Correlations between scaffold/matrix attachment region (S/MAR) binding activity and DNA duplex destabilization energy

Scaffold or matrix-attachment regions (S/MARs) are thought to be involved in the organization of eukaryotic chromosomes and in the regulation of several DNA functions. Their characteristics are conserved between plants and humans, and a variety of biological activities have been associated with them. The identification of S/MARs within genomic sequences has proved to be unexpectedly difficult, as they do not appear to have consensus sequences or sequence motifs associated with them. We have shown that S/MARs do share a characteristic structural property, they have a markedly high predicted propensity to undergo strand separation when placed under negative superhelical tension. This result agrees with experimental observations, that S/MARs contain base-unpairing regions (BURs). Here, we perform a quantitative evaluation of the association between the ease of stress-induced DNA duplex destabilization (SIDD) and S/MAR binding activity. We first use synthetic oligomers to investigate how the arrangement of localized unpairing elements within a base-unpairing region affects S/MAR binding. The organizational properties found in this way are applied to the investigation of correlations between specific measures of stress-induced duplex destabilization and the binding properties of naturally occurring S/MARs. For this purpose, we analyze S/MAR and non-S/MAR elements that have been derived from the human genome or from the tobacco genome. We find that S/MARs exhibit long regions of extensive destabilization. Moreover, quantitative measures of the SIDD attributes of these fragments calculated under uniform conditions are found to correlate very highly (r2>0.8) with their experimentally measured S/MAR-binding strengths. These results suggest that duplex destabilization may be involved in the mechanisms by which S/MARs function. They suggest also that SIDD properties may be incorporated into an improved computational strategy to search genomic DNA sequences for sites having the necessary attributes to function as S/MARs, and even to estimate their relative binding strengths.     

Diversity visualization by endonuclease: a rapid assay to monitor diverse nucleotide libraries

Many experiments require a fast and cost-effective method to monitor nucleic acid sequence diversity. Here we describe a method called diversity visualization by endonuclease (DiVE) that allows rapid visualization of sequence diversity of polymerase chain reaction (PCR) products based on DNA hybridization kinetics coupled with the activity of a single-strand specific nuclease. The assay involves only a limited number of steps and can be performed in less than 4 h, including the initial PCR. After PCR, the homoduplex double-stranded DNA (dsDNA) is denatured and reannealed under stringent conditions. During the reannealing process, incubation with S1 nuclease removes single-stranded loops of formed heteroduplexes and the resulting digest is visualized on agarose gel. The sequence diversity is inversely proportional to the band intensities of S1 nuclease surviving dsDNA molecules of expected size. As an example, we employed DiVE to monitor the diversity of panning rounds from a single-framework, semisynthetic single-chain antibody fragment (scFv) phage display library. The results are in good agreement with the observed decrease in diversity in phage display panning rounds toward the selection of monoclonal scFv. We conclude that the DiVE assay allows rapid and cost-effective monitoring of diversities of various nucleotide libraries and proves to be particularly suitable for scaffold-based randomized libraries.     

NanoDrop fluorometry adopted for microassays of proteasomal enzyme activities

Cell death by apoptosis triggers the engagement of a conserved intracellular machinery of execution, involving mainly the activation of the caspase family of cysteine proteases. Caspase-3 is a common effector of most of the apoptotic pathways and is able to cleave several target proteins whose degradation will contribute to the execution phase of the cell demise program. Here we present a modification of the Western blot protocol to improve sensitivity of caspase-3 detection, providing a valuable tool to access its activation in biological specimens.     

Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes

We previously demonstrated that tumour necrosis factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation. The mechanism of TNF-mediated A-SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression. The active forms of all three enzymes, caspase-8, caspase-7 and A-SMase, but not caspase-3, colocalize in internalized TNF receptosomes. While caspase-8 and caspase-3 are unable to induce activation of purified pro-A-SMase, we found that caspase-7 mediates A-SMase activation by direct interaction resulting in proteolytic cleavage of the 72-kDa pro-A-SMase zymogen at the non-canonical cleavage site after aspartate 253, generating an active 57 kDa A-SMase molecule. Caspase-7 down modulation revealed the functional link between caspase-7 and A-SMase, confirming proteolytic cleavage as one further mode of A-SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase-8 and caspase-7 for induction of A-SMase activation by proteolytic cleavage of pro-A-SMase.     

