Inhibition of the myotoxic activity of Bothrops jararacussu venom and its two major myotoxins, BthTX-I and BthTX-II, by the aqueous extract of Tabernaemontana catharinensis A. DC. (Apocynaceae)

Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA2 activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 microg/ml) plus AE (400 microg/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 microg/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 microg/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 microg/ml, 1 h) did not reveal any change in muscle fibers, but severe necrosis induced by BV or toxins (20 microg/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA2 activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 microg of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 microg), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with 1 h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors.     

Are dioxetanes chemiluminescent intermediates in lipoperoxidation?

Ultraweak chemiluminescence arising from lipoperoxidation has been attributed by several authors to the radiative deactivation of singlet oxygen and triplet carbonyl products. The latter emitters have been suggested to come from annihilation of RO. and ROO. radicals as well as from the thermolysis of dioxetane intermediates formed by (2 + 2) cycloaddition of 1O2 to polyunsaturated fatty acids. This article questions possible dioxetane intermediacy in lipoperoxidation, as the literature clearly states that addition of 1O2 to alpha-hydrogen-containing alyphatic olefins yields only the corresponding allylic hydroperoxides. These compounds may undergo dark thermal or Lewis acid-assisted decomposition to the same product obtained from dioxetane cleavage. Here, reexamining the chemiluminescence properties of dioxygenated tetramethylethylene and linoleic acid and comparing them with those of tetraethyldioxetane, a hindered dioxetane, we corroborate the literature information that only steric hindrance leads to dioxetane formation upon singlet oxygen addition to electron-poor olefins, albeit in very low yields. Proton nuclear magnetic resonance (1H-NMR) analysis, quenching by dioxygen and energy transfer studies to 9,10-dibromoanthracene, as well as gas chromatography (GC) analysis of triphenylphosphine-treated and untreated photo- and chemically dioxygenated olefins support our final conclusion that dioxetane formation during lipoperoxidation can be safely excluded on the basis of the data presently available.     

Rhipicephalus (Boophilus) microplus: distinct acute phase proteins vary during infestations according to the genetic composition of the bovine hosts, Bos taurus and Bos indicus

Tick bites may trigger acute phase responses. Positive and negative acute phase proteins were measured in infested cattle genetically resistant and susceptible to ticks. During heavier infestations levels of haptoglobin increased significantly in susceptible bovines; levels of serum amyloid A increased in resistant bovines; levels of alpha-1-acid glycoprotein decreased significantly in resistant bovines; levels of transferrin decreased significantly in susceptible bovines. In conclusion, tick infestations trigger acute phase responses and enhancement of specific acute phase proteins differs according to the genetic composition of hosts. Acute phase proteins may constitute useful biological signatures for monitoring the stress induced by tick infestations.     

Dynamic compartmentalization of protein tyrosine phosphatase receptor Q at the proximal end of stereocilia: implication of myosin VI-based transport

Hair cell stereocilia are apical membrane protrusions filled with uniformly polarized actin filament bundles. Protein tyrosine phosphatase receptor Q (PTPRQ), a membrane protein with extracellular fibronectin repeats has been shown to localize at the stereocilia base and the apical hair cell surface, and to be essential for stereocilia integrity. We analyzed the distribution of PTPRQ and a possible mechanism for its compartmentalization. Using immunofluorescence we demonstrate that PTPRQ is compartmentalized at the stereocilia base with a decaying gradient from base to apex. This distribution can be explained by a model of transport directed toward the stereocilia base, which counteracts diffusion of the molecules. By mathematical analysis, we show that this counter transport is consistent with the minus end-directed movement of myosin VI along the stereocilia actin filaments. Myosin VI is localized at the stereocilia base, and exogenously expressed myosin VI and PTPRQ colocalize in the perinuclear endosomes in COS-7 cells. In myosin VI-deficient mice, PTPRQ is distributed along the entire stereocilia. PTPRQ-deficient mice show a pattern of stereocilia disruption that is similar to that reported in myosin VI-deficient mice, where the predominant features are loss of tapered base, and fusion of adjacent stereocilia. Thin section and freeze-etching electron microscopy showed that localization of PTPRQ coincides with the presence of a dense cell surface coat. Our results suggest that PTPRQ and myosin VI form a complex that dynamically maintains the organization of the cell surface coat at the stereocilia base and helps maintain the structure of the overall stereocilia bundle.     

