Chemical characterization of a hormone that promotes cell arrest in g2 in complex tissues.

A G(2|)factor in the cotyledons of Pisum sativum, which arrests the growth of cells in both roots and shoots in the G(2) stage of the cell cycle, has been isolated and identified as trigonelline (N-methylnicotinic acid). To our knowledge, trigonelline is the first hormone that effects cell arrest in complex tissues of plants and animals to be chemically identified.

Furin mediates enhanced production of fibrillogenic ABri peptides in familial British dementia.

The genetic lesion underlying familial British dementia (FBD), an autosomal dominant neurodegenerative disorder, is a T-A transversion at the termination codon of the BRI gene. The mutant gene encodes BRI-L, the precursor of ABri peptides that accumulate in amyloid deposits in FBD brain. We now report that both BRI-L and its wild-type counterpart, BRI, were constitutively processed by the proprotein convertase, furin, resulting in the secretion of carboxyl-terminal peptides that encompass all or part of ABri. Elevated levels of peptides were generated from the mutant BRI precursor. Electron microscopic studies revealed that synthetic ABri peptides assembled into irregular, short fibrils. Collectively, our results support the view that enhanced furin-mediated processing of mutant BRI generates fibrillogenic peptides that initiate the pathogenesis of FBD.

At the maize/Agrobacterium interface: natural factors limiting host transformation.

BACKGROUND: Agrobacterium tumefaciens has been successfully harnessed as the only natural vector for the incorporation of foreign genes into higher plants, but its use in the grain crops is often limited. Low transformation efficiency has been partly attributed to a failure in the initial events in the transformation process, specifically in the capacity of the VirA/VirG two-component system to induce expression of the virulence genes. RESULTS: Here we show that the root exudate of Zea mays seedlings specifically inhibits virulence gene expression, determine that 2-hydroxy-4,7-dimethoxybenzoxazin-3-one (MDIBOA), which constitutes > 98% of the organic exudate of the roots of these seedlings, is the most potent and specific inhibitor of signal perception in A. tumefaciens-mediated gene transfer yet discovered, and develop a model that is able to predict the MDIBOA concentration at any distance from the root surface. Finally, variants of A. tumefaciens resistant to MDIBOA-mediated inhibition of vir gene expression have been selected and partially characterized. CONCLUSIONS: These results suggest a strategy in which a plant may resist pathogen invasion by specifically blocking virulence gene activation and yet ensure that the 'resistance factor' does not accumulate to levels sufficient to impose toxicity and selection pressure on the pathogen. The data further establish that naturally occurring inhibitors directed against signal perception by the VirA/VirG two-component regulatory system can play an important role in host defense. Finally, selected variants resistant to specific MDIBOA inhibition may now be used to extend the transformation efficiency of maize and possibly other cereals.

On becoming a parasite: evaluating the role of wall oxidases in parasitic plant development.

BACKGROUND: The temporal and spatial control of the transition from vegetative to parasitic growth is critical to any parasite, but is essential to the sessile parasitic plants. It has been proposed that this transition in Striga spp. is controlled simply by an exuded oxidase that converts host cell-surface phenols into benzoquinones which act as developmental signals that mediate the transition. An understanding of this mechanism may identify the critical molecular events that made possible the evolution of parasitism in plants. RESULTS: PoxA and PoxB are identified as the only apoplastic phenol oxidases in Striga asiatica seedlings, and the genes encoding them have been cloned and sequenced. These peroxidase enzymes are capable of oxidizing the 60 known inducing phenols into a small set of benzoquinones, and it is these quinones that induce parasitic development. Analysis of the reaction requirements and comparisons to host enzymes, however, lead us to argue that PoxA and PoxB are not necessary for host recognition. CONCLUSIONS: A new model is proposed where constitutive production of an activated oxygen species (in the case of Striga, H2O2) mediates host recognition. This strategy would allow a parasite to exploit abundant host enzymes to produce the diffusible recognition signals by converting a standard host defense into a parasitic offense.

Xenognosin sensing in virulence: is there a phenol receptor in Agrobacterium tumefaciens?

BACKGROUND: The mechanisms of signal perception and transmission in the 'two-component' autokinase transmitters/response regulators are poorly understood, especially considering the vast number of such systems now known. Virulence induction from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens represents one of the best understood systems with regard to the chemistry of the activating signal, and yet the existing data does not support a receptor-mediated perception event for the xenognostic phenols. RESULTS: Here we provide the first conclusive evidence that a specific receptor must be involved in xenognostic phenol perception, detail structural requirements of the xenognosins necessary for perception by this receptor, and develop a genetic strategy that demonstrates critical components of the phenol recognition system are not encoded on the Ti plasmid. CONCLUSIONS: Although the basic elements of the two-component system required for phenol-mediated induction of virulence gene expression are encoded on the Ti plasmid, they are dependent on the chromosomal background for even the very first stage of signal perception. This discovery suggests a curious evolutionary history, and also provides functional insight into the mechanisms of two-component signal detection and transmission in general.

