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[
J Biol Chem,
2007]
The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.
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[
Worm Breeder's Gazette,
1990]
We have been studying C. elegans microtubules from cellular and genetic aspects. Since scanning tunneling microscopy (STM) can provide atomic resolution of metals and semiconductive surfaces; our objective is to obtain images of C. elegans microtubules resolved at the atomic level. However, application of STM to biomolecules is faced with a variety of problems, including poor electrical conductivity, flexible stability under STM imaging conditions. In spite of limitations, recently, impressive images of DNA biomolecules have been obtained at atomic resolution. Microtubules (MT) were isolated from 30g of freshly grown N2 worms according to the procedure of Aamdot and Culotti 1986, and Siddiqui et al 1989, using cycles of temperature dependent assembly and disassembly (Shelanski, et al. 1973). Assembled MT were taken in MT isolation buffer (containing 0.1mM GTP, 20 M taxol, and 2.0 mM DTE), diluted tenfold and fixed in glutaraldehyde (0.1%). The samples were kept frozen at -20 C, for 4-5 days. The STM imaging samples were thawed, and a drop was smeared onto a chip of freshly cleaved HOPG ( highly oriented pyrolitic graphite, Union Carbide), and allowed to air dry at room temperature. The STM images were obtained using 'Nanascope II Digital Instruments, California (Sam Howell and C. Lee, kindly helped with the STM settings). All STM imaging was conducted at atmospheric pressure and at room temperature (22 C). Two different STM images of C. elegans MT are shown here. [See Figure 1] Most of our STM scans of MT correspond to one or two microtubules, but they appear flattened and bent along the seams of the protofilaments. The dimensions of alpha and -tubulin dimer subunits are about 8 nm, and each protofilament has a width of 4.6 nm. The flattening and buckeling of protofilaments along the seams could arise due to the fixation conditions, or caused by the STM current flow through the specimen. At present, we are unable to attribute this observation to any single factor, and the images of MT do not provide any higher resolution than conventional electron microscopy using negative staining. But it is increasingly possible that one may be able to resolve individual amino acids or even atoms using STM. We plan to continue looking for improved conditions for better STM or AFM images of C. elegans MT. Brief description of STM: Scanning tunneling microscopy (STM) traces three dimensional images of surfaces - even at the level of individual atoms. Gerd Binnig & Heinrich Rohrer of IBM Zurich received the Nobel Prize in 1986 for STM (Refer, e.g.: Scientific American, August,1985). Briefly in STM, the 'aperture' is a small tungston probe, its tip extremely fine (about 0. 2 nm in width, and may consist of a single atom). Piezoelectric controls move the tip within a nanometer or two of the surface of the conducting sample, allowing an overlap between the electron clouds of the atom at the probe tip and the closest atom of the sample placed on the surface. As a small voltage is applied to the tip, electrons 'tunnel' across this gap, creating a tiny tunneling current. The strength of the tunneling current is exponentially sensitive to the width of the gap (about an order of magnitude per angstrom). Depending on the substrate, typical currents and voltages are in the range of nanoamperes and millivolts. A servo system uses a feedback control that keeps the tip to substrate gap constant, by modulating the voltage across a piezoelectric positioning system (Piezoceramic materials expand or shrink extremely small distances (i.e. angstroms ) in response to the applied voltage. As the tip scans the surface, variations in this voltage, when plotted correspond to surface structure. I wish to thank members of Ward lab (Alicia, Andrea, Bill, Bonnie, Bruce, Jacob, John and Paul); Hameroff Lab (Dolly, Larry, Mohammad, and Richard); and Sarid Lab (Sam and Lee).
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Lou Y, Haque A, Freyzon Y, Farese RV, Terry-Kantor E, Hofbauer HF, Termine D, Welte MA, Barrasa MI, Imberdis T, Noble T, Lindquist S, Clish CB, Jaenisch R, Pincus D, Nuber S, Sandoe J, Kohlwein SD, Kim TE, Ho GPH, Ramalingam N, Walther TC, Baru V, Selkoe D, Srinivasan S, Landgraf D, Soldner F, Dettmer U, Fanning S, Becuwe M, Newby G
[
Mol Cell,
2018]
In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.