Iron mobilization by succinylacetone methyl ester in rats. A model study for hereditary tyrosinemia and porphyrias characterized by 5-aminolevulinic acid overload

Accumulation of 5-aminolevulinic acid (ALA) is an event characteristic of porphyrias that may contribute to their pathological manifestations. To investigate effects of ALA independent of porphyrin accumulation we treated rats with the methyl ester of succinylacetone, an inhibitor of 5-aminolevulinic acid dehydratase that accumulates in the porphyric-like syndrome hereditary tyrosinemia. Acute 2-day treatment of fasted rats with succinylacetone methyl ester (SAME) promoted a 27% increase in plasma ALA. This increase in plasma ALA was accompanied by augmentation of the level of total nonheme iron in liver (37%) and brain (20%). Mobilization of iron was also indicated by 49% increase in plasma iron and a 77% increase in plasma transferrin saturation. Liver responded with a mild (12%) increase in ferritin. Under these acute conditions, some indications of oxidative stress were evident: a 15% increase in liver reactive protein carbonyls, and a 42% increase in brain subcellular membrane TBARS. Brain also showed a 44% increase in CuZnSOD activity, consistent with observations in treatment with ALA. Overall, the data indicate that SAME promotes ALA-driven changes in iron metabolism that could lead to increased production of free radicals. The findings support other evidence that accumulation of ALA in porphyrias and hereditary tyrosinemia may induce iron-dependent biological damage that contributes to neuropathy and hepatoma.     

The lipid environment of Escherichia coli Aquaporin Z

In this study, we have investigated the lipids surrounding AqpZ, and the effects of a destabilizing mutation W₁₄A (Schmidt and Sturgis, 2017) on lipid protein interactions. In a first approach, we used Styrene Maleic Acid copolymer to prepare AqpZ containing nanodiscs, and these were analyzed for their lipid content, investigating both the lipid head-group and acyl-chain compositions. These results were complemented by native mass spectrometry of purified AqpZ in the presence of lipids, to give insights of variations in lipid binding at the surface of AqpZ. In an effort to gain molecular insights, to aid interpretation of these results, we performed a series of coarse grained molecular dynamics simulations of AqpZ, in mixed lipid membranes, and correlated our observations with the experimental measurements. These various results are then integrated to give a clearer picture of the lipid environment of AqpZ, both in the native membrane, and in lipid nanodiscs. We conclude that AqpZ contains a lipid binding-site, at the interface between the monomers of the tetramer, that is specific for cardiolipin. Almost all the cardiolipin, in AqpZ containing nanodiscs, is probably associated with this site. The SMA 3:1 nanodiscs we obtained contain a rather high proportion of lipid, and in the case of nanodiscs containing AqpZ cardiolipin is depleted. This is possibly because, in the membrane, there is little cardiolipin not associated with binding sites on the surface of the different membrane proteins. Surprisingly, we see no evidence for lipid sorting based on acyl chain length, even in the presence of a large hydrophobic mismatch, suggesting that conformational restrictions are energetically less costly than lipid sorting.     

The FMRP/GRK4 mRNA interaction uncovers a new mode of binding of the Fragile X mental retardation protein in cerebellum

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by the silencing of the FMR1 gene encoding an RNA-binding protein (FMRP) mainly involved in translational control. We characterized the interaction between FMRP and the mRNA of GRK4, a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase super-family, both in vitro and in vivo. While the mRNA level of GRK4 is unchanged in the absence or in the presence of FMRP in different regions of the brain, GRK4 protein level is increased in Fmr1-null cerebellum, suggesting that FMRP negatively modulates the expression of GRK4 at the translational level in this brain region. The C-terminal region of FMRP interacts with a domain of GRK4 mRNA, that we called G4RIF, that is folded in four stem loops. The SL1 stem loop of G4RIF is protected by FMRP and is part of the S1/S2 sub-domain that directs translation repression of a reporter mRNA by FMRP. These data confirm the role of the G4RIF/FMRP complex in translational regulation. Considering the role of GRK4 in GABAB receptors desensitization, our results suggest that an increased GRK4 levels in FXS might contribute to cerebellum-dependent phenotypes through a deregulated desensitization of GABAB receptors.     

Simultaneous ectopic adrenocorticotropic hormone syndrome and adrenal metastasis of a medullary thyroid carcinoma causing paraneoplastic Cushing's syndrome

Medullary thyroid carcinomas (MTC) constitute about 5 to 7 % of thyroid neoplasms. They originate from parafollicular C-cells which can secrete adrenocorticotropic hormone (ACTH) and/or corticotropin-releasing factor (CRF) in abnormally high concentrations, potentially causing paraneoplastic Cushing's Syndrome (CS).We report on a 42-year-old male patient with a ten year history of metastatic medullary thyroid carcinoma suffering from paraneoplastic Cushing's Syndrome caused by ectopic hypersecretion of ACTH and a simultaneous Cortisol producing adrenal metastasis.     

Chemiluminescent aerobic oxidation of protein adducts with glycolaldehyde catalyzed by horseradish peroxidase

Horseradish peroxidase (EC 1.11.1.7) is shown to catalyze the aerobic oxidation of lysozyme, bovine serum albumin, and protamine adducts with glycolaldehyde at physiological pH. This reaction is accompanied by light emission, which is attributed to the generation of triplet species. The intensity of chemiluminescence is enhanced by addition of chlorophyll alpha solubilized in Brij 35. A role of electronically excited species in deleterious and pathological processes associated with formation of Schiff-type adducts is suggested, with emphasis on the case of alcohol-induced liver injury.