Reciprocal regulation of protein kinase C isoforms results in differential cellular responsiveness

Immunological homeostasis is often maintained by counteractive functions of two different cell types or two different receptors signaling through different intermediates in the same cell. One of these signaling intermediates is protein kinase C (PKC). Ten differentially regulated PKC isoforms are integral to receptor-triggered responses in different cells. So far, eight PKC isoforms are reported to be expressed in macrophages. Whether a single receptor differentially uses PKC isoforms to regulate counteractive effector functions has never been addressed. As CD40 is the only receptor characterized to trigger counteractive functions, we examined the relative role of PKC isoforms in the CD40-induced macrophage functions. We report that in BALB/c mouse macrophages, higher doses of CD40 stimulation induce optimum phosphorylation and translocation of PKCα, βI, βII, and ε whereas lower doses of CD40 stimulation activates PKCδ, ζ, and λ. Infection of macrophages with the protozoan parasite Leishmania major impairs PKCα, βI, βII, and ε isoforms but enhances PKCδ, ζ, and λ isoforms, suggesting a reciprocity among these PKC isoforms. Indeed, PKCα, βI, βII, and ε isoforms mediate CD40-induced p38MAPK phosphorylation, IL-12 expression, and Leishmania killing; PKCδ and ζ/λ mediate ERK1/2 phosphorylation, IL-10 production, and parasite growth. Treatment of the susceptible BALB/c mice with the lentivirally expressed PKCδ- or ζ-specific short hairpin RNA significantly reduces the infection and reinstates host-protective IFN-γ-dominated T cell response, defining the differential roles for PKC isoforms in immune homeostasis and novel PKC-targeted immunotherapeutic and parasite-derived immune evasion strategies.     

Differential expression of caveolin-1 and caveolin-3: potential marker for cardiac toxicity subsequent to chronic ozone inhalation

Earlier studies from our laboratory have shown myocardial dysfunction subsequent to chronic O(3) exposure in rats may be associated with a decrease in antioxidant reserve and increased activity of inflammatory mediators. The present study tested the hypothesis that O(3)-induced cardiac dysfunction in healthy adult rats may be due to changes in caveolin-1 and caveolin-3 levels. Sprague-Dawley rats were exposed 8?h/day for 28 and 56?days to filtered air or 0.8?ppm O(3). In order to assess the chronic effects to O(3), in vivo cardiac function was assessed by measuring LVDP, 24?h after termination of O(3) exposure. Compared to rats exposed to filtered air, LVDP values significantly decreased in all O(3)-exposed animals. This attenuation of cardiac function was associated with increased myocardial TNF-α levels and decreased myocardial activities of superoxidase dismutase. Progressive increases in the expression of myocardial TNF-α in 28?days and 56?days O(3)-exposed animals were followed by decreases in cardiac caveolin-1 levels. On the other hand, differential changes in the expression of caveolin-3 in hearts from 28 and 56?days O(3)-exposed animals were independent of intra-cardiac TNF-α levels. These novel findings suggest the interesting possibility that a balance between caveolin-1 and caveolin-3 may be involved in O(3)-mediated cardiac toxicity.     

Cardiac dysfunction subsequent to chronic ozone exposure in rats

A number of advancements have been made toward identifying the risk factors associated with cardiovascular disease (CVD) and have resulted in a decline in mortality. However, many patients with cardiac disease show no established previous risk. Thus, it appears that other unknown factors contribute to the pathophysiology of CVD. Out of 350,000 sudden cardiac deaths each year in the United States, 60,000 deaths have been linked to air pollution, suggesting a detrimental role of environmental pollutants in the development of CVD. This study tested the hypothesis that chronic ozone (O(3)) exposure diminishes myocardial function in healthy population. Male Sprague-Dawley rats were exposed 8 h/day for 28 and 56 days to filtered air or 0.8 ppm O(3). In vivo cardiac function was assessed by measuring LVDP, +dP/dt, -dP/dt, and LVEDP 24 h after termination of the O(3) exposure. Compared to rats exposed to filtered air, LVDP, +dP/dt, and -dP/dt were significantly decreased, and LVEDP was significantly increased in O(3) exposed animals. This attenuation of cardiac function was associated with increased myocardial TNF-alpha levels and lipid peroxidation as well as decreased myocardial activities of superoxidase dismutase and interleukin-10 levels. These novel findings suggest myocardial dysfunction subsequent to chronic O(3) exposure in normal adult rats may be associated with a decrease in antioxidant reserve and with an increased production of inflammatory mediators.     

Mast cells are required for the development of renal fibrosis in the rodent unilateral ureteral obstruction model

Mast cells are associated with inflammation and fibrosis. Whether they protect against or contribute to renal fibrosis is unclear. Based on our previous findings that mast cells can express and secrete active renin, and that angiotensin (ANG II) is profibrotic, we hypothesized that mast cells play a critical role in tubulointerstitial fibrosis. We tested this hypothesis in the 14-day unilateral ureteral obstruction (UUO) model in rats and mast cell-deficient (MCD) mice (WBB6F1-W/Wv) and their congenic controls (CC). In the 14-day UUO rat kidney, mast cell number is increased and they express active renin. Stabilizing mast cells in vivo with administration of cromolyn sodium attenuated the development of tubulointerstitial fibrosis, which was confirmed by measuring newly synthesized pepsin-soluble collagen and blind scoring of fixed trichrome-stained kidney sections accompanied by spectral analysis. Fibrosis was absent in UUO kidneys from MCD mice unlike that observed in the CC mice. Losartan treatment reduced the fibrosis in the CC UUO kidneys. The effects of mast cell degranulation and renin release were tested in the isolated, perfused kidney preparation. Mast cell degranulation led to renin-dependent protracted flow recovery. This demonstrates that mast cell renin is active in situ and the ensuing ANG II can modulate intrarenal vascular resistance in the UUO kidney. Collectively, the data demonstrate that mast cells are critical to the development of renal fibrosis in the 14-day UUO kidney. Since renin is present in human kidney mast cells, our work identifies potential targets in the treatment of renal fibrosis.     