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[
PLoS One,
2017]
In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.
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[
Pathog Dis,
2014]
Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added.
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Haass C, Hegermann J, Giese A, Eimer S, Kamp F, Lutz AK, Nuscher B, Wender N, Brunner B, Winklhofer KF, Exner N, Beyer K, Bartels T
[
EMBO J,
2010]
Aggregation of -synuclein (S) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of S is largely unknown. We demonstrate with in vitro vesicle fusion experiments that S has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, S binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous S. In contrast, siRNA-mediated downregulation of S results in elongated mitochondria in cell culture. S can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, S prevents fusion of differently labelled mitochondrial populations. Thus, S inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of S is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin 1-79 or by DJ-1 C106A.
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[
MicroPubl Biol,
2021]
For El Mouridi, S; AlHarbi, S; Frkjr-Jensen, C (2021). A histamine-gated channel is an efficient negative selection marker for C. elegans transgenesis. microPublication Biology. 10.17912/micropub.biology.000349.
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[
Mol Cell Biol,
1997]
The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae
dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a
dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe
dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe
dbr1::
leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe
dbr1::
leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.
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[
International Worm Meeting,
2009]
Amyloids are protein fibers rich in beta sheet structure that are associated with numerous neurodegenerative diseases, including Alzheimer disease. Amyloids have two major biological properties. First, they can be infectious--meaning that they can spread the altered amyloid conformation to the same protein or even among proteins of distinct primary sequence (the latter phenomenon is known as cross-polymerization). Infectious amyloids are called prions. Second, amyloids can be toxic, whether they are infectious or not. Both properties have tremendous fundamental and biomedical impact. [Het-s] is a prion of the fungus Podospora anserina involved in a defense cell suicide mechanism named heterokaryon incompatibility, which disables mixing of different strains by somatic fusion, an ubiquitous feature in filamentous fungi. The HET-s protein exists in a soluble state or in an aggregated, prion state named [Het-s]. The [Het-s] prion is not toxic by itself in P. anserina or in yeast. However cell death is rapidly triggered by the lethal interaction between 2 alleles of the het-s locus: het-S (het-"large S") and het-s (het-"small s") when the HET-s protein adopts the prion form. This prion has been extensively characterized and it is the only prion for which tridimensional structure is available to date. The critical point is that in the [Het-s] system, transgenic expression of het-s allows propagating a non-toxic prion while co-expression of het-s and het-S generates toxicity. We constructed strains expressing het-s or the prion domain only het-s(218-289) (the 218-289 fragment of the protein) as GFP fusions under the control of the ubiquitous promoter
dpy-30. The fluorescence of
pdpy-30het-s::gfp appears to be mainly diffuse in the cytoplasm while it shows strong aggregation in
pdpy-30het-s(218-289)::gfp strains. We constructed strains expressing het-s(218-289)::gfp in the touch neurons using
mec-4 promoter and observed that the fusion protein is mostly aggregated into a large fiber spanning the cell body and the proximal part of the axon. These aggregates remain to be shown as infectious [Het-s] amyloids. In future experiments, we will aim to generate neuronal toxicity through the co-expression of het-s(218-289) and het-S. This C. elegans model for prion studies, filling a gap between simple fungal systems and highly complex mammalian systems, might allow a better understanding of the molecular and cellular basis of amyloid infectivity and toxicity.
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[
Am J Trop Med Hyg,
1988]
A comprehensive study of the transmission of Onchocerca volvulus at 4 locations in Guatemala with different prevalence rates of onchocerciasis included observations on potential secondary vectors, the most prevalent of which were Simulium metallicum, S. callidum, and S. gonzalezi. Filariae encountered in S. metallicum were primarily of a Dipetalonema-like species, but third-stage larvae indistinguishable from O. volvulus were found in 4 flies of this species. Our findings suggest that O. volvulus may occasionally be transmitted by S. metallicum, but such transmission is likely limited to areas having both a high parasite prevalence maintained by S. ochraceum and a relatively high host-seeking density of S. metallicum. Two third-stage larvae that could not be differentiated from O. volvulus were found once in S. gonzalezi; however, transmission by this species appears to be inconsequential.