Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis

The MUTYH DNA glycosylase specifically removes adenine misincorporated by replicative polymerases opposite the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoG). A defective protein activity results in the accumulation of G > T transversions because of unrepaired 8-oxoG:A mismatches. In humans, MUTYH germline mutations are associated with a recessive form of familial adenomatous polyposis and colorectal cancer predisposition (MUTYH-associated polyposis, MAP). Here we studied the repair capacity of the MUTYH variants R171W, E466del, 137insIW, Y165C and G382D, identified in MAP patients. Following expression and purification of human proteins from a bacterial system, we investigated MUTYH incision capacity on an 8-oxoG:A substrate by standard glycosylase assays. For the first time, we employed the surface plasmon resonance (SPR) technology for real-time recording of the association/dissociation of wild-type and MUTYH variants from an 8-oxoG:A DNA substrate. When compared to the wild-type protein, R171W, E466del and Y165C variants showed a severe reduction in the binding affinity towards the substrate, while 137insIW and G382D mutants manifested only a slight decrease mainly due to a slower rate of association. This reduced binding was always associated with impairment of glycosylase activity, with adenine removal being totally abrogated in R171W, E466del and Y165C and only partially reduced in 137insIW and G382D. Our findings demonstrate that SPR analysis is suitable to identify defective enzymatic behaviour even when mutant proteins display minor alterations in substrate recognition.     

Shewanella sp. GA-22, a psychrophilic hydrocarbonoclastic antarctic bacterium producing polyunsaturated fatty acids

AIMS: The effects of different growth media and temperature on production of polyunsaturated fatty acids (PUFA) by Shewanella sp. GA-22 were investigated. The attempts to characterize the GA-22 genes, homologous to those of PUFA biosynthesis gene cluster, was performed. METHODS AND RESULTS: Physiological and phylogenetic characterization of new Antarctic isolate GA-22 was performed. Total fatty acids were isolated from the cells growing under different conditions and analysed by gas chromatography-mass spectrometry (GC-MS). Using degenerated primers derived from the conserved regions within PUFA fatty acid synthase operons, five fragments of homological genes were amplified from GA-22 DNA, and two of them corresponding to pfaA and pfaC synthase subunits were sequenced. CONCLUSIONS: Strain GA-22 was shown to be able to produce three different PUFA: linoleic, arachidonic and eicosapentaenoic acids. The PUFA production was temperature- and carbon source-dependent. The deduced gene products exhibited high similarity to corresponding fatty acid synthases PfaA and PfaC. SIGNIFICANCE AND IMPACT OF STUDY: The PUFA production was detected on media supplemented with crude oil, gasoline and n-tetradecane. The apparent conservation of PUFA genes may point to the potential utilization of designed primers as functional markers in culture-independent ecological studies, and for initial screening in biotechnological fields.     

The mammalian mismatch repair pathway removes DNA 8-oxodGMP incorporated from the oxidized dNTP pool

Mismatch repair (MMR) corrects replication errors. It requires the MSH2, MSH6, MLH1, and PMS2 proteins which comprise the MutSalpha and MutLalpha heterodimers. Inactivation of MSH2 or MLH1 in human tumors greatly increases spontaneous mutation rates. Oxidation produces many detrimental DNA alterations against which cells deploy multiple protective strategies. The Ogg-1 DNA glycosylase initiates base excision repair (BER) of 8-oxoguanine (8-oxoG) from 8-oxoG:C pairs. The Myh DNA glycosylase removes mismatched adenines incorporated opposite 8-oxoG during replication. Subsequent BER generates 8-oxoG:C pairs, a substrate for excision by Ogg-1. MTH1-an 8-oxodGTPase which eliminates 8-oxodGTP from the dNTP pool-affords additional protection by minimizing 8-oxodGMP incorporation during replication. Here we show that the dNTP pool is, nevertheless, an important source of DNA 8-oxoG and that MMR provides supplementary protection by excising incorporated 8-oxodGMP. Incorporated 8-oxodGMP contributes significantly to the mutator phenotype of MMR-deficient cells. Thus, although BER of 8-oxoG is independent of Msh2, both steady-state and H(2)O(2)-induced DNA 8-oxoG levels are higher in Msh2-defective cells than in their repair-proficient counterparts. Increased expression of MTH1 in MMR-defective cells significantly reduces steady-state and H(2)O(2)-induced DNA 8-oxoG levels. This reduction dramatically diminishes the spontaneous mutation rate of Msh2(-/-) MEFs.     

Ultrastructural and immunohistological characterization of a cell culture model for the study of neuronal-glial interactions.