Phorbol ester mediated activation of inducible nitric oxide synthase results in platelet profilin nitration.

Nitric oxide is an important precursor for peroxynitrite production under in vivo conditions leading to cell injury and cell death. In platelets, a number of cytosolic and actin binding proteins were shown to be nitrated [K.M. Naseem, S.Y. Low, M. Sabetkar, N.J. Bradley, J. Khan, M. Jacobs, K.R. Bruckdorfer, The nitration of platelet cytosolic proteins during agonist-induced activation of platelets. FEBS Lett. 473 (1) (2000) 199-122 and M. Sabetkar, S.Y. Low, K.M. Naseem, K.R. Bruckdorfer, The nitration of proteins in platelets: significance in platelet function, Free Radic. Biol. Med. 33 (6) (2002) 728-736]. We investigated the possible mechanism that regulates profilin (an actin binding protein) nitration in platelets. Activation of bovine platelets with arachidonic acid, thrombin, and phorbol 12,13-dibutyrate resulted in nitration of profilin on tyrosine residue. In vivo profilin nitration showed a four- and eight-fold increase in the presence of thrombin and phorbol 12,13-dibutyrate, respectively. Analysis of nitroprofilin levels in the presence of NOS inhibitors (1400W and EGTA), indicated that profilin nitration in phorbol 12,13-dibutyrate treated platelets is mediated by inducible nitric oxide synthase. Phorbol ester treated platelets exhibited higher levels by inducible nitric oxide synthase (491% over control), while total nitric oxide synthase activity increased by 5% over control. Higher levels of peroxynitrite in platelets treated with phorbol 12,13-dibutyrate indicated that profilin nitration is mediated by peroxynitrite. Increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity in platelets treated with thrombin and phorbol 12,13-dibutyrate indicates that nitration of platelet profilin could be mediated by PI 3-kinase. A decrease in the level of nitroprofilin in PDBu treated platelets in the presence of inducible nitric oxide synthase inhibitor, 1400W, was observed suggesting that profilin nitration is mediated by PI 3-kinase dependent activation of inducible nitric oxide synthase.     

Impact of oxygation on soil respiration,yield and water use efficiency of three crop species

Aims Oxygation refers to irrigation of crops with aerated water, through air injection using the venturi principle or the supply of hydrogen peroxide in the root zone, both using subsurface drip irrigation (SDI) system. Oxygation improves water use efficiency (WUE), producing more yield and, and therefore, optimizes the use of drip and SDI. But the efficiency of oxygation is quite possibly dependent on a number of factors. The primary objective of this study was, therefore, to quantify the effects of oxygation, emitter depths and soil type on crop root zone oxygen content, soil respiration, plant physiological response, biomass yield, quality and WUE of three crop species.Methods This study investigated the potential of oxygation to enhance soil respiration, plant growth, yield and water use efficiencies (WUE) of cotton and wheat in experiments in enclosed heavy-duty concrete troughs (tubs) and pineapple and cotton in field experiments. Experimental treatments in tubs for wheat included comparisons between two soil types (vertisol and ferrosol) and superimposed were two oxygation methods (Mazzei air injector and Seair Diffusion System) compared to a control, and for cotton, emitters at two depths using Mazzei air injectors were compared to a control. The field experiments compared Mazzei air injectors and a control for cotton in Emerald and pineapple in Yeppoon, both in central Queensland, Australia.Important findings In all experiments, soil oxygen content and soil respiration markedly increased in response to the oxygation treatments. The O 2 concentration in the crop root zone increased by 2.4–32.6%, for oxygation compared to control at the same depth. The soil respiration increased by 42–100%. The number of wheat ears, leaf dry weight and total dry matter were significantly greater in Mazzei and Seair oxygation compared to the control. Fresh biomass of wheat increased by 11 and 8%, and dry weight of wheat increased by 8 and 3% in Mazzei and Seair oxygation treatments compared to the control, respectively. Likewise, the irrigation water use efficiency increased with oxygation compared to the control in wheat. The yield, WUE and number of other physiological parameters in wheat were enhanced in vertisol compared to ferrosol. The seed cotton yield in the tub experiment increased with oxygation by 14%, and significant differences for fresh biomass, dry matter and yield were also noted between oxygation and the control in the field. Lint yield and WUE both increased by 7% using Mazzei in the cotton field trial during 2008–09. There were significant effects of oxygation on pineapple fresh biomass, and dry matter weight, industry yield and a number of quality parameters were significantly improved. The total fruit yield and marketable increased by 17 and 4% and marketable WUE increased by 3% using Mazzei. Our data suggest that the benefits of oxygation are notable not only for dicotyledonous cotton but also for monocotyledonous wheat and pineapple representing different rooting morphologies and CO₂ fixation pathways.     

Identification of PDC-109-like protein(s) in buffalo seminal plasma

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.