Detection of fos protein during osteogenesis by monoclonal antibodies.

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.     

Cyclic AMP inhibition of phosphoinositide turnover in human neutrophils

The effect of increased intracellular levels of cyclic AMP on phosphoinositide metabolism was studied in human neutrophils stimulated with fMet-Leu-Phe. Intracellular cyclic AMP was raised by preincubation either with dibutyryl cyclic AMP and theophylline or with prostaglandin E1. Concentrations of dibutyryl cyclic AMP and theophylline fully inhibitory for the metabolic responses inhibited phosphoinositide breakdown and phosphatidic acid formation to a large extent. The accumulation of the water-soluble inositol phosphates was also measured. In agreement with the data obtained on the phospholipids, inositol phosphate generation was found to be severely, though not completely, reduced. Treatment with dibutyryl cyclic AMP and theophylline also inhibited resynthesis of membrane inositol lipids. Treatment with prostaglandin E1 had a similar, though less, marked effect on inositol lipid turnover, which was parallel with a smaller inhibition of metabolic responses. We therefore suggest that the elevation of intracellular cyclic AMP mainly affects neutrophil responses by inhibiting the phosphoinositide cycle.     

Inhibition by verapamil of neutrophil responses to formylmethionylleucylphenylalanine and phorbol myristate acetate. Mechanisms involving Ca2+ changes, cyclic AMP and protein kinase C

Verapamil inhibits in human neutrophils the respiratory burst, the secretion and the change of transmembrane potential induced by formylmethionylleucylphenylalanine, a Ca2+-dependent stimulus, and by phorbol myristate acetate, a Ca2+-independent stimulus. Besides the blocking of Ca2+ channels, many mechanisms are responsible for the inhibition of neutrophil responses. In fact, verapamil (i) increases the intracellular cAMP concentration, potentiates the cAMP response induced by the chemotactic peptide and induces the appearance of a cAMP response also when the stimulant is phorbol myristate acetate; (ii) causes a decrease of Ca2+ association to cell membranes, so depleting the pools of exchangeable Ca2+ and depressing the 'Ca2+ response' in terms of rise in [Ca2+]i monitored with Quin 2 and of rapid mobilization from cell membranes monitored by chlorotetracycline fluorescence change; (iii) inhibits the Ca2+-activated phospholipid-dependent protein kinase C. The data, discussed in relation to the biochemical mechanisms of the stimulus-response coupling, are compatible with the hypothesis of an involvement of the activation of protein kinase C as key step in the sequence of transduction events for the induction of many neutrophil functions.     

Studies on stimulus-response coupling in human neutrophils. II. Relationships between the effects of changes of external ionic composition on the properties of N-formylmethionylleucylphenylalanine receptors and on the respiratory and secretory responses

Studies were carried out on the mechanism responsible for the enhancement of the respiratory and secretory responses to N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) exhibited by human neutrophils suspended in Na+-free, high-K+ buffered solution. The results demonstrate that: (a) the variation of Na+ concentration in the suspending solution induces in human neutrophils a marked modification of the recognition apparatus for the chemotactic peptide fMet-Leu-Phe, the lack of or low concentration of this ion increasing the number of the receptors and their specific affinity for the ligand; (b) the greater respiratory burst and secretion induced by fMet-Leu-Phe in human neutrophils suspended in Na+-free, high-K+ medium are due to the increased formation of receptor-ligand complexes at the cell membrane; (c) the greater respiratory response is partially due also to a higher efficiency of these receptor-ligand complexes. The molecular mechanism by which Na+ exerts a regulative role on the properties of the recognition apparatus for the chemotactic peptide and its possible significance are discussed.     

Multiplicity of genes encoding secreted aspartic proteinases in Candida species

The secreted aspartic proteinases (SAP) of Candida sp. are presumed to be potential virulence factors. In the opportunistic pathogen Candida albicans the proteinase genes identified to date, SAP1, SAP2, SAP3 and SAP4, constitute a multigene family. Before addressing the possible role of each proteinase in virulence, we sought to isolate all the members of this multigene family by screening a genomic library with a SAP1 probe for additional C. albicans SAP genes using low-stringency hybridization conditions. Three putative new members, SAP5, SAP6 and SAP7 were isolated and sequenced. The N-terminal segments of the deduced amino acid sequences of SAP5 and SAP6 contained secretion signal sequences similar to those of other Candida SAPs. Upon comparison and alignment with the other reported SAP amino acid sequences, SAP7 is not only the most divergent protein but also exhibits a much longer putative pro-sequence with a single Lys-Lys putative processing site. Using SAP1 to SAP7 as probes, the overall number of SAP genes in C. albicans was tentatively estimated by low-stringency hybridization to EcoRI-digested genomic DNA. While each isolated SAP gene could be assigned to distinct EcoRI bands, the existence of two additional genes not isolated after screening of the C. albicans gene library was inferred. Furthermore, evidence was obtained for the existence of SAP muttigene families in other Candida species such as C. tropicalis, C. parapsilosis and C. guiller-mondii.     

