Ligand-induced association of the type I interferon receptor components.

Two transmembrane polypeptides, IFNAR and IFN-alpha/Beta R, were previously identified as essential components of the type I interferon (IFN) receptor, but their interrelationship and role in ligand binding were not clear. To study these issues, we stably expressed and characterized the two polypeptides in host murine cells. In human cells, native IFN-alpha/beta R is a 102-kDa protein but upon reduction only a 51-kDa protein is detected. In host murine cells human IFN-alpha/beta R was expressed as a 51-kDa protein. Host cells expressing IFN-alpha/beta R bound IFN-alpha 2 with a high affinity (Kd of 3.6 nM), whereas cells expressing IFNAR exhibited no ligand binding. Upon coexpression of IFNAR and the 51-kDa IFN-alpha/beta R, the affinity for IFN-alpha 2 was increased 10-fold, approaching that of the native receptor. We show by cross-linking that both the cloned (51-kDa) and native (102-kDa) IFN-alpha/beta R bind IFN-alpha 2 to form an intermediate product, while IFNAR associates with this product to form a ternary complex. Hence, IFNAR and IFN-alpha/beta R are components of a common type I IFN receptor, cooperating in ligand binding. Ligand-induced association of IFNAR and IFN-alpha/beta R probably triggers transmembrane signaling.     

Phenology and polyploidy in annual Brachypodium species (Poaceae) along the aridity gradient in Israel

Local adaptation of plants along environmental gradients provides strong evidence for clinal evolution mediated by natural selection. Plants have developed diverse strategies to mitigate stress, for example, drought escape is a phenological strategy to avoid drought stress, while polyploidy was proposed as a genomic adaptation to stress. Polyploidy as an adaptation to aridity (an environmental parameter integrating temperature and precipitation) was previously documented in annual Brachypodium spp. (Poaceae) in the Western Mediterranean. Here, we examined whether polyploidy or phenology are associated with aridity in annual Brachypodium spp. along the aridity gradient in the Eastern Mediterranean. Using flow cytometry, we determined ploidy levels of plants from natural populations along the Israeli gradient, spanning ∼424 km from mesic Mediterranean to extreme desert climates. In a common garden we recorded time of seedling emergence, flowering and senescence. We tested whether the proportion of allotetraploids in the populations and phenological traits were associated with aridity. Contrary to a previous study in the Western Mediterranean, we found no effect of aridity on the proportion of allotetraploids and diploids within populations. Interestingly, phenology was associated with aridity: time of emergence was later, while flowering and senescence were earlier in desert plants. Our results indicate that in the Eastern Mediterranean, adaptation of Brachypodium to aridity is mediated mainly by phenology, rather than ploidy level. Therefore, we suggest that genome duplication is not the main driver of adaptation to environmental stress; rather, phenological change as a drought escape mechanism may be the major adaptation.     

ALFY-Controlled DVL3 Autophagy Regulates Wnt Signaling,Determining Human Brain Size

Primary microcephaly is a congenital neurodevelopmental disorder of reduced head circumference and brain volume, with fewer neurons in the cortex of the developing brain due to premature transition between symmetrical and asymmetrical cellular division of the neuronal stem cell layer during neurogenesis. We now show through linkage analysis and whole exome sequencing, that a dominant mutation in ALFY, encoding an autophagy scaffold protein, causes human primary microcephaly. We demonstrate the dominant effect of the mutation in drosophila: transgenic flies harboring the human mutant allele display small brain volume, recapitulating the disease phenotype. Moreover, eye-specific expression of human mutant ALFY causes rough eye phenotype. In molecular terms, we demonstrate that normally ALFY attenuates the canonical Wnt signaling pathway via autophagy-dependent removal specifically of aggregates of DVL3 and not of Dvl1 or Dvl2. Thus, autophagic attenuation of Wnt signaling through removal of Dvl3 aggregates by ALFY acts in determining human brain size.     

Intercellular transfer of oncogenic H-Ras at the immunological synapse

Immune cells establish dynamic adhesive cell-cell interactions at a specific contact region, termed the immunological synapse (IS). Intriguing features of the IS are the formation of regions of plasma membrane fusion and the intercellular exchange of membrane fragments between the conjugated cells. It is not known whether upon IS formation, intact intracellular proteins can transfer from target cells to lymphocytes to allow the transmission of signals across cell boundaries. Here we show by both FACS and confocal microscopy that human lymphocytes acquire from the cells they scan the inner-membrane protein H-Ras, a G-protein vital for common lymphocyte functions and a prominent participant in human cancer. The transfer was cell contact-dependent and occurred in the context of cell-conjugate formation. Moreover, the acquisition of oncogenic H-RasG12V by natural killer (NK) and T lymphocytes had important biological functions in the adopting lymphocytes: the transferred H-RasG12V induced ERK phosphorylation, increased interferon-gamma and tumor necrosis factor-alpha secretion, enhanced lymphocyte proliferation, and augmented NK-mediated target cell killing. Our findings reveal a novel mode of cell-to-cell communication-allowing lymphocytes to extend the confines of their own proteome-which may moreover play an important role in natural tumor immunity.     

A Survey of Heavy Metal Levels in Eels (Anguilla anguilla) from some Rivers in East Anglia,England: the Use of Eels as Pollution Indicators

A survey was made of mercury, cadmium and lead in the liver and flesh of 517 eels from eleven rivers in East Anglia. Eels from some sites showed high metal levels, indicating sources of metal contamination. Eel liver is a suitable indicator of inorganic pollution and the flesh is a suitable indicator of organic pollution.     

Electrostatic attraction by surface charge does not contribute to the catalytic efficiency of acetylcholinesterase.

Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.     

Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains.

An extended PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to lepidopteran, coleopteran, and dipteran pests (Ben-Dov et al., Appl. Environ. Microbiol. 63:4883-4890, 1997). To optimize identification of all reported cry genes, this methodology needs a complete PCR set of primers. In the study reported here, a set of universal (Un9) and specific primers for multiplex rapid screening for all four known genes from the cry9 group was designed. PCR analyses were performed for cry9 genes on 16 standard strains and 215 field isolates of B. thuringiensis. Among the standard strains, only B. thuringiensis subsp. aizawai HD-133, which harbors cry1 and cry2 genes, was positive with Un9 but negative to all four specific primers for cry9 genes. DNA of 22 field-collected isolates was also found to be positive with Un9. These isolates were classified into three cry9 profiles using specific primers; all of them harbor cry1 and cry2. This newly designed set of primers complements the existing PCR methodology for most currently known cry genes.     

The Erwinia chrysanthemi type III secretion system is required for multicellular behavior

Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a sigma54-dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36 degrees C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.