Cellular distribution of phosphofructokinase activity and implications to metabolic regulation in human breast cancer

Neoplastic cells generally present profound changes in glucose metabolism. The mechanisms underlying such process are numerous and all may involve altered cellular hormonal responses. Here we report the first evidence that cellular location of phosphofructokinase activity in human breast cancer tissues is different from the one observed in control tissues and that this phenomenon may be involved in the increased glycolytic flux observed in those cells. Through co-sedimentation techniques, we observed that 60% of phosphofructokinase activity in neoplastic tissues is located in an actin-enriched fraction, against 36% in control tissues. Additionally, metastatic tumor tissues presented a two fold increase in this particulate activity when compared to non-metastatic tumor samples. We propose that the alteration in cellular distribution of phosphofructokinase activity in human breast cancer tissues is a mechanism associated to the process of cell transformation and may be a consequence of the altered hormonal milieu observed in several types of cancer.     

A-kinase anchoring proteins in amygdala are involved in auditory fear memory

A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that bind the regulatory subunits of protein kinase A (PKA). AKAP binding to PKA regulates the phosphorylation of various proteins, some of which have been implicated in synaptic plasticity and memory consolidation. Here we show that the regulatory subunits of PKA are colocalized with AKAP150 (an AKAP isoform that is expressed in the brain) in the lateral amygdala (LA) and that infusion to the LA of the peptide St-Ht31, which blocks PKA anchoring onto AKAPs, impairs memory consolidation of auditory fear conditioning.     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.     

An analytical model for the dissolution of different particle size samples of Bioglass in TRIS-buffered solution

We analyzed the early stages of reactivity of three different particle size samples of Bioglass 45S5 and a bulk sample in TRIS-buffered solution at pH 8. Ion release, measured with ion-coupled plasma emission spectroscopy, and pH variations are reported. It was demonstrated that differences in the initial surface area influence the increase in pH, the rate of elemental release, and the rate of calcium phosphate reprecipitation. In particular, a thicker Ca/P layer was obtained on larger particles. The equilibrium value of Si in solution was independent of sample form and amount of sample dissolved, and was always close to the value observed when bulk silica is dissolved at pH 8. An analytical model is proposed for cation release, based on a two-step mechanism. It was found that the early stage of dissolution was nearly diffusion controlled for larger particles and bulk samples. The second stage was similar to a first-order homogeneous dissolution. The influence of sample surface area/solution volume ratio seemed to be more complex than that proposed in the early works presented in the literature. It is suggested that variation of surface area has a significant impact on the course of the dissolution.     

Prevalence of transmitted HIV-1 drug resistance and the role of resistance algorithms: data from seroconverters in the CASCADE collaboration from 1987 to 2003

OBJECTIVES: To examine factors influencing the rate of transmitted drug resistance (TDR) among seroconverters, with particular emphasis on 3 widely used genotypic drug resistance algorithms. METHODS: The study used data from CASCADE (Concerted Action on Seroconversion to AIDS and Death in Europe), a collaboration of seroconverter cohorts in Europe and Canada. Genotypic resistance data were derived within 18 months of the last seronegative test or date of laboratory evidence of acute infection and before the initiation of antiretroviral therapy. The Stanford algorithm was used to analyze each individual's nucleotide sequence. A multivariate logistic model was used to assess independent relationships between the presence of TDR and exposure category, sex, age at seroconversion, and year of seroconversion. The paper also describes 3 alternative definitions of resistance: the Stanford algorithm, the key resistance mutations defined by the International AIDS Society, and the Agence Nationale de Recherches sur le Sida (ANRS) algorithm. RESULTS: Forty-five of 438 patients (10.3%) seroconverting between 1987 and 2003 were infected with a drug-resistant HIV-1 variant. Forty patients (9.1%) showed resistance mutations to only 1 class of antiretroviral drugs, 2 (0.5%) to 2 classes, and 3 (0.7%) to 3 classes of antiretroviral therapy. It was suggested that individuals seroconverting later in calendar time were more likely to have TDR (relative risk 3.89 and 95% CI: 0.84 to 18.02, and relative risk 4.69 and 95% CI: 1.03 to 21.31, for 1996-1999 and 2000-2003, respectively, compared with pre-1996; P trend = 0.08). This trend was apparent regardless of the definition of TDR used. The total estimated proportion of individuals with TDR varied between 10.3% and 15.5% according to which definition was used. CONCLUSIONS: Evidence was found for the rise of TDR over time. A specific definition of what constitutes TDR rather than a simple list of mutations is needed.     

Up-regulation of tRNA biosynthesis affects translational readthrough in maf1-delta mutant of Saccharomyces cerevisiae

Maf1p is a negative effector of RNA polymerase III in yeast. The maf1-delta mutation caused an increase in the level of cellular tRNAs, but a decrease of translational readthrough at nonsense codons. Using the lacZ- luc dual gene reporter system, we detected an almost twofold diminution of UAA and UAG readthrough in maf1-delta compared with the parental strain. The maf1-delta mutation did not affect the rate of protein biosynthesis and growth at standard conditions, but resulted in temperature-sensitive growth on non-fermentable carbon sources. We examined the correlation of the temperature sensitive and antisuppression phenotypes of maf1- Delta using a colour phenotype assay in the ade2-1 SUP11 strain. Antisuppression, but not the temperature-sensitive growth defect, was compensated either by increased dosage of SUP11or by [PSI(+)], the prion form of the translation termination factor Sup35p. Summarizing, the elevated tRNA levels in maf1- Delta increase translational fidelity and, independently, affect growth under special conditions.     

An IFN-beta-albumin fusion protein that displays improved pharmacokinetic and pharmacodynamic properties in nonhuman primates.

The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-beta (IFN-beta ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-beta induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 microg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 microg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-beta, and the terminal half-life was 36-40 h compared with 8 h for IFN-beta. Importantly, Albuferon beta induced sustained increases in serum neopterin levels and 2',5' mRNA expression. At a molar dose equivalent to one-half the dose of IFN-beta, Albuferon beta elicited comparable neopterin responses and significantly higher 2',5'-OAS mRNA levels in rhesus monkeys. The enhanced in vivo pharmacologic properties of IFN-beta when fused to serum albumin suggest a clinical opportunity for improved IFN-beta therapy.     

A new CARD15 mutation in Blau syndrome

The caspase recruitment domain gene CARD15/NOD2, encoding a cellular receptor involved in an NF-kappaB-mediated pathway of innate immunity, was first identified as a major susceptibility gene for Crohn's disease (CD), and more recently, as responsible for Blau syndrome (BS), a rare autosomal-dominant trait characterized by arthritis, uveitis, skin rash and granulomatous inflammation. While CARD15 variants associated with CD are located within or near the C-terminal leucine-rich repeat domain and cause decreased NF-kappaB activation, BS mutations affect the central nucleotide-binding NACHT domain and result in increased NF-kappaB activation. In an Italian family with BS, we detected a novel mutation E383K, whose pathogenicity is strongly supported by cosegregation with the disease in the family and absence in controls, and by the evolutionary conservation and structural role of the affected glutamate close to the Walker B motif of the nucleotide-binding site in the NACHT domain. Interestingly, substitutions at corresponding positions in another NACHT family member cause similar autoinflammatory phenotypes.