Samita R. Patel

Phytochrome E Influences Internode Elongation and Flowering Time in Arabidopsis

From a screen of M2 seedlings derived from γ-mutagenesis of seeds doubly null for phytochromes phyA and phyB, we isolated a mutant lacking phyE. The PHYE gene of the selected mutant, phyE-1, was found to contain a 1-bp deletion at a position equivalent to codon 726, which is predicted to result in a premature stop at codon 739. Immunoblot analysis showed that the phyE protein was undetectable in the phyE-1 mutant. In the phyA- and phyB-deficient background, phyE deficiency led to early flowering, elongation of internodes between adjacent rosette leaves, and reduced petiole elongation. This is a phenocopy of the response of phyA phyB seedlings to end-of-day far-red light treatments. Furthermore, a phyE deficiency attenuated the responses of phyA phyB seedlings to end-of-day far-red light treatments. Monogenic phyE mutants were indistinguishable from wild-type seedlings. However, phyB phyE double mutants flowered earlier and had longer petioles than did phyB mutants. The elongation and flowering responses conferred by phyE deficiency are typical of shade avoidance responses to the low red/far-red ratio. We conclude that in conjunction with phyB and to a lesser extent with phyD, phyE functions in the regulation of the shade avoidance syndrome.

Analysis of promoter region polymorphism in the aldosterone synthase gene (CYP11B2) as a risk factor for myocardial infarction

Several polymorphisms in genes of the reninangiotensin-aldosterone system have been found to have pleiotropic effects on cardiovascular disorders. Recently, a polymorphism (-344 C/T) in the promoter region of the aldosterone synthase gene (CYP11B2), which may influence plasma aldosterone levels, has been reported to strongly influence left ventricular diameters and mass in young adults and arterial stiffness in essential hypertensives. We investigated any association with risk of myocardial infarction (MI). CYP11B2 -344 polymorphism genotypes were determined by polymerase chain reaction (PCR) in 542 acute MI cases and 500 control subjects without history of coronary disease. All subjects were white and <75 years old. There was no significant difference in either genotype distributions (cases CC 17%, CT 52%, TT 31%; controls CC 22%, CT 47%, TT 31%, P = .10) or allele frequencies (cases C/T 0.43/0.57, controls C/T 0.46/0.54, P = .39) between cases and controls. The odds ratio (OR) for MI associated with the CC genotype was 0.75 (0.54-1.05), and remained insignificant when analysis was restricted to the 129 (24%) cases and 193 (37%) controls < 55 years of age (OR 0.68 [0.36-1.27], P = .20). In further analyses, there was no interaction of the polymorphism with other cardiovascular risk factors (smoking, hypertension, diabetes, body mass index, or cholesterol level) in determining MI risk, and the polymorphism did not influence the frequency of these risk factors in either cases or controls. In the case cohort, age at MI was not significantly different in subjects with the three genotypes (CC 61.2 +/- 9.8 years, CT 61.8 +/- 9.1 years, TT 62.2 +/- 9.0 years, P = .69). We conclude that the aldosterone synthase -344 promoter region polymorphism does not significantly influence the risk of MI either directly or via interaction with other risk factors.

Activation and developmental regulation of an Arabidopsis anther-specific promoter in microspores and pollen of Nicotiana tabacum

SummarySeven cytologically distinct stages during micro spore development were identified and used to define the activation of an Arabidopsis anther-specific gene (apg) in transgenic tobacco plants containing an apg promoter-gus fusion. Histochemical analysis of GUS activity showed that the apg promoter was activated in miduninucleate microspores prior to equatorial and polar nuclear migration. Quantitative analysis in isolated spores showed that GUS activity per milligram of protein decreased progressively during pollen development, but on a per spore basis showed a second peak of activity in mid-bicellular pollen. Activation of the apg promoter in isolated gametophytic cells during development was also investigated following DNA delivery by microprojectile bombardment. Levels of transient expression were detectable in uninucleate microspores, but peaked in mid-bicellular pollen, in contrast to the progressive increase in activity of the promoter of the ‘late’ pollen gene lat52. These data show that the apg promoter is activated in a biphasic pattern, and indicate that the activity of transcription factors which mediate apg promoter activity persist through microspore mitosis, but decrease during pollen maturation.

