Effect of antiandrogen flutamide on measures of hepatic regeneration in rats

Male rat liver undergoes a process of demasculinization during hepatic regeneration following partial hepatectomy. The possibility that antiandrogens might potentiate this demasculinization process and in so doing augment the hepatic regenerative response was investigated. Adult male Wistar rats were treated with the antiandrogen flutamide (2 mg/rat/day or 5 mg/rat/day subcutaneously) or vehicle for three days prior to and daily after a 70% partial hepatectomy. At various times after hepatectomy, the liver remnants were removed and weighed. Rates of DNA and polyamine synthesis were assessed by measuring thymidine kinase and ornithine decarboxylase activities, respectively. Hepatic estrogen receptor status and the activity of alcohol dehydrogenase, an androgen-sensitive protein, were measured. Prior to surgery, the administration of 5 mg/day flutamide reduced the hepatic cytosolic androgen receptor activity by 98% and hepatic cytosolic estrogen receptor content by 92% compared to that present in vehicle-treated controls. After hepatectomy, however, all differences in sex hormone receptor activity between the treatment groups were abolished. The rate of liver growth after partial hepatectomy in the three groups was identical. Moreover, hepatectomy-induced increases in ornithine decarboxylase activity and thymidine kinase activity were comparable. These data demonstrate that, although flutamide administration initially alters the sex hormone receptor status of the liver, these affects have no effect on the hepatic regenerative response following a partial hepatectomy. © 1989 Plenum Publishing Corporation

Destabilization of The Ornithine Decarboxylase mRNA Transcript by the RNA-Binding Protein Tristetraprolin

Ornithine decarboxylase (ODC) is the first and usually rate-limiting enzyme in the polyamine biosynthetic pathway. In a normal physiological state, ODC is tightly regulated. However, during neoplastic transformation, ODC expression becomes upregulated. The studies described here show that the ODC mRNA transcript is destabilized by the RNA-binding protein tristetraprolin (TTP). We show that TTP is able to bind to the ODC mRNA transcript in both non-transformed RIE-1 cells and transformed Ras12V cells. Moreover, using mouse embryonic fibroblast cell lines that are devoid of a functional TTP protein, we demonstrate that in the absence of TTP both ODC mRNA stability and ODC enzyme activity increase when compared to wild-type cells. Finally, we show that the ODC 3′ untranslated region contains cis acting destabilizing elements that are affected by, but not solely dependent on, TTP expression. Together, these data support the hypothesis that TTP plays a role in the post-transcriptional regulation of the ODC mRNA transcript

Neisseria oralis sp. nov., isolated from healthy gingival plaque and clinical samples

A polyphasic analysis was undertaken of seven independent isolates of Gram-negative cocci collected from pathological clinical samples from New York, Louisiana, Florida and Illinois and healthy subgingival plaque from a patient in Virginia, USA. The 16S rRNA gene sequence similarity among these isolates was 99.7–100 %, and the closest species with a validly published name was Neisseria lactamica (96.9 % similarity to the type strain). DNA–DNA hybridization confirmed that these isolates are of the same species and are distinct from their nearest phylogenetic neighbour, N. lactamica. Phylogenetic analysis of 16S and 23S rRNA gene sequences indicated that the novel species belongs in the genus Neisseria. The predominant cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C18 : 1ω7c. The cellular fatty acid profile, together with other phenotypic characters, further supports the inclusion of the novel species in the genus Neisseria. The name Neisseria oralis sp. nov. (type strain 6332T = DSM 25276T = LMG 26725T) is proposed

On the Role of Polyamines and Microvesicles in Tumour Development. Regulation by Hypoxia and Implications for Therapeutic Intervention of Cancer.

Novel strategies for specific tumour cell targeting are necessary in order to improve survival rates and to reduce side effects of current therapies in cancer patients. Hypoxia is a hallmark of solid tumours and one of the major driving forces for tumour progression. Targeting of the adaptive responses of cancer cells to hypoxia offers opportunities for tumour specific therapies. The aim of this thesis was to study the hypoxic regulation of polyamines and tumour cell-derived microvesicles (MVs), both of which have been associated with tumour development. It was initially demonstrated that epitope-specific interference of cell surface heparan sulphate proteoglycans (HSPGs) by anti-HS antibodies inhibits the bioavailability of exogenous polyamines, and that combined targeting of polyamine biosynthesis by α-difluoromethylornithine (DFMO) and HSPG-dependent uptake by a HIV-1 trans-activator of transcription (TAT) peptide, Tat, results in reduced tumour growth. We found a novel role for the polyamine system in the hypoxia-induced adaptive response of tumour cells; depletion of polyamines sensitizes tumour cells to hypoxic stress and enhances the tumour inhibiting effect of anti-angiogenic therapy. Finally, tumour cell-derived MVs are shown to elicit a hypoxic, pro-angiogenic response in endothelial cells (ECs) and the upregulation of a specific subset of microvesicular proteins and mRNAs by hypoxia implicates MVs as novel biomarkers for hypoxic signalling in cancer cells. This thesis implicates the targeting of hypoxia-induced adaptive responses as a means of obtaining tumour cell specific therapies and suggests the polyamine system and tumour-derived MVs as promising candidates for therapeutic intervention or as biomarkers of cancer disease

Regulation of polyamine biosynthesis in Saccharomyces cerevisiae

Polyamines are essential organic cations with multiple cellular functions. Their synthesis is controlled by a feedback regulation whose main target is ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. In mammals, ODC has been shown to be inhibited and targeted for ubiquitin-independent degradation by ODC antizyme. The synthesis of mammalian antizyme was reported to involve a polyamine-induced ribosomal frameshifting mechanism. High levels of polyamine therefore inhibit new synthesis of polyamines by inducing ODC degradation. In this work, a previously unrecognized sequence in the genome of Saccharomyces cerevisiae encoding an orthologue of mammalian antizyme was identified. Synthesis of yeast antizyme (Oaz1) involves polyamine-regulated frameshifting. New elements, termed OFRE (OAZ1 frameshifting repressor element) and OPRE (OAZ1 polyamine responsive element) that are necessary for the polyamines to regulate frameshifting were mapped in the OAZ1 mRNA. Degradation of yeast ODC by the proteasome depends on Oaz1. Oaz1 mediates the degradation by binding to ODC thereby exposing a degradation signal at the N-terminus of ODC. Using the novel transplantable yeast ODC degradation signal (ODS) identified in this work a new possible role of the shuttle factor Rad23 in ODC degradation was identified. In addition, ODS is shown to interact with multiple 19S lid subunits in the proteasome. Using this novel model system for polyamine regulation another level of its control was discovered. Oaz1 itself is subject to ubiquitin-mediated proteolysis by the proteasome. Degradation of Oaz1, however, is efficiently inhibited by polyamines. I propose a model, in which polyamines inhibit their ODC-mediated biosynthesis by two mechanisms, the control of Oaz1 synthesis and inhibition of its degradation. In a second part of the work, peptide aptamers were isolated that inhibit the ubiquitin-dependent turnover of test substrates of the proteasome. These aptamers appear to either inhibit ubiquitylation or the proteasome and thereby lead to a stabilization of test substrates. I Propose that ODS due to its ubiquitin-independent mode of degradation can be used as a tool in aptamer screens that are aimed at identifying additional peptide inhibitors of the proteasome with potential clinical relevance