Residue Gln4863 within a predicted transmembrane sequence of the Ca2+ release channel (ryanodine receptor) is critical for ryanodine interaction

Despite the pivotal role of ryanodine in ryanodine receptor (RyR) research, the molecular basis of ryanodine-RyR interaction remains largely undefined. We investigated the role of the proposed transmembrane helix TM10 in ryanodine interaction and channel function. Each amino acid residue within the TM10 sequence, 4844IIFDITFFFFVIVILLAIIQGLII4867, of the mouse RyR2 was mutated to either alanine or glycine. Mutants were expressed in human embryonic kidney 293 cells, and their properties were assessed. Mutations D4847A, F4850A, F4851A, L4858A, L4859A, and I4866A severely curtailed the release of intracellular Ca2+ in human embryonic kidney 293 cells in response to extracellular caffeine and diminished [3H]ryanodine binding to cell lysates. Mutations F4846A, T4849A, I4855A, V4856A, and Q4863A eliminated or markedly reduced [3H]ryanodine binding, but cells expressing these mutants responded to extracellular caffeine by releasing stored Ca2+. Interestingly these two groups of mutants, each with similar properties, are largely located on opposite sides of the predicted TM10 helix. Single channel analyses revealed that mutation Q4863A dramatically altered the kinetics and apparent affinity of ryanodine interaction with single RyR2 channels and abolished the effect of ryanodol, an analogue of ryanodine, whereas the single channel conductance of the Q4863A mutant and its responses to caffeine, ATP, and Mg2+ were comparable to those of the wild type channels. Furthermore the effect of ryanodine on single Q4863A mutant channels was influenced by the transmembrane holding potential. Together these results suggest that the TM10 sequence and in particular the Q4863 residue constitute an important determinant of ryanodine interaction.     

The origin and evolution of Eragrostis tef (Poaceae) and related polyploids: evidence from nuclear waxy and plastid rps16

Tef (Eragrostis tef; Poaceae) is an allotetraploid (2n = 4x = 40) cereal crop whose origin within the large genus Eragrostis is unknown. Previous studies have suggested a total of 14 wild Eragrostis species as potential progenitors. Phylogenetic analysis of sequence data from the nuclear gene waxy and the plastid locus rps16 strongly supports the widely held hypothesis of a close relationship between tef and E. pilosa, a wild allotetraploid. Eragrostis heteromera, another previously proposed progenitor, is shown by the waxy data to be a close relative of one of the tef genomes. Other putative progenitors included in the taxon sample are not supported as closely related to tef. Plastid sequences from five varieties of tef and four E. pilosa accessions are identical and therefore are uninformative with respect to the question of multiple origins of these polyploids. The waxy phylogeny also resolves the relationships among other allopolyploids, supporting a close relationship between the morphologically similar allotetraploids E. macilenta, E. minor, and E. mexicana. Eragrostis cilianensis, another morphologically similar allopolyploid, appears to have shared one diploid progenitor with these species but derived its other genome from an unrelated diploid.     

Autosomal dominant stapes ankylosis with broad thumbs and toes,hyperopia, and skeletal anomalies is caused by heterozygous nonsense and frameshift mutations in NOG,the gene encoding noggin

Although fixation of the stapes is usually progressive and secondary to otosclerosis, it may present congenitally, with other skeletal manifestations, as an autosomal dominant syndrome-such as proximal symphalangism (SYM1) or multiple-synostoses syndrome (SYNS1), both of which are caused by mutations in NOG, the gene encoding noggin. We describe a family that was ascertained to have nonsyndromic otosclerosis but was subsequently found to have a congenital stapes ankylosis syndrome that included hyperopia, a hemicylindrical nose, broad thumbs and great toes, and other minor skeletal anomalies but lacked symphalangism. A heterozygous nonsense NOG mutation-c.328C-->T (Q110X), predicted to truncate the latter half of the protein-was identified, and a heterozygous insertion in NOG-c.252-253insC, in which the frameshift is predicted to result in 96 novel amino acids before premature truncation-was identified in a previously described second family with a similar phenotype. In contrast to most NOG mutations that have been reported in kindreds with SYM1 and SYNS1, the mutations observed in these families with stapes ankylosis without symphalangism are predicted to disrupt the cysteine-rich C-terminal domain. These clinical and molecular findings suggest that (1) a broader range of conductive hearing-loss phenotypes are associated with NOG mutations than had previously been recognized, (2) patients with sporadic or familial nonsyndromic otosclerosis should be evaluated for mild features of this syndrome, and (3) NOG alterations should be considered in conductive hearing loss with subtle clinical and skeletal features, even in the absence of symphalangism.     

