Wnt5a induces ROR1 to associate with 14-3-3ζ for enhanced chemotaxis and proliferation of chronic lymphocytic leukemia cells.

Wnt5a can activate Rho GTPases in chronic lymphocytic leukemia (CLL) cells by inducing the recruitment of ARHGEF2 to ROR1. Mass spectrometry on immune precipitates of Wnt5a-activated ROR1 identified 14-3-3ζ, which was confirmed by co-immunoprecipitation. The capacity of Wnt5a to induce ROR1 to complex with 14-3-3ζ could be blocked in CLL cells by treatment with cirmtuzumab, a humanized mAb targeting ROR1. Silencing 14-3-3ζ via small interfering RNA impaired the capacity of Wnt5a to: (1) induce recruitment of ARHGEF2 to ROR1, (2) enhance in vitro exchange activity of ARHGEF2 and (3) induce activation of RhoA and Rac1 in CLL cells. Furthermore, CRISPR/Cas9 deletion of 14-3-3ζ in ROR1-negative CLL cell-line MEC1, and in MEC1 cells transfected to express ROR1 (MEC1-ROR1), demonstrated that 14-3-3ζ was necessary for the growth/engraftment advantage of MEC1-ROR1 over MEC1 cells. We identified a binding motif (RSPS857SAS) in ROR1 for 14-3-3ζ. Site-directed mutagenesis of ROR1 demonstrated that serine-857 was required for the recruitment of 14-3-3ζ and ARHGEF2 to ROR1, and activation of RhoA and Rac1. Collectively, this study reveals that 14-3-3ζ plays a critical role in Wnt5a/ROR1 signaling, leading to enhanced CLL migration and proliferation.

Wnt5a induces ROR1 to complex with HS1 to enhance migration of chronic lymphocytic leukemia cells.

ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL). We found that ROR1 associates with hematopoietic-lineage-cell-specific protein 1 (HS1) in freshly isolated CLL cells or in CLL cells cultured with exogenous Wnt5a. Wnt5a also induced HS1 tyrosine phosphorylation, recruitment of ARHGEF1, activation of RhoA and enhanced chemokine-directed migration; such effects could be inhibited by cirmtuzumab, a humanized anti-ROR1 mAb. We generated truncated forms of ROR1 and found its extracellular cysteine-rich domain or kringle domain was necessary for Wnt5a-induced HS1 phosphorylation. Moreover, the cytoplamic, and more specifically the proline-rich domain (PRD), of ROR1 was required for it to associate with HS1 and allow for F-actin polymerization in response to Wnt5a. Accordingly, we introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1 PRD at positions 784, 808, 826, 841 or 850 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1P→︀A mutants, ROR1P(841)A had impaired capacity to recruit HS1 and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate HS1 or activate ARHGEF1, and was unable to enhance CLL-cell motility. Collectively, these studies indicate HS1 plays an important role in ROR1-dependent Wnt5a-enhanced chemokine-directed leukemia-cell migration.

Proteomic divergence in Arabidopsis autopolyploids and allopolyploids and their progenitors.

Autopolyploidy and allopolyploidy are common in many plants and some animals. Rapid changes in genomic composition and gene expression have been observed in both autopolyploids and allopolyploids, but the effects of polyploidy on proteomic divergence are poorly understood. Here, we report quantitative analysis of protein changes in leaves of Arabidopsis autopolyploids and allotetraploids and their progenitors using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry. In more than 1000 proteins analyzed, the levels of protein divergence were relatively high (~18%) between Arabidopsis thaliana and Arabidopsis arenosa, relatively low (~6.8%) between an A. thaliana diploid and autotetraploid and intermediate (~8.3 and 8.2%) in F(1)- and F(8)-resynthesized allotetraploids relative to mid-parent values, respectively. This pattern of proteomic divergence was consistent with the previously reported gene expression data. In particular, many non-additively accumulated proteins (61-62%) in the F(1) and F(8) allotetraploids were also differentially expressed between the parents. The differentially accumulated proteins in functional categories of abiotic and biotic stresses were overrepresented between an A. thaliana autotetraploid and diploid and between two Arabidopsis species, but not significantly different between allotetraploids and their progenitors. Although the trend of changes is similar, the percentage of differentially accumulated proteins that matched previously reported differentially expressed genes was relatively low. Western blot analysis confirmed several selected proteins with isoforms the cumulative levels of which were differentially expressed. These data suggest high protein divergence between species and rapid changes in post-transcriptional regulation and translational modifications of proteins during polyploidization.

