Regulation of cell behaviour by plant receptor kinases: Pattern recognition receptors as prototypical models

In this review we focus on pattern recognition receptors in plants that detect extracellular signals indicative for pathogen attack and injury. We start out with a discussion on FLS2, which binds and responds to bacterial flagellin, and then concentrate on ligand–receptor interactions as initial steps in the molecular receptor activation process. Comparison with other receptor kinases, whether involved in plant immunity or regulation of other cellular programs, might indicate common principles of receptor activation.     

Progressive changes in adherens junction structure during intestinal adenoma formation in Apc mutant mice

The C57BL/6J-Min/+ (Min/+) mouse bears a mutant Apc gene and therefore is an important in vivo model of intestinal tumorigenesis. Min/+ mice develop adenomas that exhibit loss of the wild-type Apc allele (Apc(Min/-)). Previously, we found that histologically normal enterocytes bearing a truncated Apc protein (Apc(Min/+)) migrated more slowly in vivo than enterocytes with either wild-type Apc (Apc(+/+)) or with heterozygous loss of Apc protein (Apc(1638N)). To study this phenotype further, we determined the effect of the Apc(Min) mutation upon cell-cell adhesion by examining the components of the adherens junction (AJ). We observed a reduced association between E-cadherin and beta-catenin in Apc(Min/+) enterocytes. Subcellular fractionation of proteins from Apc(+/+), Apc(Min/+), and Apc(Min/-) intestinal tissues revealed a cytoplasmic localization of intact E-cadherin only in Apc(Min/+), suggesting E-cadherin internalization in these enterocytes. beta-Catenin tyrosine phosphorylation was also increased in Apc(Min/+) enterocytes, consistent with its dissociation from E-cadherin. Furthermore, Apc(Min/+) enterocytes showed a decreased association between beta-catenin and receptor protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), and Apc(Min/-) cells demonstrated an association between beta-catenin and receptor protein-tyrosine phosphatase gamma. In contrast to the Apc(Min/+) enterocytes, Apc(Min/-) adenomas displayed increased expression and association of E-cadherin, beta-catenin, and alpha-catenin relative to Apc(+/+) controls. These data show that Apc plays a role in regulating adherens junction structure and function in the intestine. In addition, discovery of these effects in initiated but histologically normal tissue (Apc(Min/+)) defines a pre-adenoma stage of tumorigenesis in the intestinal mucosa.     

Evolutionary transition from single to multiple mating in fungus-growing ants

Queens of leafcutter ants exhibit the highest known levels of multiple mating (up to 10 mates per queen) among ants. Multiple mating may have been selected to increase genetic diversity among nestmate workers, which is hypothesized to be critical in social systems with large, long-lived colonies under severe pressure of pathogens. Advanced fungus-growing (leafcutter) ants have large numbers (104-106 workers) and long-lived colonies, whereas basal genera in the attine tribe have small (< 200 workers) colonies with probably substantially shorter lifespans. Basal attines are therefore expected to have lower queen mating frequencies, similar to those found in most other ants. We tested this prediction by analysing queen mating frequency and colony kin structure in three basal attine species: Myrmicocrypta ednaella, Apterostigma collare and Cyphomyrmex longiscapus. Microsatellite marker analyses revealed that queens in all three species were single mated, and that worker-to-worker relatedness in these basal attine species is very close to 0.75, the value expected under exclusively single mating. Fungus growing per se has therefore not selected for multiple queen mating. Instead, the advanced and highly productive social structure of the higher attine ants, which is fully dependent on the rearing of an ancient clonal fungus, may have necessitated high genetic diversity among nestmate workers. This is not the case in the lower attines, which rear fungi that were more recently derived from free-living fungal populations.     

Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis

Mitochondria are particularly vulnerable to oxidative stress, and mitochondrial swelling and vacuolization are among the earliest pathologic features found in two strains of transgenic amyotrophic lateral sclerosis (ALS) mice with SOD1 mutations. Mice with the G93A human SOD1 mutation have altered electron transport enzymes, and expression of the mutant enzyme in vitro results in a loss of mitochondrial membrane potential and elevated cytosolic calcium concentration. Mitochondrial dysfunction may lead to ATP depletion, which may contribute to cell death. If this is true, then buffering intracellular energy levels could exert neuroprotective effects. Creatine kinase and its substrates creatine and phosphocreatine constitute an intricate cellular energy buffering and transport system connecting sites of energy production (mitochondria) with sites of energy consumption, and creatine administration stabilizes the mitochondrial creatine kinase and inhibits opening of the mitochondrial transition pore. We found that oral administration of creatine produced a dose-dependent improvement in motor performance and extended survival in G93A transgenic mice, and it protected mice from loss of both motor neurons and substantia nigra neurons at 120 days of age. Creatine administration protected G93A transgenic mice from increases in biochemical indices of oxidative damage. Therefore, creatine administration may be a new therapeutic strategy for ALS.     

TROSY-based HNCO pulse sequences for the measurement of 1HN-15N, 15N-13CO, 1HN-13CO, 13CO-13Cα and 1HN-13Cα dipolar couplings in 15N, 13C, 2H-labeled proteins

HNCO-based 3D pulse schemes are presented for measuring ¹HN-¹⁵N,¹⁵N-¹³CO, ¹HN-¹³CO,¹³CO-¹³C and ¹HN-¹³C dipolar couplings in ¹⁵N,¹³C,²-labeled proteins. The experiments are based on recently developed TROSY methodology for improving spectral resolution and sensitivity. Data sets recorded on a complex of Val, Leu, Ile (1 only) methyl protonated ¹⁵N,¹³C,²H-labeled maltose binding protein and -cyclodextrin as well as ¹⁵N,¹³C,²H-labeled human carbonic anhydrase II demonstrate that precise dipolar couplings can be obtained on proteins in the 30–40 kDa molecular weight range. These couplings will serve as powerful restraints for obtaining global folds of highly deuterated proteins.     

