Purification and Characterization of p68/70, Regeneration-Associated Proteins from Goldfish Brain

Two acidic proteins (p68/70) previously shown to be associated with regeneration of the goldfish optic nerve were purified 887-fold from brain homogenates of Carassius auratus. Purification to homogeneity was achieved by sequential chromatography of a 100,000 g brain supernatant fraction on DEAE-Sephacel, Cu 2+ -charged iminodiacetic acid agarose, and gel filtration. The Stokes radius of the doublet was determined to be 5.8 nm, and the sedimentation coefficient calculated to be 5 2. From these values a molecular mass of 128 kDa and a frictional coefficient ratio of 1.6 were calculated. Chromatofocusing on a high-resolution DEAE column resolved the protein doublet into three dimeric species of p68, p68/70, and p70. These results indicate that the proteins are highly elongated and associate as homodimers or as a hetero-dimer. Subcellular localization and membrane extraction experiments indicated p68/70 to be a component of the plasma membrane associated primarily through hydro-phobic interactions. p68/70 demonstrated biphasic behavior in phase partition experiments using Triton 114. Analysis of hydrolytic products indicated p68/70 to be a glyco-protein, containing 11% carbohydrate.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65206/1/j.1471-4159.1994.62031182.x.pd

Purification and characterization of the goldfish regeneration-associated proteinsgp68/70.

Successful regeneration of nerves in the central nervous system in cold-blooded vertebrates is an intriguing phenomenon, in that warm-blooded vertebrates show only a limited capacity for CNS regeneration. By studying regeneration in the goldfish retinotectal projection, strategies may be developed to stimulate regeneration in species that are presently incapable of CNS regeneration. Two acidic proteins (gp68/70) previously shown to be associated with regeneration were purified 887-fold from brain homogenates of Carassius auratus. Purification to homogeneity was achieved by sequential chromatography of a 100,000 g brain supernatant fraction on DEAE-Sephacel, Cu\sp{2+}-charged iminodiacetic acid agarose, and gel filtration. The Stokes radius of the doublet was determined to be 5.8 nm, and the sedimentation coefficient calculated to be 5.2 S. From these values a molecular mass of 128 kDa and a frictional coefficient ratio of 1.6 were calculated. Chromatofocusing on a high resolution DEAE column resolved the protein doublet into three dimeric species of gp68, gp68/70, and gp70. These results indicate that the proteins are highly elongated and associate as either homodimers or as a heterodimer. Sequence analysis of gp68/70 identified homologies to three known proteins. gp68/70 was found to possess 2\sp\prime,3\sp\prime-cyclic nucleotide 3\sp\prime-phosphodiesterase activity. Subcellular localization and membrane extraction experiments indicated gp68/70 to be a component of the plasma membrane associated primarily through hydrophobic interactions. Carbohydrate analysis of gp68/70 indicated it to contain 26% carbohydrate by weight, with fucose, N-acetylgalactosamine, galactose, N-acetylglucosamine, glucose, mannose and sialic acid present in a 1/0.4/0.9/1.0/0.7/0.3/0.1 ratio. Modification of the purification procedure enabled similar proteins to be purified from the eggs of both goldfish and carp. The results presented in this thesis, together with the results of Wilmot et al (1993), support a dynamic role for gp68/70 in neurite extension and in non-neuronal membrane function.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103633/1/9332118.pdfDescription of 9332118.pdf : Restricted to UM users only

The CTLA4 -819 C/T and +49 A/G dimorphisms are associated with Type 1 diabetes in Egyptian children author

