Neomycin: An Effective Inhibitor of Jasmonate-Induced Reactions in Plants

Jasmonates are important phytohormones involved in both plant developmental processes as well as defense reactions. Many JA-mediated plant defense responses have been studied in model plants using mutants of the jasmonate signaling pathway. However, in plant species where JA-signaling mutants are not accessible, the availability of a tool targeting JA signaling is crucial to investigate jasmonate-dependent processes. Neomycin is a poly-cationic aminoglycoside antibiotic that blocks the release of Ca²⁺ from internal stores. We examined the inhibitory activities of neomycin on different jasmonate-inducible responses in eight different plant species: Intracellular calcium measurements in Nicotiana tabacum cell culture, Sporamin gene induction in Ipomoea batatas, PDF2.2 gene expression in Triticum aestivum, Nepenthesin protease activity measurement in Nepenthes alata, extrafloral nectar production in Phaseolus lunatus, nectary formation in Populus trichocarpa, terpene accumulation in Picea abies, and secondary metabolite generation in Nicotiana attenuata. We are able to show that neomycin, an easily manageable and commercially available compound, inhibits JA-mediated responses across the plant kingdom.

     

Methylglyoxal accumulation de-regulates HoxA5 expression,thereby impairing angiogenesis in glyoxalase 1 knock-down mouse aortic endothelial cells

Impaired angiogenesis leads to long-term complications and is a major contributor of the high morbidity in patients with Diabetes Mellitus (DM). Methylglyoxal (MGO) is a glycolysis byproduct that accumulates in DM and is detoxified by the Glyoxalase 1 (Glo1). Several studies suggest that MGO contributes to vascular complications through mechanisms that remain to be elucidated. In this study we have clarified for the first time the molecular mechanism involved in the impairment of angiogenesis induced by MGO accumulation.Angiogenesis was evaluated in mouse aortic endothelial cells isolated from Glo1-knockdown mice (Glo1KD MAECs) and their wild-type littermates (WT MAECs). Reduction in Glo1 expression led to an accumulation of MGO and MGO-modified proteins and impaired angiogenesis of Glo1KD MAECs. Both mRNA and protein levels of the anti-angiogenic HoxA5 gene were increased in Glo1KD MAECs and its silencing improved both their migration and invasion. Nuclear NF-?B-p65 was increased 2.5-fold in the Glo1KD as compared to WT MAECs. Interestingly, NF-?B-p65 binding to HoxA5 promoter was also 2-fold higher in Glo1KD MAECs and positively regulated HoxA5 expression in MAECs. Consistent with these data, both the exposure to a chemical inhibitor of Glo1 “SpBrBzGSHCp2” (GI) and to exogenous MGO led to the impairment of migration and the increase of HoxA5 mRNA and NF-?B-p65 protein levels in microvascular mouse coronary endothelial cells (MCECs).This study demonstrates, for the first time, that MGO accumulation increases the antiangiogenic factor HoxA5 via NF-?B-p65, thereby impairing the angiogenic ability of endothelial cells.     

Loss of calcineurin from the medial preoptic area of primiparous rats

Western blot analyses reveal that calcineurin A (CNA), which is present in the hippocampus, basolateral amygdala, parietal cortex, and MPOA of virgin males and females, is undetectable only in the MPOA of primiparous females regardless of whether they had postpartum pup contact or not. In contrast, CNB was expressed at unchanging levels in the PC and MPOA. Similarly, G(alphao) and PKA(RI) were expressed at high levels in all of the brain regions of virgin males, virgin females, and primiparous females, supporting the concept that this loss of CNA is a specific event. Understanding how and why the expression of CNA, the sole neuronal Ca2+/CaM-dependent protein phosphatase, is down-regulated specifically in the MPOA of primiparous females may yield some insight into the signal transduction events that mediate the onset of mammalian maternal behavior.     

