Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 11 de 11
Filter
Add more filters










Publication year range
1.
Biol Methods Protoc ; 5(1): bpaa004, 2020.
Article in English | MEDLINE | ID: mdl-32395633

ABSTRACT

Despite its widespread value to molecular biology, the polymerase chain reaction (PCR) encounters modes that unproductively consume PCR resources and prevent clean signals, especially when high sensitivity, high SNP discrimination, and high multiplexing are sought. Here, we show how "self-avoiding molecular recognition systems" (SAMRS) manage such difficulties. SAMRS nucleobases pair with complementary nucleotides with strengths comparable to the A:T pair, but do not pair with other SAMRS nucleobases. This should allow primers holding SAMRS components to avoid primer-primer interactions, preventing primer dimers, allowing more sensitive SNP detection, and supporting higher levels of multiplex PCR. The experiments here examine the PCR performances of primers containing different numbers of SAMRS components placed strategically at different positions, and put these performances in the context of estimates of SAMRS:standard pairing strengths. The impact of these variables on primer dimer formation, the overall efficiency and sensitivity of SAMRS-based PCR, and the value of SAMRS primers when detecting single nucleotide polymorphisms (SNPs) are also evaluated. With appropriately chosen polymerases, SNP discrimination can be greater than the conventional allele-specific PCR, with the further benefit of avoiding primer dimer artifacts. General rules guiding the design of SAMRS-modified primers are offered to support medical research and clinical diagnostics products.

2.
Nucleic Acids Res ; 46(12): 5902-5910, 2018 07 06.
Article in English | MEDLINE | ID: mdl-29800323

ABSTRACT

Described here are the synthesis, enzymology and some applications of a purine nucleoside analog (H) designed to have two tautomeric forms, one complementary to thymidine (T), the other complementary to cytidine (C). The performance of H is compared by various metrics to performances of other 'biversal' analogs that similarly rely on tautomerism to complement both pyrimidines. These include (i) the thermodynamic stability of duplexes that pair these biversals with various standard nucleotides, (ii) the ability of the biversals to support polymerase chain reaction (PCR), (iii) the ability of primers containing biversals to equally amplify targets having polymorphisms in the primer binding site, and (iv) the ability of ligation-based assays to exploit the biversals to detect medically relevant single nucleotide polymorphisms (SNPs) in sequences flanked by medically irrelevant polymorphisms. One advantage of H over the widely used inosine 'universal base' and 'mixed sequence' probes is seen in ligation-based assays to detect SNPs. The need to detect medically relevant SNPs within ambiguous sequences is especially important when probing RNA viruses, which rapidly mutate to create drug resistance, but also suffer neutral drift, the second obstructing simple methods to detect the first. Thus, H is being developed to detect variants of viruses that are rapidly mutating.


Subject(s)
Nucleosides/chemistry , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , DNA Primers , Isomerism , Mutation , Nucleosides/chemical synthesis , Oligonucleotides/chemical synthesis , Oligonucleotides/chemistry , Purines/chemistry , Temperature
3.
Chembiochem ; 16(9): 1365-70, 2015 Jun 15.
Article in English | MEDLINE | ID: mdl-25953623

ABSTRACT

Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low-resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off-target priming, and other artifacts. Here, we show how a "self avoiding molecular recognition system" (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase-dependent isothermal amplification (SAMRS-HDA). We also show that incorporating SAMRS into the 3'-ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS-HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS-HDA is a more versatile approach than standard HDA, with a broader applicability for xNA-targeted diagnostics and research.


Subject(s)
DNA Helicases/metabolism , DNA/genetics , Nucleic Acid Amplification Techniques/methods , RNA/genetics , Base Sequence , DNA/analysis , DNA Primers/chemistry , DNA Primers/genetics , HIV Infections/virology , HIV-1/genetics , Humans , Male , Point-of-Care Systems , Polymerase Chain Reaction , RNA/analysis , RNA, Viral/analysis , RNA, Viral/genetics , Temperature
4.
Neurobiol Dis ; 22(2): 404-20, 2006 May.
Article in English | MEDLINE | ID: mdl-16439141

ABSTRACT

Sporadic Parkinson's disease (PD) is most likely caused by a combination of environmental exposures and genetic susceptibilities, although there are rare monogenic forms of the disease. Mitochondrial impairment at complex I, oxidative stress, alpha-synuclein aggregation, and dysfunctional protein degradation, have been implicated in PD pathogenesis, but how they are related to each other is unclear. To further evaluated PD pathogenesis here, we used in vivo and in vitro models of chronic low-grade complex I inhibition with the pesticide rotenone. Chronic rotenone exposure in vivo caused oxidative modification of DJ-1, accumulation of alpha-synuclein, and proteasomal impairment. Interestingly, the effects become more regionally restricted such that systemic complex I inhibition eventually results in highly selective degeneration of the nigrostriatal pathway. DJ-1 modifications, alpha-synuclein accumulation, and proteasomal dysfunction were also seen in vitro and these effects could be prevented with alpha-tocopherol. Thus, chronic exposure to a pesticide and mitochondrial toxin brings into play three systems, DJ-1, alpha-synuclein, and the ubiquitin-proteasome system, and implies that mitochondrial dysfunction and oxidative stress link environmental and genetic forms of the disease.


