AT4 receptor structure-binding relationship: N-terminal-modified angiotensin IV analogues

The effect of structural changes in the N-terminal amino acid of AIV, with respect to AT₄ receptor binding, was examined by competition with [¹²⁵I]AIV in bovine adrenal membranes. Analogues with modifications of the first residue α-amino group possessed lower affinities than the primary amine-containing parent compound. Peptides with a residue 1 α-carbon in the d conformation exhibited poor affinity for the AT₄ receptor. Modifications of the residue 1 R-group demonstrate that a straight chain aliphatic moiety containing four carbons is optimal for receptor-ligand binding, as evidenced by the extremely high affinity of [Nle¹]AIV (K_i = 3.59±0.51 pM). Replacement of the 1–2 peptide bond of AIV with the methylene bond isostere Ψ (CH₂-NH), increased the K_i approximately fivefold, indicating that the peptide bond may be replaced wihle maintaining relatively high-affinity receptor binding.     

Irbesartan attenuates ischemic brain damage by inhibition of MCP-1/CCR2 signaling pathway beyond AT1 receptor blockade

Angiotensin II type 1 (AT₁) receptor blockers (ARBs) are known to prevent the onset of stroke and to attenuate neural damage. Additional beneficial effects of ARBs, independent of AT₁ receptor blockade, have been highlighted. Irbesartan is reported to act as an antagonist of the monocyte chemoattractant protein-1 (MCP-1) receptor, C–C chemokine receptor 2 (CCR2), due to its molecular structure. We examined the possible synergistic effects of co-administration of irbesartan with propagermanium, a CCR2 antagonist, on ischemic brain damage. Administration of propagermanium decreased ischemic brain area after middle cerebral artery occlusion (MCAO). To study the possible synergistic effects of propagermanium with ARBs, we employed non-effective lower doses of irbesartan and losartan. Administration of irbesartan with propagermanium decreased the ischemic brain area more markedly compared with propagermanium alone, but co-administration of losartan did not. MCP-1 mRNA level was significantly increased on the ipsilateral side after MCAO, and administration of irbesartan with propagermanium decreased the MCP-1 level, whereas co-administration of losartan did not. Similar results were obtained for MCP-1 protein level. CCR2 mRNA expression was significantly elevated on the ipsilateral side; however, no significant difference was observed between each group. mRNA levels of other inflammatory cytokines such as TNF-α and IL-1β also significantly increased on the ipsilateral side, but the expression levels were not changed by each drug treatment. Taking these findings together, irbesartan exerts more beneficial effects on ischemic brain damage with an MCP-1 receptor blocker, at least due to its inhibitory effects on MCP-1/CCR2 signaling beyond AT₁ receptor blockade.     

Differential effects of prazosin and rauwolsin on the release of prostaglandins I2 and E2 from the perfused mesenteric arterial bed of the rabbit following nerve stimulation

Sympathetic nerve stimulation of the perfused mesenteric arterial bed of the rabbit, , increase the secretion of prostaglandin (PG)I₂ and PGE₂. Prazosin (4.8 × 10⁻⁶), and α₁ adrenergic receptor antagonist, inhibited this inrease in release of PGI₂ but not of PGE₂ whereas rauwolsin (10⁻⁷ M), an α₂ adrenergic receptor antagonist, inhibited the increase in release of PGE₂ but not of PGI₂. Prazosin (10⁻⁶ M) completely blocked the vasoconstrictor response to nerve stimulation, and to norepinephrine and phenylephrine administration, suggesting there to be little of an α₂ adrenergic receptor component in this response. It is concluded that the increase in PGI₂ release follows the activation of α₁ adrenergic receptors and is therefore post-junctional in origin, whereas the increase in PGE₂ release follows the activation of α₂ adrenergic receptors and may be pre- and/or post-junctional in origin.Indomethacin (2.8 × 10⁻⁷, 5.6 × 10⁻⁷ and 1.12 × 10^{−6 M} did not affect the vasoconstrictor responses to nerve stimulation at 10 Hz, whereas rauwolsin (10⁻⁷ M) in the presence of indomethacin substantially increased them. These results indicate that PGE₂ does not regulate norepinephrine release following nerve stimulation at 10 Hz to rabbit mesenteric arteries, and that the inhibition of norepinephrine release following stimulation of α₂ pre-junctional receptors is independent of PG involvement.     

