Pigmentary system of the adult alpine salamander Salamandra atra aurorae (Trevisan, 1982)

The pigmentary system of skin from adult specimens of the amphibian urodele Salamandra atra aurorae was investigated by light microscope, electron microscope, and biochemical studies. Yellow (dorsum and head) and black (flank and belly) skin was tested. Three chromatophore types are present in yellow skin: xanthophores, iridophores, and melanophores. Xanthophores are located in the epidermis whereas iridophores and melanophores are found in the dermis. Xanthophores contain types I, II, and III pterinosomes. Some pterinosomes are very electron-dense. Black skin has a single type of chromatophore: the melanophores. Some melanophores are located in the epidermis. In contrast to the dermal melanophores, these present, in addition to typical melanosomes, organelles with different morphology and vesicles having a limiting membrane and containing little amorphous material. Both skin types present some pteridines and flavins, though they are qualitatively and quantitatively more abundant in yellow skin extracts.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

DNA repair investigations in nine Italian patients affected by trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by brittle hair, mental and growth retardation, peculiar face, ichthyosis, and in 20% of the reported cases photosensitivity. Cellular photosensitivity due to the same genetic defect present in xeroderma pigmentosum group D (XP-D) has been described in several patients. Nine patients with clinical symptoms diagnostic for TTD have been identified in Italy to date. We report the results of DNA repair investigations performed in cultured fibroblasts from these patients and 8 TTD parents. Survival, DNA repair synthesis and RNA synthesis following UV irradiation were all normal in the 8 TTD heterozygous cell strains. Among the 9 TTD-affected individuals, normal cellular UV sensitivity was observed in the 2 patients without signs of clinical photosensitivity. In contrast, the other 7 TTD cell strains showed a notable reduction in UV-induced DNA repair synthesis (UDS) levels, ranging between 40% and 5-15% of normal values. Complementation analysis indicated that in the repair-deficient TTD cell strains the genetic defect is the same as that present in XP-D cells. The biochemical heterogeneity of the XP-D defect in TTD patients characterized by different degrees of defective UDS results in different patterns of response to the killing effect of UV light in non-proliferating cells.     

Pigmentary system of the adult alpine salamander Salamandra atra atra (Laur., 1768).

The pigmentary system of the skin from adult specimens of the black alpine salamander Salamandra atra atra was investigated by light microscope, electron microscope, and biochemical studies. Results were compared with those obtained in previous study of the subspecies Salamandra atra aurorae. Unlike Salamandra atra aurorae, which presents epidermal xanthophores and iridophores, Salamandra atra atra is completely melanized, presenting only epidermal and dermal melanophores. The melanosomes in both the epidermis and the dermis appear to derive from a multivesicular premelanosome similar to that in the goldfish, and the epidermal melanosomes are smaller than those in the dermis. Premelanosomes with an internal lamellar matrix were not observed. The biochemical results have shown that in the ethanol extracts obtained from the skin in toto and from the melanosomes, pteridines and flavins are always present and are the same as those extracted from the black skin areas of Salamandra atra aurorae.     

Rapid Communication Chronic Ingestion of Ethanol Up-Regulates NMDAR1 Receptor Subunit Immunoreactivity in Rat Hippocampus

We examined the effects of chronic ethanol exposure on the levels of N -methyl-D-aspartate receptor subunit 1 (NMDAR1) protein, an essential component of N -methyl-D-aspar- tate glutamate receptors, in rat brain. By immunoblotting procedures using a specific antibody for the NMDAR1 subunit, we found that ethanol dramatically up-regulated (by 65%) NMDAR1 immunoreactivity in the hippocampus but not in the nucleus accumbens, cerebral cortex, or striatum. In contrast, ethanol did not alter the levels of glutamate receptor subunit (GLUR) 1 or GLUR2 protein, subunits that make up the α-amino-3-hydroxy-5-methy4-isoxazole propionic acid glutamate receptor, in the hippocampus. Because ethanol can potentially influence many different neurotransmitter systems, we examined whether chronic treatment with several psychotropic drugs with different pharmacological profiles (cocaine, haloperidol, SCH 23390, imipramine, and morphine) could mimic the effect of ethanol. None of these agents increased hippocampal NMDAR1 subunit immunoreactivity after chronic administration. Increased NMDAR1 subunit levels in the hippocampus after chronic ethanol exposure may represent an important neurochemical substrate for some of the features associated with ethanol dependence and withdrawal.     

