CrFK feline kidney cells produce an RD114-like endogenous virus that can package murine leukemia virus-based vectors

The feline kidney cell line CrFK is used extensively for viral infectivity assays and for study of the biology of various retroviruses and derived vectors. We demonstrate the production of an endogenous, RD114-like, infectious retrovirus from CrFK cells. This virus also is shown to efficiently package Moloney murine leukemia virus vectors.     

Sulfasalazine in the treatment of juvenile chronic arthritis: a randomized, double-blind, placebo-controlled, multicenter study. Dutch Juvenile Chronic Arthritis Study Group

AIMS: To determine whether left ventricular volumes and ejection fractions calculated from single plane two-dimensional echocardiograms using the algorithm (0.85A2L) correlate with those calculated using the biplane Simpson's method, and whether small changes in volumes and ejection fraction occurring post-infarction could be detected from single-plane as well as from biplane two-dimensional echocardiograms. METHODS AND RESULTS: Serial two-dimensional echocardiograms were obtained in 371 patients from the DEFIANT II trial a mean of 2 days, 1 week and 6 months post-infarction. Single plane volumes from the apical four chamber and apical long axis correlated closely with biplane Simpson's left ventricular volumes. Both single-plane left ventricular volumes significantly over-estimated biplane Simpson's volumes. Biplane Simpson's ejection fractions were consistently slightly under-estimated from the single-plane images. Differences between biplane Simpson's and single-plane volumes increased independently with increasing left ventricular size and distortion. The small changes in left ventricular volumes and ejection fraction over time were as reliably detected from single plane as from biplane images. CONCLUSION: Single-plane left ventricular volumes over-estimate biplane Simpson's volumes and under-estimate ejection fraction, and these discrepancies are amplified in dilated hearts with abnormal shape.     

Calphostin C triggers calcium-dependent apoptosis in human acute lymphoblastic leukemia cells

Recent studies have demonstrated that the naturally occurring perylenequinone antibiotic calphostin C is a potent inhibitor of protein kinase C and can induce apoptosis in some tumor cell lines by an as yet unknown mechanism. Here we demonstrate that calphostin C induces dose-dependent apoptosis in DT40 chicken lymphoma B-cells, and targeted disruption of lyn, syk, btk, PLCgamma2, or IP3R genes does not prevent or attenuate its cytotoxicity. In our study, calphostin C also induced rapid apoptosis in human acute lymphoblastic leukemia (ALL) cell lines ALL-1 (BCR-ABL+ pre-pre-B ALL), RS4;11 (MLL-AF4+ pro-B ALL), NALM-6 (pre-B ALL), DAUDI (Burkitt's/B-cell ALL), MOLT-3 (T-ALL), and JURKAT (T-ALL), whereas other potent PKC inhibitors did not. In biochemical studies, calphostin C was discovered to induce rapid calcium mobilization from intracellular stores of ALL cell lines, and its cytotoxicity against ALL cell lines was well correlated with the magnitude of this calcium signal. Calphostin C-induced apoptosis was markedly suppressed by BAPTA/AM, a cell-permeable Ca2+ chelator as well as NiCl2, an inhibitor of Ca2+/Mg2+-dependent endonucleases. Inhibition of the Ca2+/calmodulin-dependent phosphatase calcineurin with perfluoreperazine dimadeate (a calmodulin antagonist) or cyclosporin A (a specific inhibitor of calcineurin) also reduced the magnitude of calphostin C-induced apoptosis in ALL cell lines. Calphostin C was capable of inducing calcium mobilization and apoptosis in freshly obtained primary leukemic cells from children with ALL. Taken together, our results provide unprecedented evidence that calphostin C triggers a Ca2+-dependent apoptotic signal in human ALL cells.     