Bitrophic toxicity of Cry1Ac to Cycloneda sanguinea,a predator in Brazilian cotton

Insect predators are exposed to the Cry1Ac toxin in Bt cotton fields through several pathways. In this study, we investigated the effects of activated Cry1Ac added to a diet on Cycloneda sanguinea (L.) (Coleoptera: Coccinellidae), which is one of the main predators of non‐target pests in Brazilian cotton. Direct bitrophic exposure of C. sanguinea to Cry1Ac was done by feeding beetles with Aphis gossypii (Glover) (Hemiptera: Aphidae) sprayed with 500 μg per ml Cry1Ac solution. Larval and pupal survival, development time, aphid consumption, and adult longevity were recorded daily. Couples within the same experimental treatment were paired and numbers of eggs laid and hatched per female were recorded daily. Net replacement rate was calculated for each female. During development, a C. sanguinea larva consumed on average 1.8 μg of activated Cry1Ac. No significant differences due to Cry1Ac were observed for any of the response variables, except aphid consumption. Larvae receiving Cry1Ac consumed more aphids than larvae receiving distilled water alone. Additional statistical analyses were conducted to evaluate independence of responses, and for the independent responses, a simple meta‐analysis was conducted to test the null hypothesis that all responses were zero. Nearly all of the response variables were statistically independent. Two pairs of responses were not independent, but the associated multivariate tests were not significant. The meta‐analysis suggested that all effects were not different from random variation around zero and no cumulative effects could be detected. Our results indicated that bitrophic exposure to activated Cry1Ac is likely to have little or no adverse ecological effect on C. sanguinea.     

The Value of In Vitro Diagnostic Testing in Medical Practice: A Status Report

BackgroundIn vitro diagnostic (IVD) investigations are indispensable for routine patient management. Appropriate testing allows early-stage interventions, reducing late-stage healthcare expenditure (HCE).AimTo investigate HCE on IVDs in two developed markets and to assess the perceived value of IVDs on clinical decision-making. Physician-perceived HCE on IVD was evaluated, as well as desired features of new diagnostic markers.MethodsPast and current HCE on IVD was calculated for the US and Germany. A total of 79 US/German oncologists and cardiologists were interviewed to assess the number of cases where: physicians ask for IVDs; IVDs are used for initial diagnosis, treatment monitoring, or post-treatment; and decision-making is based on an IVD test result. A sample of 201 US and German oncologists and cardiologists was questioned regarding the proportion of HCE they believed to be attributable to IVD testing. After disclosing the actual IVD HCE, the physician’s perception of the appropriateness of the amount was captured. Finally, the association between physician-rated impact of IVD on decision-making and perceived contribution of IVD expenditure on overall HCE was assessed.ResultsIVD costs account for 2.3% and 1.4% of total HCE in the US and Germany. Most physicians (81%) believed that the actual HCE on IVDs was >5%; 19% rated the spending correctly (0–4%, p<0.001). When informed of the actual amount, 64% of physicians rated this as appropriate (p<0.0001); 66% of decision-making was based on IVD. Significantly, more physicians asked for either additional clinical or combined clinical/health economic data than for the product (test/platform) alone (p<0.0001).ConclusionsOur results indicate a poor awareness of actual HCE on IVD, but a high attributable value of diagnostic procedures for patient management. New markers should deliver actionable and medically relevant information, to guide decision-making and foster improved patient outcomes.     

Design and optimisation of orally active TLR7 agonists for the treatment of hepatitis C virus infection

The synthesis and structure-activity relationships of a series of novel interferon inducers are described. Pharmacokinetic studies and efficacy assessment of a series of 8-oxo-3-deazapurine analogues led to the identification of compound 33, a potent and selective agonist of the TLR7 receptor with an excellent in vivo efficacy profile in a mouse model.     

Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase

The assembly‐line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6‐deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein–protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃‐S‐ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein–protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.