).36-38 It is therefore conceivable that the loss of parkin function may lead to the accumulation of a nonubiquitinated substrate, which is deleterious to the dopaminergic cell, but, due to its nonubiquitinated nature, does not form typical Lewy bodies. Several proteins have been shown to interact, with parkin and Inhibitors,research,lifescience,medical could possibly be its relevant partner with regard to neurodegeneration: an O-glycosylated form of oc-synuclein.39; a protein associated with synaptic vesicles, CDCrel-1.37;

a transmembrane protein, called the pael receptor40; and synphillin-1.41 However, other pathogenetic mechanisms could also be important. Recently, it has been shown that, in parkin knockout mice, a number of Inhibitors,research,lifescience,medical genes related to the oxidative metabolism in mitochondria are downregulated. Oxidative damage and dysfunction of components of the mitochondrial respiratory chain could be demonstrated, implicating this pathway in the pathogenesis of PD.42 Figure 1 Parkin and its function in the ubiquitin-proteasome system (UPS).

Parkin functions as a ubiquitin ligase, by adding ubiquitin, a small signal Inhibitors,research,lifescience,medical polypeptide, to target proteins. The polyubiquitinated protein is directed to the proteasome, a multi-subunit … PARK6: parkinsonism caused by selleck screening library mutations in the gene for PINK1 Very recently, mutations in the gene for PINK1 (PTEN-induced kinase 1) have been found to cause another autosomal-recessive variant of early-onset PD,39 PARK6, which has previously been mapped to chromosomal region lp36.43 Two homozygous mutations affecting Inhibitors,research,lifescience,medical the PINK1 kinase domain were identified in three consanguineous PARK6 families: Inhibitors,research,lifescience,medical a truncating nonsense mutation and a missense mutation at a highly conserved amino acid. Cell culture studies suggested that PINK1 is a mitochondrially located protein and may exert a protective effect on the cell upon oxidative stress, which is abrogated by the mutations.9

As in families with parkin mutations, PINK1 -associated parkinsonism is of early onset of and takes a relatively benign cause. PARK7: parkinsonism caused by mutations in the gene for DJ-1 Another recessive locus was mapped to chromosome 1 in a consanguineous Italian family.44 These patients had disease onset in their mid-thirties with L-dopa responsiveness, slow progression, and focal dystonic symptoms, such as blepharospasm. Pathogenic mutations were identified in the gene for DJ-1.10 A missense mutation resulting in the substitution of a highly conserved leucine for a proline at position 166 (L166P mutation) and a large homozygous deletion of several exons (exons 1 to 5) were detected in an Italian and a Dutch family, respectively.

5 M EDTA (Becton Dickinson, ) solution, carried on ice and kept a

5 M EDTA (Becton Dickinson, ) solution, carried on ice and kept at -70 ºC for DNA extraction. Because G6PD Mediterranean (C563T) is reported as the most common mutation in Middle East and some provinces of Iran, at first we analyzed all samples for this mutation.16 Finally 64 (55 males and 9 females) out of 231 samples were recognized without Mediterranean mutation, which were then analyzed to identify Cosenza mutation.Genomic DNA was extracted from leukocytes by using “PicoPure ” DNA extraction kit #AZD0530 keyword# from Molecular Devices (San Diego, CA). mutation site is located

in exon 12 of G6PD gene. For detection of the Cosenza mutation, exon 11-13 of G6PD gene was selectively amplified by PCR method using F-cos (5´-GCA GCC AGT GGC ATC AGC AAG-3´) and R-cos (5´-GGG AAG GAG GGT GGC CGT GG-3´) primers.14 Inhibitors,research,lifescience,medical Polymerase chain reaction (PCR) assay was

performed in final volume of 25 μl. PCR reaction mix contained 10X PCR buffer, 10 mM of each deoxynucleotide triphosphate, 25 pmol of each primer, 0.5 μg genomic DNA, 2 U/ml of Taq DNA polymerase and 50 mM MgCl2. The PCR reaction was carried out for 30 cycles as follows: 10 cycles (94 ºC for 30 seconds, 68 ºC for 1 min and 72 Inhibitors,research,lifescience,medical ºC for 30 seconds) and 20 cycles (95 ºC, 65 ºC, 72 ºC each temperature for 1 min). In order to certify the fidelity of PCR, amplified segments were run on 1.5% agarose gel (figure 1). Since the mutation creates an Eco81I recognition site (figure 2), this endonuclease was used to perform

Restriction fragment length polymorphism (RFLP) analysis. Cozenza PCR products were digested by Eco81I enzyme (Fermentas GmbH, ) at 37 ºC, overnight. The digested fragments were Inhibitors,research,lifescience,medical tested on 2% agarose gel. Figure 1 Polymerase chain reaction (PCR) products related to glucose-6-phosphate dehydrogenase Cosenza mutation on 1.5 % agarose gel. Lane 1: Size Marker 1 Kbp, Lane 2: negative control, Lanes 3, 4, 5, 6, 7, 8 and 9: Cosenza PCR products with 548 bp length Figure 2 Oligonucleotide Inhibitors,research,lifescience,medical primers F-cos and R-cos amplify a 548pb fragment across exon 11 and 13 of the glucose-6-phosphate dehydrogenase gene that after digestion by Eco81I appeared as two fragments with 232 bp and 316 bp Results Among the 231 G6PD deficient individuals (a total of 267 alleles), 195 (84.1%) were males and 36 were females. Only 64 samples (55 males and Electron transport chain 9 females) out of 231 deficient subjects did not have G6PD Mediterranean. They were analyzed to characterize G6PD Cosenza Mutation, using PCR-RFLP method. Cosenza PCR product was a 548 bp fragment, which appeared as two fragments with 232 bp and 316 bp lengths after digestion by Eco81I on 2% agarose gel in mutant subjects (figure 3). The result showed that 6 males out of 231 samples had the Cosenza mutation. Therefore the mutation relative rate and allele frequency in Khuzestanian deficient subjects are 2.6% and 0.023, respectively. Fifty eight samples did not have Mediterranean and mutations.