Sialic acid attenuates puromycin aminonucleoside-induced desialylation and oxidative stress in human podocytes

Sialoglycoproteins make a significant contribution to the negative charge of the glomerular anionic glycocalyx-crucial for efficient functioning of the glomerular permselective barrier. Defects in sialylation have serious consequences on podocyte function leading to the development of proteinuria. The aim of the current study was to investigate potential mechanisms underlying puromycin aminonucleosisde (PAN)-induced desialylation and to ascertain whether they could be corrected by administration of free sialic acid. PAN treatment of podocytes resulted in a loss of sialic acid from podocyte proteins. This was accompanied by a reduction, in the expression of sialyltransferases and a decrease in the key enzyme of sialic acid biosynthesis N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). PAN treatment also attenuated expression of the antioxidant enzyme superoxide dismutase (mSOD) and concomitantly increased the generation of superoxide anions. Sialic acid supplementation rescued podocyte protein sialylation and partially restored expression of sialyltransferases. Sialic acid also restored mSOD mRNA expression and quenched the oxidative burst. These data suggest that PAN-induced aberrant sialylation occurs as a result of modulation of enzymes involved sialic acid metabolism some of which are affected by oxidative stress. These data suggest that sialic acid therapy not only reinstates functionally important negative charge but also acts a source of antioxidant activity.

Pharmacological Enhancement of the Kallikrein-Kinin System Promotes Anti-Fibrotic Responses in Human Mesangial Cells

The aim of the present study was to investigate whether pharmacological enhancement of the renal kallikrein-kinin system using the vasopeptidase inhibitor omapatrilat plays a direct role in modulating the fibrotic responses of human mesangial cells to injury. Treatment with 40µmol/L omapatrilat was able to reduce macrophage-conditioned medium (MPCM)-induced fibronectin levels without affecting mRNA expression. MPCM injury also suppressed kallikrein and low molecular weight kininogen mRNA. Omapatrilat was able to attenuate this suppression. Bradykinin levels in contrast were increased by MPCM and treatment with omapatrilat further augmented levels. Co-incubation with the bradykinin B2 receptor antagonist HOE 140 attenuated the omapatrilat-induced lowering of fibronectin. Moreover, inhibition of cGMP release had a similar effect. Paradoxically, RT-PCR and Southern blotting demonstrated that bradykinin B2 receptor mRNA levels were down regulated in response to omapatrilat. Western blotting supported this data. Supernatant levels of tissue plasminogen activator (tPA), a product of bradykinin stimulation, were decreased by omapatrilat while cell associated tPA levels were increased. Matrix metalloproteinase-9 (MMP-9) mRNA expression was up regulated by omapatrilat treament, although no difference in active zymogen levels was observed. In conclusion enhancement of kallikrein-kinin system appears to play a direct role in promoting anti-fibrotic responses in MPCM-injured human mesangial cells.

A novel transient assay system demonstrates that DT-Atsm is a temperature-sensitive toxin in plant tissues

Abstract We describe a rapid transient assay system for analysing the efficacy of diphtheria toxin A chain (DT-A) derivatives as agents of genetic cell ablation in plant tissues. Because of the extreme cytotoxic effects of DT-A in eukaryotic cells we are investigating the use of temperature sensitive (DT-A tsm ) and attenuated (tox 176) derivatives of DT-A to generate more intermediate phenotypes. The relative toxicities of DT-A, DT-A tsm and tox 176 were measured by determining their effects on the expression of a co-transfected luciferase reporter gene construct, using microprojectile bombardment into plant tissues. In pollen and leaves incubated at 16°C, the expression of DT-A or DT-A tsm resulted in a significant reduction in luciferase (LUC) activity. However, at 30°C the expression of DT-A tsm had no effect on the level of LUC activity, in contrast to the DT-A gene, which abolished LUC activity. Furthermore, at the lower permissive temperature DT-A tsm was between 50- and 200-fold less toxic than DT-A. Similar co-bombardment experiments showed that, in contrast to the mammalian systems, the toxicity of tox 176 was not attenuated relative to DT-A.