A porphyrin pathway impairment is responsible for the phenotype of a dominant disease lesion mimic mutant of maize.

The maize lesion mimic gene Les22 is defined by dominant mutations and characterized by the production of minute necrotic spots on leaves in a developmentally specified and light-dependent manner. Phenotypically, Les22 lesions resemble those that are triggered during a hypersensitive disease resistance response of plants to pathogens. We have cloned Les22 by using a Mutator-tagging technique. It encodes uroporphyrinogen decarboxylase (UROD), a key enzyme in the biosynthetic pathway of chlorophyll and heme in plants. Urod mutations in humans are also dominant and cause the metabolic disorder porphyria, which manifests itself as light-induced skin morbidity resulting from an excessive accumulation of photoexcitable uroporphyrin. The phenotypic and genetic similarities between porphyria and Les22 along with our observation that Les22 is also associated with an accumulation of uroporphyrin revealed what appears to be a case of natural porphyria in plants.

Regulation of leaf initiation by the terminal ear 1 gene of maize.

Higher plants elaborate much of their architecture post-embryonically through development initiated at the tips of shoots. During vegetative growth, leaf primordia arise at predictable sites to give characteristic leaf arrangements, or phyllotaxies. How these sites are determined is a long-standing question that bears on the nature of pattern-formation mechanisms in plants. Fate-mapping studies in several species indicate that each leaf primordium becomes organized from a group of 100-200 cells on the flank of the shoot apex. Although molecular studies indicate that the regulated expression of specific homeobox genes plays some part in this determination process, mechanisms that regulate the timing and position of leaf initiation are less well understood. Here we describe a gene from maize, terminal ear 1. Patterns of expression of this gene in the shoot and phenotypes of mutants indicate a role for terminal ear 1 in regulating leaf initiation. The tel gene product contains conserved RNA-binding motifs, indicating that it may function through an RNA-binding activity.

Plant-pathogen microevolution: molecular basis for the origin of a fungal disease in maize.

A new and severe disease of maize caused by a previously unknown fungal pathogen, Cochliobolus carbonum race 1, was first described in 1938. The molecular events that led to the sudden appearance of this disease are described in this paper. Resistance to C. carbonum race 1 was found to be widespread in maize and is conferred by a pair of unlinked duplicate genes, Hm1 and Hm2. Here, we demonstrate that resistance is the wild-type condition in maize. Two events, a transposon insertion in Hm1 and a deletion in Hm2, led to the loss of resistance, resulting in the origin of a new disease. None of the other plant species tested is susceptible to C. carbonum race 1, and they all possess candidate genes with high homology to Hm1 and Hm2. In sorghum and rice, these homologs map to two chromosomal regions that are syntenic with the maize Hm1 and Hm2 loci, indicating that they are related to the maize genes by vertical descent. These results suggest that the Hm-encoded resistance is of ancient origin and probably is conserved in all grasses.

Plant genomics: more than food for thought.

In all but the poorest countries of South Asia and Africa, the supply and quality of food will rise to meet the demand. Biotechnology, accelerated by genomics, will create wealth for both producers and consumers by reducing the cost and increasing the quality of food. Famine and malnutrition in the poorest countries may be alleviated by applying genomics or other tools of biotechnology to improving subsistence crops. The role of the public sector and the impact of patent law both could be great, but government policies on these issues are still unclear.

Analysis of a chemical plant defense mechanism in grasses.

In the Gramineae, the cyclic hydroxamic acids 2,4-dihydroxy-1, 4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1, 4-benzoxazin-3-one (DIMBOA) form part of the defense against insects and microbial pathogens. Five genes, Bx1 through Bx5, are required for DIBOA biosynthesis in maize. The functions of these five genes, clustered on chromosome 4, were demonstrated in vitro. Bx1 encodes a tryptophan synthase alpha homolog that catalyzes the formation of indole for the production of secondary metabolites rather than tryptophan, thereby defining the branch point from primary to secondary metabolism. Bx2 through Bx5 encode cytochrome P450-dependent monooxygenases that catalyze four consecutive hydroxylations and one ring expansion to form the highly oxidized DIBOA.

A novel suppressor of cell death in plants encoded by the Lls1 gene of maize.

