Failure to Achieve PSA Level ≤I ng/ml Following Neo-Adjuvant LHRHa Therapy Predicts for a Lower Biochemical Control Rate and Overall Survival in Localized Prostate Cancer Treated with Radiotherapy

PURPOSE: To investigate whether failure to suppress the prostate-specific antigen (PSA) level to /=2 months of neoadjuvant luteinizing hormone-releasing hormone agonist therapy in patients scheduled to undergo external beam radiotherapy for localized prostate carcinoma is associated with reduced biochemical failure-free survival. METHODS AND MATERIALS: A retrospective case note review of consecutive patients with intermediate- or high-risk localized prostate cancer treated between January 2001 and December 2002 with neoadjuvant hormonal deprivation therapy, followed by concurrent hormonal therapy and radiotherapy was performed. Patient data were divided for analysis according to whether the PSA level in Week 1 of radiotherapy was 1 ng/mL in 52. At a median follow-up of 49 months, the 4-year actuarial biochemical failure-free survival rate was 84% vs. 60% (p = 0.0016) in favor of the patients with a PSA level after neoadjuvant hormonal deprivation therapy of 1 ng/mL at the beginning of external beam radiotherapy after >/=2 months of neoadjuvant luteinizing hormone-releasing hormone agonist therapy have a significantly greater rate of biochemical failure and lower survival rate compared with those with a PSA level of

Biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases.

The arterivirus equine arteritis virus nonstructural protein 10 (nsp10) has previously been predicted to contain a Zn finger structure linked to a superfamily 1 (SF1) helicase domain. A recombinant form of nsp10, MBP-nsp10, was produced in Escherichia coli as a fusion protein with the maltose-binding protein. The protein was partially purified by affinity chromatography and shown to have ATPase activity that was strongly stimulated by poly(dT), poly(U), and poly(dA) but not by poly(G). The protein also had both RNA and DNA duplex-unwinding activities that required the presence of 5' single-stranded regions on the partial-duplex substrates, indicating a 5'-to-3' polarity in the unwinding reaction. Results of this study suggest a close functional relationship between the arterivirus nsp10 and the coronavirus helicase, for which NTPase and duplex-unwinding activities were recently demonstrated. In a number of biochemical properties, both arterivirus and coronavirus SF1 helicases differ significantly from the previously characterized RNA virus SF1 and SF2 enzymes. Thus, the combined data strongly support the idea that nidovirus helicases may represent a separate group of RNA virus-encoded helicases with distinct properties.

Biochemical characterization of arterivirus nonstructural protein 11 reveals the nidovirus-wide conservation of a replicative endoribonuclease

Nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand RNA viruses that includes important human and animal pathogens. Nidovirus RNA synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral RNA polymerase, but also unusual and poorly understood RNA-processing functions. Of these, a conserved endoribonuclease (NendoU) is a major genetic marker that is unique to nidoviruses. NendoU activity was previously verified in vitro for the coronavirus nsp15, but not for any of its distantly related orthologs from other nidovirus lineages, like the arterivirus nsp11. Here, we show that the bacterially expressed nsp11 proteins of two arteriviruses, equine arteritis virus and porcine respiratory and reproductive syndrome virus, possess pyrimidine-specific endoribonuclease activity. RNA cleavage was independent of divalent cations in vitro and was greatly reduced by replacement of residues previously implicated in catalysis. Comparative characterization of the NendoU activity in arteriviruses and severe acute respiratory syndrome coronavirus revealed common and distinct features of their substrate requirements and reaction mechanism. Our data provide the first biochemical evidence of endoribonuclease activity associated with arterivirus nsp11 and support the conclusion that this remarkable RNA-processing enzyme, whose substrate in the infected cell remains to be identified, distinguishes nidoviruses from all other RNA viruses.

Functional and biochemical characterisation of the Escherichia coli major facilitator superfamily multidrug transporter MdtM

Multidrug resistance (MDR) occurs when bacteria simultaneously acquire resistance to a broad spectrum of structurally dissimilar compounds to which they have not previously been exposed. MDR is principally a consequence of the active transport of drugs out of the cell by proteins that are integral membrane transporters. We characterised and purified the putative Escherichia coli MDR transporter, MdtM, a 410 amino acid residue protein that belongs to the large and ubiquitous major facilitator superfamily. Functional characterisation of MdtM using growth inhibition and whole cell transport assays revealed its role in intrinsic resistance of E. coli cells to the antimicrobials ethidium bromide and chloramphenicol. Site-directed mutagenesis studies implied that the MdtM aspartate 22 residue and the highly conserved arginine at position 108 play a role in proton recognition. MdtM was homologously overexpressed and purified to homogeneity in dodecyl maltopyranoside detergent solution and the oligomeric state and stability of the protein in a variety of detergent solutions was investigated using size-exclusion HPLC. Purified MdtM is monomeric and stable in dodecyl maltopyranoside solution and binds chloramphenicol with nanomolar affinity in the same detergent. This work provides a firm foundation for structural studies on this class of multidrug transporter protein.