Rapid enzymatic test for phenotypic HIV protease drug resistance

A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid onestep procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIVinfected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for highthroughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (K-i). The Michaelis constant (K-m) and the inhibition constant (K-i) of recombinant wildtype protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIVpositive patients scored from sensitive to highly resistant according to mutations detected by genotypic analysis. The measured K-i values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The noninfectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays

Rapid enzymatic test for phenotypic HIV protease drug resistance

A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid onestep procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIVinfected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for highthroughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (K-i). The Michaelis constant (K-m) and the inhibition constant (K-i) of recombinant wildtype protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIVpositive patients scored from sensitive to highly resistant according to mutations detected by genotypic analysis. The measured K-i values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The noninfectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays

The relationship between the presence of anti-cyclic citrullinated peptide antibodies and clinical phenotype in very early rheumatoid arthritis

<p>Abstract</p> <p>Background</p> <p>Anti-cyclic citrullinated peptide (anti-CCP) antibodies are highly specific for RA, but are not detectable in all RA patients. The aim of this study was to establish whether the clinical phenotypes of anti-CCP positive and negative disease are distinct at the earliest clinically apparent phase of disease.</p> <p>Methods</p> <p>Patients were recruited from the Birmingham early inflammatory arthritis clinic. Participants were included in the current study if they presented within 3 months of symptom onset and fulfilled 1987 ACR criteria for RA at some point during an 18 month follow-up. Data were collected on demographic variables, joint symptoms and tender (n = 68) and swollen (n = 66) joint counts. CRP, ESR, rheumatoid factor and anti-CCP2 status were measured.</p> <p>Results</p> <p>92 patients were included (48 anti-CCP positive). The anti-CCP positive and negative groups were comparable in terms of demographic variables, inflammatory markers, joint counts and 1987 ACR classification criteria, except that more anti-CCP positive patients were rheumatoid factor positive (83.3% vs. 11.4%, p < 0.01). There was no significant difference in the pattern of joint involvement, except for an increased prevalence of knee joint swelling in anti-CCP positive patients (42.9% vs. 22.2%, p = 0.03).</p> <p>Conclusions</p> <p>Patients with and without anti-CCP antibodies present in a similar way, even within three months of clinically apparent disease that eventually develops into RA.</p

An Assay to Monitor HIV-1 Protease Activity for the Identification of Novel Inhibitors in T-Cells

The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR). The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP) gene only in the presence of an effective PR Inhibitor (PI). Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells

Ligand-Receptor Interactions

The formation and dissociation of specific noncovalent interactions between a variety of macromolecules play a crucial role in the function of biological systems. During the last few years, three main lines of research led to a dramatic improvement of our understanding of these important phenomena. First, combination of genetic engineering and X ray cristallography made available a simultaneous knowledg of the precise structure and affinity of series or related ligand-receptor systems differing by a few well-defined atoms. Second, improvement of computer power and simulation techniques allowed extended exploration of the interaction of realistic macromolecules. Third, simultaneous development of a variety of techniques based on atomic force microscopy, hydrodynamic flow, biomembrane probes, optical tweezers, magnetic fields or flexible transducers yielded direct experimental information of the behavior of single ligand receptor bonds. At the same time, investigation of well defined cellular models raised the interest of biologists to the kinetic and mechanical properties of cell membrane receptors. The aim of this review is to give a description of these advances that benefitted from a largely multidisciplinar approach

Translation of rous sarcoma virus rna in a cell-free system from ascites krebs ii cells.

The template activities of the 60-70S RNA complex and of the 30-40S subunit RNA species of Rous sarcoma virus were tested in a cell-free protein-synthesizing system from mouse ascites Krebs II cells. Stimulation of protein synthesis over the endogenous background was about 2-fold with 30-40S viral RNA and about 1.3-fold with 60-70S viral RNA as template. Analysis by sodium dodecyl sulfate-gel electrophoresis showed that the predominant polypeptide synthesized in vitro in response to 30-40S RNA of Rous sarcoma virus had a molecular weight of 75,000-80,000. This polypeptide could be precipitated by antiserum against the group-specific antigens of the virus, although its molecular weight is higher than that of virion group-specific antigen proteins. Analysis of tryptic digests of the protein made in vitro indicates similarity to tryptic digests from authentic virion group-specific proteins. It is concluded that part of the RNA from Rous sarcoma virus is translated in vitro into a high-molecular-weight protein, perhaps a precursor of the virion group-specific proteins