Susceptibility of European sheatfish Silurus glanis to a panel of ranaviruses

The current study was undertaken in order to assess the risk that different ranaviruses might impose on European sheatfish aquaculture. As the European sheatfish virus (ESV) is a known pathogen causing losses in European sheatfish aquaculture, it was assumed that closely related exotic ranaviruses might also be able to infect European sheatfish and probably cause disease and mortality in this species. The differential susceptibility of European sheatfish (Silurus glanis) to various ranavirus isolates was assessed at two different temperatures (15°C and 25°C) in a recirculation system. Fish were infected experimentally with a panel of ranavirus isolates including ESV, European catfish virus (ECV), European catfish virus isolate 24 (ECV‐24), Epizootic haematopoietic necrosis virus (EHNV), Rana esculenta virus isolate Italy 282/ I02 (REV), short‐finned eel virus (SERV), Bohle iridovirus (BIV), guppy virus 6 (GV6), doctor fish virus (DFV) and Frog virus 3 (FV3). Significant mortalities were observed, as expected, in fish infected with ESV at 15°C (100%) as well as at 25°C (86/83%). Fish infected with ECV at 15°C showed no clinical signs of disease (8% mortality), whereas those fish infected at 25°C exhibited a cumulative mortality of 54%. Fatal disease was also induced by Italian isolate ECV‐24 at 25°C (81%). Virus isolates ESV, ECV and ECV‐24, generally the most genetically closely related viruses, were successfully isolated from dead fish by cell culture with subsequent identification by polymerase chain reaction (PCR) and sequence analysis. However, no mortality or clinical signs of disease were observed in the groups of sheatfish infected with the other ranaviruses investigated in the study, and none of those viruses were re‐isolated in cell culture or identified by PCR. It was concluded that European sheatfish are susceptible to infection with ESV, ECV and ECV‐24 under laboratory conditions, but not to infection with EHNV, REV, SERV, BIV, GV6, DFV or FV3. For ESV, the incubation period was shorter at 25°C compared to 15°C water temperature, but whereas all fish died after ESV infection at 15°C, some fish survived the infection at 25°C. Futhermore, the very young sheatfish were susceptible to ECV and ECV‐24 at 25°C, whereas there was no significant mortality in the group of older sheatfish challenged with ECV at 15°C. Therefore, the clinical characteristics of the disease seem to depend on the age of the fish as well as on the water temperature.     

Acid Sphingomyelinase Serum Activity Predicts Mortality in Intensive Care Unit Patients after Systemic Inflammation: A Prospective Cohort Study

Introduction

Acid sphingomyelinase is involved in lipid signalling pathways and regulation of apoptosis by the generation of ceramide and plays an important role during the host response to infectious stimuli. It thus has the potential to be used as a novel diagnostic marker in the management of critically ill patients. The objective of our study was to evaluate acid sphingomyelinase serum activity (ASM) as a diagnostic and prognostic marker in a mixed intensive care unit population before, during, and after systemic inflammation.

Methods

40 patients admitted to the intensive care unit at risk for developing systemic inflammation (defined as systemic inflammatory response syndrome plus a significant procalcitonin [PCT] increase) were included. ASM was analysed on ICU admission, before (PCT_before), during (PCT_peak) and after (PCT_low) onset of SIRS. Patients undergoing elective surgery served as control (N = 8). Receiver-operating characteristics curves were computed.

Results

ASM significantly increased after surgery in the eight control patients. Patients from the intensive care unit had significantly higher ASM on admission than control patients after surgery. 19 out of 40 patients admitted to the intensive care unit developed systemic inflammation and 21 did not, with no differences in ASM between these two groups on admission. In patients with SIRS and PCT peak, ASM between admission and PCT_before was not different, but further increased at PCT_peak in non-survivors and was significantly higher at PCT_low compared to survivors. Survivors exhibited decreased ASM at PCT_peak and PCT_low. Receiver operating curve analysis on discrimination of ICU mortality showed an area under the curve of 0.79 for ASM at PCT_low.

Conclusions

In summary, ASM was generally higher in patients admitted to the intensive care unit compared to patients undergoing uncomplicated surgery. ASM did not indicate onset of systemic inflammation. In contrast to PCT however, it remained high in non-surviving ICU patients after systemic inflammation.     

Human herpes virus 8 interleukin-6 homologue triggers gp130 on neuronal and hematopoietic cells.