The Lls1 (lethal leaf spot1) locus of maize is defined by a recessive mutation characterized by the initiation, in a developmentally programmed manner, of necrotic lesions that expand to kill leaves cell autonomously. The loss-of-function nature of all Lls1 mutants implies that the Lls1 gene is required to limit the spread of cell death in mature leaves. We have cloned the Lls1 gene by tagging with Mutator, a transposable element system in maize, and we show that it encodes a novel protein highly conserved in plants. Two consensus binding motifs of aromatic ring-hydroxylating dioxygenases are present in the predicted LLS1 protein, suggesting that it may function to degrade a phenolic mediator of cell death.

Diversification of C-function activity in maize flower development.

The Arabidopsis gene AGAMOUS is required for male and female reproductive organ development and for floral determinacy. Reverse genetics allowed the isolation of a transposon-induced mutation in ZAG1, the maize homolog of AGAMOUS. ZAG1 mutants exhibited a loss of determinacy, but the identity of reproductive organs was largely unaffected. This suggested a redundancy in maize sex organ specification that led to the identification and cloning of a second AGAMOUS homolog, ZMM2, that has a pattern of expression distinct from that of ZAG1. C-function organ identity in maize (as defined by the A, B, C model of floral organ development) may therefore be orchestrated by two closely related genes, ZAG1 and ZMM2, with overlapping but nonidentical activities.

The efficacy of conjugated linoleic acid in mammary cancer prevention is independent of the level or type of fat in the diet.

The objective of the present study was to investigate whether the anticarcinogenic activity of conjugated linoleic acid (CLA) is affected by the amount and composition of dietary fat consumed by the host. Because the anticancer agent of interest is a fatty acid, this approach may provide some insight into its mechanism of action, depending on the outcome of these fat feeding experiments. For the fat level experiment, a custom formulated fat blend was used that simulates the fatty acid composition of the US diet. This fat blend was present at 10, 13.3, 16.7 or 20% by weight in the diet. For the fat type experiment, a 20% (w/w) fat diet containing either corn oil (exclusively) or lard (predominantly) was used. Mammary cancer prevention by CLA was evaluated using the rat dimethylbenz[a]anthracene model. The results indicated that the magnitude of tumor inhibition by 1% CLA was not influenced by the level or type of fat in the diet. It should be noted that these fat diets varied markedly in their content of linoleate. Fatty acid analysis showed that CLA was incorporated predominantly in mammary tissue neutral lipids, while the increase in CLA in mammary tissue phospholipids was minimal. Furthermore, there was no evidence that CLA supplementation perturbed the distribution of linoleate or other fatty acids in the phospholipid fraction. Collectively these carcinogenesis and biochemical data suggest that the cancer preventive activity of CLA is unlikely to be mediated by interference with the metabolic cascade involved in converting linoleic acid to eicosanoids. The hypothesis that CLA might act as an antioxidant was also examined. Treatment with CLA resulted in lower levels of mammary tissue malondialdehyde (an end product of lipid peroxidation), but failed to change the levels of 8-hydroxydeoxyguanosine (a marker of oxidatively damaged DNA). Thus while CLA may have some antioxidant function in vivo in suppressing lipid peroxidation, its anticarcinogenic activity cannot be accounted for by protecting the target cell DNA against oxidative damage. The finding that the inhibitory effect of CLA maximized at 1% (regardless of the availability. of linoleate in the diet) could conceivably point to a limiting step in the capacity to metabolize CLA to some active product(s) which is essential for cancer prevention.

Modification of a Specific Class of Plasmodesmata and Loss of Sucrose Export Ability in the sucrose export defective1 Maize Mutant.

We report on the export capability and structural and ultrastructural characteristics of leaves of the sucrose export defective1 (sed1; formerly called sut1) maize mutant. Whole-leaf autoradiography was combined with light and transmission electron microscopy to correlate leaf structure with differences in export capacity in both wild-type and sed1 plants. Tips of sed1 blades had abnormal accumulations of starch and anthocyanin and distorted vascular tissues in the minor veins, and they did not export sucrose. Bases of sed1 blades were structurally identical to those of the wild type and did export sucrose. Electron microscopy revealed that only the plasmodesmata at the bundle sheath-vascular parenchyma cell interface in sed1 minor veins were structurally modified. Aberrant plasmodesmal structure at this critical interface results in a symplastic interruption and a lack of phloem-loading capability. These results clarify the pathway followed by photosynthates, the pivotal role of the plasmodesmata at the bundle sheath-vascular parenchyma cell interface, and the role of the vascular parenchyma cells in phloem loading.

Plant disease resistance. Grand unification theory in sight.

Molecular characterization of the components of signalling pathways that mediate disease resistance is at last providing a unified picture of how plants fight disease.

Cloning and characterization of the maize An1 gene.