Effects of Echinostoma caproni infections on metallic ions in the intestinal mucosa of ICR mice

Inductively coupled plasma atomic emission spectrometry (ICP-AES) was used to study metallic ions in the intestinal mucosa of ICR mice infected with Echinostoma caproni and the mucosa of uninfected control mice. Infected mucosa (n = 9 with about 100 mg wet weight per sample) were examined at 2 weeks p.i. in mice that were infected with about 25 worms per host. Uninfected mucosa (n = 9 with about 100 mg wet weight per sample) were examined in the same time frame as the infected mucosa. Five metals were measured in the mucosa by ICP-AES analysis, as follows: calcium, potassium, magnesium, sodium and zinc. There were no significant differences (Student's t-test, P > 0.05) in the concentrations of calcium, potassium or zinc in infected versus uninfected mucosa. The concentration of sodium was significantly greater (P < 0.05) in the mucosa of infected versus uninfected mucosa, but the situation was reversed in regard to magnesium.     

Expression of receptor tyrosine phosphatases during development of the retinotectal projection of the chick

Receptor tyrosine kinases and receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including axon growth and guidance. Here, we focus on the possible roles of RPTPs in the developing avian retinotectal system. Using both in situ hybridization analysis and immunohistochemistry, we show for the first time that five RPTP genes--CRYPalpha, CRYP-2, PTPmu, PTPgamma, and PTPalpha--have different but overlapping expression patterns throughout the retina and the tectum. PTPalpha is restricted to Muller glia cells and radial glia of the tectum, indicating a possible function in controlling neuronal migration. PTPgamma expression is restricted to amacrine neurons. CRYPalpha and CRYP-2 mRNAs in contrast are expressed throughout the retinal ganglion cell layer from where axons grow out to their tectal targets. PTPmu is expressed in a subset of these ganglion cells. CRYPalpha, CRYP-2, and PTPmu proteins are also localized in growth cones of retinal ganglion cell axons and are present in defined laminae of the tectum. Thus, the spatial and temporal expression of three distinct RPTP subtypes--CRYPalpha, CRYP-2, and PTPmu--are consistent with the possibility of their involvement in axon growth and guidance of the retinotectal projection.     

Noncleavable transmembrane mouse tumor necrosis factor-alpha (TNFalpha) mediates effects distinct from those of wild-type TNFalpha in vitro and in vivo

Tumor necrosis factor-alpha (TNFalpha) exists in two biologically active forms, a 26-kDa transmembrane form and a proteolytically cleaved and secreted form. We sequentially inactivated all three known cleavage sites of mouse TNFalpha by mutating the corresponding DNA sequences. A murine T cell hybridoma transfected with the nonsecretable mutant TNFalpha efficiently lysed L929 target cells in a cell contact-dependent manner and induced expression of vascular cell adhesion molecule-1 on mouse endothelioma cells. A genomic mouse TNFalpha clone encoding this mutant was subsequently introduced as a transgene into TNFalpha(-/-) lymphotoxin-alpha(-/-) mice. The 3' AU-rich regulatory elements of the TNF locus were maintained in the transgene to assure adequate gene regulation. Transmembrane TNFalpha transgenic mice were fully protected from endotoxic shock, and no TNFalpha bioactivity was detectable in the serum after stimulation with lipopolysaccharide. Activated CD4 T cells from these animals, however, lysed L929 cells in a cell contact-dependent way. After administration of lipopolysaccharide, transmembrane TNFalpha transgenic mice produced significantly higher levels of interleukin-12 than wild-type mice or TNF-deficient mice. This indicates that transmembrane TNFalpha may greatly affect the course of a cellular immune responses in vivo and exerts quantitatively and qualitatively distinct functions from secreted TNFalpha in vitro and in vivo.     

SAG, a novel zinc RING finger protein that protects cells from apoptosis induced by redox agents

SAG (sensitive to apoptosis gene) was cloned as an inducible gene by 1,10-phenanthroline (OP), a redox-sensitive compound and an apoptosis inducer. SAG encodes a novel zinc RING finger protein that consists of 113 amino acids with a calculated molecular mass of 12.6 kDa. SAG is highly conserved during evolution, with identities of 70% between human and Caenorhabditis elegans sequences and 55% between human and yeast sequences. In human tissues, SAG is ubiquitously expressed at high levels in skeletal muscles, heart, and testis. SAG is localized in both the cytoplasm and the nucleus of cells, and its gene was mapped to chromosome 3q22-24. Bacterially expressed and purified human SAG binds to zinc and copper metal ions and prevents lipid peroxidation induced by copper or a free radical generator. When overexpressed in several human cell lines, SAG protects cells from apoptosis induced by redox agents (the metal chelator OP and zinc or copper metal ions). Mechanistically, SAG appears to inhibit and/or delay metal ion-induced cytochrome c release and caspase activation. Thus, SAG is a cellular protective molecule that appears to act as an antioxidant to inhibit apoptosis induced by metal ions and reactive oxygen species.