Diversity and Functionality of Arbuscular Mycorrhizal Fungi in Three Plant Communities in Semiarid Grasslands National Park, Canada

Septate endophytes proliferating in the roots of grasslands’ plants shed doubts on the importance of arbuscular mycorrhizal (AM) symbioses in dry soils. The functionality and diversity of the AM symbioses formed in four replicates of three adjacent plant communities (agricultural, native, and restored) in Grasslands National Park, Canada were assessed in periods of moisture sufficiency and deficiency typical of early and late summer in the region. The community structure of AM fungi, as determined by polymerase chain reaction-denaturing gradient gel electrophoresis, varied with sampling time and plant community. Soil properties other than soil moisture did not change significantly with sampling time. The DNA sequences dominating AM extraradical networks in dry soil apparently belonged to rare taxa unreported in GenBank. DNA sequences of Glomus viscosum, Glomus mosseae, and Glomus hoi were dominant under conditions of moisture sufficiency. In total, nine different AM fungal sequences were found suggesting a role for the AM symbioses in semiarid areas. Significant positive linear relationships between plant P and N concentrations and active extraradical AM fungal biomass, estimated by the abundance of the phospholipid fatty acid marker 16:1ω5, existed under conditions of moisture sufficiency, but not under dry conditions. Active extraradical AM fungal biomass had significantly positive linear relationship with the abundance of two early season grasses, Agropyron cristatum (L.) Gaertn. and Koeleria gracilis Pers., but no relationship was found under dry conditions. The AM symbioses formed under conditions of moisture sufficiency typical of early summer at this location appear to be important for the nutrition of grassland plant communities, but no evidence of mutualism was found under the dry conditions of late summer.     

TMKP1 is a novel wheat stress responsive MAP kinase phosphatase localized in the nucleus

The regulation of plant signalling responses by Mitogen-Activated Protein Kinases (MAPKs)-mediated protein phosphorylation is well recognized. MAP kinase phosphatases (MKPs) are negative regulators of MAPKs in eukaryotes. We report here the identification and the characterization of TMKP1, the first wheat MKP (Triticum turgidum L. subsp. Durum). Expression profile analyses performed in two durum wheat cultivars showing a marked difference in salt and drought stress tolerance, revealed a differential regulation of TMKP1. Under salt and osmotic stress, TMKP1 is induced in the sensitive wheat variety and repressed in the tolerant one. A recombinant TMKP1 was shown to be an active phosphatase and capable to interact specifically with two wheat MAPKs (TMPK3 and TMPK6). In BY2 tobacco cells transiently expressing GFP::TMKP1, the fusion protein was localized into the nucleus. Interestingly, the deletion of the N-terminal non catalytic domain results in a strong accumulation of the truncated fusion protein in the cytoplasm. In addition, when expressed in BY2 cells, TMPK3 and TMPK6 fused to red fluorescent protein (RFP) were shown to be present predominantly in the nucleus. Surprisingly, when co-expressed with the N-terminal truncated TMKP1 fusion protein; both kinases are excluded from the nuclear compartment and accumulate in the cytoplasm. This strongly suggests that TMKP1 interacts in vivo with TMPK3 and TMPK6 and controls their subcellular localization. Taken together, our results show that the newly isolated wheat MKP might play an active role in modulating the plant cell responses to salt and osmotic stress responses.     

Deletion 17q12 is a recurrent copy number variant that confers high risk of autism and schizophrenia

Autism spectrum disorders (ASD) and schizophrenia are neurodevelopmental disorders for which recent evidence indicates an important etiologic role for rare copy number variants (CNVs) and suggests common genetic mechanisms. We performed cytogenomic array analysis in a discovery sample of patients with neurodevelopmental disorders referred for clinical testing. We detected a recurrent 1.4 Mb deletion at 17q12, which harbors HNF1B, the gene responsible for renal cysts and diabetes syndrome (RCAD), in 18/15,749 patients, including several with ASD, but 0/4,519 controls. We identified additional shared phenotypic features among nine patients available for clinical assessment, including macrocephaly, characteristic facial features, renal anomalies, and neurocognitive impairments. In a large follow-up sample, the same deletion was identified in 2/1,182 ASD/neurocognitive impairment and in 4/6,340 schizophrenia patients, but in 0/47,929 controls (corrected p = 7.37 × 10⁻⁵). These data demonstrate that deletion 17q12 is a recurrent, pathogenic CNV that confers a very high risk for ASD and schizophrenia and show that one or more of the 15 genes in the deleted interval is dosage sensitive and essential for normal brain development and function. In addition, the phenotypic features of patients with this CNV are consistent with a contiguous gene syndrome that extends beyond RCAD, which is caused by HNF1B mutations only.     

