Activation of the CPP32 protease in apoptosis induced by 1-beta-D- arabinofuranosylcytosine and other DNA-damaging agents

The response of human myeloid leukemia cells to treatment with 1-beta- arabinofuranosylcytosine (ara-C) includes the induction of apoptosis. Ara-C induced apoptosis is associated with proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and protein kinase C (PKC) delta. However, the signals involved in this response are unknown. The present studies show that ara-C treatment of U-937 cells is associated with induction of a protease activity that cleaves the tetrapeptides Ac-DEVD- pNA and Ac-DMOD-pNA found at the cleavage sites of PARP and PKC delta, respectively. The ara-C-induced protease activity was sensitive to overexpression of the anti-apoptotic protein Bcl-xL and the baculovirus protein p35. By contrast, overexpression of the cowpox virus protein CrmA blocked apoptosis induced by engagement of the Fas receptor but not that induced by ara-C. CrmA overexpression also had no detectable effect on ara-C-induced cleavage of PKC delta. The results further show that ara-C induces activation of the CPP32 protease by a CrmA- insensitive and p35-sensitive mechanism. Similar results were obtained with cisplatinum, etoposide, and camptothecin. These findings indicate that ara-C and other DNA-damaging agents activate a CrmA-insensitive apoptotic pathway involving CPP32 and that these signals differ from those associated with apoptosis induced by the Fas receptor.     

Expression and occupancy of BAFF-R on B cells in systemic lupus erythematosus

OBJECTIVE: To determine whether receptors for B lymphocyte stimulator (BLyS) are altered on B cells of patients with systemic lupus erythematosus (SLE). METHODS: Total available receptors for BLyS were measured by analysis of binding of recombinant soluble BLyS to peripheral blood B cells in 36 SLE patients, 29 healthy controls, and 10 disease controls. Antibodies to the receptors BAFF-R, BCMA, and TACI were used to define expression of the individual BLyS receptors on subsets of B cells in blood, spleen, and tonsils. Two different antibodies to BAFF-R, which were differentially sensitive to BAFF-R occupancy, were used to compare BAFF-R on B cells in an additional 20 healthy subjects and 25 SLE patients. Assays of B cell survival after stimulation in vitro were used to determine the sensitivity of B cells to exogenous BLyS. RESULTS: Total available receptors for BLyS were decreased in patients with SLE, independent of changes of subsets in the blood in these patients. The decrease correlated with changes in disease activity. Although total surface BAFF-R was not significantly different between healthy controls and SLE patients, BAFF-R was occupied in SLE patients. B cells from these patients were less responsive to exogenous BLyS. CONCLUSION: BAFF-R is consistently occupied on blood B cells in SLE. Occupancy of BAFF-R on blood B cells is likely to contribute to disease mechanisms in SLE and could serve as a biomarker of disease activity. Targeting BLyS as a therapeutic strategy will require overcoming the persistent binding of BLyS to BAFF-R.     

Lysis of fibrillar collagen by neutrophils in synovial fluid. A role for surface-bound immunoglobulins

Synovial fluids (SF) contain inhibitors capable of neutralizing the activity of proteases secreted by inflammatory cells and fibroblasts. To further define a potential role for SF polymorphonuclear neutrophils (PMN) in mediating joint destruction, peripheral blood PMN were suspended in SF and incubated with reconstituted collagen fibrils. Incubation of PMN-SF mixtures with collagen fibrils precoated with monomeric IgG resulted in significant lysis of the underlying fibrils relative to that seen with uncoated fibrils. Augmented fibril lysis by PMN-SF mixtures in which the PMN were activated with fluid-phase ligands such as phorbol myristate acetate or heat-aggregated IgG was not seen. Lysis of IgG-coated fibrils by PMN-SF was inhibited in the presence of EDTA or sodium azide. PMN-mediated resorption of fibrillar collagen occurred despite the presence of protease inhibitors in the SF at a concentration capable of neutralizing human neutrophil collagenase. These results suggest that the focal release and activation of human neutrophil collagenase during PMN stimulation by tissue-bound immunoglobulins may mediate the resorption of joint tissue collagens in rheumatoid arthritis, even in the presence of protease inhibitors.     

Fixed Load Incremental Respiratory Muscle Training: A pilot study

The purpose of this pilot study was to examine the use of a new incremental test of respiratory endurance (the ‘TIRE’) as a method of fixed load respiratory muscle training (RMT) in normal volunteers. Ten subjects completed ten weeks of RMT with training levels set at 80% of peak. Using the related t-test to analyse the data significant increases were seen in respiratory muscle strength (p < 0.005), and respiratory muscle endurance (p < 0.005). Subjects reported anecdotally that after four to six weeks of RMT they experienced reduced levels of dyspnoea and improved exercise performance during regular sporting activities.We conclude that fixed load incremental RMT, set at a level consistent with systemic training regimes, increases the strength and endurance of respiratory muscles. Further controlled investigation in a larger population is required. This should include training level manipulation by the TIRE to obtain strength or endurance effect and formal assessment of the dyspnoea perception and systemic performance reported anecdotally during this study.     

Guidelines for international collaborative research

OBJECTIVE: As the global village becomes a reality, there is an increasing need to conduct international collaborative studies in family practice. A workshop at the WONCA meeting in Hong Kong used international attendees to produce a set of guidelines for international research. METHODS: At the workshop four completed international projects, each using a different strategy, were presented so that common themes might become apparent. The themes were then discussed and guidelines emerged from the process. RESULTS: Seven guidelines emerged for consideration before embarking on an international collaborative research project in family medicine. The guidelines deal with the characteristics of the research question and the importance of communication. The need for simple, brief methods of data collection, funding and pilot testing were identified. CONCLUSION: The question must be relevant to all participants to maintain interest and measurement tools must be validated to understand the impact of cultural differences in understanding.      

31P NMR of mammalian cells encapsulated in alginate gels utilizing a new phosphate-free perfusion medium

A simple technique permitting long term 31P magnetic resonance spectroscopy of cultured mammalian cells is described. Cells are encapsulated in calcium alginate gels and perfused with a new phosphate-fre medium designed for maintenance of high cell viability. Intracellular pH and beta-NTP/Pi ratios, studied in three cell types, remained stable over a 16-20 h NMR monitoring period. The techniques described may be useful for monitoring cell metabolism with a variety of cell types.     

Quantitative proton spectroscopy and histology of a canine brain tumor model

Quaintitative, single voxel proton nuclear magnetic resonance (NMR) spectroscopy and histological analysis was performed in eight dogs implanted with the transplantable canine glioma model of Wodinsky (Proc. Am. Assoc. Cancer Res. 10, 99 (1969). Signals from choline, creatine, N-Acetyl Aspartate (NAA) and lactate were converted to molar concentration units and correlated with the quantitative analysis of histologically determined tissue types within the localized volume selected for NMR spectroscopy. In general, compared with normal brain, the lesions were associated with reductions in all metabolite concentrations, with the exception of lactate, which was increased. NAA and creatine decreases were most significantly correlated with the total lesion volume (P < 0.01), suggesting that these compounds are present in normal brain only. Changes in choline levels did not correlate strongly with any particular tissue type. Lactate was found to increase with increasing total lesion volume (P < 0.01), but not with increasing percent tumor, suggesting that it accumulates in abnormal tissue other than the tumor. The spectra reported were similar to those observed in human glioblastomas, with the exception that elevations of choline were not observed. The transplant-able canine gliosarcoma system appears to be a suitable tumor model for evaluation by clinical radiological techniques such as magnetic resonance imaging (MRI) and proton NMR spectroscopy.