Meconium ileus caused by mutations in GUCY2C, encoding the CFTR-activating guanylate cyclase 2C

Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage.     

Novel Evolutionary-conserved Role for the Activity-dependent Neuroprotective Protein (ADNP) Family That Is Important for Erythropoiesis

Activity-dependent neuroprotective protein (ADNP) and its homologue ADNP2 belong to a homeodomain, the zinc finger-containing protein family. ADNP is essential for mouse embryonic brain formation. ADNP2 is associated with cell survival, but its role in embryogenesis has not been evaluated. Here, we describe the use of the zebrafish model to elucidate the developmental roles of ADNP and ADNP2. Although we expected brain defects, we were astonished to discover that the knockdown zebrafish embryos were actually lacking blood and suffered from defective hemoglobin production. Evolutionary conservation was established using mouse erythroleukemia (MEL) cells, a well studied erythropoiesis model, in which silencing of ADNP or ADNP2 produced similar results as in zebrafish. Exogenous RNA encoding ADNP/ADNP2 rescued the MEL cell undifferentiated state, demonstrating phenotype specificity. Brg1, an ADNP-interacting chromatin-remodeling protein involved in erythropoiesis through regulation of the globin locus, was shown here to interact also with ADNP2. Furthermore, chromatin immunoprecipitation revealed recruitment of ADNP, similar to Brg1, to the mouse β-globin locus control region in MEL cells. This recruitment was apparently diminished upon dimethyl sulfoxide (DMSO)-induced erythrocyte differentiation compared with the nondifferentiated state. Importantly, exogenous RNA encoding ADNP/ADNP2 significantly increased β-globin expression in MEL cells in the absence of any other differentiation factors. Taken together, our results reveal an ancestral role for the ADNP protein family in maturation and differentiation of the erythroid lineage, associated with direct regulation of β-globin expression.     

Insights into physiological traits of Bifidobacterium animalis subsp. lactis BB-12 through membrane proteome analysis

Bifidobacterium animalis subsp. lactis BB-12 is a widely used probiotic strain associated with a variety of health-promoting traits. There is, however, only limited knowledge available regarding the membrane proteome and the proteins involved in oligosaccharide transport in BB-12. We applied two enrichment strategies to improve the identification of membrane proteins from BB-12 cultures grown on glucose and on xylo-oligosaccharides, the latter being an emerging prebiotic substrate recently reported to be fermented by BB-12. Our approach encompassed consecutive steps of detergent- and carbonate-treatment in order to generate inside-out membrane vesicles and to interfere with binding of membrane-associated proteins to the membrane, respectively. Proteins in the enriched membrane fraction and membrane-associated fraction were digested by lysyl endopeptidase and trypsin followed by peptide sequencing by LC-ESI-Q-TOF MS/MS. Ninety of a total of 248 identified unique proteins were predicted to possess transmembrane segments (TMSs), and 56 of these have more than one TMS. Seventy-nine of the identified proteins are annotated to be involved in transport of amino acids, oligosaccharides, inorganic ions, nucleotides, phosphate or exopolysaccharides, or to belong to the F1F0-ATP-synthetase complex and the protein translocation machinery, respectively.     

Recurrent sublethal warming reduces embryonic survival,inhibits juvenile growth,and alters species distribution projections under climate change

The capacity to tolerate climate change often varies across ontogeny in organisms with complex life cycles. Recently developed species distribution models incorporate traits across life stages; however, these life‐cycle models primarily evaluate effects of lethal change. Here, we examine impacts of recurrent sublethal warming on development and survival in ecological projections of climate change. We reared lizard embryos in the laboratory under temperature cycles that simulated contemporary conditions and warming scenarios. We also artificially warmed natural nests to mimic laboratory treatments. In both cases, recurrent sublethal warming decreased embryonic survival and hatchling sizes. Incorporating survivorship results into a mechanistic species distribution model reduced annual survival by up to 24% compared to models that did not incorporate sublethal warming. Contrary to models without sublethal effects, our model suggests that modest increases in developmental temperatures influence species ranges due to effects on survivorship.     

Photothermal control of the imposition of summer dormancy in Poabulbosa, a perennial grass geophyte

Poa bulbosa L., like many other Mediterranean geophytes, grows in the winter and enters a phase of summer dormancy in the spring. Summer dormancy enables these plants to survive the hot and dry summer. Long days are the main environmental factor active in the induction of summer dormancy in P . bulbosa and elevated temperatures accelerate dormancy development. P . bulbosa becomes dormant earlier than most other species that grow actively in the winter. Previous studies suggested that pre-exposure of P . bulbosa to short days and low temperatures during the autumn and early winter increased its sensitivity to photoperiodic induction in late winter, and thus enabled the early imposition of dormancy. To study this hypothesis, experiments were carried out under controlled photothermal conditions in the phytotron, under natural daylight extended with artificial lighting. The critical photoperiod for induction of summer dormancy at an optimal temperature (22/17°C day/night) was between 11 and 12 h. Photoperiods shorter than 12 h were noninductive, while 14- and 16-h days were fully inductive. A night break of 1 h of light given at the middle of the dark period of an 8-h photoperiod also resulted in full induction of dormancy. Pre-exposure to either low temperature (chilling at 5°C) or to short days of 8 h (SD) enhanced the inductive effect of subsequent 16-h long days (LD). The enhancing effect of chilling and SD increased with longer duration, i.e. fewer LDs were required to impose dormancy. However, the day-length during the low-temperature pretreatment had no effect on the level of induction at the following LD. Chilling followed by SD did not induce dormancy. The relevance of these responses to the development and survival of P . bulbosa in its natural habitat is discussed.     

