The Ras antagonist, farnesylthiosalicylic acid (FTS), decreases fibrosis and improves muscle strength in dy/dy mouse model of muscular dystrophy

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy(2J)/dy(2J) mouse model of merosin deficient congenital muscular dystrophy. The dy(2J)/dy(2J) mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy(2J)/dy(2J) mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy(2J)/dy(2J) mouse model of congenital muscular dystrophy.     

Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene

In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh ‘Charentais’ type melon line that accumulates β-carotene. One mutagenized M₂ family segregated for a novel recessive trait, a yellow–orange fruit flesh (‘yofI’). HPLC analysis revealed that ‘yofI’ accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of ‘yofI’ is associated with a significant change of the fruit aroma since cleavage of β-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to β-carotene in melon fruit. Cloning and sequencing of ‘yofI’ CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A–T transversion in ‘yofI’ which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.     

Rapid Kill—Novel Endodontic Sealer and Enterococcus faecalis

With growing concern over bacterial resistance, the identification of new antimicrobial means is paramount. In the oral cavity microorganisms are essential to the development of periradicular diseases and are the major causative factors associated with endodontic treatment failure. As quaternary ammonium compounds have the ability to kill a wide array of bacteria through electrostatic interactions with multiple anionic targets on the bacterial surface, it is likely that they can overcome bacterial resistance. Melding these ideas, we investigated the potency of a novel endodontic sealer in limiting Enterococcus faecalis growth. We used a polyethyleneimine scaffold to synthesize nano-sized particles, optimized for incorporation into an epoxy-based endodontic sealer. The novel endodontic sealer was tested for its antimicrobial efficacy and evaluated for biocompatibility and physical eligibility. Our results show that the novel sealer foundation affixes the nanoparticles, achieving surface bactericidal properties, but at the same time impeding nanoparticle penetration into eukaryotic cells and thereby mitigating a possible toxic effect. Moreover, adequate physical properties are maintained. The nanosized quaternary amine particles interact within minutes with bacteria, triggering cell death across wide pH values. Throughout this study we demonstrate a new antibacterial perspective for endodontic sealers; a novel antibacterial, effective and safe antimicrobial means.     

Catabolism of l–methionine in the formation of sulfur and other volatiles in melon (Cucumis melo L.) fruit

Sulfur‐containing aroma volatiles are important contributors to the distinctive aroma of melon and other fruits. Melon cultivars and accessions differ in the content of sulfur‐containing and other volatiles. l –methionine has been postulated to serve as a precursor of these volatiles. Incubation of melon fruit cubes with ¹³C‐ and ²H‐labeled l –methionine revealed two distinct catabolic routes into volatiles. One route apparently involves the action of an l ‐methionine aminotransferase and preserves the main carbon skeleton of l ‐methionine. The second route apparently involves the action of an l ‐methionine‐γ–lyase activity, releasing methanethiol, a backbone for formation of thiol‐derived aroma volatiles. Exogenous l ‐methionine also generated non‐sulfur volatiles by further metabolism of α–ketobutyrate, a product of l ‐methionine‐γ–lyase activity. α–Ketobutyrate was further metabolized into l –isoleucine and other important melon volatiles, including non‐sulfur branched and straight‐chain esters. Cell‐free extracts derived from ripe melon fruit exhibited l ‐methionine‐γ–lyase enzymatic activity. A melon gene (CmMGL) ectopically expressed in Escherichia coli, was shown to encode a protein possessing l ‐methionine‐γ–lyase enzymatic activity. Expression of CmMGL was relatively low in early stages of melon fruit development, but increased in the flesh of ripe fruits, depending on the cultivar tested. Moreover, the levels of expression of CmMGL in recombinant inbred lines co‐segregated with the levels of sulfur‐containing aroma volatiles enriched with +1 m/z unit and postulated to be produced via this route. Our results indicate that l ‐methionine is a precursor of both sulfur and non‐sulfur aroma volatiles in melon fruit.     

The expanded supraclavicular flap, prefabricated with thoracoacromial vessels, for reconstruction of postburn anterior cervical contractures

Mentosternal contractures are well-known complications after burns, scald injuries, and injuries with acid or lye. These contractures may cause severe deformities that are both functionally and aesthetically crippling. Reconstruction of the neck requires the transfer of large flaps of thin, pliable skin to optimally match the texture and color of the recipient region. With the introduction of free tissue transfer, the availability of flaps for reconstruction of large neck defects has greatly increased. Unfortunately, many of these flaps are bulky and are not well matched to the thin and pliable skin of the neck. This article introduces the expanded supraclavicular flap prefabricated with the thoracoacromial vessels for reconstruction of anterior cervical contractures. Their anatomic location, length, and arc of rotation make the thoracoacromial vessels an excellent choice for prefabricating the supraclavicular skin for its subsequent interpolation into the anterior neck. Skin expansion in the donor region not only allows coverage of the larger unit of the anterior neck but also modifies the morphologic characteristics of the transferred flap through capsule formation and fatty tissue atrophy, which is beneficial for obtaining an optimal neck reconstruction.     

