A long insertion reverts the functional effect of a substitution in acetylcholinesterase

Proteins are thought to undertake single substitutions, deletions and insertions to explore the fitness landscape. Nevertheless, the ways in which these different kind of mutations act together to alter a protein phenotype remain poorly described. We introduced incrementally the single substitution W290A and a 26 amino acid long insertion at the 297 location in the Nippostrongylus brasiliensis acetylcholinesterase B sequence and analysed in vitro the induced changes in the hydrolysis rate of three hemi-substrates: pirimicarb, paraoxon methyl and omethoate. The substitution decreased the hydrolysis rate of the three hemi-substrates. The insertion did not influence this kinetic alteration induced by the substitution for the former hemi-substrate, but reverted it for the two others. These results show that two different kinds of mutations can interact together to influence the direction of a protein's adaptative walk on the fitness landscape.     

Phosphatidylinositol 4,5-bisphosphate (PIP2) modulation of ATP and pH sensitivity in Kir channels. A tale of an active and a silent PIP2 site in the N terminus

Phosphatidylinositol polyphosphates (PIPs) are potent modulators of Kir channels. Previous studies have implicated basic residues in the C terminus of Kir6.2 channels as interaction sites for the PIPs. Here we examined the role of the N terminus and identified an arginine (Arg-54) as a major determinant for PIP(2) modulation of ATP sensitivity in K(ATP) channels. Mutation of Arg-54 to the neutral glutamine (R54Q) and, in particular, to the negatively charged glutamate (R54E) impaired PIP(2) modulation of ATP inhibition, while mutation to lysine (R54K) had no effect. These data suggest that electrostatic interactions between PIP(2) and Arg-54 are an essential step for the modulation of ATP sensitivity. This N-terminal PIP(2) site is highly conserved in Kir channels with the exception of the pH-gated channels Kir1.1, Kir4.1, and Kir5.1 that contain a neutral residue at the corresponding positions. Introduction of an arginine at this position in Kir1.1 channels rendered the N-terminal PIP(2) site functional largely increasing the PIP(2) affinity. Moreover, Kir1.1 channels lose the ability to respond to physiological changes of the intracellular pH. These results explain the need of a silent N-terminal PIP(2) site in pH-gated channels and highlight the N terminus as an important region for PIP(2) modulation of Kir channel gating.     

Growth analysis in patients with 21-hydroxylase deficiency influence of glucocorticoid dosage,age at diagnosis,phenotype and genotype on growth and height outcome

OBJECTIVE: To evaluate the impact of hydrocortisone dosage, age at diagnosis, compliance, genotype and phenotype on growth and height outcome in 21-hydroxylase-deficient patients. METHODS: We analyzed 37 patients with 21-hydroxylase deficiency (17 had completed growth, 20 still growing). Final (FH)/predicted final height (pFH) and loss of height potential related to target height (TH) were calculated and the impact of 4 hydrocortisone (HC) dosage regimens on height outcome and growth velocities was evaluated. Mean FH SDS and pFH SDS were analyzed in accordance to age at diagnosis, compliance, genotype and phenotype. RESULTS: Mean (FH SDS, pFH SDS) was -1.8+/-1.06 SD, with 35.1% of all 37 patients exhibiting short stature. Doses >20 mg/m2/day during the first year and >15 mg/m2/day during age 1-5 and at puberty resulted in significantly lower FH SDS, pFH SDS and greater height losses. Age at diagnosis, compliance, genotype and phenotype played only a minor role in growth development. CONCLUSIONS: Hydrocortisone substitution in 21-hydroxylase-deficient patients should be kept at the lowest efficient level, if possible <20 during the first year and <15 mg/m2/day until age 5 and during puberty. Normal growth and not complete androgen suppression should be aimed for.     

Identification of the domains of the fibronectin-binding protein I of Streptococcus pyogenes responsible for adjuvanticity

Intranasal administration of antigens coupled to full-length fibronectin-binding protein I (SfbI) of Streptococcus pyogenes results in the elicitation of improved humoral and cellular immune responses, at both systemic and mucosal levels. We want to evaluate if SfbI also exhibits adjuvant properties when co-administered with the antigen, as well as identify the minimal domain responsible for its adjuvanticity. To achieve this aim, mice were immunized by the intranasal route with the model antigen beta-galactosidase (beta-gal) co-administered with recombinant proteins spanning different portions of the SfbI protein. The obtained results demonstrated that the adjuvant properties of SfbI were maintained intact when admixed to the model antigen. Similar kinetics and absolute titers of beta-gal-specific IgG antibodies as well as a dominant IgG(1) isotype response pattern were observed using SfbI derivatives spanning either the aromatic and proline-rich (H10) or the fibronectin-binding (H12) domains, respectively. The use of all tested derivatives also stimulated the elicitation of efficient beta-gal-specific IgA responses in lung lavages (23-25% of the total IgA). The obtained results suggest that different sub-domains of the SfbI protein can be used as adjuvants for the development of mucosal vaccines.     