Subject(s)
Nerve Degeneration/chemically induced , Oncogene Proteins/drug effects , Parkinsonian Disorders/chemically induced , Proteasome Endopeptidase Complex/drug effects , Rotenone/toxicity , Ubiquitin/drug effects , alpha-Synuclein/drug effects , Animals , Cell Line, Tumor , Disease Models, Animal , Electron Transport Complex I/drug effects , Electron Transport Complex I/physiology , Energy Metabolism/drug effects , Energy Metabolism/physiology , Humans , Insecticides/toxicity , Male , Mitochondria/drug effects , Mitochondria/metabolism , Nerve Degeneration/metabolism , Nerve Degeneration/physiopathology , Neurons/drug effects , Neurons/metabolism , Oncogene Proteins/metabolism , Oxidative Stress/drug effects , Oxidative Stress/physiology , Parkinsonian Disorders/metabolism , Parkinsonian Disorders/physiopathology , Peroxiredoxins , Proteasome Endopeptidase Complex/metabolism , Protein Deglycase DJ-1 , Rats , Rats, Inbred Lew , Signal Transduction/drug effects , Signal Transduction/physiology , Substantia Nigra/drug effects , Substantia Nigra/metabolism , Substantia Nigra/physiopathology , Ubiquitin/metabolism , alpha-Synuclein/metabolism
5.
Proc Natl Acad Sci U S A ; 101(24): 9103-8, 2004 Jun 15.
Article in English | MEDLINE | ID: mdl-15181200

ABSTRACT

Loss-of-function DJ-1 mutations can cause early-onset Parkinson's disease. The function of DJ-1 is unknown, but an acidic isoform accumulates after oxidative stress, leading to the suggestion that DJ-1 is protective under these conditions. We addressed whether this represents a posttranslational modification at cysteine residues by systematically mutating cysteine residues in human DJ-1. WT or C53A DJ-1 was readily oxidized in cultured cells, generating a pI 5.8 isoform, but an artificial C106A mutant was not. We observed a cysteine-sulfinic acid at C106 in crystalline DJ-1 but no modification of C53 or C46. Oxidation of DJ-1 was promoted by the crystallization procedure. In addition, oxidation-induced mitochondrial relocalization of DJ-1 and protection against cell death were abrogated in C106A but not C53A or C46A. We suggest that DJ-1 protects against neuronal death, and that this is signaled by acidification of the key cysteine residue, C106.


Subject(s)
Cysteine/analogs & derivatives , Cysteine/metabolism , Mitochondria/metabolism , Neuroprotective Agents/metabolism , Oncogene Proteins/metabolism , 1-Methyl-4-phenylpyridinium/toxicity , Amino Acid Substitution , Cell Line, Tumor , Cysteine/chemistry , Cysteine/genetics , Humans , Intracellular Membranes/metabolism , Intracellular Signaling Peptides and Proteins , Models, Molecular , Neurotransmitter Agents , Oncogene Proteins/chemistry , Oncogene Proteins/genetics , Oxidation-Reduction , Oxidative Stress , Protein Deglycase DJ-1 , Protein Isoforms , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity , Transfection
6.
Brain ; 127(Pt 2): 420-30, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14662519

ABSTRACT

Two mutations in the DJ-1 gene on chromosome1p36 have been identified recently to cause early-onset, autosomal recessive Parkinson's disease. As no information is available regarding the distribution of DJ-1 protein in the human brain, in this study we used a monoclonal antibody for DJ-1 to map its distribution in frontal cortex and substantia nigra, regions invariably involved in Parkinson's disease. Western blotting of human frontal cortex showed DJ-1 to be an abundant protein in control, idiopathic Parkinson's disease, cases with clinical and pathological phenotypes of Parkinson's disease with R98Q polymorphism for DJ-1, and in progressive supranuclear palsy (PSP) brains. We also showed that DJ-1 immunoreactivity (IR) was particularly prominent in astrocytes and astrocytic processes in both control and Parkinson's disease frontal cortex, whereas neurons showed light or no DJ-1 IR. Only occasional Lewy bodies (LBs), the pathological hallmarks of Parkinson's disease, showed faint DJ-1 IR, localized to the outer halo. In preclinical studies we showed that DJ-1 is expressed in primary hippocampal and astrocyte cultures of mouse brain. By 2D gel analysis we also showed multiple pI isoforms for DJ-1 ranging between 5.5-6.6 in both control and Parkinson's disease brains, whilst exposure of M17 cells to the oxidizing agent paraquat was manifested as a shift in pI of endogenous DJ-1 towards more acidic isoforms. We conclude that DJ-1 is not an essential component of LBs and Lewy neurites, is expressed mainly by astrocytes in human brain tissue and is sensitive to oxidative stress conditions. These results are consistent with the hypothesis that neuronal-glial interactions are important in the pathophysiology of Parkinson's disease.