Construction of an efficient expression system for Agrobacterium tumefaciens based on the coliphage T5 promoter.

A versatile expression vector utilizing a promoter of coliphage T5, P(N25) (Gentz and Bujard, 1985. J. Bacteriol. 164, 70-77) and a derivative of the IncW broad-host-range plasmid pJB20 (Beaupré et al., 1997. J. Bacteriol. 179, 78-89) has been developed. This vector successfully expresses virulence proteins of Agrobacterium tumefaciens encoded by virG and a mutant allele of virA, virA (delta1-284, G665D) in Escherichia coli as well as in A. tumefaciens. The signal transduction proteins VirA (delta1-284, G665D) and VirG are fully functional when expressed in Agrobacterium, and the P(N25) driven expression overrides the complex transcriptional regulation present with the native promoters. This expression system will enable a more detailed analysis of the activation events in signal transduction in A. tumefaciens, and we expect it to be useful in other prokaryotes.

Familial British dementia: expression and metabolism of BRI.

Vidal et al. (1999. Nature 399: 776-778) discovered that the underlying genetic lesion in familial British dementia (FBD) is a T-A transversion at the termination codon of a membrane protein, termed BRI. The mutation creates an arginine codon; translational read-through generates a novel protein, termed BRI-L, that is extended by 11 amino acids at the carboxyl-terminus. BRI-L is the precursor of the ABri peptide, a component of amyloid deposits in FBD brain. We demonstrate that both BRI and its mutant counterpart are constitutively processed by furin, resulting in the secretion of carboxyl-terminal peptide derivatives that correspond to all, or part of, ABri. Notably, elevated levels of peptides are generated from the mutant BRI precursor, suggesting that subtle conformational alterations at the carboxyl-terminus may influence furin-mediated processing. We have examined BRI/BRI-L processing by other members of the prohormone convertase (PC) family (PACE4, LPC, PC 5/6) and found that these enzymes also process BRI, albeit inefficiently. Moreover, BRI-L processing by the other PC members is severely compromised. Finally, our electron microscopic studies reveal that synthetic ABri peptides assemble into insoluble beta-pleated fibrils. Collectively, our results support the view that enhanced furin-mediated processing of mutant BRI generates amyloidogenic peptides that initiate the pathogenesis of FBD.

Source and function of keto elaidic acids from lungs of cotton workers.

A series of cytotoxic oxygenated derivatives of oleic acid, 8-oxo-9, 9-oxo-10, 10-oxo-8-, and 11-oxo-9 trans octadecenoic acid, uniquely found at post-mortem in airway cells of cotton workers, were synthesized and shown to be cytotoxic, i.e., inhibitory of growth for several cell lines, including HL-60 and U-937 promyelocytes and Eagle's KB carcinoma cells. At microM concentrations, the 8- and 11- keto acids: are chemokinetic for human neutrophils; activate production of O2-. and H2O2; stimulate promyelocytes in culture to differentiate into neutrophils; and increase diglyceride metabolism in inflammatory cells. These results indicate that these four monooxygenated fatty acids, which are found in airways of cotton workers and initiate both inflammation and differentiation in vitro, may be etiologic in the abnormal differentiation and inflammation seen in small airways of cotton workers. This abnormal differentiation in bronchi may result from altered diglyceride metabolism with resultant activation of phosphoprotein kinase C initiated by the keto fatty acids.

Biosynthesis of dehydrodiconiferyl alcohol glucosides: implications for the control of tobacco cell growth.

The dehydrodiconiferyl alcohol glucosides A and B are factors isolated from transformed Vinca rosea tumor cells that can replace the cytokinin requirement for growth of tobacco (Nicotiana tabacum) pith and callus cells in culture. These factors, present in tobacco pith cells, have their concentrations elevated approximately 2 orders of magnitude after cytokinin exposure. Biosynthesis experiments showed that these compounds are not cell wall fragments, as previously suggested, but are produced directly from coniferyl alcohol. Their synthesis is probably associated with the existing pathway for cell wall biosynthesis in both Vinca tumors and tobacco pith explants. The pathway requires only two steps, the dimerization of coniferyl alcohol by a soluble intracellular peroxidase and subsequent glycosylation. Biosynthetic experiments suggested that dehydrodiconiferyl alcohol glucoside breakdown was very slow and control of its concentration was exerted through restricted availability of coniferyl alcohol.

Reading DNA differently.