Specificity of receptors for leukotrienes A4, B4, C4, D4, E4 and histamine on the guinea-pig lung parenchyma. Effect of FPL-55712 and desensitization of the myotropic activity

The novel metabolites of arachidonic acid, leukotriene (LT) A₄, B₄, C₄, D₄ and E₄ have potent myotropic activity on guinea-pig lung parenchymal strip . The receptors responsible for their action were characterized using desensitization experiments and the selective SRS-A antagonist, FPL-55712. During the continuous infusion of LTB₄, the tissues became desensitized to LTB₄ but were still responsive to histamine, LTA₄, LTC₄, LTD₄ and LTE₄. When LTD₄ was infused continuously, the lung strips contracted to LTB₄ and histamine but were no longer responsive to LTA₄, LTC₄, LTD₄ and LTE₄. Furthermore, FPL-55712 (10 ng ml⁻¹− 10 ug ml⁻¹) produced dose-dependent inhibitions of LTA₄, LTC₄, LTD₄ and LTE₄ without inhibiting the contraction to LTB₄ and histamine. On the basis of these results, it appears that the guinea-pig lung parenchyma may have one type of receptor for LTB₄ and another for LTD₄; LTA₄, LTC₄ and LTE₄ probably act on the LTD₄ receptor.     

V1 receptor in ENS mediates the excitatory effect of vasopressin on circular muscle strips of gastric body in vitro in rats

The purpose of this study was to localize vasopressin (VP) V_1a receptor in stomach and to characterize the role of VP in the regulation of gastric motility in rats. Double staining was used to locate the V_1a receptor in the gastric body of the rat. The contraction of the circular muscle strips of gastric body was monitored by a polygraph. V_1a receptor was expressed on the neurons of myenteric plexus of the gastric body. VP (10^− 10–10^− 6 M) caused a concentration-dependent contractile effect on the circular muscle strips of gastric body in vitro. V-1880 ([deamino-Pen¹, O-Me-Tyr², Arg⁸]-Vasopressin, 10^− 7 M), a V₁ receptor antagonist, inhibited the spontaneous contraction of the strips. Tetradotoxin (TTX, 10^− 6 M) and V-1880 (10^− 7 M) abolished the excitatory effect of VP. Atropine (10^− 6 M) partially inhibited VP-induced excitatory effect on the muscle strips but hexamethonium (10^− 4 M) did not influence it. These results suggest that V_1a receptor was expressed on the neurons of myenteric nerves. The cholinergic nerve was involved in the excitatory effect of VP on the contraction of gastric body.     

Na-ATPase in spontaneous hypertensive rats: Possible AT1 receptor target in the development of hypertension

Clinical and experimental data show an increase in sodium reabsorption on the proximal tubule (PT) in essential hypertension. It is well known that there is a link between essential hypertension and renal angiotensin II (Ang II). The present study was designed to examine ouabain-insensitive Na⁺-ATPase activity and its regulation by Ang II in spontaneously hypertensive rats (SHR). We observed that Na⁺-ATPase activity was enhanced in 14-week-old but not in 6-week-old SHR. The addition of Ang II from 10^− 12 to 10^− 6 mol/L decreased the enzyme activity in SHR to a level similar to that obtained in WKY. The Ang II inhibitory effect was completely reversed by a specific antagonist of AT₂ receptor, PD123319 (10^− 8 mol/L) indicating that a system leading to activation of the enzyme in SHR is inhibited by AT₂-mediated Ang II. Treatment of SHR with losartan for 10 weeks (weeks 4-14) prevents the increase in Na⁺-ATPase activity observed in 14-week-old SHR. These results indicate a correlation between AT₁ receptor activation in SHR and increased ouabain-insensitive Na⁺-ATPase activity. Our results open new possibilities towards our understanding of the pathophysiological mechanisms involved in the increased sodium reabsorption in PT found in essential hypertension.     