Internal transcribed spacer repeat-specific primers and the analysis of hybridization in the Glycine tomentella (Leguminosae) polyploid complex

Polyploid and diploid hybridization is a ubiquitous and evolutionarily important phenomenon in the plant world. Determining the parental species of a hybrid, however, is difficult. Molecular markers such as the nuclear ribosomal DNA gene complex, particularly its internal transcribed spacer (ITS) region, have proved powerful in determining hybrid parentage. In some cases, population and genomic phenomena, such as genetic drift and concerted evolution, result in the loss of all or many of the tandemly repeated copies derived from one parental species, making the recovery of hybrid history difficult or impossible. Methods such as direct sequencing and cloning are typically used to find ITS sequences contributed from parental species, but are limited in their ability to detect rare repeat types. Here we report that repeat-specific polymerase chain reaction primers can recover rare parental ITS sequences in the Glycine tomentella polyploid complex. In three allopolyploid lineages of this complex, repeat-specific primers reliably detected rare repeats that both direct sequencing and the screening of many cloned sequences failed to detect. Other strategies, such as the use of exclusion primers, may detect rare parental repeat types in hybrids when previous hypotheses regarding the second parental species are lacking.     

A decaepitope polypeptide primes for multiple CD8+ IFN-gamma and Th lymphocyte responses: evaluation of multiepitope polypeptides as a mode for vaccine delivery

Proteins are generally regarded as ineffective immunogens for CTL responses. We synthesized a 100-mer decaepitope polypeptide and tested its capacity to induce multiple CD8(+) IFN-gamma and Th lymphocyte (HTL) responses in HLA transgenic mice. Following a single immunization in the absence of adjuvant, significant IFN-gamma in vitro recall responses were detected for all epitopes included in the construct (six A2.1-, three A11-restricted CTL epitopes, and one universal HTL epitope). Immunization with truncated forms of the decaepitope polypeptide was used to demonstrate that optimal immunogenicity was associated with a size of at least 30-40 residues (3-4 epitopes). Solubility analyses of the truncated constructs were used to identify a correlation between immunogenicity for IFN-gamma responses and the propensity of these constructs to form particulate aggregates. Although the decaepitope polypeptide and a pool of epitopes emulsified in IFA elicited similar levels of CD8(+) responses using fresh splenocytes, we found that the decaepitope polypeptide more effectively primed for in vitro recall CD8(+) T cell responses. Finally, immunogenicity comparisons were also made between the decaepitope polypeptide and a corresponding gene encoding the same polypeptide delivered by naked DNA immunization. Although naked DNA immunization induced somewhat greater direct ex vivo and in vitro recall responses 2 wk after a single immunization, only the polypeptide induced significant in vitro recall responses 6 wk following the priming immunization. These studies support further evaluation of multiepitope polypeptide vaccines for induction of CD8(+) IFN-gamma and HTL responses.     

Production of granulocyte colony-stimulating factor in the nonspecific acute phase response enhances host resistance to bacterial infection

Mice mounting an acute phase response, induced by sterile inflammation after a single s.c. injection of casein 24 h beforehand, were remarkably protected against lethal infection with Gram-positive or Gram-negative bacteria. This was associated with enhanced early clearance of bacteremia, greater phagocytosis and oxidative burst responses by neutrophils, and enhanced recruitment of neutrophils into tissues compared with control, nonacute phase mice. Casein-induced inflammation was also associated with increased concentrations of G-CSF in serum, and administration of neutralizing Ab to this cytokine completely abrogated protection against Escherichia coli infection after casein pretreatment. Injection of recombinant murine G-CSF between 3 and 24 h before infection conferred the same protection as casein injection. In contrast, the casein-induced acute phase response affected neither serum values of TNF-alpha, IL-1 beta, or IL-6 after E. coli infection nor susceptibility to LPS toxicity. Furthermore, protection against infection was unaffected in IL-1R knockout mice, which have deficient acute phase plasma protein responses, or after nonspecific inhibition of acute phase protein synthesis by D-galactosamine or specific depletion of complement C3 by cobra venom factor. Increased production of G-CSF in the acute phase response is thus a key physiological component of host defense, and pretreatment with G-CSF to prevent bacterial infection in at-risk patients now merits further study, especially in view of increasing bacterial resistance to antibiotics.