Enrichment of Anammox biomass from municipal activated sludge: experimental and modelling results

Anaerobic Ammonia Oxidising (Anammox) biomass was enriched from sludge collected at a municipal wastewater treatment plant, employing a Sequential Batch Reactor (SBR). After 60 days Anammox activity started to be detected, by consumption of stoichiometric amounts of NO₂^? and NH₄⁺ in the system. Fluorescence In Situ Hybridisation analysis confirmed the increase of Anammox bacteria concentration with time. A final concentration of enriched biomass of 3–3.5 gVSS dm^?3 was obtained, showing a Specific Anammox Activity of 0.18 gNH₄⁺‐N gVSS^?1 d^?1 The reactor was able to treat nitrogen loading rates of up to 1.4 kgN m^?3 d^?1, achieving a removal efficiency of 82 %. On the other hand, the start‐up and operation of the Anammox SBR reactor were consequentially modelled with the Activated Sludge Model nr 1, extended for Anammox. The simulations predicted quite well the experimental data in relation to the concentrations of nitrogenous compounds and can be used to estimate the evolution of Anammox and heterotrophic biomass in the reactor. These simulations reveal that heterotrophs still remain in the system after the start‐up of the reactor and can protect the Anammox microorganisms from a negative effect of the oxygen. Copyright © 2004 Society of Chemical Industry     

Cell-surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S-layer of Caulobacter crescentus

The paracrystalline surface (S)-layer of Caulobacter crescentus is composed of a single secreted protein (RsaA) that interlocks in a hexagonal pattern to completely envelop the bacterium. Using a genetic approach, we inserted a 12 amino acid peptide from Pseudomonas aeruginosa strain K pilin at numerous semirandom positions in RsaA. We then used an immunological screen to identify those sites that presented the inserted pilin peptide on the C. crescentus cell surface as a part of the S-layer. Eleven such sites (widely separated in the primary sequence) were identified, demonstrating for the first time that S-layers can be readily exploited as carrier proteins to display 'epitope-size' heterologous peptides on bacterial cell surfaces. Whereas intact RsaA molecules carrying a pilin peptide could always be found on the surface of C. crescentus regardless of the particular insertion site, introduction of the pilin peptide at 9 of the 11 sites resulted in some proteolytic cleavage of RsaA. Two types of proteolytic phenomena were observed. The first was characterized by a single cleavage within the pilin peptide insert with both fragments of the S-layer protein remaining anchored to the outer membrane. The other proteolytic phenomenon was characterized by cleavage of the S-layer protein at a point distant from the site of the pilin peptide insertion. This cleavage always occurred at the same location in RsaA regardless of the particular insertion site, yielding a surface-anchored 26 kDa proteolytic fragment bearing the RsaA N-terminus; the C-terminal cleavage product carrying the pilin peptide was released into the growth medium. When the results of this work were combined with the results of a previous study, the RsaA primary sequence could be divided into three regions with respect to the location of a peptide insertion and its effect on S-layer biogenesis: (i) insertions in the extreme N-terminus of RsaA either produce no apparent effect on S-layer biogenesis or disrupt surface-anchoring of the protein; (ii) insertions in the extreme C-terminus either produce no apparent effect on S-layer biogenesis or disrupt protein secretion; and (iii) insertions more centrally located in the protein either have no apparent effect on S-layer biogenesis or result in proteolytic cleavage of RsaA. These data are discussed in relation to our previous assignment of the RsaA N- and C-terminus as regions that are important for surface anchoring and secretion respectively.     

Expression of an antigen homologous to the human CO17-1A/GA733 colon cancer antigen in animal tissues

The CO17-1A/GA733 antigen is associated with human carcinomas and some normal epithelial tissues. This antigen has shown promise as a target in approaches to passive and active immunotherapy of colorectal cancer. The relevance of animal models for studies of immunotherapy targeting this antigen in patients is dependent on the expression of the antigen on normal animal tissues. Immunohistoperoxidase staining with polyclonal rabbit antibodies to the human antigen revealed the human homologue on normal small intestine, colon and liver of mice, rats and non-human primates, whereas mouse monoclonal antibodies to the CO17-1A or GA733 epitopes on the human antigen did not detect the antigen. Polyclonal rabbit antibodies, elicited by the murine antigen homologue derived from recombinant baculovirus-infected insect cells, immunoprecipitated the antigen from mouse small intestine, colon, stomach, kidney and lung. The isolated recombinant murine protein bound polyclonal, but not monoclonal, antibodies to the human CO17-1A/GA733 antigen, and recombinant human antigen bound polyclonal antibodies elicited by the murine antigen homologue. Thus, the antigen homologue expressed by animal tissues is similar, but not identical, to the human antigen. These results have important implications for experimental active and passive immunotherapy targeting the CO17-1A/GA733 antigen.