Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance

The recently cloned NPR1 gene of Arabidopsis thaliana is a key regulator of acquired resistance responses. Upon induction, NPR1 expression is elevated and the NPR1 protein is activated, in turn inducing expression of a battery of downstream pathogenesis-related genes. In this study, we found that NPR1 confers resistance to the pathogens Pseudomonas syringae and Peronospora parasitica in a dosage-dependent fashion. Overexpression of NPR1 leads to enhanced resistance with no obvious detrimental effect on the plants. Thus, for the first time, a single gene is shown to be a workable target for genetic engineering of nonspecific resistance in plants.

Accumulation of Salicylic Acid and 4-Hydroxybenzoic Acid in Phloem Fluids of Cucumber during Systemic Acquired Resistance Is Preceded by a Transient Increase in Phenylalanine Ammonia-Lyase Activity in Petioles and Stems1

Cucumber (Cucumis sativa) leaves infiltrated with Pseudomonas syringae pv. syringae cells produced a mobile signal for systemic acquired resistance between 3 and 6 h after inoculation. The production of a mobile signal by inoculated leaves was followed by a transient increase in phenylalanine ammonia-lyase (PAL) activity in the petioles of inoculated leaves and in stems above inoculated leaves; with peaks in activity at 9 and 12 h, respectively, after inoculation. In contrast, PAL activity in inoculated leaves continued to rise slowly for at least 18 h. No increases in PAL activity were detected in healthy leaves of inoculated plants. Two benzoic acid derivatives, salicylic acid (SA) and 4-hydroxybenzoic acid (4HBA), began to accumulate in phloem fluids at about the time PAL activity began to increase, reaching maximum concentrations 15 h after inoculation. The accumulation of SA and 4HBA in phloem fluids was unaffected by the removal of all leaves 6 h after inoculation, and seedlings excised from roots prior to inoculation still accumulated high levels of SA and 4HBA. These results suggest that SA and 4HBA are synthesized de novo in stems and petioles in response to a mobile signal from the inoculated leaf.

Obese gene expression: reduction by fasting and stimulation by insulin and glucose in lean mice, and persistent elevation in acquired (diet-induced) and genetic (yellow agouti) obesity.

Mutations in the obese (ob) gene lead to obesity. This gene has been recently cloned, but the factors regulating its expression have not been elucidated. To address the regulation of the ob gene with regard to body weight and nutritional factors, Northern blot analysis was used to assess ob mRNA in adipose tissue from mice [lean, obese due to diet, or genetically (yellow agouti) obese] under different nutritional conditions. ob mRNA was elevated in both forms of obesity, compared to lean controls, correlated with elevations in plasma insulin and body weight, but not plasma glucose. In lean C57BL/6J mice, but not in mice with diet-induced obesity, ob mRNA decreased after a 48-hr fast. Similarly, in lean C57BL/6J controls, but not in obese yellow mice, i.p. glucose injection significantly increased ob mRNA. For up to 30 min after glucose injection, ob mRNA in lean mice significantly correlated with plasma glucose, but not with plasma insulin. In a separate study with only lean mice, ob mRNA was inhibited >90% by fasting, and elevated approximately 2-fold 30 min after i.p. injection of either glucose or insulin. These results suggest that in lean animals glucose and insulin enhance ob gene expression. In contrast to our results in lean mice, in obese animals ob mRNA is elevated and relatively insensitive to nutritional state, possibly due to chronic exposure to elevated plasma insulin and/or glucose.

Null mutation of endothelin receptor type B gene in spotting lethal rats causes aganglionic megacolon and white coat color.

Mutations in the gene encoding the endothelin receptor type B (EDNRB) produce congenital aganglionic megacolon and pigment abnormalities in mice and humans. Here we report a naturally occurring null mutation of the EDNRB gene in spotting lethal (sl) rats, which exhibit aganglionic megacolon associated with white coat color. We found a 301-bp deletion spanning the exon 1-intron 1 junction of the EDNRB gene in sl rats. A restriction fragment length polymorphism caused by this deletion perfectly cosegregates with the sl phenotype. The deletion leads to production of an aberrantly spliced EDNRB mRNA that lacks the coding sequence for the first and second putative transmembrane domains of the G-protein-coupled receptor. Radioligand binding assays revealed undetectable levels of functional EDNRB in tissues from homozygous sl/sl rats. We conclude that EDNRB plays an essential role in the normal development of two neural crest-derived cell lineages, epidermal melanocytes and enteric neurons, in three mammalian species--humans, mice, and rats. The EDNRB-deficient rat may also prove valuable in defining the postnatal physiologic role of this receptor.

