Effect of zinc and calcium ions on the rat kidney membrane-bound form of dipeptidyl peptidase IV

Dipeptidyl peptidase IV (DPP-IV) is an ectopeptidase with many roles, and a target of therapies for different pathologies. Zinc and calcium produce mixed inhibition of porcine DPP-IV activity. To investigate whether these results may be generalized to mammalian DPP-IV orthologues, we purified the intact membrane-bound form from rat kidney. Rat DPP-IV hydrolysed Gly-Pro-p-nitroanilide with an average V_max of 0.86±0.01 μmol min^–1mL^–1 and K_M of 76±6 μM. The enzyme was inhibited by the DPP-IV family inhibitor l-threo-Ile-thiazolidide (K_i=64.0±0.53 nM), competitively inhibited by bacitracin (K_i=0.16±0.01 mM) and bestatin (K_i=0.23±0.02 mM), and irreversibly inhibited by TLCK (IC₅₀ value of 1.20±0.11 mM). The enzyme was also inhibited by divalent ions like Zn²⁺ and Ca²⁺, for which a mixed inhibition mechanism was observed (K_i values of the competitive component: 0.15±0.01 mM and 50.0±1.05 mM, respectively). According to bioinformatic tools, Ca²⁺ ions preferentially bound to the β-propeller domain of the rat and human enzymes, while Zn²⁺ ions to the α-β hydrolase domain; the binding sites were essentially the same that were previously reported for the porcine DPP-IV. These data suggest that the cationic susceptibility of mammalian DPP-IV orthologues involves conserved mechanisms.     

Evidence for Nerve Growth Factor-Potentiating Activities of the Nonpeptidic Compound SR 57746A in PC12 Cells

Abstract: SR 57746A {1-[2-(naphth-2-yl)ethyl]-4-(3-trifluoromethylphenyl)-1,2,5,6-tetrahydropyridine hydrochloride} exhibits neurotrophic activities in vivo and in vitro. We used the rat pheochromocytoma PC12 cell line to investigate in vitro cellular changes induced by SR 57746A. A significant increase in the percentage of cells bearing neurite-like processes was obtained in cells treated by SR 57746A and nerve growth factor (NGF) compared with NGF treatment alone. SR 57746A added alone, however, had no effect on morphogenesis or on survival of cells in serum-free medium. In contrast, SR 57746A induced a "priming" effect on PC12 cells for neurite outgrowth within 6 h of addition of the protein tyrosine kinase inhibitor genistein. An increase in α-actinin content resulted from treatment with SR 57746A. Expression of NGF-mediated acetylcholinesterase and choline acetyltransferase was enhanced within 5 days by SR 57746A. The molecule also induced rapid F-actin redistribution. Within 2 min of incubation, outgrowth of F-actin-containing filopodia was clearly visible at the cell periphery, as previously shown with NGF. It is interesting that this effect of SR 57746A could be mimicked by protein tyrosine kinase inhibitors and abolished by preincubation with sodium orthovanadate, a protein tyrosine phosphatase inhibitor.     

Introduced Drosophila subobscura populations perform better than native populations during an oviposition choice task due to increased fecundity but similar learning ability

The success of invasive species is tightly linked to their fitness in a putatively novel environment. While quantitative components of fitness have been studied extensively in the context of invasive species, fewer studies have looked at qualitative components of fitness, such as behavioral plasticity, and their interaction with quantitative components, despite intuitive benefits over the course of an invasion. In particular, learning is a form of behavioral plasticity that makes it possible to finely tune behavior according to environmental conditions. Learning can be crucial for survival and reproduction of introduced organisms in novel areas, for example, for detecting new predators, or finding mates or oviposition sites. Here we explored how oviposition performance evolved in relation to both fecundity and learning during an invasion, using native and introduced Drosophila subobscura populations performing an ecologically relevant task. Our results indicated that, under comparable conditions, invasive populations performed better during our oviposition task than did native populations. This was because invasive populations had higher fecundity, together with similar cognitive performance when compared to native populations, and that there was no interaction between learning and fecundity. Unexpectedly, our study did not reveal an allocation trade‐off (i.e., a negative relationship) between learning and fecundity. On the contrary, the pattern we observed was more consistent with an acquisition trade‐off, meaning that fecundity could be limited by availability of resources, unlike cognitive ability. This pattern might be the consequence of escaping natural enemies and/or competitors during the introduction. The apparent lack of evolution of learning may indicate that the introduced population did not face novel cognitive challenges in the new environment (i.e., cognitive “pre‐adaptation”). Alternatively, the evolution of learning may have been transient and therefore not detected.     

High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall

The heptapeptide Tyr-Gly- Arg-Gly-Asp- Ser-Pro containing the sequence Arg-Gly-Asp (RGD – the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (K_d1 1 nM, and K_d2 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.     

Variation in Resistance to High Hydrostatic Pressure and rpoS Heterogeneity in Natural Isolates of Escherichia coli O157:H7

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.     

Solving man-induced large-scale conservation problems: the Spanish imperial eagle and power lines

Background

Man-induced mortality of birds caused by electrocution with poorly-designed pylons and power lines has been reported to be an important mortality factor that could become a major cause of population decline of one of the world rarest raptors, the Spanish imperial eagle (Aquila adalberti). Consequently it has resulted in an increasing awareness of this problem amongst land managers and the public at large, as well as increased research into the distribution of electrocution events and likely mitigation measures.

Methodology/Principal Findings

We provide information of how mitigation measures implemented on a regional level under the conservation program of the Spanish imperial eagle have resulted in a positive shift of demographic trends in Spain. A 35 years temporal data set (1974–2009) on mortality of Spanish imperial eagle was recorded, including population censuses, and data on electrocution and non-electrocution of birds. Additional information was obtained from 32 radio-tracked young eagles and specific field surveys. Data were divided into two periods, before and after the approval of a regional regulation of power line design in 1990 which established mandatory rules aimed at minimizing or eliminating the negative impacts of power lines facilities on avian populations. Our results show how population size and the average annual percentage of population change have increased between the two periods, whereas the number of electrocuted birds has been reduced in spite of the continuous growing of the wiring network.

Conclusions

Our results demonstrate that solving bird electrocution is an affordable problem if political interest is shown and financial investment is made. The combination of an adequate spatial planning with a sustainable development of human infrastructures will contribute positively to the conservation of the Spanish imperial eagle and may underpin population growth and range expansion, with positive side effects on other endangered species.     

On the use of the quenching of luminol luminescence to evaluate sod activity

Addition of horseradish peroxidase to a luminol solution (pH = 9.4) produces a burst of light followed by a steady luminescence that lasts for several minutes. This steady-state luminescence is readily quenched by SOD, with a concentration (the additive concentration needed to decrease by one-half the emitted luminescence intensity) of c.a. 4 ng/ml (14 mU/ml). The luminescence intensity decrease can then be employed to evaluate SOD activity in SOD-containing samples. However, the light intensity can also be quenched by additives, such as Trolox, that are able to trap luminol-derived intermediates. It is proposed that double quenching experiments must be performed in order to be able to relate the observed effect of an additive to its SOD-like activity.     

Cardiovascular effects of peptide kinin B2 receptor antagonists in rats

Bradykinin (BK) is a vasoactive peptide reputed to play an important role in cardiovascular homeostasis. In this study, we describe the cardiovascular changes (mean blood pressure (BP) and heart rate (HR)) induced by the i.v. administration (left jugular vein) of two selective kinin B2 receptor antagonist, namely icatibant (0.1-1 micromol/kg as a bolus) and MEN1 1270 (0.1-1 micromol/kg as a bolus or 1 micromol/kg infused in 15 or 60 min), in urethane-anaesthetized or conscious rats with an indwelling catheter implanted in the right carotid artery for BP measurements. In conscious rats, icatibant at 0.1 or 0.3 micromol/kg did not change BP but at 0.1 micromol/kg increased HR at 30 min from administration. MEN1 1270 at 0.1 or 0.3 micromol/kg induced a dose-related increase in BP and a concomitant bradycardia (significant at 0.3 micromol/kg) lasting for 5 or 30 min, respectively. Icatibant at 1 micromol/kg induced a slight (P < 0.05) increase in BP that resolved in 5 min and a biphasic tachycardia (peaks at 30 and 90 min from administration). MEN1 1270 at 1 micromol/kg induced a triphasic change in HR (tachycardia in the first 5 min, bradycardia at 30 min, and tachycardia at 90 and 120 min) and a biphasic change in BP (hypotension at 15 min and hypertension at 30 min). The i.v. infusion of MEN1 1270 (1 micromol/kg in 15 or 60 min) produced hypertension, whereas HR was increased only following the 15-min infusion. In urethane-anaesthetized rats, both icatibant and MEN1 1270 (0.1 micromol/kg as a bolus) increased BP and the onset for this effect was correlated with the time course of the antagonism of BK-induced hypotension, where the effect of MEN1 1270 was more rapid than that of icatibant. These results indicate that kinin B2 receptor antagonists can induce acute cardiovascular effects, and the reason for the different haemodynamic profile between icatibant and MEN1 1270 could be putatively attributed to kinetic characteristics.     

Fine needle aspiration of peripheral neuroepithelioma of soft tissues.

Peripheral neuroepithelioma of soft tissue is a malignant primitive neuroectodermal tumor that appears both in children and adults and usually has a poor outcome. Fine needle aspiration on two patients with tumors in the lower limbs showed small round cells with unipolar processes and a tendency to form Homer-Wright rosettes. The cells had a round to oval nucleus with fine chromatin, up to four small, conspicuous nucleoli and vacuolated, periodic acid-Schiff-positive cytoplasm. The diagnosis was supported by electron microscopic study of the aspirate, which showed features of neuroblastic differentiation (i.e., neurosecretory granules), and by histologic, immunohistochemical and cytogenetic study of the resected tumors.