The maintenance of neuromuscular function requires UBC-25 in Caenorhabditis elegans

Caenorhabditis elegans gene ubc-25 encodes a novel type of an E2 ubiquitin transferase domain (UBCc) protein, which is highly conserved in multicellular animals, but which is not present in the genomes of fungi or plants. To identify the cellular localization of UBC-25 during the development of C. elegans, we used a ubc-25::gfp reporter gene construct. These experiments showed that ubc-25 expression starts during embryogenesis and that it is restricted to neurons and muscle cells in all later stages of development as well as in adult animals. RNA interference with ubc-25 caused late-onset paralysis of most muscular functions such as locomotion, egg laying, and defecation. We therefore propose that ubc-25 in C. elegans is required for the maintenance (homeostasis) of neuromuscular functions by contributing to a tissue specific protein modification pathway, and we speculate that the adult onset phenotype results from the accumulation of target proteins which fail to be degraded.     

A high-density genetic recombination map of sequence-tagged sites for sorghum, as a framework for comparative structural and evolutionary genomics of tropical grains and grasses

We report a genetic recombination map for Sorghum of 2512 loci spaced at average 0.4 cM ( approximately 300 kb) intervals based on 2050 RFLP probes, including 865 heterologous probes that foster comparative genomics of Saccharum (sugarcane), Zea (maize), Oryza (rice), Pennisetum (millet, buffelgrass), the Triticeae (wheat, barley, oat, rye), and Arabidopsis. Mapped loci identify 61.5% of the recombination events in this progeny set and reveal strong positive crossover interference acting across intervals of 相似文献   

Genomewide scan and fine-mapping linkage studies in four European samples with bipolar affective disorder suggest a new susceptibility locus on chromosome 1p35-p36 and provides further evidence of loci on chromosome 4q31 and 6q24

We present the findings of a large linkage study of bipolar affective disorder (BPAD) that involved genomewide analysis of 52 families (448 genotyped individuals) of Spanish, Romany, and Bulgarian descent and further fine mapping of the 1p34-p36, 4q28-q31, and 6q15-q24 regions. An additional sample of 56 German families (280 individuals) was included for this fine-mapping step. The highest nonparametric linkage scores obtained in the fine mapping were 5.49 for 4q31 and 4.87 for 6q24 in the Romany families and 3.97 for 1p35-p36 in the Spanish sample. MOD-score (LOD scores maximized over genetic model parameters) analysis provided significant evidence of linkage to 4q31 and at least borderline significance for the 1p and 6q regions. On the basis of these results and previous positive research findings, 4q31 and 6q24 should now be considered confirmed BPAD susceptibility loci, and 1p35-p36 is proposed as a new putative locus that requires confirmation in replication studies.     

Expansion and contraction of major histocompatibility complex genes: a teleostean example

The MHC is a multigene family that has arisen through recurrent expansion and contraction of genes, and a continuum of the evolutionary process is observed in the teleost fishes. The number of duplicated genes observed in different phylogenetic groups of teleost fish varies from one to 42, with only a few genes observed in the primitive euteleost species, and greater numbers of genes observed in the more advanced neoteleost species. In this study, an attempt is made to isolate all of the Mhc class I genes of an early neoteleost species, Atlantic cod (Gadus morhua L.), in the superorder Paracanthopterygii. Eighty-three sequences were isolated from the cDNA of an individual G. morhua. The level of gene duplication observed within each of the lineages and sublineages was similar, and most contained an estimated two to four duplicated genes. Mhc class I gene duplication in G. morhua was independent of, and possibly more recent than, extensive duplication in the Acanthopterygian superorder. Only limited contraction of Mhc genes is observed in G. morhua. A low level of haplotype diversity is observed, with most individuals containing at least one copy of each of the lineages tested. Divergence of the conserved N- and C-terminal residues of the antigen recognition site is observed, indicative of the initial stage of degeneration from classical to non-classical genes. However, most or all of the lineages are still polymorphic, and degeneration is present both within and among lineages. Thus, the outcome (i.e., which genes will remain classical) is as yet undetermined.     

Stable biocompatible adjuvants--a new type of adjuvant based on solid lipid nanoparticles: a study on cytotoxicity,compatibility and efficacy in chicken

A new type of adjuvant was tested for its ability to initiate antibody production in chickens, and its cellular and tissue compatibility were assessed. The stable biocompatible adjuvants tested are based on surface-modified solid lipid nanoparticles (SLNs), made from paraffin or biodegradable glycerides, and are simply admixed to the antigens before administration. The tissue-damaging potency of four formulations of the new adjuvants (H1, H2, H3 and H4) were first tested in vitro by using human foreskin fibroblasts and RAW 264.7 macrophages. The adjuvants were well tolerated by both cell types. Immunisation studies in chickens were performed by using a Mycoplasma bovis antigen and mouse immunoglobulin G (IgG). The resulting antibodies were non-invasively extracted from egg yolk. The use of the various adjuvant formulations resulted in a significant production of specific antibodies after the first and second booster immunisations. Freund's complete adjuvant (FCA), considered until now to be the "gold standard" among the adjuvants, revealed the highest antibody titre against mouse IgG. SLNs with a particle size of more than 100 nm exhibited a clear adjuvant activity, whereas SLNs with a particle size below 100 nm, in various concentrations, revealed a lower adjuvant activity. Immunisation of chickens with the mouse IgG alone, dissolved in phosphate-buffered saline, resulted in a slow antibody titre development. At the end of the experiment, the chickens were examined for vaccination-associated tissue damage. In contrast to FCA, the SLN formulations caused only minor tissue irritation at the injection sites. In conclusion, SLNs seem to be a promising alternative to FCA for antibody production in chickens, and potentially in other animals.