Studies of group B streptococcal infection in mice deficient in complement component C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity.

Group B streptococci (GBS) cause sepsis and meningitis in neonates and serious infections in adults with underlying chronic illnesses. Specific antibodies have been shown to be an important factor in protective immunity for neonates, but the role of serum complement is less well defined. To elucidate the function of the complement system in immunity to this pathogen, we have used the approach of gene targeting in embryonic stem cells to generate mice totally deficient in complement component C3. Comparison of C3-deficient mice with mice deficient in complement component C4 demonstrated that the 50% lethal dose for GBS infection was reduced by approximately 50-fold and 25-fold, respectively, compared to control mice. GBS were effectively killed in vitro by human blood leukocytes in the presence of specific antibody and C4-deficient serum but not C3-deficient serum. The defective opsonization by C3-deficient serum in vitro was corroborated by in vivo studies in which passive immunization of pregnant dams with specific antibodies conferred protection from GBS challenge to normal and C4-deficient pups but not C3-deficient pups. These results indicate that the alternative pathway is sufficient to mediate effective opsonophagocytosis and protective immunity to GBS in the presence of specific antibody. In contrast, the increased susceptibility to infection of non-immune mice deficient in either C3 or C4 implies that the classical pathway plays an essential role in host defense against GBS infection in the absence of specific immunity.

Animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors.

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is the major target for antiretroviral therapy of the acquired immunodeficiency syndrome (AIDS). While some inhibitors exhibit activity against most retroviral RTs, others are specific for the HIV-1 enzyme. To develop an animal model for the therapy of the HIV-1 infection with RT inhibitors, the RT of the simian immunodeficiency virus (SIV) was replaced by the RT of HIV-1. Macaques infected with this SIV/HIV-1 hybrid virus developed AIDS-like symptoms and pathology. The HIV-1-specific RT inhibitor LY300046.HCl, but not zidovudine [3'-azido-3'-deoxythymidine (AZT)] delayed the appearance of plasma antigenemia in macaques infected with a high dose of the chimeric virus. Infection of macaques with the chimeric virus seems to be a valuable model to study the in vivo efficacy of new RT inhibitors, the emergence and reversal of drug resistance, the therapy of infections with drug-resistant viruses, and the efficacy of combination therapy.

Signal transduction in systemic acquired resistance.

Systemic acquired resistance (SAR) is an important component of plant defense against pathogen infection. Accumulation of salicylic acid (SA) is required for the induction of SAR. However, SA is apparently not the translocated signal but is involved in transducing the signal in target tissues. Interestingly, SA accumulation is not required for production and release of the systemic signal. In addition to playing a pivotal role in SAR signal transduction, SA is important in modulating plant susceptibility to pathogen infection and genetic resistance to disease. It has been proposed that SA inhibition of catalase results in H2O2 accumulation and that therefore H2O2 serves as a second messenger in SAR signaling. We find no accumulation of H2O2 in tissues expressing SAR; thus the role of H2O2 in SAR signaling is questionable.

Methodology based on registration techniques for representing subjects and their deformations acquired from general purpose 3D sensors

In this thesis a methodology for representing 3D subjects and their deformations in adverse situations is studied. The study is focused in providing methods based on registration techniques to improve the data in situations where the sensor is working in the limit of its sensitivity. In order to do this, it is proposed two methods to overcome the problems which can difficult the process in these conditions. First a rigid registration based on model registration is presented, where the model of 3D planar markers is used. This model is estimated using a proposed method which improves its quality by taking into account prior knowledge of the marker. To study the deformations, it is proposed a framework to combine multiple spaces in a non-rigid registration technique. This proposal improves the quality of the alignment with a more robust matching process that makes use of all available input data. Moreover, this framework allows the registration of multiple spaces simultaneously providing a more general technique. Concretely, it is instantiated using colour and location in the matching process for 3D location registration.