Kinetics and mechanism of CO substitution of [η-CpFe(CO)3]PF6 with PPh3 in the presence of substituted pyridine N-oxide

The kinetics of substitution reactions of [η-CpFe(CO)₃]PF₆ with PPh₃ in the presence of R-PyOs have been studied. For all the R-PyOs (R = 4-OMe, 4-Me, 3,4-(CH)₄, 4-Ph, 3-Me, 2,3-(CH)₄, 2,6-Me₂, 2-Me), the reactions yeild the same product [η⁵-CpFe(CO)₂PPh₃]PF₆, according to a second-order rate law that is first order in concentrations of [η⁵-CpFe(CO)₃]PF₆ and of R-PyO but zero order in PPh₃ concentration. These results, along with the dependence of the reaction rate on the nature of R-PyO, are consistent with an associative mechanism. Activation parameters further support the bimmolecular nature of the reactions: ΔH^≠ = 13.4 ± 0.4 kcal mol⁻¹, ΔS^≠ = −19.1 ± 1.3 cal k⁻¹ mol⁻¹ for 4-PhPyO; ΔH^≠ = 12.3 ± 0.3 kcal mol⁻¹, ΔS^≠ = 24.7 ±1.0 cal K⁻¹ mol⁻¹ for 2-MePyO. For the various substituted pyridine N-oxides studied in this paper, the rates of reaction increase with the increasing electron-donating abilities of the substituents on the pyridine ring or N-oxide basicities, but decrease with increasing ¹⁷O chemical shifts of the N-oxides. Electronic and steric factors contributing to the reactivity of pyridine N-oxides have been quantitatively assessed.     

Acute modulation of myocardial function by angiotensin 1–7

Angiotensin 1–7 is a bioactive heptapeptide of the renin–angiotensin system. Its cardiovascular actions have recently acquired growing relevance, mainly due to its counter-regulatory actions in the angiotensin cascade. The aim of the present study was to evaluate the actions of angiotensin 1–7 on myocardial function. Increasing concentrations of angiotensin 1–7 (10⁻⁹ to 10⁻⁵ M) were added to rabbit right papillary muscles: (1) in baseline conditions with intact endocardial endothelium (EE); (2) after selective removal of the EE with Triton X-100 (1 s, 0.01%); (3) with intact EE in the presence of the Mas receptor antagonist A-779, the AT₁ receptor antagonist ZD-7155, the AT₂ receptor antagonist PD-123,319 or the nitric oxide synthesis inhibitor NG-nitro-l-arginine (l-NA). Concerning the effects on contractility, we observed a significant decrease on active tension, dT/dt_max, peak shortening and dL/dt_max of −10.5 ± 3.6%, −8.0 ± 3.0%, −5.3 ± 2.6% and −5.7 ± 2.3%, respectively. There was no change on relaxation parameters, namely dT/dt_min or dL/dt_min. Time to half relaxation was significantly decreased. The presence of ZD-7155 or PD-123,319 did not change these effects. However, angiotensin 1–7 effects on myocardial properties were abolished after selective EE removal and in the presence of A-779 or l-NA. In conclusion, in this animal species, angiotensin 1–7 through its binding to Mas receptor induces a negative inotropic effect modulated by the EE and nitric oxide and independent of AT₁ or AT₂ receptors activation. As the effects described in the present work were influenced by the endocardial endothelium, they may be disrupted in situations associated to endothelial dysfunction, as in heart failure or myocardial ischemia.     