Subject(s)
Brain/metabolism , Oncogene Proteins/metabolism , Parkinson Disease/metabolism , Aged , Aged, 80 and over , Animals , Antibodies, Monoclonal/immunology , Astrocytes/metabolism , Blotting, Western , Cells, Cultured , Female , Frontal Lobe/metabolism , Hippocampus/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Lewy Bodies/metabolism , Lewy Bodies/ultrastructure , Male , Mice , Neurons/metabolism , Oncogene Proteins/immunology , Oxidative Stress , Parkinson Disease/pathology , Protein Deglycase DJ-1 , Protein Isoforms/metabolism , Substantia Nigra/metabolism
7.
Hum Mol Genet ; 12(22): 2957-65, 2003 Nov 15.
Article in English | MEDLINE | ID: mdl-14519684

ABSTRACT

The Parkin gene (PRKN) encodes an E3 protein-ubiquitin ligase for which loss of function is associated with autosomal-recessive juvenile (<20 years) and early-onset Parkinsonism (<45 years). Although detailed pathological reports are scarce, brains from patients with homozygous exonic deletions demonstrate neuronal loss in the substantia nigra, albeit without the Lewy body pathology characteristic of idiopathic Parkinson's disease. However, there are rare descriptions of more florid pathology, including Lewy bodies and tau positive astrocytes in individuals with compound heterozygous mutations. In the present study we examined whether PRKN point mutations, leading to amino acid substitutions, may alter the cellular distribution of the protein produced. Wild-type Parkin was homogeneously distributed throughout the cytoplasm with a small amount of protein in the nucleus after transfection into human embryonic kidney cells. Mutant isoforms with A82E, G328E and C431F amino acid substitutions were also normally distributed. However, two mutant isoforms, R256C and R275W, within RING finger 1 of the Parkin protein (238-293 amino acids), produced an unusual distribution of the protein, with large cytoplasmic and nuclear inclusions. We have replicated this observation in primary cultured neurons and demonstrate, by the accumulation/co-localization of cytoskeletal protein vimentin, that the inclusion bodies are aggresomes, a cellular response to misfolded protein.


Subject(s)
Point Mutation , Ubiquitin-Protein Ligases/genetics , Zinc Fingers/genetics , Amino Acid Sequence , Amino Acid Substitution , Cell Line , Cell Nucleus/chemistry , Cells, Cultured , Cloning, Molecular , Cytoplasm/chemistry , Hippocampus/cytology , Humans , Lewy Bodies/metabolism , Mutagenesis, Site-Directed , Neurons/cytology , Protein Folding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Vimentin/biosynthesis
8.
J Biol Chem ; 278(38): 36588-95, 2003 Sep 19.
Article in English | MEDLINE | ID: mdl-12851414

ABSTRACT

Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, earlyonset Parkinson's disease. While one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point mutation, L166P, whose precise effects on protein function are unclear. In the present study, we show that L166P destabilizes DJ-1 protein and promotes its degradation through the ubiquitin-proteasome system. A double mutant (K130R, L166P) was more stable than L166P, suggesting that this lysine residue contributes to stability of the protein. Subcellular localization was broadly similar for both wild type and L166P forms of the protein, indicating that the effect of the mutation is predominantly on protein stability. These observations are reminiscent of other recessive gene mutations that produce an effective loss of function. The L166P mutation has the simple effect of promoting DJ-1 degradation, thereby reducing net DJ-1 protein within the cell.