Human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase. A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity

The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Gel filtration studies performed on human enzyme suggested that this enzyme is monomeric in solution. However, cross-linking studies performed on both yeast and avian ATIC indicated that this enzyme might be dimeric. To determine the oligomeric state of this protein in solution, we carried out sedimentation equilibrium analysis of ATIC over a broad concentration range. We find that ATIC participates in a monomer/dimer equilibrium with a dissociation constant of 240 +/- 50 nM at 4 degrees C. To determine whether the presence of substrates affects the monomer/dimer equilibrium, further ultracentrifugation studies were performed. These showed that the equilibrium is only significantly shifted in the presence of both AICAR and a folate analog, resulting in a 10-fold reduction in the dissociation constant. The enzyme concentration dependence on each of the catalytic activities was studied in steady state kinetic experiments. These indicated that the transformylase activity requires dimerization whereas the cyclohydrolase activity only slightly prefers the dimeric form over the monomeric form.     

Structure of the cell-adhesion fragment of intimin from enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) induce gross cytoskeletal rearrangement within epithelial cells, immediately beneath the attached bacterium. The C-terminal 280 amino acid residues of intimin (Int280; 30.1 kDa), a bacterial cell-adhesion molecule, mediate the intimate bacterial host-cell interaction. Recently, interest in this process has been stimulated by the discovery that the bacterial intimin receptor protein (Tir) is translocated into the host cell membrane, phosphorylated, and after binding intimin triggers the intimate attachment. Using multidimensional nuclear magnetic resonance (NMR) and combining perdeuteration with site-specific protonation of methyl groups, we have determined the global fold of Int280. This represents one of the largest, non-oligomeric protein structures to be determined by NMR that has not been previously resolved by X-ray crystallography. Int280 comprises three domains; two immunoglobulin-like domains and a C-type lectin-like module, which define a new family of bacterial adhesion molecules. These findings also imply that carbohydrate recognition may be important in intimin-mediated cell adhesion.     

A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK⁻ and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.     

A genomic analysis of Drosophila somatic sexual differentiation and its regulation

In virtually all animals, males and females are morphologically, physiologically and behaviorally distinct. Using cDNA microarrays representing one-third of Drosophila genes to identify genes expressed sex-differentially in somatic tissues, we performed an expression analysis on adult males and females that: (1) were wild type; (2) lacked a germline; or (3) were mutant for sex-determination regulatory genes. Statistical analysis identified 63 genes sex-differentially expressed in the soma, 20 of which have been confirmed by RNA blots thus far. In situ hybridization experiments with 11 of these genes showed they were sex-differentially expressed only in internal genital organs. The nature of the products these genes encode provides insight into the molecular physiology of these reproductive tissues. Analysis of the regulation of these genes revealed that their adult expression patterns are specified by the sex hierarchy during development, and that doublesex probably functions in diverse ways to set their activities.     

The role of the renin-angiotensin system in malignant vascular injury affecting the systemic and cerebral circulations

Malignant hypertension is a rare but serious syndrome complicating 1% of essential hypertension and causing neurological, renal and cardiac complications. Despite improved anti-hypertensive medication, the incidence of this condition fails to decline. In the first part of this review, we discuss transgenic rat models of malignant hypertension, generated by over-expressing renin, to illustrate the role of the renin–angiotensin system in the development of systemic hypertensive vascular remodelling and hypertension. In the second part, we focus on the cerebrovascular response to hypertension and discuss new data using a conditional, transgenic model of malignant hypertension, the inducible hypertensive rat (IHR). Cerebral infarction associates strongly with hypertension in man and the mechanisms by which hypertension predisposes to different types of stroke remains poorly understood. Rats have similar cerebrovascular anatomy and structure to humans and as such provide a good experimental tool. To date, such models lack controllability and blood-pressure matched controls. Using the IHR, we have manipulated dietary salt and water intake to generate a novel, controllable stroke phenotype. Hypertensive small-vessel stroke develops over a predictable time period, permitting the study of developing cerebrovascular lesions. Systemic end-organ injury and hypertension are not affected. Dissociation of the systemic and central vascular responses in this way, will allow for comparative study of animals with equivalent hypertension, genetic background and systemic features of hypertension with or without stroke.