The dual face of endogenous alpha-aminoketones: pro-oxidizing metabolic weapons

Amino metabolites with potential prooxidant properties, particularly alpha-aminocarbonyls, are the focus of this review. Among them we emphasize 5-aminolevulinic acid (a heme precursor formed from succinyl-CoA and glycine), aminoacetone (a threonine and glycine metabolite), and hexosamines and hexosimines, formed by Schiff condensation of hexoses with basic amino acid residues of proteins. All these metabolites were shown, in vitro, to undergo enolization and subsequent aerobic oxidation, yielding oxyradicals and highly cyto- and genotoxic alpha-oxoaldehydes. Their metabolic roles in health and disease are examined here and compared in humans and experimental animals, including rats, quail, and octopus. In the past two decades, we have concentrated on two endogenous alpha-aminoketones: (i) 5-aminolevulinic acid (ALA), accumulated in acquired (e.g., lead poisoning) and inborn (e.g., intermittent acute porphyria) porphyric disorders, and (ii) aminoacetone (AA), putatively overproduced in diabetes mellitus and cri-du-chat syndrome. ALA and AA have been implicated as contributing sources of oxyradicals and oxidative stress in these diseases. The end product of ALA oxidation, 4,5-dioxovaleric acid (DOVA), is able to alkylate DNA guanine moieties, promote protein cross-linking, and damage GABAergic receptors of rat brain synaptosome preparations. In turn, methylglyoxal (MG), the end product of AA oxidation, is also highly cytotoxic and able to release iron from ferritin and copper from ceruloplasmin, and to aggregate proteins. This review covers chemical and biochemical aspects of these alpha-aminoketones and their putative roles in the oxidative stress associated with porphyrias, tyrosinosis, diabetes, and cri-du-chat. In addition, we comment briefly on a side prooxidant behaviour of hexosamines, that are known to constitute building blocks of several glycoproteins and to be involved in Schiff base-mediated enzymatic reactions.     

Cutaneous hypersensitivity induced in dogs and guinea-pigs by extracts of the tick Rhipicephalus sanguineus (Acari: Ixodidae)

The cutaneous hypersensitivity induced by Rhipicephalus sanguineus tick extract in dogs (natural host) and guinea-pigs (laboratory host) was evaluated. The left ear of infested and control (tick-bite naive) dogs and guinea-pigs was injected intradermally with an extract from unfed adult ticks and the right ear with phosphate buffered saline (PBS). Ear thickness variations were then measured after 10 min and 1, 2, 6, 18, 24, 48, 72 and 96 h post-injection. Results were expressed as percentual changes in the ear thickness in relation to pre-inoculation values. The final variation in ear thickness induced by the extract was given by subtracting, in each animal, the right ear percentual increase from that of the left ear. Guinea-pigs were tested at two different times following infestation and with two different doses of extract. Infested guinea-pigs from the three experiments developed an immediate (within the first 2 h post-inoculation) and a strong delayed reaction (24 h) to the extract. Dogs, unlike guinea-pigs, developed only a strong immediate reaction whereby an 80% increase in ear thickness was observed. Control animals, with the exception of one dog, did not develop any significant reaction to the extract. Only mild reactions were induced by PBS in the right ear of all animals. The correlation between the absence of a strong delayed type reaction to tick extract and the lack of resistance of the natural host to R. sanguineus tick is discussed.     

Calcium-dependent mitochondrial oxidative damage promoted by 5-aminolevulinic acid

Swelling of isolated rat liver mitochondria is shown to be induced by metal-catalyzed 5-aminolevulinic acid (ALA) aerobic oxidation, a putative endogenous source of reactive species (ROS), at concentrations as low as 50–100 μM. In this concentration range, ALA is estimated to occur in the liver of acute intermittent porhyria patients. Removal of Ca²⁺ (10 μM) from the suspension of isolated rat liver mitochondria by added EGTA abolishes both the ALA-induced transmembrane-potential collapse and mitochondrial swelling. Prevention of the ALA-induced swellling by addition of ruthenium red prior to mitochondrial energization by succinate demonstrates the deleterious involvement of internal Ca²⁺. Addition of MgCl₂ at concentrations higher than 2.5 mM, prevents the ALA-induced mitochondrial swelling, transmembrane potential collapse and Ca²⁺ efflux. This indicates that Mg²⁺ protects against the mitochondrial damage promoted by ALA-generated ROS. The ALA-induced mitochondrial damage might be a key event in the liver mitochondrial damage of acute intermittent porphyria patients reported elsewhere.     

The structural origin and biological function of pH-sensitivity in firefly luciferases.