Scn3b knockout mice exhibit abnormal ventricular electrophysiological properties

We report for the first time abnormalities in cardiac ventricular electrophysiology in a genetically modified murine model lacking the Scn3b gene (Scn3b^−/−). Scn3b^−/− mice were created by homologous recombination in embryonic stem (ES) cells. RT-PCR analysis confirmed that Scn3b mRNA was expressed in the ventricles of wild-type (WT) hearts but was absent in the Scn3b^−/− hearts. These hearts also showed increased expression levels of Scn1b mRNA in both ventricles and Scn5a mRNA in the right ventricles compared to findings in WT hearts. Scn1b and Scn5a mRNA was expressed at higher levels in the left than in the right ventricles of both Scn3b^−/− and WT hearts. Bipolar electrogram and monophasic action potential recordings from the ventricles of Langendorff-perfused Scn3b^−/− hearts demonstrated significantly shorter ventricular effective refractory periods (VERPs), larger ratios of electrogram duration obtained at the shortest and longest S₁–S₂ intervals, and ventricular tachycardias (VTs) induced by programmed electrical stimulation. Such arrhythmogenesis took the form of either monomorphic or polymorphic VT. Despite shorter action potential durations (APDs) in both the endocardium and epicardium, Scn3b^−/− hearts showed ΔAPD₉₀ values that remained similar to those shown in WT hearts. The whole-cell patch-clamp technique applied to ventricular myocytes isolated from Scn3b^−/− hearts demonstrated reduced peak Na⁺ current densities and inactivation curves that were shifted in the negative direction, relative to those shown in WT myocytes. Together, these findings associate the lack of the Scn3b gene with arrhythmic tendencies in intact perfused hearts and electrophysiological features similar to those in Scn5a^+/− hearts.     

Modulation of human myometrial PGE2 receptor by GTP characterization of receptor subtype

We studied PGE₂ specific binding sites in human myometrial microsomes prepared from uterine specimens obtained by hysterectomy (women between 38 and 55 years of age). Competition experiments showed that the potency order for various prostaglandins (PGs) was : PGE₂ ≥ PGE₁ PGF_2α > Iloprost ≥ Carbacyclin ZK 110841 (PGD₂ analogue). These relative affinities indicated that the receptor was of the EP type.In kinetic experiments GTP, GppNHp and GTPγS increased the rate of PGE₂ binding (steady state was reached more rapidly in the presence of nucleotides) but maximal specific binding was not significantly different. Complete dissociation could not be obtained, even in the presence of GTP. Only 50% of maximal binding was readily dissociable. The dissociation rate was 4.56.10⁻⁴ sec⁻¹ (half time of about 660 sec) and in the presence of GTP analogues it was slightly increased (k−1 = 7.16 10⁻⁴ sec⁻¹ half time 420 sec.). Scatchard analysis of saturation curves showed an increase in ligand receptor affinity in the presence of GTP or nucleotide analogues: the Kd shifted from 9.66 ± 2.8.10⁻⁹ M to 4.96 ± 1.25.10⁻⁹M, but the number of binding sites did not change significantly (310 ± 37 to 350 ± 17 fmol/mgP). The effect of GTP was observed at a concentration of 5.10⁻⁴M. GppNHp and GTPγS were effective at 1.10⁻⁵M. Pretreatment of myometrial membranes with pertussis or cholera toxins had no effect on PGE₂ binding to membrane sites. Our conclusion is that GTP induced conversion of a population of low affinity sites into a population of higher affinity sites. This effect of guanine nucleotides was described in adipocytes and kidney medulla.Competition studies with PGE₂ analogues (sulprostone, 17-phenyl-ω-trinor PGE₂, M&B 28,767, misoprostol, butaprost) showed that this receptor mediates a contractile response and is probably an EP₃ subtype.     