The Anther ear1 (An1) gene product is involved in the synthesis of ent-kaurene, the first tetracyclic intermediate in the gibberellin (GA) biosynthetic pathway. Mutations causing the loss of An1 function result in a GA-responsive phenotype that includes reduced plant height, delayed maturity, and development of perfect flowers on normally pistillate ears. The an1::Mu2-891339 allele was recovered from a Mutator (Mu) F2 family. Using Mu elements as molecular probes, an An1-containing restriction fragment was identified and cloned. The identity of the cloned gene as An1 was confirmed by using a reverse genetics screen for maize families that contain a Mu element inserted into the cloned gene and then by demonstrating that the insertion causes an an1 phenotype. The predicted amino acid sequence of the An1 cDNA shares homology with plant cyclases and contains a basic N-terminal sequence that may target the An1 gene product to the chloroplast. The sequence is consistent with the predicted subcellular localization of AN1 in the chloroplast and with its biochemical role in the cyclization of geranylgeranyl pyrophosphate, a 20-carbon isoprenoid, to ent-kaurene. The semidwarfed stature of an1 mutants is in contrast with the more severely dwarfed stature of GA-responsive mutants at other loci in maize and may be caused by redundancy in this step of the GA biosynthetic pathway. DNA gel blot analysis indicated that An1 is a single-copy gene that lies entirely within the deletion of the an1-bz2-6923 mutant. However, homozygous deletion mutants accumulated ent-kaurene to 20% of the wild-type level, suggesting that the function of An1 is supplemented by an additional activity.

Effect of excess dietary iron on the promotion stage of 1-methyl-1-nitrosourea-induced mammary carcinogenesis: pathogenetic characteristics and distribution of iron.

The effect of feeding a 10-fold excess of dietary iron on the promotion stage of MNU-induced mammary carcinogenesis was investigated. Rats fed excess iron in the diet had more mammary carcinomas than rats fed the recommended level of iron. A significantly greater proportion of carcinomas in rats fed the excess iron diet had the normal Ha-ras gene rather than the mutated form (G-->A transition mutation in codon 12). In non-tumor bearing rats, mammary epithelial cells in lobules were the primary site of iron accumulation. However, in mammary carcinomas, a shift in the distribution of iron from the epithelial cells to the stroma was noted. Iron was predominantly found in tumor stroma; malignant epithelial cells failed to accumulate comparable levels of iron. These observations indicate that in the presence of excess iron there is an increase in the number of mammary carcinomas that do not bear the mutant Ha-ras gene. Whether changes in the distribution of iron within the mammary gland contribute to the altered pathogenetic characteristics of these tumors is being investigated.

Genetic patterns of plant host-parasite interactions.

A parasite's ability to infect and a host's ability to resist infection can be heritable traits. Patterns of inheritance suggest how host genes interact with parasite genes to determine whether or not infection occurs. Recent progress in the isolation and characterization of these genes in plants sheds new light on parasitism.

Antioxidant status and dietary lipid unsaturation modulate oxidative DNA damage.

High performance liquid chromatography with electrochemical detection (HPLC-EC) was used to measure 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker for oxidative DNA damage, in mammary gland isolated from tumor-bearing and tumor-free rats fed diets of varied fatty acid composition and vitamin E and selenium content. A method for tissue preparation and analysis is reported and a significant positive correlation shown between degree of unsaturation of dietary fatty acids and 8-OHdG concentration, regardless of antioxidant status. The increase in 8-OHdG concentration with greater fatty acid unsaturation was more pronounced in the absence of adequate dietary vitamin E and selenium. The implications of these data for defining the role of dietary lipid in the process of mammary carcinogenesis are discussed.

Reductase activity encoded by the HM1 disease resistance gene in maize.

The HM1 gene in maize controls both race-specific resistance to the fungus Cochliobolus carbonum race 1 and expression of the NADPH (reduced form of nicotinamide adenine dinucleotide phosphate)-dependent HC toxin reductase (HCTR), which inactivates HC toxin, a cyclic tetrapeptide produced by the fungus to permit infection. Several HM1 alleles were generated and cloned by transposon-induced mutagenesis. The sequence of wild-type HM1 shares homology with dihydroflavonol-4-reductase genes from maize, petunia, and snap-dragon. Sequence homology is greatest in the beta alpha beta-dinucleotide binding fold that is conserved among NADPH- and NADH (reduced form of nicotinamide adenine dinucleotide)-dependent reductases and dehydrogenases. This indicates that HM1 encodes HCTR.