Human herpes virus-8 (HHV8) encodes a cytokine named viral interleukin-6 (vIL-6) that shares 25% amino-acid identity with its human homologue. Human IL-6 is known to be a growth and differentiation factor of lymphatic cells and plays a potential role in the pathophysiology of various lymphoproliferative diseases. vIL-6 is expressed in HHV8-associated-diseases including Kaposi's sarcoma, Body-cavity-based-lymphoma and Castleman's disease, suggesting a pathogenetic involvement in the malignant growth of B-cell associated diseases and other malignant tumours. We expressed vIL-6 in Escherichia coli as a fusion protein with recombinant periplasmic maltose binding protein. After cleavage from the maltose binding protein moiety and purification, vIL-6 was shown to be correctly folded using circular dichroism spectroscopy. A rabbit antiserum was raised against the recombinant vIL-6 protein. vIL-6 turned out to be active on cells that expressed gp130 but no IL-6 receptor (IL-6-R) suggesting that, in contrast to human IL-6, vIL-6 stimulated gp130 directly. Accordingly, vIL-6 activity could be inhibited by a soluble gp130 Fc Fusion protein. vIL-6 was shown to induce neuronal differentiation of rat pheochromocytoma cells and to stimulate colony formation of human hematopoietic progenitor cells. Thus, vIL-6 exhibits biologic activity that has only been observed for the IL-6/soluble IL-6-R complex but not for IL-6 alone. These properties are important for the evaluation of the pathophysiological potential of vIL-6.     

Tumor necrosis factor-induced changes of gene expression in U937 cells. Differentiation-dependent plasticity of the responsive state

TNF-alpha alone or in combination with IFN-gamma differentially affects the proliferation and differentiation of the human leukemic cell line U937 and two derivatives C27 and G3. All three cell lines express similar numbers of functional, high affinity receptors for both TNF-alpha and IFN-gamma. In C27 and G3 cells, TNF-alpha as well as IFN-gamma induced changes in steady state levels of specific mRNA, which appear to be associated with TNF-alpha and IFN-gamma diverse effects on cell growth and differentiation. Constitutive differences in membrane phosphorylation patterns suggest that altered transduction of TNF-alpha signals may account for the differential response of these three cell lines. Several lines of evidence indicate that C27 and G3 cells, when compared with parental U937 cells represent discretely higher stages of monocytic differentiation, suggesting that cellular differentiation may contribute to the development of resistance to the action of TNF-alpha.     

Effects of clinically relevant alumina ceramic, zirconia ceramic and titanium particles of different sizes and concentrations on TNF-alpha release in a human macrophage cell line]

INTRODUCTION: Aseptic loosening is considered to be the main problem of modern endoprothesis. Tumor necrosis factor alpha (TNFalpha) seems to be the initiator protein of particle disease. The aim of our study was to investigate the TNFalpha response of macrophage like cells (MLC) after stimulation with periprosthetic particles, typically found during revision surgery. For this purpose alumina ceramic (Al2O3), zirconia ceramic (ZrO2) and titanium (Ti) particles of different sizes and concentrations were used. Important was to study the effects of different sizes due to TNFalpha secretion and the comparison of the biological effects of alumina ceramic and titanium. METHOD: To obtain an TNFalpha profile we used an established macrophage model (Rader et al.) with THP-1 cells (human monocytic cell line). Therefore 106 MLC were incubated with different particle concentrations and sizes for 6 h. The supernatant was then investigated for TNF using ELISA assay. RESULTS: Ti-particles provoked in both sizes (0.2 microm and 2.5 microm) the greatest TNFalpha response, 8 times and 17 times as high in comparison with control. But substantially more 0.2 microm sized Ti-particles were necessary to get the above mentioned results. Al2O3-particles were not as effective as Ti, but they released fourfold more TNFalpha compared to control. There was no difference in TNFalpha-secretion comparing Al2O3-particles of different sizes (0.6 microm and 2 microm), but a 1000 times greater concentration of the 0.6 microm sized particles were needed. Using Al2O3- and Ti-particles of the same size and concentration, Ti provoked a significant higher TNFalpha response. ZrO2 showed no effects on TNFalpha release. CONCLUSION: Because of our results we recommend ceramic articulating surfaces, which are superior to metal on metal matings ion term of biological reactions. Additionally bigger wear particles should be avoided. Revisionoperation should be done early to avoid huge amount of wear particles and to minimize local osteolysis.     

Bone marrow transplantation for the treatment of leukaemia-results of the Munich Cooperative Group

This report summarizes the results of marrow transplantation from HLA-identical siblings and syngeneic twins for treatment of acute myelogenous leukaemia, chronic myelogenous leukaemia, acute lymphoblastic and undifferentiated leukaemia from 1975 until December 1986. Three conditioning regimens and treatment of the marrow graft in vitro with absorbed antithymocyte globulin or the monoclonal antibody "Campath 1" for prophylaxis of graft-versus-host disease (GVHD) have been studied and analyzed retrospectively. The regimen of total body irradiation in large fractions of 4 Gy and of cyclosphosphamide (200 mg/kg) has achieved the most favorable results. Inactivation of T-cells by treatment of the marrow "in vitro" has decreased the severity of GVHD without improving survival. The antileukaemic effect of the graft may be important for control of the disease and may be improved by better immunosuppression of the recipient.