Product inhibition has provided the limiting barrier to efficient template-directed ligation and polymerization reactions. Here we review the attempts to circumvent this limitation and outline a translation strategy that does overcome the barrier and allows the information encoded in DNA to be read and amplified into backbone-modified oligonucleotides.

The haustorium and the chemistry of host recognition in parasitic angiosperms.

Two parasitic angiosperms,Agalinis purpurea (Scrophulariaceae) andStriga asiatica (Scrophulariaceae), are compared as to the chemical recognition events involved in host selection.Agalinis is a hemiparasite which can mature to seed-set without a host, whereasStriga is a holoparasite and survives for only a very limited time without a host. Both parasites, however, attach to a host through a specialized organ known as the haustorium and regulate the development of this organ through the recognition of chemical factors from host plants. We now describe the discovery of 2,6-dimethoxy-p-benzoquinone (2,6-DMBQ) as an haustoria-inducing principle fromSorghum root extracts. Our investigation of this compound has led us to suggest that one level of host recognition in these parasitic plants is mediated through their enzymatic digestion of the host root surface. Degradation of surface components liberates quinonoid compounds, such as 2,6-DMBQ, which in turn trigger haustorial development.

Template-directed ligation: from DNA towards different versatile templates.

A systematic approach evaluating template-directed ligation reactions has now resulted in a simple outline for a two-stage replication cycle. This cycle builds on an efficient method for reading the information encoded in DNA into an amplified translation product. It is further demonstrated that the translation product strand is capable of catalyzing the synthesis of the original DNA strand. We propose that this cycle represents just one of many possible solutions; other chemical ligation or polymerization reactions could be accommodated with different templates. In that context, a new template, derived by modest changes to the DNA backbone, has been developed and has been shown to hybridize under reaction conditions different than those accessible to DNA. Therefore, the conceptual groundwork has been laid for extending this approach to encoding and reading stored information in molecules other than the natural biopolymers at the densities found in biology.

Review: model peptides and the physicochemical approach to beta-amyloids.

beta-Amyloid peptides are the main protein components of neuritic plaques and may be important in the pathogenesis of Alzheimer's Disease. The determination of the structure of beta-amyloid fibrils poses a challenge because of the limited solubility of beta-amyloid peptides and the noncrystalline nature of fibrils formed from these peptides. In this paper, we describe several physicochemical approaches which have been used to examine fibrils and the fibrillogenesis of peptide models of beta-amyloid. Recent advances in solid state NMR, such as the DRAWS pulse sequence, have made this approach a particularly attractive one for peptides such as beta-amyloid, which are not yet amenable to high-resolution solution phase NMR and crystallography. The application of solid state NMR techniques has yielded information on a model peptide comprising residues 10-35 of human beta-amyloid and indicates that in fibrils, this peptide assumes a parallel beta-strand conformation, with all residues in exact register. In addition, we discuss the use of block copolymers of Abeta peptides and polyethylene glycol as probes for the pathways of fibrillogenesis. These methods can be combined with other new methods, such as high-resolution synchrotron X-ray diffraction and small angle neutron and X-ray scattering, to yield structural data of relevance not only to disease, but to the broader question of protein folding and self-assembly.

Metabolic thermotolerance: magnetic resonance detected protection of glutamate synthase.

Metabolism in maize meristem cultures exposed to different heat treatments has been analyzed by 13C-NMR spectroscopy of tissue extracts. The effects of a 40 degrees C permissive stress were compared with a 45 degrees C lethal stress, and the metabolism of glutamate and glutamine were markedly altered by both temperatures. Changes in the incorporation of labeled precursors, alterations due to the in vivo application of enzyme inhibitors, and differences in the activity of enzymes in cell free extracts have confirmed that glutamate synthase (GluS) is partially inactivated by the lethal thermal exposure. This enzyme is quantitatively protected by the induction of thermotolerance. The time dependence for the protection correlates with the appearance of a set of late-arising heat shock proteins (hsps). The function of these late-arising proteins is not yet known, but only one of them, a 67-kDa protein, is spatially correlated with GluS protection. Therefore, the quantitative protection of a key metabolic enzyme has been correlated with the in vivo function of a specific hsp.

Control of Germination in Striga asiatica: Chemistry of Spatial Definition.

Striga asiatica (Scrophulariaceae), a member of a heterogeneous group known as the parasitic plants, is totally dependent on host root attachment for survival. In agar, Striga seeds germinated in high percentages within 5 millimeters of a sorghum (Sorghum bicolor (L.) Moench) host root surface, and no germination was observed at distances greater than 1 centimeter. This spatially restricted germination may be explained by the chemistry of a single compound, 2-hydroxy-5-methoxy-3-[8'Z, 11'Z)-8', 11', 14' -pentadecatriene]-p-hydroquinone, structure 1, which is exuded by sorghum roots. The presence of the compound was chemically imaged with pigments such as methylene blue. The use of methylene blue suggested that structure 1 was exuded along the entire surface of the root for long periods. This exudation and the inherent instability of structure 1 together establish an apparent steady state concentration gradient of the germination stimulant around the sorghum root. The Striga seed must be exposed to micromolar concentrations of 1 for >/=5 hours before high germination percentages were observed. Such a requirement for a long term exposure to a steady state concentration of an inherently labile, exuded compound would provide an extra degree of resolution to signal detection and host commitment in Striga parasitism.