Host Physiology and Pathogenic Variation of Cochliobolus heterostrophus Strains with Mutations in the G Protein Alpha Subunit, CGA1

Conserved eukaryotic signaling proteins participate in development and disease in plant-pathogenic fungi. Strains with mutations in CGA1, a heterotrimeric G protein G alpha subunit gene of the maize pathogen Cochliobolus heterostrophus, are defective in several developmental pathways. Conidia from CGA1 mutants germinate as abnormal, straight-growing germ tubes that form few appressoria, and the mutants are female sterile. Nevertheless, these mutants can cause normal lesions on plants, unlike other filamentous fungal plant pathogens in which functional homologues of CGA1 are required for full virulence. Δcga1 mutants of C. heterostrophus were less infective of several maize varieties under most conditions, but not all, as virulence was nearly normal on detached leaves. This difference could be related to the rapid senescence of detached leaves, since delaying senescence with cytokinin also had differential effects on the virulence of the wild type and the Δcga1 mutant. In particular, detached leaves may provide a more readily available nutrient source than attached leaves. Decreased fitness of Δcga1 as a pathogen may reflect conditions under which full virulence requires signal transduction through CGA1-mediated pathways. The virulence of these signal transduction mutants is thus affected differentially by the physiological state of the host.     

Lizards fail to plastically adjust nesting behavior or thermal tolerance as needed to buffer populations from climate warming

Although observations suggest the potential for phenotypic plasticity to allow adaptive responses to climate change, few experiments have assessed that potential. Modeling suggests that Sceloporus tristichus lizards will need increased nest depth, shade cover, or embryonic thermal tolerance to avoid reproductive failure resulting from climate change. To test for such plasticity, we experimentally examined how maternal temperatures affect nesting behavior and embryonic thermal sensitivity. The temperature regime that females experienced while gravid did not affect nesting behavior, but warmer temperatures at the time of nesting reduced nest depth. Additionally, embryos from heat‐stressed mothers displayed increased sensitivity to high‐temperature exposure. Simulations suggest that critically low temperatures, rather than high temperatures, historically limit development of our study population. Thus, the plasticity needed to buffer this population has not been under selection. Plasticity will likely fail to compensate for ongoing climate change when such change results in novel stressors.     

Taxol-induced apoptosis in human SKOV3 ovarian and MCF7 breast carcinoma cells is caspase-3 and caspase-9 independent

Taxol is used in chemotherapy regimens against breast and ovarian cancer. Treatment of tumor model cell lines with taxol induces apoptosis, but exact mechanism is not sufficiently understood. Our results demonstrate that in response to taxol, various cell types differentially utilize distinct apoptotic pathways. Using MCF7 breast carcinoma cells transfected with caspase-3 gene, we showed that taxol-induced apoptosis occurred in the absence of caspase-3 and caspase-9 activation. Similar results were obtained with ovarian SKOV3 carcinoma cells, expressing high level of endogenous caspase-3. In contrast, staurosporine-induced apoptosis in these cells was accompanied by proteolytic cleavage of pro-caspase-3 and induction of caspase-3 enzymatic activity. The effect of taxol appears to be cell type-specific, since taxol-induced apoptosis in leukemia U937 cells involved caspase-3 activation step. We conclude that a unique caspase-3 and caspase-9 independent pathway is elicited by taxol to induce apoptosis in human ovarian and breast cancinoma cells.     

Alteration of a cyclic AMP-dependent protein kinase phosphorylation site in the c-Fos protein augments its transforming potential.

We have studied the phosphorylation of the nuclear oncoprotein Fos by cyclic AMP-dependent protein kinase (PKA). We demonstrate that the human c-Fos protein, phosphorylated either in vitro with purified PKA or in vivo in JEG3 cells following treatment with forskolin, has similar phosphotryptic peptide maps. Serine 362, which constitutes part of a canonical PKA phosphorylation site (RKGSSS), is phosphorylated both in vivo and in vitro. A mutant of Fos protein in which serine residues 362 to 364 have been altered to alanine residues is not efficiently phosphorylated in vitro. Furthermore, Fos protein in which serines 362 to 364 have been altered to alanine shows increased transforming potential. We propose that phosphorylation of Fos by PKA is an important regulatory step in controlling its activity in normal cell growth and differentiation.