Human Papillomavirus Type 16 E6 and E7 Cause Polyploidy in Human Keratinocytes and Up-Regulation of G2-M-phase Proteins

Human papillomavirus type 16 proteins E6 and E7 have been shown to cause centrosome amplification and lagging chromosomes during mitosis. These abnormalities during mitosis can result in missegregation of the chromosomes, leading to chromosomal instability. Genomic instability is thought to be an essential part of the conversion of a normal cell to a cancer cell. We now show that E6 and E7 together cause polyploidy in primary human keratinocytes soon after these genes are introduced into the cells. Polyploidy seems to result from a spindle checkpoint failure arising from abrogation of the normal functions of p53 and retinoblastoma family members by E6 and E7, respectively. In addition, E6 and E7 cause deregulation of cellular genes such as Plk1, Aurora-A, cdk1, and Nek2, which are known to control the G2-M-phase transition and the ordered progression through mitosis.

Cochlin in the eye: Functional implications

Aqueous humor is actively produced in the ciliary epithelium of the anterior chamber and has important functions for the eye. Under normal physiological conditions, the inflow and outflow of the aqueous humor are tightly regulated, but in the pathologic state this balance is lost. Aqueous outflow involves structures of the anterior chamber and experiences most resistance at the level of the trabecular meshwork (TM) that acts as a filter. The modulation of the TM structure regulates the filter and its mechanism remains poorly understood. Proteomic analyses have identified cochlin, a protein of poorly understood function, in the glaucomatous TM but not in healthy control TM from human cadaver eyes. The presence of cochlin has subsequently been confirmed by Western and immunohistochemical analyses. Functionally, cochlin undergoes multimerization induced by shear stress and other changes in the microenvironment. Cochlin along with mucopolysaccharide deposits have been found in the TM of glaucoma patients and in the inner ear of subjects affected by the hearing disorder DNFA9, a late onset, progressive disease that also involves alterations in fluid shear regimes. In vitro, cochlin induces aggregation of primary TM cells suggesting a role in cell adhesion, possibly in mechanosensation, and in modulation of the TM filter.

Review: formation and properties of amyloid-like fibrils derived from alpha-synuclein and related proteins.

Synucleins are small proteins that are highly expressed in brain tissue and are localised at presynaptic terminals in neurons. alpha-Synuclein has been identified as a component of intracellular fibrillar protein deposits in several neurodegenerative diseases, and two mutant forms of alpha-synuclein have been associated with autosomal-dominant Parkinson's Disease. A fragment of alpha-synuclein has also been identified as the non-Abeta component of Alzheimer's Disease amyloid. In this review we describe some structural properties of alpha-synuclein and the two mutant forms, as well as alpha-synuclein fragments, with particular emphasis on their ability to form beta-sheet on ageing and aggregate to form amyloid-like fibrils. Differences in the rates of aggregation and morphologies of the fibrils formed by alpha-synuclein and the two mutant proteins are highlighted. Interactions between alpha-synuclein and other proteins, especially those that are components of amyloid or Lewy bodies, are considered. The toxicity of alpha-synuclein and related peptides towards neurons is also discussing in relation to the aetiology of neurodegenerative diseases.

Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units.

Transcription from morbillivirus genomes commences at a single promoter in the 3' non-coding terminus, with the six genes being transcribed sequentially. The 3' and 5' untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5' UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5' UTRs. Insertions into the 5' UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5' UTR, govern this decreased expression from TU2.

Conformational changes during proteolytic processing of a picornavirus capsid proteins

We have used synthetic peptide antibodies to probe conformational changes that occur during the cleavage cascade which generates the capsid proteins of a picornavirus. The initial translation product of 97 kDa, the precursor of all four structural proteins, is cleaved to form a 63 kDa fragment which, we show, has significantly different folding characteristics to both its larger parent and its products. We demonstrate that proteolytic cleavages as distant as 520 residues from epitopes confer sufficiently large conformational changes as to render them unrecognisable. To our knowledge, this is the first demonstration of this phenomenon in the picornavirus system.

Novel approaches to the analysis of the Maillard reaction of proteins

The Maillard reaction comprises a complex network of reactions which has proven to be of great importance in both food science and medicine. The majority of methods developed for studying the Maillard reaction in food have focused on model systems containing amino acids and monosaccharides. In this study, a number of electrophoretic techniques, including two-dimensional gel electrophoresis and capillary electrophoresis, are presented. These have been developed specifically for the analysis of the Maillard reaction of food proteins, and are giving important insights into this complex process.