Down-regulation of ether-a-go-go-related gene potassium channel protein through sustained stimulation of AT1 receptor by angiotensin II

We investigated the effects of AT₁ receptor stimulation by angiotensin II (Ang II) on human ether-a-go-go-related gene (hERG) potassium channel protein in a heterogeneous expression system with the human embryonic kidney (HEK) 293 cells which stably expressed hERG channel protein and were transiently transfected with the human AT₁ receptors (HEK293/hERG). Western-blot analysis showed that Ang II significantly decreased the expression of mature hERG channel protein (155-kDa band) in a time- and dose-dependent manner without affecting the level of immature hERG channel protein (135-kDa band). The relative intensity of 155-kDa band was 64.7 ± 6.8% of control (P < 0.01) after treatment of Ang II at 100 nM for 24 h. To investigate the effect of Ang II on the degradation of mature hERG channel protein, we blocked forward trafficking from ER to Golgi with a Golgi transit inhibitor brefeldin A (10 μM). Ang II significantly enhanced the time-dependent reduction of mature hERG channel protein. In addition, the proteasomal inhibitor lactacystin (5 μM) inhibited Ang II-mediated the reduction of mature hERG channel protein, but the lysosomal inhibitor bafilomycin A1 (1 μM) had no effect on the protein. The protein kinase C (PKC) inhibitor bisindolylmaleimide 1 (1 μM) antagonized the reduction of mature hERG channel protein induced by Ang II. The results indicate that sustained stimulation of AT₁ receptors by Ang II reduces the mature hERG channel protein via accelerating channel proteasomal degradation involving the PKC pathway.     

Candidate Agtr2 influenced genes and pathways identified by expression profiling in the developing brain of Agtr2 mice

Intellectual disability (ID) is a common developmental disability observed in 1 to 3% of the human population. A possible role for the Angiotensin II type 2 receptor (AGTR2) in brain function, affecting learning, memory, and behavior, has been suggested in humans and rodents. Mice lacking the Agtr2 gene (Agtr2^−/y) showed significant impairment in their spatial memory and exhibited abnormal dendritic spine morphology. To identify Agtr2 influenced genes and pathways, we performed whole genome microarray analysis on RNA isolated from brains of Agtr2^−/y and control male mice at embryonic day 15 (E15) and postnatal day one (P1). The gene expression profiles of the Agtr2^−/y brain samples were significantly different when compared to profiles of the age-matched control brains. We identified 62 differently expressed genes (p ≤ 0.005) at E15 and in P1 brains of the Agtr2^−/y mice. We verified the differential expression of several of these genes in brain samples using quantitative RT-PCR. Differentially expressed genes encode molecules involved in multiple cellular processes including microtubule functions associated with dendritic spine morphology. This study provides insight into Agtr2 influenced candidate genes and suggests that expression dysregulation of these genes may modulate Agtr2 actions in the brain that influences learning and memory.     

Involvement of the AT1 receptor in the venoconstriction induced by angiotensin II in both the inferior vena cava and femoral vein

Although angiotensin II-induced venoconstriction has been demonstrated in the rat vena cava and femoral vein, the angiotensin II receptor subtypes (AT₁ or AT₂) that mediate this phenomenon have not been precisely characterized. Therefore, the present study aimed to characterize the pharmacological receptors involved in the angiotensin II-induced constriction of rat venae cavae and femoral veins, as well as the opposing effects exerted by locally produced prostanoids and NO upon induction of these vasomotor responses. The obtained results suggest that both AT₁ and AT₂ angiotensin II receptors are expressed in both veins. Angiotensin II concentration-response curves were shifted toward the right by losartan but not by PD 123319 in both the vena cava and femoral vein. Moreover, it was observed that both 10⁻⁵ M indomethacin and 10⁻⁴ M L-NAME improve the angiotensin II responses in the vena cava and femoral vein. In conclusion, in the rat vena cava and femoral vein, angiotensin II stimulates AT₁ but not AT₂ to induce venoconstriction, which is blunted by vasodilator prostanoids and NO.     

