Crystal structure of the C1 domain of cardiac myosin binding protein-C: implications for hypertrophic cardiomyopathy

C-protein is a major component of skeletal and cardiac muscle thick filaments. Mutations in the gene encoding cardiac C-protein [cardiac myosin binding protein-C (cMyBP-C)] are one of the principal causes of hypertrophic cardiomyopathy. cMyBP-C is a string of globular domains including eight immunoglobulin-like and three fibronectin-like domains termed C0-C10. It binds to myosin and titin, and probably to actin, and may have both a structural and a regulatory role in muscle function. To help to understand the pathology of the known mutations, we have solved the structure of the immunoglobulin-like C1 domain of MyBP-C by X-ray crystallography to a resolution of 1.55 Å. Mutations associated with hypertrophic cardiomyopathy are clustered at one end towards the C-terminus, close to the important C1C2 linker, where they alter the structural integrity of this region and its interactions.     

ULTRASTRUCTURAL STUDIES OF VASOPRESSIN EFFECT ON ISOLATED PERFUSED RENAL COLLECTING TUBULES OF THE RABBIT

Isolated cortical collecting tubules from rabbit kidney were studied during perfusion with solutions made either isotonic or hypotonic to the external bathing medium. Examination of living tubules revealed a reversible increase in thickness of the cellular layer, prominence of lateral cell membranes, and formation of intracellular vacuoles during periods of vasopressin-induced osmotic water transport. Examination in the electron microscope revealed that vasopressin induced no changes in cell structure in collecting tubules in the absence of an osmotic difference and significant bulk water flow across the tubule wall. In contrast, tubules fixed during vasopressin-induced periods of high osmotic water transport showed prominent dilatation of lateral intercellular spaces, bulging of apical cell membranes into the tubular lumen, and formation of intracellular vacuoles. It is concluded that the ultrastructural changes are secondary to transepithelial bulk water flow and not to a direct effect of vasopressin on the cells, and that vasopressin induces osmotic flow by increasing water permeability of the luminal cell membrane. The lateral intercellular spaces may be part of the pathway for osmotically induced transepithelial bulk water flow.     

Quantitative Determination and Location of Newly Synthesized Virus-Specific Ribonucleic Acid in Chicken Cells Infected with Rous Sarcoma Virus

A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively labeled avian tumor virus-specific RNA in infected chicken cells has been developed. In our experiments we made use of the fact that DNA synthesized by virions of avian myeloblastosis virus in the presence of actinomycin D (AMV DNA) is complementary to at least 35% of the sequences of 70S RNA from the Schmidt-Ruppin strain (SRV) of Rous sarcoma virus. Annealing of radioactive RNA (either SRV RNA or RNA extensively purified from SRV-infected chicken cells) with AMV DNA followed by ribonuclease digestion and Sephadex chromatography yielded products which were characterized as avian tumor virus-specific RNA-DNA hybrids by hybridization competition with unlabeled 70S AMV RNA, equilibrium density-gradient centrifugation in Cs(2)SO(4) gradients, and by analysis of their ribonucleotide composition. The amount of viral RNA synthesized during pulse labeling with (3)H-uridine could be quantitated by the addition of an internal standard consisting of (32)P-labeled SRV RNA prior to purification and hybridization. This quantitative assay was used to determine that, in SRV-infected chicken cells labeled for increasing lengths of time with (3)H-uridine, labeled viral RNA appeared first in a nuclear fraction, then in a cytoplasmic fraction, and still later in mature virions. This observation is consistent with the hypothesis that RNA tumor virus RNA is synthesized in the nucleus of infected cells.     

