady-state rate of uptake were 15.0 and 31.8 nmol/g for these ratios of palmitate/albumin of 2/1 and 4/1.