Firefly luciferases are called pH-sensitive because their bioluminescence spectra display a typical red-shift at acidic pH, higher temperatures, and in the presence of heavy metal cations, whereas other beetle luciferases (click beetles and railroadworms) do not, and for this reason they are called pH-insensitive. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. This subject is revised in view of recent results. Some substitutions of amino-acid residues influencing pH-sensitivity in firefly luciferases have been identified. Sequence comparison, site-directed mutagenesis and modeling studies have shown a set of residues differing between pH-sensitive and pH-insensitive luciferases which affect bioluminescence colors. Some substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). A network of hydrogen bonds and salt bridges involving the residues N229-S284-E311-R337 was found to be important for affecting bioluminescence colors. It is suggested that these structural elements may affect the benzothiazolyl side of the luciferin-binding site affecting bioluminescence colors. Experimental evidence suggest that the residual red light emission in pH-sensitive luciferases could be a vestige that may have biological importance in some firefly species. Furthermore, the potential utility of pH-sensitivity for intracellular biosensing applications is considered.     

Dynamic length regulation of sensory stereocilia

Stereocilia, the mechanosensory organelles of hair cells, are a distinctive class of actin-based cellular protrusions with an unparalleled ability to regulate their lengths over time. Studies on actin turnover in stereocilia, as well as the identification of several deafness-related proteins essential for proper stereocilia structure and function, provide new insights into the mechanisms and molecules involved in stereocilia length regulation and long-term maintenance. Comparisons of ongoing investigations on stereocilia with studies on other actin protrusions offer new opportunities to further understand common principles for length regulation, the diversity of its mechanisms, and how the specific needs of each cell are met.     

Protein localization by actin treadmilling and molecular motors regulates stereocilia shape and treadmilling rate

We present a physical model that describes the active localization of actin-regulating proteins inside stereocilia during steady-state conditions. The mechanism of localization is through the interplay of free diffusion and directed motion, which is driven by coupling to the treadmilling actin filaments and to myosin motors that move along the actin filaments. The resulting localization of both the molecular motors and their cargo is calculated, and is found to have an exponential (or steeper) profile. This localization can be at the base (driven by actin retrograde flow and minus-end myosin motors), or at the stereocilia tip (driven by plus-end myosin motors). The localization of proteins that influence the actin depolymerization and polymerization rates allow us to describe the narrow shape of the stereocilia base, and the observed increase of the actin polymerization rate with the stereocilia height.     

Morphological characterization of the ovary and oocytes vitellogenesis of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

This study presents the morphology of the ovary, as well as the process of the vitellogenesis in oocytes of the tick Rhipicephalus sanguineus. The ovary of these individuals is of the panoistic type; therefore, it lacks nurse cells. This organ consists of a single tubular structure, continuous, and composed of a wall formed by small epithelial cells with rounded nuclei which delimit the lumen. The oocytes in the different developmental stages in this tick species were classified into five stages (I-V). They remain attached to the ovary during vitellogenesis by a cellular pedicel and afterwards the mature oocytes (stage V) are released into the ovary lumen.     

Evidence and implications of inhomogeneity in tectorial membrane elasticity

The motion of the tectorial membrane (TM) with respect to the reticular lamina subserves auditory function by bending the outer hair cell bundles and inducing fluid flows that shear the inner hair bundles in response to sound energy. Little is currently known about its intrinsic elasticity or about the relation between the mechanical properties and function of the membrane. Here we subdivide the TM into three longitudinal regions and five radial zones and map the shear modulus of the TM using atomic force microscopy, and present evidence that the TM elasticity varies radially, after the distribution of type A collagen fibrils. This is seen most dramatically as a decrease in shear modulus in the neighborhood of the sensory hair cells; we argue that this inhomogeneity of properties not only protects the hair bundles but also increases the energy efficiency of the vibrational shearing during sound transduction.     

An actin molecular treadmill and myosins maintain stereocilia functional architecture and self-renewal

We have previously shown that the seemingly static paracrystalline actin core of hair cell stereocilia undergoes continuous turnover. Here, we used the same approach of transfecting hair cells with actin-green fluorescent protein (GFP) and espin-GFP to characterize the turnover process. Actin and espin are incorporated at the paracrystal tip and flow rearwards at the same rate. The flux rates (approximately 0.002-0.04 actin subunits s(-1)) were proportional to the stereocilia length so that the entire staircase stereocilia bundle was turned over synchronously. Cytochalasin D caused stereocilia to shorten at rates matching paracrystal turnover. Myosins VI and VIIa were localized alongside the actin paracrystal, whereas myosin XVa was observed at the tips at levels proportional to stereocilia lengths. Electron microscopy analysis of the abnormally short stereocilia in the shaker 2 mice did not show the characteristic tip density. We argue that actin renewal in the paracrystal follows a treadmill mechanism, which, together with the myosins, dynamically shapes the functional architecture of the stereocilia bundle.