Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers.

To determine the role of myosin regulatory light chain (RLC) in modulating contraction in skeletal muscle, we examined the rate of tension development in bundles of skinned skeletal muscle fibers as a function of the level of Ca(2+) activation after UV flash-induced release of Ca(2+) from the photosensitive Ca(2+) chelator DM-nitrophen. In control fiber bundles, the rate of tension development was highly dependent on the concentration of activator Ca(2+) after the flash. There was a greater than twofold increase in the rate of tension development when the post-flash [Ca(2+)] was increased from the lowest level tested (which produced a steady tension that was 42% of maximum tension) to the highest level (producing 97% of maximum tension). However, when 40-70% of endogenous myosin RLC was extracted from the fiber bundles, tension developed at the maximum rate, regardless of the post-flash concentration of Ca(2+). Thus, the Ca(2+) dependence of the rate of tension development was eliminated by partial extraction of myosin RLC, an effect that was partially reversed by recombination of RLC back into the fiber bundles. The elimination of the Ca(2+) dependence of the kinetics of tension development was specific to the extraction of RLC rather than an artifact of the co-extraction of both RLC and Troponin C, because the rate of tension development was still Ca(2+) dependent, even when nearly 50% of endogenous Troponin C was extracted from fiber bundles fully replete with RLC. Thus, myosin RLC appears to be a key component in modulating Ca(2+) sensitive cross-bridge transitions that limit the rate of force development after photorelease of Ca(2+) in skeletal muscle fibers.     

In vivo assay for protein-protein interactions using Drosophila chromosomes

The ability of a chimeric HP1-Polycomb (Pc) protein to bind both to heterochromatin and to euchromatic sites of Pc protein binding was exploited to detect stable protein-protein interactions in vivo. Previously, we showed that endogenous Pc protein was recruited to ectopic heterochromatic binding sites by the chimeric protein. Here, we examine the association of other Pc group (Pc-G) proteins. We show that Posterior sex combs (Psc) protein also is recruited to heterochromatin by the chimeric protein, demonstrating that Psc protein participates in direct protein-protein interaction with Pc protein or Pc-associated protein. In flies carrying temperature-sensitive alleles of Enhancer of zeste[E(z)] the general decondensation of polytene chromosomes that occurs at the restrictive temperature is associated with loss of binding of endogenous Pc and chimeric HP1-Polycomb protein to euchromatin, but binding of HP1 and chimeric HP1-Polycomb protein to the heterochromatin is maintained. The E(z) mutation also results in the loss of chimera-dependent binding to heterochromatin by endogenous Pc and Psc proteins at the restrictive temperature, suggesting that interaction of these proteins is mediated by E(z) protein. A myc-tagged full-length Suppressor 2 of zeste [Su(z)2] protein interacts poorly or not at all with ectopic Pc-G complexes, but a truncated Su(z)2 protein is strongly recruited to all sites of chimeric protein binding. Trithorax protein is not recruited to the heterochromatin by the chimeric HP1-Polycomb protein, suggesting either that this protein does not interact directly with Pc-G complexes or that such interactions are regulated. Ectopic binding of chimeric chromosomal proteins provides a useful tool for distinguishing specific protein-protein interactions from specific protein-DNA interactions important for complex assembly in vivo.     

Identification of single tiny seeds ofOrobanche using RAPD analysis

The tiny seeds of parasitic weeds of the genusOrobanche can be identified by using RAPD markers. A simple procedure for DNA extraction from single seeds, 10 μg each, followed by RAPD-PCR and using specific DNA markers, leads to species identification. Seeds of five different species could be identified using this method.     

Vegetative incompatibility in filamentous fungi: het genes begin to talk

Somatic or vegetative incompatibility is widespread in filamentous fungi. It prevents the coexistence of genetically different nuclei within a common cytoplasm. Cloning the het genes that control this process has been achieved in several species. This has provided essential information on the function of the genes in the biology of fungi and has also led to the formulation of models that may explain similar phenomena in other organisms.     

Global temperature stability by rule induction: An interdisciplinary bridge

Rules incorporating influences on global temperature, an estimate of radiation balance, were induced from astronomical, geophysical, and anthropogenic variables. During periods of intermediate global temperatures (generally like the present century), the influences assume cancelingroles; influences cancel the effects of extreme states potentially imposed by other influences because they are, in aggregate, most likely to be assuming opposite values. This imparts an overall stability to the global temperature. To achieve cold or hot global temperature, influences assume reinforcingroles. CO ₂ is an active influence on global temperature. By virtue of its constancy in the atmosphere, it can be expected to sponsor frequent hot years in combination with the other influences as they cycle through their periods. If measures were implemented to maintain warm or cool global temperatures, it could retain the status quoof present global agricultural regions. They are probably more productive than hot world regions would be because of narrow storm tracks.     