Vegetative/Parasitic transition: control and plasticity in striga development.

Striga asiatica (Scrophulariaceae), an obligate parasite of grasses including many of the world's major grain crops, switches from vegetative to parasitic development by the differentiation of the root meristem into the host attachment organ, the haustorium. This change was induced in culture by the exposure to a single, low molecular weight signal molecule, 2,6-dimethoxy-p-benzo-quinone. A concentration of 10(-6) molar quinone and an exposure time of >/=6 hours were required before the developmental process could be completed. With shorter exposure times, haustorial development was prematurely aborted and meristematic elongation was reestablished. The new meristem was capable of developing a second haustorium if reexposed to the signal molecule. These results are discussed in terms of the transition to the parasitic phase and the general control of plant cellular development.

Expansin message regulation in parasitic angiosperms: marking time in development.

Parasitic strategies are widely distributed across the angiosperms and are estimated to have evolved at least eight different times. Within the obligate hemiparasitic and holoparasitic members, elaborate strategies for host selection have emerged. Here, we demonstrate that in the parasitic Scrophulariceae Striga asiatica, for which signal-mediated host detection is critical, expansin mRNA provides a reliable and accurate downstream molecular marker for the transition to the parasitic mode. Three different expansin genes, saExp1, saExp2, and saExp3, are regulated by xenognostic quinones. saExp3 appears to function as a seedling expansin, and its mRNA is depleted within minutes after induction of the host attachment organ. saExp1 and saExp2 share less homology with the known expansins, and their transcripts accumulate linearly over a critical induction period. The regulation of these genes suggests that the resources for developmental commitment must accumulate to a defined threshold before commitment to organogenesis is terminal. When the induction signal is removed prematurely, the accumulated message decays with a time constant that correlates with the time required for additional signal exposures to reinduce parasitic development. These results suggest that sophisticated controls exist for the accumulation of the necessary components for terminal commitment to the parasitic mode. Furthermore, building on the redox dependence of the inducing signal, they suggest a model akin to a "molecular capacitor" for clocking organogenesis in S. asiatica.

Strategies in pathogenesis: mechanistic specificity in the detection of generic signals.

The virulence genes of the plant pathogen Agrobacterium tumefaciens are induced by more than 40 low-molecular-weight phenolic compounds. The prevailing opinion is that (i) wound-derived phenols produced on breach of the integrity of the cell wall act as the initiating signal in a series of events which results in host cell transformation, and (ii) a classical membrane receptor, putatively VirA, is responsible for the recognition of all such phenolic inducers. Here, we argue that the discovery of the subset of inducers that are relatives of the dehydrodiconiferyl alcohol glucoside (DCG) growth factors redirects our attention to work on the plant wound as a site of cell division, and suggests that we further explore the implications of early work on the relationship between transformation efficiency and the status of the cell cycle of the host. In addition, we argue that the significant structural diversity allowed in the para position of the phenol ring of inducers suggests that a receptor-ligand interaction based solely on structural recognition is insufficient, but that recognition followed by a specific proton transfer event may be sufficient to explain vir induction activity. Hence, the specificity of the response of A. tumefaciens may be a consequence of the features required for a chemical reaction to occur on the receptor surface. Finally, we review affinity labelling studies which exploit this phenol detection mechanism and which provide evidence that the phenol receptor may be other than VirA, the sensory kinase of the two component regulatory system implicated in Agrobacterium virulence.(ABSTRACT TRUNCATED AT 250 WORDS)

The structural characterization of endogenous factors from Vinca rosea crown gall tumors that promote cell division of tobacco cells.

The ability of two compounds, a cytokinin and an auxin, to stimulate tobacco cell growth and differentiation has been known for greater than 30 years, but the molecular mechanism of this activation is still unknown. Previous reports of factors endogenous in crown gall tumors of Vinca rosea that could replace the cytokinin requirement in tobacco cell culture has motivated an investigation of these tissues. The optimization of a reverse-phase isolation scheme has led to the purification of sufficient material to allow for the identification of six different related compounds. The structures of two of these compounds have been assigned as a set of epimeric dehydrodiconiferyl alcohol beta-D-glycosides. The structure of these compounds suggests that they would most likely be derived from the plant cell wall.