Skeletal muscle sarcolemma in malignant hyperthermia: evidence for a defect in calcium regulation

Sarcolemmal properties implicated in the skeletal muscle disorder, malignant hyperthermia (MH), were examined using sarcolemma-membrane vesicles isolated from normal and MH-susceptible (MHS) porcine skeletal muscle. MHS and normal sarcolemma did not differ in the distribution of the major proteins, cholesterol or phospholipid content, vesicle size and sidedness, (Na+ + K+)-ATPase activity, ouabain binding, or adenylate cyclase activity (total and isoproterenol sensitivity). The regulation of the initial rates of MHS and normal sarcolemmal ATP-dependent calcium transport (calcium uptake after 1 min) by Ca2+ (K1/2 = 0.64-0.81 microM), calmodulin, and cAMP-dependent protein kinase were similar. However, when sarcolemmal calcium content was measured at either 2 or 20 min after the initiation of active calcium transport, a significant difference between MHS and normal sarcolemmal calcium uptake became apparent, with MHS sarcolemma accumulating approximately 25% less calcium than normal sarcolemma. Calcium transport by MHS and normal sarcolemma, at 2 or 20 min, had a similar calmodulin dependence (C1/2 = 150 nM), and was stimulated to a similar extent by cAMP-dependent protein kinase or calmodulin. Halothane inhibited MHS and normal sarcolemmal active calcium uptake in a similar fashion (half-maximal inhibition at 10 mM halothane), while dantrolene (30 microM) and nitrendipine (1 microM) had little effect on either MHS or normal sarcolemmal calcium transport. After 20 min of ATP-supported calcium uptake, 2 mM EGTA plus 10 microM sodium orthovanadate were added to initiate sarcolemmal calcium efflux. Following an initial rapid phase of calcium release, an extended slow phase of calcium efflux (k = 0.012 min-1) was similar for both MHS and normal sarcolemma vesicles. We conclude that although a number of sarcolemmal properties, including passive calcium permeability, are normal in MH, a small but significant defect in MHS sarcolemmal ATP-dependent calcium transport may contribute to the abnormal calcium homeostasis and altered contractile properties of MHS skeletal muscle.     

Potential secondary structure at translation-initiation sites.

Since translational start codons also occur internally, more-complex features within mRNA must determine initiation. We compare the potential secondary structure of 123 prokaryotic mRNA start regions to that of regions coding for internal methionines. The latter display an unexpectedly-uniform, almost-periodic pattern of pairing potential. In contrast, sequences 5' to start codons have little self-pairing, and do not pair extensively with the proximal coding region. Pairing potential surrounding start codons was found to be less than half of that found near internal AUGs. In groups of random sequences where the distribution of nucleotides at each position, or of trinucleotides at each in-frame codon position, matched the observed natural distribution, there was no periodicity in the pairing potential of the internal sequences. Randomized internal sequences had less pairing: the ratio of pairing intensity between internals and starts was reduced from 2.0 to 1.6 by randomization. We propose that the transition from the relatively-unstructured start domains to the highly-structured internal sequences may be an important determinant of translational start-site recognition.     

Application of a new method for detecting the phenotype of target binding cells

Summary A new method was developed for detecting the phenotype of target binding cells (TBC) in a single-cell assay system. This methodology was evaluated during a clinical trial of recombinant interferon alfa-2a (rIFN alfa-2a) for the treatment of 10 metastatic renal cell carcinoma patients. Total TBC with K562 targets, HNK-1+ TBC, and HLA-DR+ TBC were quantitated during rIFN alfa-2a therapy. A significantly increased proportion of lymphocytes bound to target cells on day 9 of therapy bore the HNK-1 marker. This proportion subsequently declined to pretreatment levels. Total TBC paralleled the rise and fall in HNK-1+ TBC. HLA-DR+ TBC binding to targets remained constant and low throughout therapy. These findings suggest that rIFN alfa-2a early in therapy (day 9) caused the recruitment of additional HNK-1+ cells into binders. However, with continued therapy, this proportion reverts to pretreatment levels. The results of this clinical trial served to illustrate the ability of the modified single-cell assay system to detect TBC phenotype.Supported in part by Hoffman-La Roche, NIH grant CA 12582, and Jonsson Comprehensive Cancer Center grant CA 15866Dr. Figlin is a recipient of an American Cancer Society Junior Faculty Fellowship-JFCF 762-A     