Subject(s)
Cysteine Endopeptidases/metabolism , Genes, Recessive , Multienzyme Complexes/metabolism , Mutation , Oncogene Proteins/genetics , Oncogene Proteins/physiology , Parkinson Disease/genetics , Ubiquitin/metabolism , Animals , Blotting, Western , COS Cells , Cell Line , Chromatography , Cytosol/metabolism , Dose-Response Relationship, Drug , Gene Deletion , Green Fluorescent Proteins , Humans , Intracellular Signaling Peptides and Proteins , Luminescent Proteins/metabolism , Lysine/chemistry , Microscopy, Fluorescence , Mitochondria/metabolism , Point Mutation , Precipitin Tests , Proteasome Endopeptidase Complex , Protein Binding , Protein Deglycase DJ-1 , Reverse Transcriptase Polymerase Chain Reaction , Subcellular Fractions/metabolism , Transfection , Two-Hybrid System Techniques
9.
Biochem Biophys Res Commun ; 305(3): 529-33, 2003 Jun 06.
Article in English | MEDLINE | ID: mdl-12763025

ABSTRACT

The characterization of the enzymes responsible for amyloid beta-peptide (Abeta) production is considered to be a primary goal towards the development of future therapeutics for the treatment of Alzheimer's disease. Inhibitors of gamma-secretase activity were critical in demonstrating that the presenilins (PSs) likely comprised at least part of the active site of the gamma-secretase enzyme complex, with two highly conserved membrane aspartates presumably acting as catalytic residues. However, whether or not these aspartates are actually the catalytic residues of the enzyme complex or are merely essential for normal PS function and/or maturation is still unknown. In this paper, we report the development of reactive inhibitors of gamma-secretase activity that are functionally irreversible. Since such inhibitors have been shown to bind catalytic residues in other aspartyl proteases (e.g., HIV protease), they might be used to determine if the transmembrane aspartates of PSs are involved directly in substrate cleavage.


Subject(s)
Endopeptidases/metabolism , Enzyme Inhibitors/pharmacology , Oligopeptides/pharmacology , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases , CHO Cells , Cricetinae , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Epoxy Compounds/pharmacology , Humans , Kinetics , Membrane Proteins/metabolism , Oligopeptides/chemistry , Receptors, Notch , Tumor Cells, Cultured
10.
FASEB J ; 17(9): 1138-40, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12692078

ABSTRACT

Gamma-secretase cleavage is the final proteolytic step that releases the amyloid beta-peptide (Abeta) from the amyloid beta-protein precursor (APP). Significant evidence indicates that the presenilins (PS) are catalytic components of a high molecular weight gamma-secretase complex. The glycoprotein nicastrin was recently identified as a functional unit of this complex based on 1) binding to PS and 2) the ability to modulate Abeta production following mutation of a conserved DYIGS region. In contrast to the initial report, we find that overexpression of wild-type (WT) nicastrin increases Abeta production, whereas DYIGS mutations (MT) have little or no effect. The increase in Abeta production is associated with an increase in gamma-secretase activity but not with a detectable increase in PS1 levels. Subcellular fractionation studies show that WT but not MT nicastrin matures into buoyant membrane fractions enriched in gamma-secretase activity. These data support the hypothesis that nicastrin is an essential component of the gamma-secretase complex. The finding that WT nicastrin overexpression can increase gamma-secretase activity without altering levels of the presumed catalytic component (PS) of the enzyme may point to a role for nicastrin in facilitating cleavage by regulating substrate interactions with the gamma-secretase complex.


Subject(s)
Amyloid beta-Peptides/biosynthesis , Membrane Glycoproteins/physiology , Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , Cell Line , Endopeptidases/chemistry , Endopeptidases/metabolism , Humans , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Models, Biological , Mutation , Peptides/metabolism , Protein Subunits , Transfection
11.
Neurobiol Dis ; 9(1): 11-23, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11848681

ABSTRACT

Buoyant membrane fractions containing presenilin 1 (PS1), an essential component of the gamma-secretase complex, and APP CTFbeta, a gamma-secretase substrate, can be isolated from cultured cells and brain by several different fractionation procedures that are compatible with in vitro gamma-secretase assays. Analysis of these gradients for amyloid beta protein (Abeta) and CTFgamma production indicated that gamma-secretase activity is predominantly localized in these buoyant membrane microdomains. Consistent with this localization, we find that gamma-secretase activity is cholesterol dependent. Depletion of membrane cholesterol completely inhibits gamma-secretase cleavage, which can be restored by cholesterol replacement. Thus, altering cholesterol levels may influence the development of Alzheimer's disease (AD) by influencing production and deposition of Abeta within cholesterol rich membrane microdomains.


Subject(s)
Cholesterol/metabolism , Endopeptidases/metabolism , Membrane Microdomains/metabolism , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases , Amyloid beta-Peptides/metabolism , Animals , CHO Cells , Cell Fractionation , Cricetinae , Enzyme Activation/physiology , Membrane Proteins/metabolism , Presenilin-1
SELECTION OF CITATIONS
SEARCH DETAIL
...