Background: Type 1 diabetes (T1D) is an organ-specific autoimmune disease characterized by T cell-mediated destruction of pancreatic islets. T cell proliferation is negatively regulated by cytotoxic lymphocyte antigen-4 (CTLA-4). CTLA-4 polymorphisms are associated with T1D in some but not all populations. Aims: The study was conducted to investigate the association of the C-819T and A+49G single nucleotide polymorphisms (SNP) of CTLA-4 gene in T1D patients in the Egyptian population. Methods: The association of the C-819T SNP in intron 1 and A+49G SNP in exon 1 of the CTLA-4 gene with T1D were investigated in 396 Egyptian patients ≤14 years old and 396 control subjects> 24 years old, with the same ratio of males to females in both groups. The diagnosis of T1D was made on the basis of ketoacidosis or ketosis with severe symptoms of acute onset at presentation and continuous dependence on insulin. Controls were negative for anti-GAD antibodies and were greater than 24 years of age. Genotyping was performed using single strand conformation polymorphism (SSCP), temperature gradient gel electrophoresis (TGGE), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: The results demonstrated an association of the C-819T and A+49G SNPs in the CTLA-4 gene with T1D patients (P=0.0047) and (P=0.000575), respectively. Moreover, this association was stratified by gender and age to female patients with age at onset 0-5 years old (P=0.0186) and (P=0.00115) more than male patient with the age at onset 0-5 years old (P= 0.3120) and (P=0.345161), respectively. Conclusion: The results support an association of the C-819T and A+49G SNPs in the CTLA-4 gene with Egyptian children, specifically, females of onset age 0-5 years old

Overview of clinical forensic services in various countries of the European Union

Examination of a person who has been a victim of a physical or sexual assault may be very important for upcoming legal proceedings. In the context of a clinical forensic examination, physical findings are recorded and biological trace material is gathered and secured. Ideally, all forensic findings are documented in a detailed report combined with photographic documentation, which employs a forensic scale to depict the size of the injuries. However, the integrity of such forensic findings depends particularly on two factors. First, the examination needs to be conducted professionally to ensure that the findings are properly admissible as court evidence. Second, the examination should take place as soon as possible because the opportunity to successfully secure biological samples declines rapidly with time. Access to low-threshold clinical forensic examinations is not evenly provided in all member states of the European Union (EU); in some states, they are not available at all. As part of the JUSTeU! (Juridical standards for clinical forensic examinations of victims of violence in Europe) project, the Ludwig Boltzmann Institute for Clinical Forensic Imaging in Graz, Austria created (in cooperation with its international partner consortium) a questionnaire: the purpose was to collect information about support for victims of physical and/or sexual assault in obtaining a low-threshold clinical forensic examination in various countries of the EU. Our paper provides a summary of the responses and an overview of the current situation concerning provided clinical forensic services

Sequence Variability and Geographic Distribution of Lassa Virus, Sierra Leone

Lassa virus (LASV) is endemic to parts of West Africa and causes highly fatal hemorrhagic fever. The multimammate rat (Mastomys natalensis) is the only known reservoir of LASV. Most human infections result from zoonotic transmission. The very diverse LASV genome has 4 major lineages associated with different geographic locations. We used reverse transcription PCR and resequencing microarrays to detect LASV in 41 of 214 samples from rodents captured at 8 locations in Sierra Leone. Phylogenetic analysis of partial sequences of nucleoprotein (NP), glycoprotein precursor (GPC), and polymerase (L) genes showed 5 separate clades within lineage IV of LASV in this country. The sequence diversity was higher than previously observed; mean diversity was 7.01% for nucleoprotein gene at the nucleotide level. These results may have major implications for designing diagnostic tests and therapeutic agents for LASV infections in Sierra Leone

Pairwise Ф_ST values between subspecies calculated from mitochondrial haplotype frequencies in Arlequin.

<p>Values significantly different in the exact tests of differentiation are in bold.</p

Number of mitochondrial haplotypes and haplotype diversity values of each population.

<p>Idaho, Quebec and Georgia were excluded from the analysis since they were represented by less than five individuals.</p

Population structure inferred in STRUCTURE based on microsatellite data.

<p>Each genetic cluster is represented by a colour. Every individual is represented by a single vertical line with coloured segments depicting the estimated proportion of ancestry from a given cluster. a) Results from the analysis where individuals were grouped into populations <i>a priori</i>; K = 5. b) Results from the analysis where individuals were grouped into subspecies <i>a priori</i>; K = 6.</p