Mapping of the 5' terminal nucleotides of Grapevine leafroll-associated virus 3 sgRNAs.

Grapevine leafroll-associated virus 3 (GLRaV-3) is a positive sense, single stranded RNA virus that has a detrimental effect on wine and table grapes worldwide. Previous studies have shown that GLRaV-3, like other closteroviruses produces subgenomic (sg) RNAs during replication and that these sgRNAs are deployed for the expression of the ORFs on the 3' half of the genome. In this study a dsRNA blot confirmed the presence of three, 3' co-terminal sgRNAs species [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] in GLRaV-3-infected plant material when using a probe directed at the coat protein. The specific 5' terminal nucleotides for these sgRNAs as well as four additional sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] were determined by RLM-RACE for GLRaV-3 isolate GP18.

Soluble CD44 secretion contributes to the acquisition of aggressive tumor phenotype in human colon cancer cells.

CD44, a widely distributed cell surface glycoprotein and a receptor for hyaluronan (HA), has been implicated in facilitating tumor growth and metastasis, antiapoptosis and directional motility of cancer cells. In order to investigate the role of soluble CD44 (CD44(sol)) in colon cancer cell growth, SW620, a human colon cancer cell line deficient in CD44 expression was stably transfected with human CD44 cDNA containing exons 1-5, 15 and 16 of the human CD44. Western blot analyses demonstrated the presence of 78 kDa soluble CD44 protein in the culture supernatant of stably transfected cell lines (CD44(sol) clones) and were not detected in the empty vector control line (clone m). The CD44(sol) transfected cells showed higher cell proliferation and clonal growth in vitro, confirmed by MTT and clonogenic assays respectively, when compared to the control cells. Cell adhesion to hyaluronan was significantly lower with CD44(sol) cells compared to the control cells. Western blot analyses were negative for cleaved PARP in lysates from CD44(sol) cells, suggesting resistance to apoptosis. These findings indicate that the secretion of soluble CD44 contributes to colon cancer growth in vitro, possibly as a decoy receptor.

CD44 regulates cell migration in human colon cancer cells via Lyn kinase and AKT phosphorylation.

Colon cancer is among the leading causes of cancer death in North America. CD44, an adhesion and antiapoptotic molecule is overexpressed in colon cancer. Cofilin is involved in the directional motility of cells. In the present study, we looked at how CD44 might modulate cell migration in human colon cancer via cofilin. We used a human colon cancer cell line, HT29, which expresses CD44, HT29 where CD44 expression was knocked down by siRNA, SW620, a human colon cancer cell line which does not express CD44, stably transfected exons of CD44 in SW620 cells and the colon from CD44 knockout and wild-type mouse. Western blot analysis of siRNA CD44 lysates showed increased level of AKT phosphorylation and decreased level of cofilin expression. Similar results were also observed with SW620 cells and CD44 knockout mouse colon lysates. Experiments using the AKT phosphorylation inhibitor LY294002 indicate that AKT phosphorylation downregulates cofilin. Immunoprecipitation studies showed CD44 complex formation with Lyn, providing an essential link between CD44 and AKT phosphorylation. LY294002 also stabilized Lyn from phosphorylated AKT, suggesting an interaction between Lyn and AKT phosphorylation. Immunocytochemistry showed that cofilin and Lyn expression were downregulated in siRNA CD44 cells and CD44 knockout mouse colon. siRNA CD44 cells had significantly less migration compared to HT29 vector. Given the well-defined roles of CD44, phosphorylated AKT in apoptosis and cancer, these results indicate that CD44-induced cell migration is dependent on its complex formation with Lyn and its consequent regulation of AKT phosphorylation and cofilin expression.

Overrepresentation of the founder PPOX gene mutation R59W in a South African patient with severe clinical manifestation of porphyria.

A patient, who presented with abdominal pain and severe photosensitivity that resulted in scarring and mutilation of the fingers, nose and ears, was referred for biochemical assessment of porphyria and DNA screening. Although these clinical manifestations were suggestive of both acute porphyria and congenital erythropoietic porphyria, the biochemical profile was consistent with variegate porphyria (VP). Analysis of the protoporphyrinogen oxidase (PPOX) gene underlying VP resulted in the identification of the founder mutation R59W in a heterozygous state in this patient. Despite extensive mutation analysis, no other potential disease-causing genetic alterations could be detected in the PPOX gene or the uroporphyrinogen III synthase gene. Slight overrepresentation of the mutant PPOX allele was however, observed repeatedly in DNA of the proband compared to other R59W heterozygotes, including his mother who also tested positive for mutation R59W using restriction enzyme analysis and direct DNA sequencing. Confirmation of this phenomenon by real-time polymerase chain reaction analysis and microsatellite analysis, using highly informative markers flanking the PPOX gene, raised the possibility of partial homozygosity for VP in this patient. This study represents the first report of overrepresentation of mutation R59W in a patient with a severe form of VP. A homozygote for the R59W mutation has never been detected, and the severe clinical manifestation observed in our patient is consistent with the hypothesis that such a genotype will not be compatible with life.