Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase δ, PCNA, RFC and RPA

<p>Abstract</p> <p>Background</p> <p>Adeno-associated virus (AAV) type 2 is an important virus due to its use as a safe and effective human gene therapy vector and its negative association with certain malignancies. AAV, a dependo-parvovirus, autonomously replicates in stratified squamous epithelium. Such tissue occurs in the nasopharynx and anogenitals, from which AAV has been clinically isolated. Related autonomous parvoviruses also demonstrate cell tropism and preferentially replicate in oncogenically transformed cells. Combining these two attributes of parvovirus tropism, squamous and malignant, we assayed if AAV might replicate in squamous cervical carcinoma cell isolates.</p> <p>Results</p> <p>Three primary isolates (PT1-3) and two established cervical cancer cell lines were compared to normal keratinocytes (NK) for their ability to replicate AAV. One isolate, PT3, allowed for high levels of AAV DNA replication and virion production compared to others. In research by others, four cellular components are known required for <it>in vitro </it>AAV DNA replication: replication protein A (RPA), replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase delta (POLD1). Thus, we examined PT3 cells for expression of these components by DNA microarray and real-time quantitative PCR. All four components were over-expressed in PT3 over two representative low-permissive cell isolates (NK and PT1). However, this super-permissiveness did not result in PT3 cell death by AAV infection.</p> <p>Conclusion</p> <p>These data, for the first time, provide evidence that these four cellular components are likely important for AAV <it>in vivo </it>DNA replication as well as <it>in vitro</it>. These data also suggest that PT3 will be a useful reagent for investigating the AAV-permissive transcriptome and AAV anti-cancer effect.</p

Mutant profilin1 transgenic mice recapitulate cardinal features of motor neuron disease

The recent identification of profilin1 mutations in 25 familial ALS cases has linked altered function of this cytoskeletonregulating protein to the pathogenesis of motor neuron disease. To investigate the pathological role of mutant profilin1 in motor neuron disease, we generated transgenic lines of mice expressing human profilin1 with a mutation at position 118 (hPFN1G118V). One of the mouse lines expressing high levels of mutant human PFN1 protein in the brain and spinal cord exhibited many key clinical and pathological features consistent with human ALS disease. These include loss of lower (ventral horn) and upper motor neurons (corticospinal motor neurons in layer V), mutant profilin1 aggregation, abnormally ubiquitinated proteins, reduced choline acetyltransferase (ChAT) enzyme expression, fragmented mitochondria, glial cell activation, muscle atrophy, weight loss, and reduced survival. Our investigations of actin dynamics and axonal integrity suggest that mutant PFN1 protein is associated with an abnormally low filamentous/globular (F/G)-actin ratio that may be the underlying cause of severe damage to ventral root axons resulting in a Wallerian-like degeneration. These observations indicate that our novel profilin1 mutant mouse line may provide a new ALS model with the opportunity to gain unique perspectives into mechanisms of neurodegeneration that contribute to ALS pathogenesis

Novel mechanisms in accelerated atherosclerosis in kidney disease

WOS: 000252812500013PubMed ID: 18089447Objective: Urea undergoes a spontaneous, nonenzymatic transformation to cyanate, the active part of which is isocyanic acid, which can cause modifications of a variety of proteins in a process called carbamylation. We postulated that, in patients with renal disease, the carbamylation of low-density lipoprotein (LDL) is a nontraditional risk factor for cardiovascular disease, and that elevated urea leads to carbamylated LDL (cLDL), which causes vascular injury and leads to atherosclerosis. Results: We showed that carbamylated LDL manifests all of the biological effects relevant to atherosclerosis, including endothelial-cell injury, the expression of adhesion molecules, and vascular smooth muscle cell proliferation. We also developed an enzyme-linked immunosorbent assay to measure carbamylated LDL in patients, and showed that cLDL is markedly elevated in dialysis patients. Conclusions: Our data indicate that cLDL may be an important nontraditional risk factor for atherosclerosis in patients with kidney disease. (c) 2008 by the National Kidney Foundation, Inc