Prostaglandin D2 metabolite stimulates collagen synthesis by human osteoblasts during calcification

We investigate the effect of the prostaglandin D₂ metabolite Δ¹²−PGD₂ (9−Deoxy−Δ⁹, Δ¹²−13,14-dihydroprostaglandin D₂) on collagen synthesis in human osteoblast. Δ¹²-PGJ₂ at 10⁻⁵M enhanced collagen synthesis in the presence of 2 mM α-glycerophosphate-2Na. The stimulative effect appeared as early as 3 days after addition and continued until 22 days. The enhancement of type I collagen synthesis was confirmed by polyacrylamide gel electrophoresis. The potency was the same as 10^1t-8M 1 α, 25 dihydroxy vitamine D₃ (1,25(OH)₂D₃). Northern blot analysis showed that 10⁻⁵M Δ ¹²-PGD₂ and 10⁻⁸M 1,25(OH)₂D₃ enhanced the transcribtion of type 1 procollagen (α₁) mRNA levels in osteoblasts.     

Emission of N2O, N2, CH4, and CO2 from constructed wetlands for wastewater treatment and from riparian buffer zones

We measured nitrous oxide (N₂O), dinitrogen (N₂), methane (CH₄), and carbon dioxide (CO₂) fluxes in horizontal and vertical flow constructed wetlands (CW) and in a riparian alder stand in southern Estonia using the closed chamber method in the period from October 2001 to November 2003. The replicates’ average values of N₂O, N₂, CH₄ and CO₂ fluxes from the riparian gray alder stand varied from −0.4 to 58 μg N₂O-N m⁻² h⁻¹, 0.02–17.4 mg N₂-N m⁻² h⁻¹, 0.1–265 μg CH₄-C m⁻² h⁻¹ and 55–61 mg CO₂-C m⁻² h⁻¹, respectively. In horizontal subsurface flow (HSSF) beds of CWs, the average N₂ emission varied from 0.17 to 130 and from 0.33 to 119 mg N₂-N m⁻² h⁻¹ in the vertical subsurface flow (VSSF) beds. The average N₂O-N emission from the microsites above the inflow pipes of the HSSF CWs was 6.4–31 μg N₂O-N m⁻² h⁻¹, whereas the outflow microsites emitted 2.4–8 μg N₂O-N m⁻² h⁻¹. In VSSF beds, the same value was 35.6–44.7 μg N₂O-N m⁻² h⁻¹. The average CH₄ emission from the inflow and outflow microsites in the HSSF CWs differed significantly, ranging from 640 to 9715 and from 30 to 770 μg CH₄-C m⁻² h⁻¹, respectively. The average CO₂ emission was somewhat higher in VSSF beds (140–291 mg CO₂-C m⁻² h⁻¹) and at the inflow microsites of HSSF beds (61–140 mg CO₂-C m⁻² h⁻¹). The global warming potential (GWP) from N₂O and CH₄ was comparatively high in both types of CWs (4.8 ± 9.8 and 6.8 ± 16.2 t CO₂ eq ha⁻¹ a⁻¹ in the HSSF CW 6.5 ± 13.0 and 5.3 ± 24.7 t CO₂ eq ha⁻¹ a⁻¹ in the hybrid CW, respectively). The GWP of the riparian alder forest from both N₂O and CH₄ was relatively low (0.4 ± 1.0 and 0.1 ± 0.30 t CO₂ eq ha⁻¹ a⁻¹, respectively), whereas the CO₂-C flux was remarkable (3.5 ± 3.7 t ha⁻¹ a⁻¹). The global influence of CWs is not significant. Even if all global domestic wastewater were treated by wetlands, their share of the trace gas emission budget would be less than 1%.     