Analytical and biological variation of biomarkers of oxidative stress during the menstrual cycle

Little information is available on the intra-individual variability of oxidative stress biomarkers in healthy individuals and even less in the context of the menstrual cycle. The objective of this study was to characterize the analytical and biological variability of a panel of 21 markers of oxidative damage, antioxidant defence and micronutrients in nine healthy, regularly menstruating women aged 18–44 years. Analyses included measurement of lipid peroxidation, antioxidant enzymes and antioxidant vitamins. Blood specimens were collected, processed and stored using standardized procedures on days 2, 7, 12, 13, 14, 18, 22 and 28 in one cycle for each subject. Replicate analyses of markers were performed and two-way nested random effects ANOVA was used to describe analytical, intra-individual and inter-individual variability. No statistically significant differences at α=0.05, or temporal effects across the menstrual cycle were observed. Analytical variability was the smallest component of variance for all variables. The ICC among replicates ranged from 0.80 to 0.98. Imprecision based on quality control materials ranged from 1 to 11%. The critical differences between serial results varied greatly between assays ranging from 6 to 216% of the mean level. These results provide important initial information on the variability of biomarkers of oxidative stress, antioxidant defence and micronutrients across the menstrual cycle.     

Mechanisms Involved in the Nociception Triggered by the Venom of the Armed Spider Phoneutria nigriventer

Background

The frequency of accidental spider bites in Brazil is growing, and poisoning due to bites from the spider genus Phoneutria nigriventer is the second most frequent source of such accidents. Intense local pain is the major symptom reported after bites of P. nigriventer, although the mechanisms involved are still poorly understood. Therefore, the aim of this study was to identify the mechanisms involved in nociception triggered by the venom of Phoneutria nigriventer (PNV).

Methodology/Principal Findings

Twenty microliters of PNV or PBS was injected into the mouse paw (intraplantar, i.pl.). The time spent licking the injected paw was considered indicative of the level of nociception. I.pl. injection of PNV produced spontaneous nociception, which was reduced by arachnid antivenin (ArAv), local anaesthetics, opioids, acetaminophen and dipyrone, but not indomethacin. Boiling or dialysing the venom reduced the nociception induced by the venom. PNV-induced nociception is not dependent on glutamate or histamine receptors or on mast cell degranulation, but it is mediated by the stimulation of sensory fibres that contain serotonin 4 (5-HT₄) and vanilloid receptors (TRPV1). We detected a kallikrein-like kinin-generating enzyme activity in tissue treated with PNV, which also contributes to nociception. Inhibition of enzymatic activity or administration of a receptor antagonist for kinin B₂ was able to inhibit the nociception induced by PNV. PNV nociception was also reduced by the blockade of tetrodotoxin-sensitive Na⁺ channels, acid-sensitive ion channels (ASIC) and TRPV1 receptors.

Conclusion/Significance

Results suggest that both low- and high-molecular-weight toxins of PNV produce spontaneous nociception through direct or indirect action of kinin B₂, TRPV1, 5-HT₄ or ASIC receptors and voltage-dependent sodium channels present in sensory neurons but not in mast cells. Understanding the mechanisms involved in nociception caused by PNV are of interest not only for better treating poisoning by P. nigriventer but also appreciating the diversity of targets triggered by PNV toxins.     

