Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco

The active defense of plants against pathogens often includes rapid and localized cell death known as hypersensitive response (HR). Protein phosphorylation and dephosphorylation are implicated in this event based on studies using protein kinase and phosphatase inhibitors. Recent transient gain-of-function studies demonstrated that the activation of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco mitogen-activated protein kinases (MAPKs) by their upstream MAPK kinase (MAPKK), NtMEK2 leads to HR-like cell death. Here, we report that the conserved kinase interaction motif (KIM) in MAPKKs is required for NtMEK2 function. Mutation of the conserved basic amino acids in this motif, or the deletion of N-terminal 64 amino acids containing this motif significantly compromised or abolished the ability of NtMEK2DD to activate SIPK/WIPK in vivo. These mutants were also defective in interacting with SIPK and WIPK, suggesting protein-protein interaction is required for the functional integrity of this MAPK cascade. To eliminate Agrobacterium that is known to activate a number of defense responses in transient transformation experiments, we generated permanent transgenic plants. Induction of NtMEK2DD expression by dexamethasone induced HR-like cell death in both T1 and T2 plants. In addition, by using PVX-induced gene silencing, we demonstrated that the suppression of all three known components in the NtMEK2-SIPK/WIPK pathway attenuated N gene-mediated TMV resistance. Together with previous report that SIPK and WIPK are activated by TMV in a gene-for-gene-dependent manner, we conclude that NtMEK2-SIPK/WIPK pathway plays a positive role in N gene-mediated resistance, possibly through regulating HR cell death.     

Structural features of piperazinyl-linked ciprofloxacin dimers required for activity against drug-resistant strains of Staphylococcus aureus

We previously demonstrated that piperazinyl-linked fluoroquinolone dimers possess potent antibacterial activity against drug-resistant strains of Staphylococcus aureus. In this study, we report the preparation and evaluation of a series of incomplete dimers toward ascertaining structural features of piperazinyl-linked ciprofloxacin dimers that render these agents refractory to fluoroquinolone-resistance mechanisms in Staphylococcus aureus.     

Modulation of cyclooxygenase-2 expression by phosphatidylcholine specific phospholipase C and D in macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) enhances the expression of cyclooxygenase 2 (COX-2) in macrophages, and stimulates production of prostaglandins that cause endothelial dysfunction in septic shock. In an effort to identify strategies for reducing LPS-inducible expression of COX-2, inhibitors of the phospholipases involved in LPS dependent over-expression of COX-2 were studied. LPS enhances expression of COX-2 mRNA and protein by activating sequentially phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC) and phosphatidylcholine-specific phospholipase D (PC-PLD). This stimulates production of phosphatidic acid (PA), which increases expression of COX-2 mRNA and protein. Inhibition of PC-PLC by D609 (tricyclodecanoyl xanthogenate), and of PC-PLD activity by 1-butanol, reduced LPS-dependent over-production of PA and suppressed the increase of COX-2 mRNA and protein. Activation of PKC, normally seen in LPS-treated cells, was mimicked with phorbol myristic acid (PMA), and this also increased PA production and enhanced COX-2 expression. Propranolol inhibition of phosphatidic acid phosphohydrolase (PPH) increased PA accumulation and enhanced LPS-dependent COX-2 protein synthesis. These results suggest that inhibitors of PC-PLC, PKC and PC-PLD, or activators of PPH could be useful in the management of LPS-induced overproduction of prostaglandins and of vascular dysfunction in septic shock.     

Down-regulation of acetate pathway through antisense strategy in Escherichia coli: improved foreign protein production

A problem with the use of Escherichia coli to produce foreign proteins is that although endogenously produced acetate is physiologically indispensable, it inhibits protein expression. Here we firstly employed an antisense RNA strategy as an elaborate metabolic engineering tool to partially block biosynthesis of two major acetate pathway enzymes, phosphotransacetylase (PTA) and acetate kinase (ACK). Three recombinant plasmids containing antisense genes targeting either or both of pta and ackA were constructed, and their effects on the acetate pathway and foreign protein productivity compared to control plasmid without any antisense genes were determined in E. coli BL21. Green fluorescent protein (GFP) was employed as a model foreign protein, and timing of antisense expression was controlled by using the intrinsic ackA promoter. We found that the antisense method partially reduced mRNA levels of target enzyme genes and, over time, lowered the concentration of acetate in culture media in all antisense-regulated strains. Notably, total production of GFP was enhanced 1.6- to 2.1-fold in antisense-regulated strains, even though the degree of acetate reduction was not significantly large. It was revealed that the acetate pathway has more critical roles in cellular physiology than expected in the previous reports. When the scale of culture was increased, enhancement of protein production became larger, demonstrating that this antisense strategy can be successfully applied to practical large-scale protein production processes.     

