Detection of a Novel Unglycosylated Form of Hepatitis C Virus E2 Envelope Protein That Is Located in the Cytosol and Interacts with PKR

The hepatitis C virus (HCV) envelope protein E2 has been shown to accumulate in the lumen of the endoplasmic reticulum (ER) as a properly folded glycoprotein as well as large aggregates of misfolded proteins. In the present study, we have identified an additional unglycosylated species, with an apparent molecular mass of 38 kDa (E2-p38). In contrast to the glycosylated E2, E2-p38 is significantly less stable and is degraded through the proteasome pathway. Correspondingly, E2-p38 is found to be ubiquitinated. E2-p38 is localized mostly in the cytosol, in contrast to the glycosylated form, which is exclusively membrane associated. Alpha interferon (IFN-alpha) treatment or overexpression of the double-stranded RNA-activated protein kinase (PKR) significantly increased the stability of E2-p38, consistent with a previous report (D. R. Taylor, S. T. Shi, P. R. Romano, G. N. Barber, and M. M. Lai, Science 285:107-110, 1999) that E2 interacts with PKR and inhibits its kinase activity. Direct interaction between PKR and E2-p38, but not the glycosylated form of E2, was also observed. These results show that E2-p38 is the form of E2 that interacts with PKR in the cytosol and may contribute to the resistance of HCV to IFN-alpha. Thus, an ER protein can exist in the cytosol as an unglycosylated species and impair cellular functions.     

Isolation and characterization of polymorphic microsatellites in European chestnut (Castanea sativa Mill.)

Thirteen polymorphic microsatellite loci were isolated from Castanea sativa (Mill.). Six contained dinucleotide repeats, six contained trinucleotide repeats, and one contained a compound microsatellite of a trinucleotide and a tetranucleotide repeat. The loci were characterized using C. sativa trees from three populations in the UK and the parents and six seedlings from a Turkish mapping population. The number of alleles revealed varied from two to 14 (mean = 5.15) per loci. Eight loci were found to be useful in the mapping family.     

Genetic mapping of meander tail, a mouse mutation affecting cerebellar development

The meander tail mouse harbors a recessive mutation on chromosome 4 that affects the anterior lobes of the cerebellum and the caudal vertebrae. Examination of the mea/mea cerebellum reveals that the complete disorganization of all cell types seen in the anterior lobes is separated by a sharp and consistent boundary from the normal cytoarchitecture of the posterior lobes. In the absence of any biochemical information regarding the affected gene product, attempts to clone the gene must rely on the strategy of reverse genetics. As an initial step in this process we have constructed a genetic linkage map spanning 68 cM of chromosome 4 using an intersubspecific phenotypic backcross. The loci included in this analysis are Calb, Ggtb, Lv, b, Ifa, mea, D4Rp1, Glut-1, Lck, Lmyc-1, and Eno-1. This analysis positions the mea phenotypic locus in the interval between Ifa and Glut1. These results also further define regions of homology between mouse chromosome 4 and human chromosomes 8, 1, and 9. This linkage map provides the means to evaluate candidate genes, and to identify tightly linked markers useful for cloning the meander tail locus.     

Pro-Tumorigenic Phosphorylation of p120 Catenin in Renal and Breast Cancer

Altered protein expression and phosphorylation are common events during malignant transformation. These perturbations have been widely explored in the context of E-cadherin cell-cell adhesion complexes, which are central in the maintenance of the normal epithelial phenotype. A major component of these complexes is p120 catenin (p120), which binds and stabilizes E-cadherin to promote its adhesive and tumor suppressing function. However, p120 is also an essential mediator of pro-tumorigenic signals driven by oncogenes, such as Src, and can be phosphorylated at multiple sites. Although alterations in p120 expression have been extensively studied by immunohistochemistry (IHC) in the context of tumor progression, little is known about the status and role of p120 phosphorylation in cancer. Here we show that tyrosine and threonine phosphorylation of p120 in two sites, Y228 and T916, is elevated in renal and breast tumor tissue samples. We also show that tyrosine phosphorylation of p120 at its N-terminus, including at the Y228 site is required for its pro-tumorigenic potential. In contrast, phosphorylation of p120 at T916 does not affect this p120 function. However, phosphorylation of p120 at T916 interferes with epitope recognition of the most commonly used p120 antibody, namely pp120. As a result, this antibody selectively underrepresents p120 levels in tumor tissues, where p120 is phosphorylated. Overall, our data support a role of p120 phosphorylation as a marker and mediator of tumor transformation. Importantly, they also argue that the level and localization of p120 in human cancer tissues immunostained with pp120 needs to be re-evaluated.     

Molecular genetic diagnosis of sickle cell disease using dried blood specimens on blotters used for newborn screening

Summary The protein-based technologies used to screen newborns for sickle cell disease require confirmation with a liquid blood specimen. We have developed a strategy for rapid and specific genotypic diagnosis using DNA extracted from a dried blood spot on the filter paper blotter used to screen newborns. DNA could be microextracted from a specimen as small as a 1/8 inch diameter punched disc representing the dried equivalent of approximately 3 μl of whole blood. We utilized the DNA from a 1/4 inch diameter specimen (12 μl equivalent) for polymerase chain reaction amplification of the β-globin region spanning the sickle cell mutation with detection by allele-specific oligonucleotide probes. Molecular confirmation of genotype from the original blotter would reduce the personnel costs associated with obtaining follow-up liquid blood specimens and would provide information to the family in a more timely and less equivocal manner.