Low-level C-reactive protein levels exert cytoprotective actions on human podocytes.

BACKGROUND Albuminuria and elevated C-reactive protein (CRP) levels are common manifestations of many inflammatory diseases. Cardiovascular-based drugs, with secondary anti-inflammatory actions, such as angiotensin-converting enzyme-inhibitors are able to reduce both proteinuria and CRP levels, raising the question of whether CRP directly influences the processes that result in proteinuria. As proteinuria is thought to be induced as a result of podocyte dysfunction, we investigated whether there is a pathomechanistic link with CRP. METHODS Podocytes were analysed for evidence of endogenous CRP production in response to inflammatory agents. In addition, they were incubated in the presence of various concentrations of exogenous CRP and analysed for evidence of a response to treatment. RESULTS Our results demonstrated that inflammatory agents such as macrophage-conditioned medium and interleukin-1β induced the expression of CRP messenger RNA in podocytes. However, they were unable to induce CRP protein. Stimulation of podocytes with exogenous CRP demonstrated that 10 μg/mL CRP induced a low but significant level of interleukin-6 secretion. Tumour necrosis factor α, however, was not detected. CRP did up-regulate the expression of the slit diaphragm proteins nephrin and CD2AP, as well as the structural proteins ezrin and podocalyxin-like protein-1, proteins known to be involved in signalling via the phosphotidylinositol-3 (PI-3) kinase pathway. CRP exposure reduced caspase-3 enzyme activity and up-regulated the expression of the anti-apoptotic protein Bcl-2. In the presence of the PI-3 kinase inhibitor LY294002, the ability of CRP to suppress caspase-3 activity was significantly reduced. CONCLUSIONS Taken together, these data suggest that rather than inducing podocyte damage, CRP may be a survival factor for podocytes by maintaining their structural integrity and initiating a survival cascade, which may facilitate podocyte recovery from injury.

Perindoprilat Modulates the Activity of Lipoprotein Receptor-related Protein in Human Mesangial Cells

Low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic receptor implicated in the modulation of a number of cellular processes, including the turnover of proteases and the degradation of extracellular matrix proteins. As such, it can play a key role in the control of fibrosis. The aim of this investigation was to ascertain whether the anti-fibrotic effects exerted by the angiotensin-converting enzyme inhibitor (ACE-I) perindoprilat on macrophage-conditioned medium (MPCM)-injured human mesangial cells can be modulated by this receptor. Addition of receptor-associated protein to MPCM-injured mesangial cells with and without ACE-I increased the amount of tissue plasminogen activator protein detected in mesangial cell culture supernatants without affecting the protein levels of plasminogen activator inhibitor-1. The ability of ACE-I to reduce fibronectin was diminished in the presence of receptor-associated protein. ACE-I induced an increase in mesangial cell MMP9 mRNA, but reduced the MMP9 enzyme activity detected in mesangial cell supernatants. Mesangial cell lysates from ACE-I-treated cells were able to bind immobilized fibronectin at higher dilutions than cell lysates from untreated cells. Flow cytometry showed that MPCM induced an increase in LRP surface expression in mesangial cells over that in control cells and that this expression was further increased by ACE-I treatment. The increase in LRP expression in response to ACE-I was also observed by Western blotting. Northern blot analysis of RNA extracted from cells following a 24-h exposure to MPCM with and without ACE-I demonstrated that there was no change in LRP mRNA expression upon ACE-I treatment. In conclusion, we show that ACE-I treatment is able to modulate mesangial cell-surface expression of LRP, providing an additional mechanism whereby ACE-Is can mediate anti-fibrotic actions independent of their hemodynamic actions.