Relationship of Adenosine 3′,5′-Monophosphate to Growth and Metabolism of Tetrahymena pyriformis

The concentration of adenosine 3',5'-monophosphate (cyclic AMP) and the activity of adenylate cyclase were determined for the first time in conjuncation with cyclic 3',5'-nucleotide phosphodiesterase (phosphodiesterase) during the growth cycle of Tetrahymena pyriformis. High levels of cyclic AMP observed during early exponential and late stationary phases were associated with elevated adenylate cyclase and decreased phosphodiesterase activities. Adenylate cyclase and cyclic AMP were decreased and phosphodiesterase was increased in cells grown in glucose-supplemented medium. In contrast to findings in mammalian liver, cyclic AMP was decreased during active gluconeogenesis in Tetrahymena. This suggests a different modulation of carbohydrate metabolism in the two species. The results illustrate that both the content of cyclic AMP and its action as a regulatory agent in Tetrahymena are uniquely suited to the metabolism of this organism.     

Lack of Nucleotide Sequence Relationship Among the Temperate Bacteriophage SPO2, Bacillus subtilis, and Virulent Bacteriophages β3 and β22

Radiolabeled phage SPO2 fragments were tested for the abiity to form interspecies deoxyribonucleic acid (DNA) duplexes with DNA from Bacillus subtilis and from phages beta3 and beta22. No reassociation, above control values, occurred between the DNA of phage SPO2 and that of its host or either of the virulent B. subtilis phages. It appears that extensive nucleotide sequence similarities between portions of host and phage DNA species are not necessary for the formation of the lysogenic state. The thermal elution profile of reassociated SPO2 DNA exhibited a normal distribution, indicating no heterogeneity in base composition. This differs from temperate enterophages whose DNA molecules contain regions of differing base composition.     

CD1a and MHC class I follow a similar endocytic recycling pathway

CD1 proteins are a family of major histocompatibility complex (MHC) class I-like antigen-presenting molecules that present lipids to T cells. The cytoplasmic tails (CTs) of all human CD1 isoforms, with the exception of CD1a, contain tyrosine-based sorting motifs, responsible for the internalization of proteins by the clathrin-mediated pathway. The role of the CD1a CT, which does not possess any sorting motifs, as well as its mode of internalization are not known. We investigated the internalization and recycling pathways followed by CD1a and the role of its CT. We found that CD1a can be internalized by a clathrin- and dynamin-independent pathway and that it follows a Rab22a- and ADP ribosylation factor (ARF)6-dependent recycling pathway, similar to other cargo internalized independent of clathrin. We also found that the CD1a CT is S-acylated. However, this posttranslational modification does not determine the rate of internalization or recycling of the protein or its localization to detergent-resistant membrane microdomains (DRMs) where we found CD1a to be enriched. We also show that plasma membrane DRMs are essential for efficient CD1a-mediated antigen presentation. These findings place CD1a closer to MHC class I in its trafficking and potential antigen-loading compartments among CD1 isoforms. Furthermore, we identify CD1a as a new marker for the clathrin- and dynamin-independent and DRM-dependent pathway of internalization as well as the Rab22a- and ARF6-dependent recycling pathway.     

Aggregation of macrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor

To examine the role of the hyaluronate receptor in cell to cell adhesion, we have employed the K-3 monoclonal antibody (MAb) which specifically binds to the hyaluronate receptor and blocks its ability to interact with hyaluronate. In the first set of experiments, we investigated the spontaneous aggregation of SV-3T3 cells, which involves two distinct mechanisms, one of which is dependent upon the presence of divalent cation and the other is independent. The divalent cation-independent aggregation was found to be completely inhibited by both intact and Fab fragments of the K-3 MAb. In contrast, the K-3 MAb had no effect on the divalent cation-dependent aggregation of cells. In a second set of experiments, we examined alveolar macrophages. The presence of hyaluronate receptors on alveolar macrophages was demonstrated by the fact that detergent extracts of these cells could bind [3H]hyaluronate, and this binding was blocked by the K-3 MAb. Immunoblot analysis of alveolar macrophages showed that the hyaluronate receptor had a Mr of 99,500, which is considerably larger than the 85,000 Mr for that on BHK cells. When hyaluronate was added to suspensions of alveolar macrophages, the cells were induced to aggregate. This effect was inhibited by the K-3 MAb, suggesting that the hyaluronate-induced aggregation was mediated by the receptor.     