Diphenyl Diselenide Reduces Mechanical and Thermal Nociceptive Behavioral Responses After Unilateral Intrastriatal Administration of 6-Hydroxydopamine in Rats

Parkinson’s disease (PD) patients, in addition to motor dysfunction, also present alterations in pain sensation. The present study characterized the antinociceptive effects of diphenyl diselenide ((PhSe)₂) in a model of nociception induced by unilateral, intrastriatal 6-hydroxydopamine (6-OHDA) injection in rats. Male adult Wistar rats received 20 μg/3 μl of 6-OHDA (in saline solution containing 0.02 % of ascorbic acid) or 3 μl of vehicle into the right striatum (1.0 mm anterior, 3.0 mm lateral, and 5.0 mm ventral—with respect to the bregma). Thirty days after injection, rats received (PhSe)₂ intragastrically at a dose of 10 mg/kg 1 h before behavioral tests (von Frey hairs, hot plate, tail immersion, formalin, and open field). Our results demonstrated that 6-OHDA injection to rats augmented the response frequency of von Frey hairs (VHF) stimulation, besides reducing the thermal withdrawal latency in the hot plate test. Importantly, the (PhSe)₂ treatment decreased the mechanical allodynia measured by the response frequency of VHF stimulation and diminished the thermal nociception in the hot plate test in 6-OHDA-injected rats. In conclusion, these results revealed that a single oral administration of (PhSe)₂ 1 h prior to the accomplishment of the behavioral tests was effective to attenuate the increased mechanical and thermal nociception caused by a single intrastriatal 6-OHDA injection to rats. Furthermore, other clarifying studies are warranted to improve the evidence bases for future clinical use of (PhSe)₂ as a new alternative therapy for the treatment of painful symptoms associated to PD.     

Myotonic dystrophy protein kinase (DMPK) prevents ROS-induced cell death by assembling a hexokinase II-Src complex on the mitochondrial surface

The biological functions of myotonic dystrophy protein kinase (DMPK), a serine/threonine kinase whose gene mutations cause myotonic dystrophy type 1 (DM1), remain poorly understood. Several DMPK isoforms exist, and the long ones (DMPK-A/B/C/D) are associated with the mitochondria, where they exert unknown activities. We have studied the isoform A of DMPK, which we have found to be prevalently associated to the outer mitochondrial membrane. The kinase activity of mitochondrial DMPK protects cells from oxidative stress and from the ensuing opening of the mitochondrial permeability transition pore (PTP), which would otherwise irreversibly commit cells to death. We observe that DMPK (i) increases the mitochondrial localization of hexokinase II (HK II), (ii) forms a multimeric complex with HK II and with the active form of the tyrosine kinase Src, binding its SH3 domain and (iii) it is tyrosine-phosphorylated by Src. Both interaction among these proteins and tyrosine phosphorylation of DMPK are increased under oxidative stress, and Src inhibition selectively enhances death in DMPK-expressing cells after HK II detachment from the mitochondria. Down-modulation of DMPK abolishes the appearance of muscle markers in in vitro myogenesis, which is rescued by oxidant scavenging. Our data indicate that, together with HK II and Src, mitochondrial DMPK is part of a multimolecular complex endowed with antioxidant and pro-survival properties that could be relevant during the function and differentiation of muscle fibers.     

Podocyte Expression of Membrane Transporters Involved in Puromycin Aminonucleoside-Mediated Injury

Several complex mechanisms contribute to the maintenance of the intricate ramified morphology of glomerular podocytes and to interactions with neighboring cells and the underlying basement membrane. Recently, components of small molecule transporter families have been found in the podocyte membrane, but expression and function of membrane transporters in podocytes is largely unexplored. To investigate this complex field of investigation, we used two molecules which are known substrates of membrane transporters, namely Penicillin G and Puromycin Aminonucleoside (PA).We observed that Penicillin G pre-administration prevented both in vitro and in vivo podocyte damage caused by PA, suggesting the engagement of the same membrane transporters by the two molecules. Indeed, we found that podocytes express a series of transporters which are known to be used by Penicillin G, such as members of the Organic Anion Transporter Polypeptides (OATP/Oatp) family of influx transporters, and P-glycoprotein, a member of the MultiDrug Resistance (MDR) efflux transporter family.Expression of OATP/Oatp transporters was modified by PA treatment. Similarly, in vitro PA treatment increased mRNA and protein expression of P-glycoprotein, as well as its activity, confirming the engagement of the molecule upon PA administration.In summary, we have characterized some of the small molecule transporters present at the podocyte membrane, focusing on those used by PA to enter and exit the cell. Further investigation will be needed to understand precisely the role of these transporter families in maintaining podocyte homeostasis and in the pathogenesis of podocyte injury.