The effect of ionic strength on the kinetics of rigor development in skinned fast-twitch skeletal muscle fibres

 Recent atomic 3-D reconstructions of the acto-myosin interface suggest that electrostatic interactions are important in the initial phase of cross-bridge formation. Earlier biochemical studies had also given strong evidence for the ionic strength dependence of this step in the cross-bridge cycle. We have probed these interactions by altering the ionic strength (Γ/2) of the medium mainly with K⁺, imidazole⁺ and EGTA^2– to vary charge shielding. We examined the effect of ionic strength on the kinetics of rigor development at low Ca²⁺ (experimental temperature 18–22°C) in chemically skinned single fast-twitch fibres of mouse extensor digitorum longus (EDL) muscle. On average the delay before rigor onset was 10 times longer, the maximum rate of rigor tension development was 10 times slower, the steady-state rigor tension was 3 times lower and the in-phase stiffness was 2 times lower at high (230 mM) compared to low (60 mM) ionic strength. These results were modelled by calculating ATP depletion in the fibre due to diffusional loss of ATP and acto-myosin Mg.ATPase activity. The difference in delay before rigor onset at low and high ionic strength could be explained in our model by assuming a 15 times higher Mg.ATPase activity and a threefold increase in K _m in relaxing conditions at low ionic strength. Activation by Ca²⁺ induced at different time points before and during onset of rigor confirmed the calculated time course of ATP depletion. We have also investigated ionic strength effects on rigor development with the activated troponin/tropomyosin complex. ATP withdrawl at maximum activation by Ca²⁺ induced force transients which led into a ”high rigor” state. The peak forces of these force transients were very similar at low and high ionic strength. The subsequent decrease in tension was only 10% slower and steady-state ”high rigor” tension was reduced by only 27% at high compared to low ionic strength. Addition of 10 mM phosphate to lower cross-bridge attachment strongly suppressed the transient increases in force at high ionic strength and reduced the steady-state rigor tension by 17%. A qualitatively similar but smaller effect of phosphate was observed at low ionic strength where steady-state rigor force was reduced by 10%. The data presented in this study show a very strong effect of ionic strength on rigor development in relaxed fibres whereas the ionic strength dependence of rigor development after thin filament activation was much less. The data confirm the importance of electrostatic interactions in cross-bridge attachment and cross-bridge-attachment-induced activation of thin filaments. Received: 3 September 1997 / Received after revision and accepted: 12 December 1997     

Bacterial colonization and infection in an intensive care unit

One hundred and one patients, nursed in an intensive care unit for at least 24h, were monitored for bacterial colonization and infection. The infection rates were similar to those in other reports. Patients were not generally colonized with common environmental strains in the unit. Bacterial dissemination between patients was uncommon. No gentamicin resistant gram negative or Staphylococcus aureus strains were observed, nor methicillin resistant Staphylococcus aureus strains. the hypothesis that these favourable conditions are partly related to the excellent isolation and barrier nursing facilities in the unit cannot be fully substantiated.     

Acute fatal myeloencephalopathy after combined intrathecal chemotherapy in a child with acute lymphoblastic leukemia

We report a case of a fatal toxic encephalomyelopathy in a 12-year-old girl due to prophylactic intrathecal injection of methotrexate and cytosine arabinoside, with a characteristic progressive symptomatology leading to death after 28 days. The location and type of neuropathological changes support the hypothesis of a direct toxic effect of methotrexate and/or cytosine arabinoside on structures directly exposed to the cerebrospinal fluid.     

Inhibition of cellular immune reactions by zymosan

Mice treated intraperitoneally with zymosan showed a strong inhibition of the DTHR (delayed-type hypersensitivity reaction) against sheep erythrocytes (SE), ovalbumin (OA), and alloantigen. Furthermore, the rejection time of skin transplants was nearly doubled while antibody formation against SE was significantly enhanced. When a DTHR against OA and an antibody formation against SE were induced at the same time in the same animals, than the suppressive and stimulating effects cancelled each other. These results are discussed with regard to the sensitivity of lymphocyte subpopulations, which may be different if exposed to phagocytosis-induced oxygen radicals.     

Recombinant human stem cell factor stimulates growth of a human glioblastoma cell line expressing c-kit protooncogene.

The growth of a panel of eight different human glioblastoma cell lines was examined in a human tumor cloning assay in agar, a tritiated thymidine uptake assay, and by counting cell numbers, in cultures performed in the absence or presence of increasing concentrations (1 to 100 ng/ml) of recombinant human stem cell factor (SCF). Growth of 7 of 8 cell lines was not significantly and reproducibly affected by recombinant human SCF. However, growth of the CRL 1620 cell line could be stimulated up to 5-fold by the cytokine. In contrast to the other cell lines investigated, CRL 1620 expressed the c-kit protooncogene assessed on the mRNA and protein level. Furthermore, SCF-induced proliferation of CRL 1620 cells was sensitive to the tyrosine kinase inhibitor erbstatin. Our data suggest that SCF can be operative in growth modulation of malignant cells outside the hematopoietic system, and this finding should be further studied for its possible clinical implications.     

Detection of the Arg702Trp, Gly908Arg and Leu1007fsinsC polymorphisms of the NOD2/CARD15 gene by real-time PCR with melting curve analysis.

Crohn's disease is a complex disorder, with multiple genetic traits. A frameshift mutation (Leu1007fsinsC) and two missense mutations (Gly908Arg and Arg702Trp) in the NOD2/CARD15 gene are strongly associated with susceptibility to Crohn's disease. The presence of one of these risk alleles confers a 2- to 4-fold increase in the risk of developing Crohn's disease, and the presence of two mutant alleles increases the risk over 20-fold. To facilitate the analysis of these polymorphisms, we developed three LightCycler assays to detect the missense mutations Arg702Trp and Gly908Arg and the frameshift mutation Leu100fsinsC in the NOD2/ CARD15 gene. All three assays can be run simultaneously on one LightCycler using identical cycling parameters. Analysis of 53 DNAs from Crohn's patients helped to identify carriers at allele frequencies similar to other Caucasian populations. The sequencing of such DNAs confirmed the accuracy of the assays. In conclusion, we present three rapid and robust assays to detect the Arg702Trp, the Gly908Arg and the Leu1007fsinsC ins mutations in the NOD2/CARD15 gene [corrected]