Analysis of HLA and Disease Susceptibility: Chromosome 6 Genes and Sex Influence Long-QT Phenotype

The long-QT (LQT) syndrome is a genetically complex disorder that is characterized by syncope and fatal ventricular arrhythmias. LQT syndrome, as defined by a prolonged electrocardiographic QT interval, has a higher incidence in females than in males and does not exhibit Mendelian transmission patterns in all families. Among those families that are nearly consistent with Mendelian transmission, linkage between a locus for LQT syndrome and the H-ras-1 locus on the short arm of chromosome 11 has been reported in some families but not in others. Earlier analyses suggesting that LQT syndrome might be caused by a gene in the HLA region of chromosome 6 were not confirmed by standard linkage analyses. Here, we present an analysis of HLA haplotype sharing among affected pedigree members, showing an excess of haplotype sharing in a previously published Japanese pedigree and possibly also in 15 families of European descent. The haplotypes shared by affected individuals derive from both affected and unaffected parents. In an analysis of independent (unrelated) HLA haplotypes, we also found a nonrandom distribution of HLA-DR genes in LQT syndrome patients compared with controls, suggesting an association between the LQT phenotype and specific HLA-DR genes. Our data indicate that DR2 has a protective effect and, particularly in males, that DR7 may increase susceptibility to the LQT syndrome. Thus, LQT syndrome may be influenced by genes on chromosomes 11 and 6, possibly with a sex-specific effect. These results provide a model for an effect of HLA-region genes inherited from either parent on the expression of an illness that may be determined principally by alleles at loci not linked to HLA.     

Rat brain Na channel mRNAs in non-excitable Schwann cells

The expression of rat brain voltage-sensitive Na⁺ channel mRNAs in Schwann cells was examined using in situ hybridization cytochemistry and RT-PCR. The mRNAs of rat brain Na⁺ channel subtype II and III, but not subtype I, were detected in cultured Schwann cells from sciatic nerve and in intact sciatic nerve, which contains Schwann cells but not neuronal cell bodies. These results indicate that rat brain Na⁺ channel mRNAs, which have been considered as mainly neuronal-type messages, are also expressed in glial cells in vitro and in vivo.     

Periodic forcing of a K+ channel at various temperatures.

The random sequence of openings and closings of single ion channels and the channel conductances have been the object of intense study over the past two decades with a view toward illuminating the underlying kinetics of the channel protein molecules. Channels that are sensitive to voltage, such as many K(+)-selective channels, have been particularly useful, because the kinetic rates can be manipulated by changing the membrane voltage. Most such studies have been performed under stationary conditions and usually at a single temperature. Here we report the results of experiments with sinusoidal modulation of the membrane potential performed at several temperatures. Dwell time and cycle histograms, objects not normally associated with ion channel experiments, are herein reported. From the last, the transition probability densities for channel opening and closing events are obtained. A new and unusual phase anticipation is observed in the cycle histograms, and its temperature dependence is measured.     

Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65.

The cytoplasmic assembly of vaccinia virus is reversibly blocked by the antibiotic rifampin, leading to the accumulation of partially membrane-delineated rifampin bodies in infected cells. Rifampin-resistant vaccinia virus mutants have point mutations in the D13L gene, which is controlled by a late promoter and expresses a 65-kDa protein, designated p65. To further characterize the mechanism of rifampin inhibition and the function of p65 in virus assembly, we raised antibodies to this protein. Immunoreactive p65 was expressed at late times of infection, and neither its expression nor its turnover was affected by rifampin. Virus-associated p65 could be extracted only with denaturing detergents from purified virions, suggesting that it is an integral viral component. Immunofluorescence studies showed that p65 is localized to the sites of virus assembly. Also, immunoelectron microscopy showed p65 to be associated with viral crescents as well as spherical, immature virions, in both cases predominantly on the inner or concave surface. In the presence of rifampin, p65 was found in large, cytoplasmic inclusion bodies that were distinct from rifampin bodies. The rifampin bodies themselves were labeled with p65 antibodies only after reversal of the rifampin block, predominantly on the viral crescents which rapidly formed following removal of the drug. We propose that p65 functions as an internal scaffold in the formation of viral crescents and immature virions, analogously to the matrix proteins of other viruses.     

Seed storage protein variation in Arachis species.

Seventy-two accessions, representing 22 species from sections Arachis, Erectoides, Extranervosae, and Triseminalae of the genus Arachis, were screened for seed storage protein polymorphism. Variation was detected between sections, between genome types, between species, and in some cases between different accessions of the same species or different seeds of the same accession. Arachis duranensis and one accession of A. cardenasii were found to have identical protein patterns. The greatest dissimilarity was found between species of the section Extranervosae and species of the section Triseminalae. Those of section Erectoides showed much similarity with some species of section Arachis. Protein polymorphism was shown to distinguish the two subspecies of A. hypogaea (fastigiata and hypogaea) in 27 of 28 cases. The seed protein profile of A. monticola was a combination of seed protein profiles from the two A. hypogaea subspecies. The relatedness between the various species was calculated and those that had the greatest similarity with A. hypogaea were A. spegazzinii and A. batizocoi.