Localization and cloning of Xp21 deletion breakpoints involved in muscular dystrophy

Summary Twenty-nine deletion breakpoints were mapped in 220 kb of the DXS164 locus relative to potential exons of the Duchenne and Becker muscular dystrophy gene. Four deletion junction fragments were isolated to acquire outlying Xp21 loci on both the terminal and centromere side of the DXS164 locus. The junction loci were used for chromosome walking, searches for DNA polymorphisms, and mapping against deletion and translocation breakpoints. Forty-four unrelated deletions were analyzed using the junction loci as hybridization probes to map the endpoints between cloned Xp21 loci. DNA polymorphisms from the DXS164 and junction loci were used to follow the segregation of a mutation in a family that represents a recombinant. Both the physical and genetic data point to a very large size for this X-linked muscular dystrophy locus.     

Study of human chromosome V

Summary The induction of fragile sites on human chromosomes has been demonstrated under various conditions that cause thymidylate stress, including exposure to uridine. In this study, we examined common fragile site expression by initially exposing peripheral lymphocytes to uridine, followed by repair of the fragile sites with media containing various concentrations of thymidine. Lymphocytes were cultured in medium 199 with 2 mM uridine. At 0.5, 1, 2, 3, 8, 10, 12, and 18 h before harvest, the uridine medium was removed and replaced by medium containing thymidine at various concentrations. Our results demonstrate that the effect of uridine on chromosome fragility can be reversed by low concentrations of thymidine (2 M up to 200 M) and the rescuing effect of thymidine can be achieved if the cells were treated prior to 2–3 h before harvest. No repair was found if thymidine was added to culture within 2 h prior to harvesting, suggesting that packing of chromosomes is also an important factor in the expression and repair of fragile sites.     

Two micromorphic bivalves from the Silurian of Gotland

Two new micromorphic bivalve species from the Silurian of Gotland are described and assigned to the new genusKenzieana, viz.Kenzieana lata andKenzieana angusta. Kenzieana exhibits morphological features characteristic of some unrelated extant species; above all a conspicuous antero-posterior flattening of the shell. Probably it was a siphonate suspension feeder with a reclining mode of life.     

Viability of Cururbita pepo pollen: biophysical and structural data

During ageing of the short-lived pollen grains of Cucurbita pepo L., water loss was examined in relation to viability using biophysical (¹H-nuclear magnetic resonance, NMR) and cytological methods (fluorochromatic reaction test, freezefracture and scanning electron microscopy). A semi-logarithmic representation of the pollen weight loss demonstrated the complexity of the dehydration process. A the study of proton loss using ¹H-NMR indicated that two major releases water of had taken place, each with different flux rates. Pulse ¹H-NMR experiments showed the occurrene of non-exponential signal decay as a function of time, indicating the existence of different fractions of water in a pollen grain sample. These fractions leave the pollen grain at different times during pollen dehydration, and one of them (that of the so-called vital water) can be related to pollen viability. The quantity of protons giving a signal during pulse ¹H-NMR experiments was very low when the pollen grains were judged to be dead according to the fluorochromatic test. Freeze-fracture replicas of these dead pollen grains (less than 25% water content) showed that the plasma membrane had become detached from the intine surface; this ultrastructural feature might therefore be involved in the loss of pollen viability.Abbreviations A initial amplitude of the NMR signal - A₂ quantity of water charcterized by T_2-2 - A₅ quantity of water characterized by T_2–5 - FCR fluorochromatic reaction - NMR nuclear magnetic resonance - T₂ transverse relaxation time - T_2-2 T₂ measured with 2 ms between each pulse of radiofrequency - T_2–5 T₂ measured with 5 ms between each pulse of radiofrequency