Heterospecific Expression of the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Cell Shape Determination Genes <Emphasis Type="Italic">mreBCD</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>*

The divIVB operon of Bacillus subtilis includes the cell shape-associated mre genes, including the membrane-associated proteins MreC and MreD. TnphoA mutagenesis was utilized to analyze a topological model for MreC. MreC has a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large carboxy terminal domain which lies externally to the outer leaflet of the cell membrane. Expression of the B. subtilis MreB protein, or the Mre C and D proteins, results in a morphological conversion of the Escherichia coli host cells from a rod to a roughly spherical cell, morphologically similar to mre-negative mutants of E. coli. Immunolocalization of the MreC protein in B. subtilis revealed that this protein is found at the midcell division site of the bacterial cells, consistent with the postulated role of the Mre proteins in the regulation of septum-specific peptidoglycan synthesis. RID= ID= <E5>Correspondence to: </E5>G.C. Stewart; <E5>email:</E5> stewart&commat;vet.ksu.edu Received: 5 August 2002 / Accepted: 7 October 2002     

Renal vascular responses to static handgrip: role of muscle mechanoreflex

During exercise, the sympathetic nervous system is activated, which causes vasoconstriction. The autonomic mechanisms responsible for this vasoconstriction vary based on the particular tissue being studied. Attempts to examine reflex control of the human renal circulation have been difficult because of technical limitations. In this report, the Doppler technique was used to examine renal flow velocity during four muscle contraction paradigms in conscious humans. Flow velocity was divided by mean arterial blood pressure to yield an index of renal vascular resistance (RVR). Fatiguing static handgrip (40% of maximal voluntary contraction) increased RVR by 76%. During posthandgrip circulatory arrest, RVR remained above baseline (2.1 +/- 0.2 vs. 2.8 +/- 0.2 arbitrary units; P < 0.017) but was only 40% of the end-grip RVR value. Voluntary biceps contraction increased RVR within 10 s of initiation of contraction. This effect was not associated with an increase in blood pressure. Finally, involuntary biceps contraction also raised RVR. We conclude that muscle contraction evokes renal vasoconstriction in conscious humans. The characteristic of this response is consistent with a primary role for mechanically sensitive afferents. This statement is based on the small posthandgrip circulatory arrest response and the vasoconstriction that was observed with involuntary biceps contraction.     

New method for the determination of anti-lactoferrin activity of microorganisms

A new method for the determination of the antilactoferrin activity (ALfA) of microorganisms, based on the detection of lactoferrin by the enzyme immunoassay, is proposed. The new method widens the spectrum of microorganisms to be tested, makes it possible to detect lactoferrin inactivation by bacteria producing antagonistically active substances (muramidases, organic acids, hydrogen peroxide, etc.), increases the reliability of the determination of the ALfA antilactoferrin activity of microorganisms due to the increased accuracy of its quantitative characterization. Testing of the culture fluid for lactoferrin following the growth of microorganisms in a medium with this protein revealed the capacity to inactivate lactoferrin in bacteria (Escherichia coli, Salmonella sp.) and yeast-like fungi (Candida sp.), isolated from feces and clinical material, of persons with carrier states, dysbiotic disturbances and pyoinflammatory diseases.     

Characterization of salicylic acid-induced genes in Chinese cabbage

Salicylic acid is a messenger molecule in the activation of defense responses in plants. In this study, we isolated four cDNA clones representing salicylic acid-induced genes in Chinese cabbage (Brassica rapa subsp. pekinensis) by subtractive hybridization. Of the four clones, the BC5-2 clone encodes a putative glucosyltransferase protein. The BC5-3 clone is highly similar to an Arabidopsis gene encoding a putative metal-binding farnesylated protein. The BC6-1 clone is a chitinase gene with similarities to a rapeseed class IV chitinase. Class IV chitinases have deletions in the chitin-binding and catalytic domains and the BC6-1 chitinase has an additional deletion in the catalytic domain. The BCP8-1 clone is most homologous to an Arabidopsis gene that contains a tandem array of two thiJ-like sequences. These four cabbage genes were barely expressed in healthy leaves, but were strongly induced by salicylic acid and benzothiadiazole. Expression of the three genes represented by the BC5-2, BC5-3 and BCP8-1 clones were also induced by Pseudomonas syringae pv. tomato, a nonhost pathogen that elicits a hypersensitive response in Chinese cabbage. None of these four genes, however, was strongly induced by methyl jasmonate or by ethylene.     

Inhibition of tryptophan hydroxylase activity and decreased 5-HT1A receptor binding in a mouse model of Huntington's disease

The pathogenic mechanisms of the mutant huntingtin protein that cause Huntington's disease (HD) are unknown. Previous studies have reported significant decreases in the levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the brains of the R6/2 transgenic mouse model of HD. In an attempt to elucidate the cause of these neurochemical perturbations in HD, the protein levels and enzymatic activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, were determined. Enzyme activity was measured in brainstem homogenates from 4-, 8-, and 12-week-old R6/2 mice and compared with aged-matched wild-type control mice. We observed a 62% decrease in brainstem TPH activity (p = 0.009) in 4-week-old R6/2 mice, well before the onset of behavioral symptoms. In addition, significant decreases in TPH activity were also observed at 8 and 12 weeks of age (61%, p = 0.02 and 86%, p = 0.005, respectively). In the 12-week-old-mice, no change in immunoreactive TPH was observed. In vitro binding showed that TPH does not bind to exon 1 of huntingtin in a polyglutamine-dependent manner. Specifically, glutathione-S-transferase huntingtin exon 1 proteins with 20, 32 or 53 polyglutamines did not interact with radiolabeled tryptophan hydroxylase. Therefore, the inhibition of TPH activity does not appear to result from a direct huntingtin/TPH interaction. Receptor binding analyses for the 5-HT1A receptor in 12-week-old R6/2 mice revealed significant reductions in 8-OH-[3H]DPAT binding in several hippocampal and cortical regions. These results demonstrate that the serotonergic system in the R6/2 mice is severely disrupted in both presymptomatic and symptomatic mice. The presymptomatic inhibition of TPH activity in the R6/2 mice may help explain the functional consequences of HD and provide insights into new targets for pharmacotherapy.