Utilization of the cyanobacteria Anabaena sp. ATCC 33047 in CO2 removal processes

In this paper the utilization of the cyanobacteria Anabaena sp. in carbon dioxide removal processes is evaluated. For this, continuous cultures of this strain were performed at different dilution rates; alternatives for the recovery of the organic matter produced being also studied. A maximum CO₂ fixation rate of 1.45 g CO₂ L⁻¹ day⁻¹ was measured experimentally, but it can be increased up to 3.0 g CO₂ L⁻¹ day⁻¹ outdoors. The CO₂ is mainly transformed into exopolysaccharides, biomass representing one third of the total organic matter produced. Organic matter can be recovered by sedimentation with efficiencies higher than 90%, the velocity of sedimentation being 2 · 10⁻⁴ s⁻¹. The major compounds were carbohydrates and proteins with productivities of 0.70 and 0.12 g L⁻¹ day⁻¹, respectively. The behaviour of the cultures of Anabaena sp. has been modelized, also the characteristics parameters requested to design separation units being reported. Finally, to valorizate the organic matter as biofertilizers and biofuels is proposed.     

Photosynthetic responses to elevated CO2and O3 in field-grown potato(Solanum tuberosum)

Potato plants (Solanum tuberosum L. cv. Bintje) were grown to maturity in open-top chambers under three carbon dioxide (CO₂; ambient and 24 h d⁻¹ seasonal mean concentrations of 550 and 680 μmol mol⁻¹) and two ozone levels (O₃; ambient and an 8 h d⁻¹ seasonal mean of 50 nmol mol⁻¹). Chlorophyll content, photosynthetic characteristics, and stomatal responses were determined to test the hypothesis that elevated atmospheric CO₂ may alleviate the damaging influence of O₃ by reducing uptake by the leaves. Elevated O₃ had no detectable effect on photosynthetic characteristics, leaf conductance, or chlorophyll content, but did reduce SPAD values for leaf 15, the youngest leaf examined. Elevated CO₂ also reduced SPAD values for leaf 15, but not for older leaves; destructive analysis confirmed that chlorophyll content was decreased. Leaf conductance was generally reduced by elevated CO₂, and declined with time in the youngest leaves examined, as did assimilation rate (A). A generally increased under elevated CO₂, particularly in the older leaves during the latter stages of the season, thereby increasing instantaneous transpiration efficiency. Exposure to elevated CO₂ and/or O₃ had no detectable effect on dark-adapted fluorescence, although the values decreased with time. Analysis of the relationships between assimilation rate and intercellular CO₂ concentration and photosynthetically active photon flux density showed there was initially little treatment effect on CO₂-saturated assimilation rates for leaf 15. However, the values for plants grown under 550 μmol mol⁻¹ CO₂ were subsequently greater than in the ambient and 680 μmol mol⁻¹ treatments, although the beneficial influence of the former treatment declined sharply towards the end of the season. Light-saturated assimilation was consistently greater under elevated CO₂, but decreased with time in all treatments. The values decreased sharply when leaves grown under elevated CO₂ were measured under ambient CO₂, but increased when leaves grown under ambient CO₂ were examined under elevated CO₂. The results obtained indicate that, although elevated CO₂ initially increased assimilation and growth, these beneficial effects were not necessarily sustained to maturity as a result of photosynthetic acclimation and the induction of earlier senescence.     

o-Phthalaldehyde–N-acetylcysteine polyamine derivatives: formation and stability in solution and in C18 supports

A comparative study of different derivatization procedures has been performed in order to improve the stability of the reaction products o-phthalaldehyde–N-acetylcysteine (OPA–NAC) polyamines. Procedures such as solution derivatization, solution derivatization followed by retention on a packing support, derivatization on different packing supports and on-column derivatization, have been optimized and compared. The degradation rate constant (k) of the derivative was dependent on the procedure used and on the analyte. For the spermine (the most unstable isoindol tested) k was 8±2×10⁻² min⁻¹ in solution versus 7.7±1.1×10⁻⁴ min⁻¹ on the (C₁₈) solid support. The results obtained showed that forming the derivative on the packing support (C₁₈) gave the best results following this procedure: conditioning the cartridges with borate buffer (1 ml, 0.5 M, pH 8), retention of the analyte, addition of 0.8 ml of OPA–NAC reagent, 0.2 ml borate buffer 0.8 M (pH 8) and elution of the isoindol with 3 ml of MeOH–borate buffer (9:1). The different derivatization procedures have been used to study the stability of the reaction products OPA–NAC polyamines formed in urine matrix using spermine as model compound. Similar results were obtained for standard solutions and urine samples.     

