The Pto kinase mediates a signaling pathway leading to the oxidative burst in tomato

The Pto gene encodes a serine/threonine kinase that confers resistance in tomato to Pseudomonas syringae pv. tomato strains that express the avirulence gene avrPto. Partial characterization of the Pto signal transduction pathway and the availability of transgenic tomato lines (± Pto) make this an ideal system for exploring the molecular basis of disease resistance. In this paper, we test two transgenic tomato cell suspension cultures (±Pto) for production of H2O2 following independent challenge with two strains of P. syringae pv. tomato (±avrPto). Only when Pto and avrPto are present in the corresponding organisms are two distinct phases of the oxidative burst seen, a rapid first burst followed by a slower and more prolonged second burst. In the remaining three plant–pathogen interactions, we observe either no burst or only a first burst, indicating that the second burst is correlated with disease resistance. Further support for this observation comes from the finding that both resistant and susceptible tomato lines produce the critical second oxidative burst when challenged with P. syringae pv. tabaci, a nonhost pathogen that elicits a hypersensitive response on both tomato lines. The Pto kinase is not required, however, for the oxidative burst initiated by non-specific elicitors such as oligogalacturonides or osmotic stress. A model describing a possible role for the Pto kinase in the overall scheme of oxidative burst signaling is proposed.

The maturity-onset diabetes of the young (MODY1) transcription factor HNF4α regulates expression of genes required for glucose transport and metabolism

Hepatocyte nuclear factor 4α (HNF4α) plays a critical role in regulating the expression of many genes essential for normal functioning of liver, gut, kidney, and pancreatic islets. A nonsense mutation (Q268X) in exon 7 of the HNF4α gene is responsible for an autosomal dominant, early-onset form of non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young; gene named MODY1). Although this mutation is predicted to delete 187 C-terminal amino acids of the HNF4α protein the molecular mechanism by which it causes diabetes is unknown. To address this, we first studied the functional properties of the MODY1 mutant protein. We show that it has lost its transcriptional transactivation activity, fails to dimerize and bind DNA, implying that the MODY1 phenotype is because of a loss of HNF4α function. The effect of loss of function on HNF4α target gene expression was investigated further in embryonic stem cells, which are amenable to genetic manipulation and can be induced to form visceral endoderm. Because the visceral endoderm shares many properties with the liver and pancreatic β-cells, including expression of genes for glucose transport and metabolism, it offers an ideal system to investigate HNF4-dependent gene regulation in glucose homeostasis. By exploiting this system we have identified several genes encoding components of the glucose-dependent insulin secretion pathway whose expression is dependent upon HNF4α. These include glucose transporter 2, and the glycolytic enzymes aldolase B and glyceraldehyde-3-phosphate dehydrogenase, and liver pyruvate kinase. In addition we have found that expression of the fatty acid binding proteins and cellular retinol binding protein also are down-regulated in the absence of HNF4α. These data provide direct evidence that HNF4α is critical for regulating glucose transport and glycolysis and in doing so is crucial for maintaining glucose homeostasis.

Detection and determination of oligonucleotide triplex formation-mediated transcription-coupled DNA repair in HeLa nuclear extracts

Transcription-coupled repair (TCR) plays an important role in removing DNA damage from actively transcribed genes. It has been speculated that TCR is the most important mechanism for repairing DNA damage in non-dividing cells such as neurons. Therefore, abnormal TCR may contribute to the development of many age-related and neurodegenerative diseases. However, the molecular mechanism of TCR is not well understood. Oligonucleotide DNA triplex formation provides an ideal system to dissect the molecular mechanism of TCR since triplexes can be formed in a sequence-specific manner to inhibit transcription of target genes. We have recently studied the molecular mechanism of triplex-forming oligonucleotide (TFO)-mediated TCR in HeLa nuclear extracts. Using plasmid constructs we demonstrate that the level of TFO-mediated DNA repair activity is directly correlated with the level of transcription of the plasmid in HeLa nuclear extracts. TFO-mediated DNA repair activity was further linked with transcription since the presence of rNTPs in the reaction was essential for AG30-mediated DNA repair activity in HeLa nuclear extracts. The involvement of individual components, including TFIID, TFIIH, RNA polymerase II and xeroderma pigmentosum group A (XPA), in the triplex-mediated TCR process was demonstrated in HeLa nuclear extracts using immunodepletion assays. Importantly, our studies also demonstrated that XPC, a component involved in global genome DNA repair, is involved in the AG30-mediated DNA repair process. The results obtained in this study provide an important new understanding of the molecular mechanisms involved in the TCR process in mammalian cells.