Partial Purification and Characterization of Detergent-Solubilized N-Sulfotransferase Activity Associated with Calf Brain Microsomes

Calf brain 3'-phosphoadenosine 5'-phosphosulfate (PAPS):proteoheparan sulfate (PHS) N-sulfotransferase activity is solubilized by extracting salt-washed microsomes with 1% Cutscum. A protocol is described for the partial purification of the sulfotransferase activity utilizing: (1) diethylaminoethyl (DEAE)-Sephacel, (2) heparin-Sepharose CL-6B, and (3) 3',5'-ADP-agarose as chromatographic supports. Sulfotransferase activity was followed by using 3'-phosphoadenosine 5'-phospho[35S]sulfate and endogenous acceptors in heat-inactivated microsomes as exogenous substrates. Two chromatographically distinct fractions (ST1 and ST2) of sulfotransferase activity are resolved on DEAE-Sephacel. Both sulfotransferase activities have been partially purified and characterized. An apparent purification of the two N-sulfotransferase fractions of 22- to 29-fold, relative to the microsomal activity, is achieved by this procedure. Since ST1 appears to represent approximately 24% of the total microsomal activity, a purification of 89-fold has been estimated for this fraction. Neither sulfotransferase activity was stimulated by MnCl2, MgCl2, or CaCl2 added at 10 mM, nor inhibited by the presence of 10 mM EDTA. ST1 and ST2 are optimally active at pH 7.5-8. Apparent Km values for PAPS of 2.3 microM and 0.9 microM have been determined for ST1 and ST2, respectively. ST1 exhibits N-sulfotransferase activity primarily and is inhibited by phosphatidylserine whereas the ST2 fraction contains a mixture of N- and O-sulfotransferase activity and is stimulated by phosphatidylserine, phosphatidylcholine, and lysophosphatidylcholine. The detection of two chromatographically distinct sulfotransferase activities raises the possibility that N-sulfation of proteoheparan sulfates could be catalyzed by more than one enzyme, and that N-sulfation and O-sulfation of proteoglycans are catalyzed by separate enzymes in nervous tissue.     

Relaxation of selective constraints causes independent selenoprotein extinction in insect genomes

Background

Selenoproteins are a diverse family of proteins notable for the presence of the 21st amino acid, selenocysteine. Until very recently, all metazoan genomes investigated encoded selenoproteins, and these proteins had therefore been believed to be essential for animal life. Challenging this assumption, recent comparative analyses of insect genomes have revealed that some insect genomes appear to have lost selenoprotein genes.

Methodology/Principal Findings

In this paper we investigate in detail the fate of selenoproteins, and that of selenoprotein factors, in all available arthropod genomes. We use a variety of in silico comparative genomics approaches to look for known selenoprotein genes and factors involved in selenoprotein biosynthesis. We have found that five insect species have completely lost the ability to encode selenoproteins and that selenoprotein loss in these species, although so far confined to the Endopterygota infraclass, cannot be attributed to a single evolutionary event, but rather to multiple, independent events. Loss of selenoproteins and selenoprotein factors is usually coupled to the deletion of the entire no-longer functional genomic region, rather than to sequence degradation and consequent pseudogenisation. Such dynamics of gene extinction are consistent with the high rate of genome rearrangements observed in Drosophila. We have also found that, while many selenoprotein factors are concomitantly lost with the selenoproteins, others are present and conserved in all investigated genomes, irrespective of whether they code for selenoproteins or not, suggesting that they are involved in additional, non-selenoprotein related functions.

Conclusions/Significance

Selenoproteins have been independently lost in several insect species, possibly as a consequence of the relaxation in insects of the selective constraints acting across metazoans to maintain selenoproteins. The dispensability of selenoproteins in insects may be related to the fundamental differences in antioxidant defense between these animals and other metazoans.