Genetic barcoding of commercial Bêche-de-mer species (Echinodermata: Holothuroidea)

There are more than 47 species of holothurians used for bêche-de-mer production, many of which are locally overfished. With three exceptions, all bêche-de-mer species are Aspidochirotida and species identification of many of these is difficult. We analysed available genetic information and newly generated sequences to determine if genetic barcoding with the mitochondrial COI gene can be used to identify bêche-de-mer species. Although genetic data were available for ～50% of bêche-de-mer species, sufficient information and within-species replication were only available for six species. We generated 96 new COI sequences extending the existing database to cover most common species. COI unambiguously identified most bêche-de-mer species providing a genetic barcode for the identification of known species. In addition, conspecific (1.3%) variation and congeneric (16.9%) divergence were well separated ('barcoding-gap') albeit with a small overlap, which may lead to some error if genetic sampling alone was applied for species discovery. In addition to identification of adults, COI sequences were useful to identify juveniles that are often morphologically different. Sequence data showed that large (deep) and small (shallow) morphotypes of Holothuria atra are the same species, but suggested potential cryptic species within this taxon. For bêche-de-mer, the COI barcode proved useful in species clarification and discovery, but further genetic and taxonomic work is essential for several species. Some bêche-de-mer clades were problematic with morphologically disparate specimens sharing the same barcode. Our study indicated the presence of undescribed species (Bohadschia sp.) and species that constitute separate species in the Indian and Pacific Ocean (e.g. Holothuria fuscogilva).     

Homoisoflavanones from Pseudoprospero firmifolium of the monotypic tribe Pseudoprospereae (Hyacinthaceae: Hyacinthoideae)

Five 3-hydroxy-type homoisoflavonoids, 3,5-dihydroxy-7,8-dimethoxy-3-(3',4'-dimethoxybenzyl)-4-chromanone, 3,5-dihydroxy-7-methoxy-3-(3',4'-dimethoxybenzyl)-4-chromanone, 3,5-dihydroxy-7,8-dimethoxy-3-(3'-hydroxy-4'-methoxybenzyl)-4-chromanone, 3,5,6-trihydroxy-7-methoxy-3-(3'-hydroxy-4'-methoxybenzyl)-4-chromanone and 3,5,7-trihydroxy-3-(3'-hydroxy-4'methoxybenzyl)-4-chromanone in addition to the nortriterpenoid, 15-deoxoeucosterol, have been isolated from the dichloromethane extract of the bulbs of Pseudoprospero firmifolium, the sole representative of the tribe Pseudoprospereae of the subfamily Hyacinthoideae of the Hyacinthaceae.     

Testosterone production in mice lacking inducible nitric oxide synthase expression is sensitive to restraint stress

Immobilization stress (IMO) induces a rapid increase in glucocorticoid secretion [in rodents, corticosterone CORT)] and this is associated with decreased circulating testosterone (T) levels. Nitric oxide (NO), a reactive free radical and neurotransmitter, has been reported to be produced at higher rates in tissues such as brain during stress. The biosynthesis of T is also known to be dramatically suppressed by NO. Specifically, the inducible isoform of nitric oxide synthase (iNOS) was directly implicated in this suppression. To assess the respective roles of CORT and NO in stress-mediated inhibition of T production, adult wild-type (WT) and inducible nitric oxide synthase knockout (iNOS(-/-)) male mice were evaluated. Animals of each genotype were assigned to either basal control or 3-h IMO groups. Basal plasma and testicular T levels were equivalent in both genotypes, whereas testicular weights of mutant mice were significantly higher compared with WT animals. Exposure to 3-h IMO increased plasma CORT and decreased T concentrations in mice of both genotypes. Testicular T levels were also affected by stress in WT and mutant males, being sharply reduced in both genotypes. However, the concentrations of nitrite and nitrate, the stable metabolites of NO measured in testicular extracts, did not differ between control and stressed WT and iNOS(-/-) mice. These results support the hypothesis that CORT, but not NO, is a plausible candidate to mediate rapid stress-induced suppression of Leydig cell steroidogenesis.     