Gas exchange of Ginkgo biloba leaves at different CO2 concentration levels

One and a half year-old Ginkgo saplings were grown for 2 years in 7 litre pots with medium fertile soil at ambient air CO₂ concentration and at 700 μmol mol⁻¹ CO₂ in temperature and humidity-controlled cabinets standing in the field. In the middle of the 2nd season of CO₂ enrichment, CO₂ exchange and transpiration in response to CO₂ concentration was measured with a mini-cuvette system. In addition, the same measurements were conducted in the crown of one 60-year-old tree in the field. Number of leaves/tree was enhanced by elevated CO₂ and specific leaf area decreased significantly.CO₂ compensation points were reached at 75–84 μmol mol⁻¹ CO₂. Gas exchange of Ginkgo saplings reacted more intensively upon CO₂ than those of the adult Ginkgo. On an average, stomatal conductance decreased by 30% as CO₂ concentration increased from 30 to 1000 μmol mol⁻¹ CO₂. Water use efficiency of net photosynthesis was positively correlated with CO₂ concentration levels. Saturation of net photosynthesis and lowest level of stomatal conductance was reached by the leaves of Ginkgo saplings at >1000 μmol mol⁻¹ CO₂. Acclimation of leaf net CO₂ assimilation to the elevated CO₂ concentration at growth occurred after 2 years of exposure. Maximum of net CO₂ assimilation was 56% higher at ambient air CO₂ concentration than at 700 μmol mol⁻¹ CO₂.     

Lysophosphatidic acid-induced arterial wall remodeling: Requirement of PPARγ but not LPA1 or LPA2 GPCR

Lysophosphatidic acid (LPA) and its ether analog alkyl-glycerophosphate (AGP) elicit arterial wall remodeling when applied intralumenally into the uninjured carotid artery. LPA is the ligand of eight GPCRs and the peroxisome proliferator-activated receptor γ (PPARγ). We pursued a gene knockout strategy to identify the LPA receptor subtypes necessary for the neointimal response in a non-injury model of carotid remodeling and also compared the effects of AGP and the PPARγ agonist rosiglitazone (ROSI) on balloon injury-elicited neointima development. In the balloon injury model AGP significantly increased neointima; however, rosiglitazone application attenuated it. AGP and ROSI were also applied intralumenally for 1 h without injury into the carotid arteries of LPA₁, LPA₂, LPA_1&2 double knockout, and Mx1Cre-inducible conditional PPARγ knockout mice targeted to vascular smooth muscle cells, macrophages, and endothelial cells. The neointima was quantified and also stained for CD31, CD68, CD11b, and α-smooth muscle actin markers. In LPA₁, LPA₂, LPA_1&2 GPCR knockout, Mx1Cre transgenic, PPARγ^fl/−, and uninduced Mx1Cre × PPARγ^fl/− mice AGP- and ROSI-elicited neointima was indistinguishable in its progression and cytological features from that of WT C57BL/6 mice. In PPARγ^−/− knockout mice, generated by activation of Mx1Cre-mediated recombination, AGP and ROSI failed to elicit neointima and vascular wall remodeling. Our findings point to a difference in the effects of AGP and ROSI between the balloon injury- and the non-injury chemically-induced neointima. The present data provide genetic evidence for the requirement of PPARγ in AGP- and ROSI-elicited neointimal thickening in the non-injury model and reveal that the overwhelming majority of the cells in the neointimal layer express α-smooth muscle actin.