Lamprey Dlx genes and early vertebrate evolution

Gnathostome vertebrates have multiple members of the Dlx family of transcription factors that are expressed during the development of several tissues considered to be vertebrate synapomorphies, including the forebrain, cranial neural crest, placodes, and pharyngeal arches. The Dlx gene family thus presents an ideal system in which to examine the relationship between gene duplication and morphological innovation during vertebrate evolution. Toward this end, we have cloned Dlx genes from the lamprey Petromyzon marinus, an agnathan vertebrate that occupies a critical phylogenetic position between cephalochordates and gnathostomes. We have identified four Dlx genes in P. marinus, whose orthology with gnathostome Dlx genes provides a model for how this gene family evolved in the vertebrate lineage. Differential expression of these lamprey Dlx genes in the forebrain, cranial neural crest, pharyngeal arches, and sensory placodes of lamprey embryos provides insight into the developmental evolution of these structures as well as a model of regulatory evolution after Dlx gene duplication events.

Direct observation of the enhancement of noncooperative protein self-assembly by macromolecular crowding: Indefinite linear self-association of bacterial cell division protein FtsZ

Recent measurements of sedimentation equilibrium and sedimentation velocity have shown that the bacterial cell division protein FtsZ self-associates to form indefinitely long rod-like linear aggregates in the presence of GDP and Mg2+. In the present study, the newly developed technique of non-ideal tracer sedimentation equilibrium was used to measure the effect of high concentrations—up to 150 g/liter—of each of two inert “crowder” proteins, cyanmethemoglobin or BSA, on the thermodynamic activity and state of association of dilute FtsZ under conditions inhibiting (−Mg2+) and promoting (+Mg2+) FtsZ self-association. Analysis of equilibrium gradients of both FtsZ and crowder proteins indicates that, under the conditions of the present experiment, FtsZ interacts with each of the two crowder proteins essentially entirely via steric repulsion, which may be accounted for quantitatively by a simple model in which hemoglobin, albumin, and monomeric FtsZ are modeled as effective spherical hard particles, and each oligomeric species of FtsZ is modeled as an effective hard spherocylinder. The functional dependence of the sedimentation of FtsZ on the concentrations of FtsZ and either crowder indicates that, in the presence of high concentrations of crowder, both the weight-average degree of FtsZ self-association and the range of FtsZ oligomer sizes present in significant abundance are increased substantially.

Neuronal death in the central nervous system demonstrates a non-fibrin substrate for plasmin

Mice deficient for plasminogen exhibit a variety of pathologies, all of which examined to date are reversed when the animals are also made fibrin(ogen) deficient. These results suggested that the predominant, and perhaps exclusive, physiological role of plasminogen is clearance of fibrin. Plasminogen-deficient mice also display resistance to excitotoxin-induced neurodegeneration, in contrast with wild-type mice, which are sensitive. Based on the genetic interaction between plasminogen and fibrinogen, we investigated whether resistance to neuronal cell death in the plasminogen-deficient mice is dependent on fibrin(ogen). Unexpectedly, mice lacking both plasminogen and fibrinogen are resistant to neurodegeneration to levels comparable to plasminogen-deficient mice. Therefore, plasmin acts on substrates other than fibrin during experimental neuronal degeneration, and may function similarly in other pathological settings in the central nervous system.