A covalent inhibitor targeting an intermediate conformation of the fusogenic subunit of the HIV-1 envelope complex

Peptide inhibitors corresponding to sequences in the six helix bundle structure of the fusogenic portion (gp41) of the HIV envelope glycoprotein have been successfully implemented in preventing HIV entry. These peptides bind to regions in HIV gp41 transiently exposed during the fusion reaction. In an effort to improve upon these entry inhibitors, we have successfully designed and tested peptide analogs composed of chemical spacers and reactive moieties positioned strategically to facilitate covalent attachment. Using a temperature-arrested state prime wash in vitro assay we show evidence for the trapping of a pre-six helix bundle fusion intermediate by a covalent reaction with the specific anti-HIV-1 peptide. This is the first demonstration of the trapping of an intermediate conformation of a viral envelope glycoprotein during the fusion process that occurs in live cells. The permanent specific attachment of the covalent inhibitor is projected to improve the pharmacokinetics of administration in vivo and thereby improve the long-term sustainability of peptide entry inhibitor therapy and help to expand its applicability beyond salvage therapy.     

A hydrophobic patch in a charged alpha-helix is sufficient to target proteins to dense core secretory granules

Many endocrine and neuroendocrine cells contain specialized secretory organelles called dense core secretory granules. These organelles are the repository of proteins and peptides that are secreted in a regulated manner when the cell receives a physiological stimulus. The targeting of proteins to these secretory granules is crucial for the generation of certain peptide hormones, including insulin and ACTH. Although previous work has demonstrated that proteins destined to a variety of cellular locations, including secretory granules, contain targeting sequences, no single consensus sequence for secretory granule-sorting signals has emerged. We have shown previously that alpha-helical domains in the C-terminal tail of the prohormone convertase PC1/3 play an important role in the ability of this region of the protein to direct secretory granule targeting (Jutras, I. Seidah, N. G., and Reudelhuber, T. L. (2000) J. Biol. Chem. 275, 40337-40343). In this study, we show that a variety of alpha-helical domains are capable of directing a heterologous secretory protein to granules. By testing a series of synthetic alpha-helices, we also demonstrate that the presence of charged (either positive or negative) amino acids spatially segregated from a hydrophobic patch in the alpha-helices of secretory proteins likely plays a critical role in the ability of these structures to direct secretory granule sorting.     

Human immunodeficiency virus-specific CD8+ T-cell activity is detectable from birth in the majority of in utero-infected infants

Human immunodeficiency virus (HIV)-infected infants in sub-Saharan Africa typically progress to AIDS or death by 2 years of life in the absence of antiretroviral therapy. This rapid progression to HIV disease has been related to immaturity of the adaptive immune response in infants. We screened 740 infants born to HIV-infected mothers and tracked development and specificity of HIV-specific CD8⁺ T-cell responses in 63 HIV-infected infants identified using gamma interferon enzyme-linked immunospot assays and intracellular cytokine staining. Forty-four in utero-infected and 19 intrapartum-infected infants were compared to 45 chronically infected children >2 years of age. Seventy percent (14 of 20) in utero-infected infants tested within the first week of life demonstrated HIV-specific CD8⁺ T-cell responses. Gag, Pol, and Nef were the principally targeted regions in chronic pediatric infection. However, Env dominated the overall response in one-third (12/36) of the acutely infected infants, compared to only 2/45 (4%) of chronically infected children (P = 0.00083). Gag-specific CD4⁺ T-cell responses were minimal to undetectable in the first 6 months of pediatric infection. These data indicate that failure to control HIV replication in in utero-infected infants is not due to an inability to induce responses but instead suggest secondary failure of adaptive immunity in containing this infection. Moreover, the detection of virus-specific CD8⁺ T-cell responses in the first days of life in most in utero-infected infants is encouraging for HIV vaccine interventions in infants.     

NPY regulates human endocardial endothelial cell function

Growing evidence suggests that endocardial endothelial cells (EECs) may play an important role in the regulation of cardiac function by releasing several cardioactive factors such as endothelin-1 (ET-1), Angiotensin II (Ang II) and nitric oxide (NO). In our laboratory, we demonstrated that similar to ET-1, EECs do possess different types of NPY receptors, specifically Y(1) and Y(2) receptors. Furthermore, activation of these receptors was found to increase the steady-state level of intracellular free Ca(2+) in EECs and the frequency of beating of cardiomyocytes. In addition, NPY was also found to be present in EECs, and an increase of steady-state intracellular free Ca(2+) induced the release of this peptide from these cells. Thus, similar to ET-1, NPY seems to be released from EECs and this peptide seems to regulate excitation-secretion of these cells as well as excitation-contraction coupling of ventricular cardiomyocytes.