Oxygen restriction of neonate rats elevates neuregulin-1alpha isoform levels: possible relationship to schizophrenia

Neuregulin-1 (NRG-1), a replicated gene in schizophrenia-association studies, exhibits six mRNA-types and two types of the EGF-like domain, alpha and beta. The beta-isoform was extensively studied, less is known about the extent and specific localization of adult brain NRG-1alpha. NRG-1alpha protein levels were reported reduced in postmortem prefrontal-cortex of schizophrenia patients. NRG-1 type I mRNA levels were found higher in postmortem brain in schizophrenia. In an attempt to decipher between a genetic or environmental involvement in the differences in NRG-1 levels in postmortem brain in schizophrenia, and since obstetric complications were suggested non-genetic risk-factors of schizophrenia, we studied the effect of perinatal hypoxia in rats on brain NRG-1alpha protein levels. Seven-day-old rats were exposed to hypoxia versus air. Frontal-cortex levels of NRG-1alpha isoform were quantified at adulthood by Western blotting. Frontal-cortex NRG-1alpha was 32% elevated in hypoxia-exposed rats. The data support the role of non-genetic factors, e.g. oxygen restriction, in the expression of genes associated with schizophrenia.     

Changing the charge distribution of beta-helical-based nanostructures can provide the conditions for charge transfer

In this work we present a computational approach to the design of nanostructures made of structural motifs taken from left-handed beta-helical proteins. Previously, we suggested a structural model based on the self-assembly of motifs taken from Escherichia coli galactoside acetyltransferase (Protein Data Bank 1krr, chain A, residues 131-165, denoted krr1), which produced a very stable nanotube in molecular dynamics simulations. Here we modify this model by changing the charge distribution in the inner core of the system and testing the effect of this change on the structural arrangement of the construct. Our results demonstrate that it is possible to generate the proper conditions for charge transfer inside nanotubes based on assemblies of krr1 segment. The electronic transfer would be achieved by introducing different histidine ionization states in selected positions of the internal core of the construct, in addition to specific mutations with charged amino acids that altogether will allow the formation of coherent networks of aromatic ring stacking, salt-bridges, and hydrogen bonds.     

Primary Coenzyme Q deficiency Due to Novel ADCK3 Variants,Studies in Fibroblasts and Review of Literature

Primary deficiency of coenzyme Q10 (CoQ10 ubiquinone), is classified as a mitochondrial respiratory chain disorder with phenotypic variability. The clinical manifestation may involve one or multiple tissue with variable severity and presentation may range from infancy to late onset. ADCK3 gene mutations are responsible for the most frequent form of hereditary CoQ10 deficiency (Q10 deficiency-4 OMIM #612016) which is mainly associated with autosomal recessive spinocerebellar ataxia (ARCA2, SCAR9). Here we provide the clinical, biochemical and genetic investigation for unrelated three nuclear families presenting an autosomal form of Spino-Cerebellar Ataxia due to novel mutations in the ADCK3 gene. Using next generation sequence technology we identified a homozygous Gln343Ter mutation in one family with severe, early onset of the disease and compound heterozygous mutations of Gln343Ter and Ser608Phe in two other families with variable manifestations. Biochemical investigation in fibroblasts showed decreased activity of the CoQ dependent mitochondrial respiratory chain enzyme succinate cytochrome c reductase (complex II?+?III). Exogenous CoQ slightly improved enzymatic activity, ATP production and decreased oxygen free radicals in some of the patient’s cells. Our results are presented in comparison to previously reported mutations and expanding the clinical, molecular and biochemical spectrum of ADCK3 related CoQ10 deficiencies.

     

Ca-mediated regulation of VDAC1 expression levels is associated with cell death induction

VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c (Cyto c) release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release. As VDAC1 transports Ca^2 + across the OMM and because Ca^2 + has been implicated in apoptosis induction, we addressed the relationship between cytosolic Ca^2 + levels ([Ca^2 +]i), VDAC1 oligomerization and apoptosis induction. We demonstrate that different apoptosis inducers elevate cytosolic Ca^2 + and induce VDAC1 over-expression. Direct elevation of [Ca^2 +]i by the Ca^2 +-mobilizing agents A23187, ionomycin and thapsigargin also resulted in VDAC1 over-expression, VDAC1 oligomerization and apoptosis. In contrast, decreasing [Ca^2 +]i using the cell-permeable Ca^2 +-chelating reagent BAPTA-AM inhibited VDAC1 over-expression, VDAC1 oligomerization and apoptosis. Correlation between the increase in VDAC1 levels and oligomerization, [Ca^2 +]i levels and apoptosis induction, as induced by H₂O₂ or As₂O₃, was also obtained. On the other hand, cells transfected to overexpress VDAC1 presented Ca^2 +-independent VDAC1 oligomerization, cytochrome c release and apoptosis, suggesting that [Ca^2 +]i elevation is not a pre-requisite for apoptosis induction when VDAC1 is over-expressed. The results suggest that Ca^2 + promotes VDAC1 over-expression by an as yet unknown signaling pathway, leading to VDAC1 oligomerization, ultimately resulting in apoptosis. These findings provide a new insight into the mechanism of action of existing anti-cancer drugs involving induction of VDAC1 over-expression as a mechanism for inducing apoptosis. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau