Allelic frequency and genotypes of prion protein at codon 136 and 171 in Iranian Ghezel sheep breeds

PrP genotypes at codons 136 and 171 in 120 Iranian Ghezel sheep breeds were studied using allele-specific PCR amplification and compared with the well-known sheep breeds in North America, the United States and Europe. The frequency of V allele and VV genotype at codon 136 of Ghezel sheep breed was significantly lower than AA and AV. At codon 171, the frequency of allele H was significantly lower than Q and R. Despite the similarities of PrP genotypes at codons 136 and 171 between Iranian Ghezel sheep breeds and some of the studied breeds, significant differences were found with others. Planning of effective breeding control and successful eradication of susceptible genotypes in Iranian Ghezel sheep breeds will not be possible unless the susceptibility of various genotypes in Ghezel sheep breeds to natural or experimental scrapie has been elucidated.Key words: scrapie, Ghezel sheep breed, PrP genotyping, allele specific amplification, codon 136, codon 171Scrapie was first described in England in 1732,¹ and it is an infectious neurodegenerative fatal disease of sheep and goats belonging to the group of transmissible subacute spongiform encephalopathies (TSEs), along with bovine spongiform encephalopathy (BSE), chronic wasting disease and Creutzfeldt-Jakob disease.^2,3 The term prion, proteinaceous infectious particles, coined by Stanley B. Prusiner, was introduced, and he presents the idea that the causal agent is a protein.⁴ Prion proteins are discovered in two forms, the wild-type form (PrP^c) and the mutant form (PrP^Sc).⁵ Although scrapie is an infectious disease, the susceptibility of sheep is influenced by genotypes of the prion protein (PrP) gene.^2,6 Researchers have found that the PrP allelic variant alanine/arginine/arginine (ARR) at codons 136, 154 and 171 is associated with resistance to scrapie in several breeds.^7–14 Most of the sheep populations in the Near East and North African Region (84% of the total population of 255 million) are raised in Iran, Turkey, Pakistan, Sudan, Algeria, Morocco, Afghanistan, Syria and Somalia.¹⁵ In 2003, the Iranian sheep population was estimated at 54,000,000 head. The Ghezel sheep breed, which also is known as Kizil-Karaman, Mor-Karaman, Dugli, Erzurum, Chacra, Chagra, Chakra, Gesel, Gezel, Kazil, Khezel, Khizel, Kizil, Qezel, Qizil and Turkish Brown, originated in northwestern Iran and northeastern Turkey. By considering sheep breeds as one of the main sources of meat, dairy products and related products, a global screening attempt is started in different areas. In compliance with European Union Decision 2003/100/EC, each member state has introduced a breeding program to select for resistance to TSEs in sheep populations to increase the frequency of the ARR allele. A similar breeding program is established in United States and Canada. The Near East and North African Region still needs additional programs to help the global plan of eradication of scrapie-susceptible genotypes. The current study was the first to assess the geographical and molecular variation of codons 136 and 171 polymorphism between Iranian Ghezel sheep breed and well-known sheep breeds.Polymorphism at codon 136 is associated with susceptibility to scrapie in both experimental and natural models.^10,11,13,16 17 and Austrian Carynthian sheep.¹⁸ Swiss White Alpine showed higher frequency of allele V at position 136 than Swiss Oxford Down, Swiss Black-Brown Mountain and Valais Blacknose.¹⁹ Comparison of polymorphism at codon 136 in the current study with some of other breeds (20 some flock of Hampshire sheep²¹ with current study, but the frequency of it is higher than that of some other breeds.

Table 1

Comparison of PrP allelic and genotype frequencies at codon 136 in different breeds

Age‐dependent alterations of the kynurenine pathway in the YAC128 mouse model of Huntington disease

Indoleamine 2,3 dioxygenase (Ido1), the first and rate‐limiting enzyme of the kynurenine pathway (KP), is a striatally enriched gene with increased expression levels in the YAC128 mouse model of Huntington disease (HD). Our objective in this study was to delineate age‐related KP alterations in this model. Three enzymes potentially catalyze the first step of the KP; Ido1 and Indoleamine 2,3 dioxygenase‐2 were highly expressed in the striatum and Tryptophan 2,3 dioxygenase (Tdo2) in the cerebellum. During development, Ido1 mRNA expression is dynamically regulated and chronically up‐regulated in YAC128 mice. Kynurenine (Kyn) to tryptophan (Trp) ratio, a measure of activity in the first step of the KP, was elevated in YAC128 striatum, but no change in Tdo2 mRNA levels or Kyn to Trp ratio was detected in the cerebellum. Ido1 induction was coincident with Trp depletion at 3 months and Kyn accumulation at 12 months of age in striatum. Changes in downstream KP metabolites of YAC128 mice generally followed a biphasic pattern with neurotoxic metabolites reduced at 3 months and increased at 12 months of age. Striatally specific induction of Ido1 and downstream KP alterations suggest involvement in HD pathogenesis, and should be taken into account in future therapeutic developments for HD.     

Optic Nerve Diffusion Tensor Imaging after Acute Optic Neuritis Predicts Axonal and Visual Outcomes

Background

Early markers of axonal and clinical outcomes are required for early phase testing of putative neuroprotective therapies for multiple sclerosis (MS).

Objectives

To assess whether early measurement of diffusion tensor imaging (DTI) parameters (axial and radial diffusivity) within the optic nerve during and after acute demyelinating optic neuritis (ON) could predict axonal (retinal nerve fibre layer thinning and multi-focal visual evoked potential amplitude reduction) or clinical (visual acuity and visual field loss) outcomes at 6 or 12 months.

Methods

Thirty-seven patients presenting with acute, unilateral ON were studied at baseline, one, three, six and 12 months using optic nerve DTI, clinical and paraclinical markers of axonal injury and clinical visual dysfunction.

Results

Affected nerve axial diffusivity (AD) was reduced at baseline, 1 and 3 months. Reduced 1-month AD correlated with retinal nerve fibre layer (RNFL) thinning at 6 (R=0.38, p=0.04) and 12 months (R=0.437, p=0.008) and VEP amplitude loss at 6 (R=0.414, p=0.019) and 12 months (R=0.484, p=0.003). AD reduction at three months correlated with high contrast visual acuity at 6 (ρ = -0.519, p = 0.001) and 12 months (ρ = -0.414, p=0.011). The time-course for AD reduction for each patient was modelled using a quadratic regression. AD normalised after a median of 18 weeks and longer normalisation times were associated with more pronounced RNFL thinning and mfVEP amplitude loss at 12 months. Affected nerve radial diffusivity (RD) was unchanged until three months, after which time it remained elevated.

Conclusions

These results demonstrate that AD reduces during acute ON. One month AD reduction correlates with the extent of axonal loss and persistent AD reduction at 3 months predicts poorer visual outcomes. This suggests that acute ON therapies that normalise optic nerve AD by 3 months could also promote axon survival and improve visual outcomes.     

Effects of the dichloromethane fraction of Dipsaci Radix on the osteoblastic differentiation of human alveolar bone marrow-derived mesenchymal stem cells

Dipsaci Radix is the dried root of Dipsacus asper Wall. It has been used in Korean herbal medicine to treat bone fractures. In this study, we examined the effect of the dichloromethane fraction of Dipsaci Radix (DR(DM)) on the osteoblastic differentiation of human alveolar bone marrow-derived MSCs (ABM-MSCs). The ABM-MSCs were isolated from healthy subjects and cultured in vitro, followed by phenotypic characterization. They showed a fibroblast-like morphology and expressed CD29, CD44, CD73, and CD105, but not CD34. Calcified nodules were generated in response to both dexamethasone (DEX) and DR(DM). There was a significant increase in the alkaline phosphatase (ALP) activity and protein expression of bone sialoprotein (BSP) and osteocalcin (OC) in response to DEX and DR(DM) as compared to control. These results provide evidence for the osteogenic potential of cultured ABM-MSCs in response to DR(DM). Also, an active single compound was additionally isolated from DR(DM). The single compound (hederagenin 3-O-(2-O-acetyl)-α-L-arabinopyranoside) also significantly increased ALP activity and the level of protein expression of BSP and OC. These results highlight the possible clinical applications of DR(DM) and hederagenin 3-O-(2-O-acetyl)-α-L-arabinopyranoside in bone regeneration.     

The F-actin-microtubule crosslinker Shot is a platform for Krasavietz-mediated translational regulation of midline axon repulsion

Axon extension and guidance require a coordinated assembly of F-actin and microtubules as well as regulated translation. The molecular basis of how the translation of mRNAs encoding guidance proteins could be closely tied to the pace of cytoskeletal assembly is poorly understood. Previous studies have shown that the F-actin-microtubule crosslinker Short stop (Shot) is required for motor and sensory axon extension in the Drosophila embryo. Here, we provide biochemical and genetic evidence that Shot functions with a novel translation inhibitor, Krasavietz (Kra, Exba), to steer longitudinally directed CNS axons away from the midline. Kra binds directly to the C-terminus of Shot, and this interaction is required for the activity of Shot to support midline axon repulsion. shot and kra mutations lead to weak robo-like phenotypes, and synergistically affect midline avoidance of CNS axons. We also show that shot and kra dominantly enhance the frequency of midline crossovers in embryos heterozygous for slit or robo, and that in kra mutant embryos, some Robo-positive axons ectopically cross the midline that normally expresses the repellent Slit. Finally, we demonstrate that Kra also interacts with the translation initiation factor eIF2beta and inhibits translation in vitro. Together, these data suggest that Kra-mediated translational regulation plays important roles in midline axon repulsion and that Shot functions as a direct physical link between translational regulation and cytoskeleton reorganization.     

De Novo Nonsense Mutations in KAT6A,a Lysine Acetyl-Transferase Gene,Cause a Syndrome Including Microcephaly and Global Developmental Delay

Chromatin remodeling through histone acetyltransferase (HAT) and histone deactylase (HDAC) enzymes affects fundamental cellular processes including the cell-cycle, cell differentiation, metabolism, and apoptosis. Nonsense mutations in genes that are involved in histone acetylation and deacetylation result in multiple congenital anomalies with most individuals displaying significant developmental delay, microcephaly and dysmorphism. Here, we report a syndrome caused by de novo heterozygous nonsense mutations in KAT6A (a.k.a., MOZ, MYST3) identified by clinical exome sequencing (CES) in four independent families. The same de novo nonsense mutation (c.3385C>T [p.Arg1129^∗]) was observed in three individuals, and the fourth individual had a nearby de novo nonsense mutation (c.3070C>T [p.Arg1024^∗]). Neither of these variants was present in 1,815 in-house exomes or in public databases. Common features among all four probands include primary microcephaly, global developmental delay including profound speech delay, and craniofacial dysmorphism, as well as more varied features such as feeding difficulties, cardiac defects, and ocular anomalies. We further demonstrate that KAT6A mutations result in dysregulation of H3K9 and H3K18 acetylation and altered P53 signaling. Through histone and non-histone acetylation, KAT6A affects multiple cellular processes and illustrates the complex role of acetylation in regulating development and disease.     

A first generation physical map of the medaka genome in BACs essential for positional cloning and clone-by-clone based genomic sequencing

In order to realize the full potential of the medaka as a model system for developmental biology and genetics, characterized genomic resources need to be established, culminating in the sequence of the medaka genome. To facilitate the map-based cloning of genes underlying induced mutations and to provide templates for clone-based genomic sequencing, we have created a first-generation physical map of the medaka genome in bacterial artificial chromosome (BAC) clones. In particular, we exploited the synteny to the closely related genome of the pufferfish, Takifugu rubripes, by marker content mapping. As a first step, we clustered 103,144 public medaka EST sequences to obtain a set of 21,121 non-redundant sequence entities. Avoiding oversampling of gene-dense regions, 11,254 of EST clusters were successfully matched against the draft sequence of the fugu genome, and 2363 genes were selected for the BAC map project. We designed 35mer oligonucleotide probes from the selected genes and hybridized them against 64,500 BAC clones of strains Cab and Hd-rR, representing 14-fold coverage of the medaka genome. Our data set is further supplemented with 437 results generated from PCR-amplified inserts of medaka cDNA clones and BAC end-fragment markers. Our current, edited, first generation medaka BAC map consists of 902 map segments that cover about 74% of the medaka genome. The map contains 2721 markers. Of these, 2534 are from expressed sequences, equivalent to a non-redundant set of 2328 loci. The 934 markers (724 different) are anchored to the medaka genetic map. Thus, genetic map assignments provide immediate access to underlying clones and contigs, simplifying molecular access to candidate gene regions and their characterization.     

Relationship between breast arterial calcification on mammography with coronary CT angiography findings

Background

Breast arterial calcification is a frequently benign finding on mammography that usually is not reported. An increasing attention has been formed to determine the association between breast arterial calcification (BAC) and Coronary artery disease. In the current study we have aimed to evaluate the relationship between BAC on mammography with coronary CT angiography findings.

Methods

The case control study was carried out on 60 women; 30 CAD and 30 healthy subjects as control, admitted to Golestan hospital radiology department. The mammography was performed in two views; Craniocaudal (CC) view and mediolateral oblique (MLO) and BAC was graded based on the severity and extent of calcifications. Coronary Arterial Calcification (CAC) were scored by Agatston criteria.

Results

Overly, 36 patients (60%) were positive for BAC. Twenty six out of them (72%) were CAD. There was a positive significant correlation between BAC and CAD. The sensitivity and specificity of BAC for CAD were 69% and 47%, respectively. Moreover, the severe BAC scores were significantly higher in CAD patients than non-CAD.

Conclusion

Our findings in line with several previous studies indicated the positive significant association between BAC and CAD occurrence. While the sensitivity and specificity of BAC in diagnosis of CAD is low, suggested the using of BAC just as a CAD risk factor. The relatively low sample size is the major limitation of the study.

     

Myosin heavy chain expression in embryonic cardiac cell cultures

Chick embryonic heart cell isolates and monolayer cultures were prepared from atria and ventricles at selected stages of cardiac development. The cardiac myocytes were assayed for myosin heavy chain (MHC) content using monoclonal antibodies (McAbs) specific in the heart for atrial (B-1), ventricular (ALD-19), or conductive system (ALD-58) isoforms. Using immunofluorescence microscopy or radioimmunoassay, MHC accumulation was measured before plating and at 48 hr or 7 days in culture. Reproducible changes in MHC antigenicity were observed by 7 days in both atrial and ventricular cultures. The changes were stage dependent and tissue specific but generally resulted in a decreased reactivity with the tissue specific MHC McAbs. In addition, the isoform recognized by ALD-58, characteristic of the conductive system cells in vivo, was never present in cultured myocytes. These results indicate that MHC isoforms produced in vivo may be replaced in monolayer cultures by an isoform(s) not recognized by our tissue specific MHC McAbs. This suggests that the intrinsic program of cardiac myogenesis, within cardiac myocytes, may not be sufficient to establish and maintain differential expression of tissue specific MHC in monolayer cell culture.