The differentiation of reaggregating cell cultures of dissociated cerebellar cells from 3- and 6-day-old mice was analyzed by electron microscopy of and immunofluorescence to the glial fibrillary acidic protein (GFA) at intervals between 8 hr and 20 days in vitro. Aggregates in culture for 8 hr were composed of 8–12 undifferentiated cells that were negative for the GFA protein and indistinguishable from each other by electron microscopy. Some cells with extended processes and a few immunofluorescent cells had already appeared after 24 hr in vitro, and the elaboration of a complex neural ultrastructure was observed during the subsequent days. After 10 days in vitro the interior of the aggregate was occupied principally by neurons, the majority of which were granule neurons, and regions of neuropil containing many synaptic complexes. Glial fibers with intense immunofluorescence to GFA were present throughout the aggregates but were mainly concentrated at the periphery. Large unidentified cells protruded from the surface. The subsequent days in culture evidenced a decline in the neuronal character of the aggregates with a concomitant increase in fibrous neuoglia. We suggest that factors controlling neuronal-glial interactions and fibrous gliosis are amenable to analysis in this tissue culture system.     

Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immunoperoxidase labeling

Glial fibrillary acidic protein was localized at the electron microscope level in the cerebellum of adult mice by indirect immunoperoxidase histology. In confirmation of previous studies at the light microscope level, the antigen was detectable in astrocytes and their processes, but not in neurons or their processes, or in oligodendroglia. Astrocytic processes were stained in white matter, in the granular layet surrounding synaptic glomerular complexes, and in the molecular layer in the form of radially oriented fibers and of sheaths surrounding Purkinje cell dendrites. Astrocytic endfeet impinging on meninges and perivascular membranes were also antigen positive. In astrocytic perikarya and processes, the immunohistochemical reaction product appears both as a diffuse cytoplasmic label and as elongated strands, which by their distribution and frequency could be considered glial filaments.     

A novel acylphloroglucinol from the brown alga Zonaria tournefortii

A novel acylphloroglucinol, (5Z,8Z11Z,13E,17Z)-2′-eicosa-15(S)-hydroxy-5,8,11,13,17-pentaenoylphloroglucinol, has been isolated from the brown alga Zonaria tournefortii and its structure proved by spectroscopic and chemical methods.     

A quinone-hydroquinone couple from the Brown alga Cystoseira stricta

2′E,6′E)-2-(10′,11′-Dihydroxygeranylgeranyl)-6-methylquinol and 2′E,6′E)-2(10′,11′-dihydroxyeranylgeranyl)-6-methyl-1,4-benzoquinone have been isolated from the brown alga Cystoseira stricta. The structures of the new algal metabolites have been elucidated by spectral analysis and chemical degradation.     

Evaluation of 2 different genetic markers for the detection of frameshift and missense mutagens in A. nidulans

21 chemicals, known to induce missense and/or frameshift mutations directly, were assayed for their ability to forward mutate a haploid strain of A. nidulans. 2 genetic markers for forward mutations were used, namely 8-azaguanine resistance and induction of meth A1 suppressors. Missense mutagens were usually active when tested with the plate-incorporation technique, whereas frameshift agents were ineffective; some of these, on the other hand, turned out to be positive when tested with a liquid-test procedure. The 2 genetic markers used showed a similar sensitivity (with only 2 exceptions) in detecting the chemical mutagens assayed.     

Molecular and histological traits of reduced lysosomal acid lipase activity in the fatty liver

Recent studies demonstrated reduced blood lysosomal acid lipase (LAL) activity in patients with nonalcoholic fatty liver disease (NAFLD). We aimed to verify hepatic LAL protein content and activity in in vitro and in vivo models of fat overload and in NAFLD patients. LAL protein content and activity were firstly evaluated in Huh7 cells exposed to high-glucose/high-lipid (HGHL) medium and in the liver of C57BL/6 mice fed with high-fat diet (HFD) for 4 and 8 months. LAL protein was also evaluated by immunohistochemistry in liver biopsies from 87 NAFLD patients and 10 controls, and correlated with hepatic histology. Huh7 cells treated with HGHL medium showed a significant reduction of LAL activity, which was consistent with reduced LAL protein levels by western blotting using an antibody towards the N-term of the enzyme. Conversely, antibodies towards the C-term of the enzyme evidenced LAL accumulation, suggesting a post-translational modification that masks the LAL N-term epitope and affects enzymatic activity. Indeed, we found a high rate of ubiquitination and extra-lysosomal localization of LAL protein in cells treated with HGHL medium. Consistent with these findings, inhibition of proteasome triggered dysfunctional LAL accumulation and affected LAL activity. Accumulation of ubiquitinated/dysfunctional LAL was also found in the liver of HFD fed mice. In NAFLD patients, hepatic levels of non-ubiquitinated/functional LAL were lower than in controls and inversely correlated with disease activity and some of the hallmarks of reduced LAL. Fat overload leads to LAL ubiquitination and impairs its function, possibly reducing hepatic fat disposal and promoting NAFLD activity.Subject terms: Non-alcoholic fatty liver disease, Translational research     

Heavier halides of transition metals by halide exchange

Examples are reported of heavier (bromides or iodides) metal halides of the d or f transition series being prepared through the halide exchange reaction from the lighter congeners (fluorides or chlorides, easily prepared by direct combination from the elements), by using gaseous hydrogen halides HX or alkyl halides RX in an anhydrous organic solvent at room temperature or even below. This represents a considerable improvement with respect to the traditional high-temperature experimental procedures from the elements. Thermodynamic data show that this synthetic route is of quite general validity.