Ferrous ion transport across chloroplast inner envelope membranes

The initial rate of Fe(2+) movement across the inner envelope membrane of pea (Pisum sativum) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Fe(2+)-sensitive fluorophore, Phen Green SK. The rate of Fe(2+) transport was rapid, coming to equilibrium within 3s. The maximal rate and concentration dependence of Fe(2+) transport in predominantly right-side-out vesicles were nearly equivalent to those measured in largely inside-out vesicles. Fe(2+) transport was stimulated by an inwardly directed electrochemical proton gradient across right-side-out vesicles, an effect that was diminished by the addition of valinomycin in the presence of K(+). Fe(2+) transport was inhibited by Zn(2+), in a competitive manner, as well as by Cu(2+) and Mn(2+). These results indicate that inward-directed Fe(2+) transport across the chloroplast inner envelope occurs by a potential-stimulated uniport mechanism.     

Hydroxamates as substrates and inhibitors for FMN-dependent 2-hydroxy acid dehydrogenases

Long-chain hydroxy acid oxydase (HAO) is a member of a flavoenzyme family with significant amino acid sequence similarity and strongly conserved three-dimensional structure; in particular, active-site amino acids involved in catalysis are invariant, with one exception, and numerous enzymatic studies suggest an identical chemical mechanism involving an intermediate carbanion for all family members. Known physiological substrates are a variety of L-2-hydroxy acids. Peroxisomal HAO differs from the other family members in that its actual physiological substrate is not known; it was first described as an L-amino acid oxidase, and recently was identified as an enzyme that converts creatol (hydroxycreatinine) to methylguanidine (a metabolite involved in a variety of uremic syndromes). Creatol (2-amino-5-hydroxy-1-methyl-4(5H)imidazolone) is not a 2-hydroxy acid. We show in this work that 2-hydroxyphenyl acetohydroxamate (HYPAH, the hydroxamate of mandelic acid), a compound that bears similarity both to mandelate (one of the best substrates known) and to creatol, is turned over by HAO, but between 10- and 100-fold less efficiently than mandelate itself. The compound also binds to the active site of homologous flavocytochrome b(2) (L-lactate dehydrogenase). Comparative pH-rate studies for mandelate and its hydroxamate suggest that HYPAH may bind in its ionized form. Both pH-rate profiles are bell-shaped curves, as are those determined for two other family members, flavocytochrome b(2) and mandelate dehydrogenase; while the group with an acid pK(a) between 5 and 6 is most likely the active-site histidine (the residue which abstracts the substrate C2 proton), the identity of the basic group is less clear. It has been proposed to be one of the active site arginines (Lehoux, I., and Mitra, B. (1999) Biochemistry38, 5836-5848); we suggest as an alternative that it could be the lysine residue that interacts with the flavin N1 and O2 positions and stabilizes the negative charge of reduced flavin. In addition to these studies, we have found that HAO is competitively inhibited by benzohydroxamate, which is one atom shorter than HYPAH; its affinity is nearly 100-fold lower than that of the substrate, in contrast to the strong inhibition it exerts on mandelate racemase (Maurice, St. M., and Bearne, S. L. (2000) Biochemistry39, 13324-13335). In the latter case, the 100-fold higher affinity compared to mandelate was proposed to arise from the fact that the hydroxamate can mimic the enolic intermediate which lies on the reaction pathway after C2 proton abstraction. Thus our results do not support the existence of a similar enolic intermediate for HAO (and probably its homologues), although they do not disprove it.     

A novel connection between the yeast Cdc42 GTPase and the Slt2-mediated cell integrity pathway identified through the effect of secreted Salmonella GTPase modulators

Modulation of host cellular GTPases through the injection of the effector proteins SopE2 and SptP is essential for Salmonella typhimurium to enter into non-phagocytic cells. Here we show that expression of the guanine nucleotide exchange factor for Cdc42 SopE2 in Saccharomyces cerevisiae leads to the activation of Fus3 and Kss1 MAPKs, which operate in the mating and filamentation pathways, causing filamentous growth in haploid yeast cells. Furthermore, it promotes the activation of the cell integrity MAPK Slt2. Cdc42 activation by removal of its putative intrinsic GTPase-activating proteins (GAPs), Rga1, Rga2, and Bem3, also results in the phosphorylation of Kss1, Fus3, and Slt2 MAPKs. These data support the role of these GAP proteins as negative regulators of Cdc42, confirm the modulating effect of this GTPase on the filamentation and mating pathways and point to a novel connection between Cdc42 and the cell integrity pathway. Cdc42-induced activation of Slt2 occurs in a mating and filamentation pathway-dependent manner, but it does not require the function of Rho1, which is the GTPase that operates in the cell integrity pathway. Moreover, we report that Salmonella SptP can act as a GAP for Cdc42 in S. cerevisiae, down-regulating MAPK-mediated signaling. Thus, yeast provides a useful system to study the interaction of bacterial pathogenic proteins with eukaryotic signaling pathways. Furthermore, these proteins can be used as a tool to gain insight into the mechanisms that regulate MAPK-mediated signaling in eukaryotes.     

The characteristics of cysteine proteinases of parasitic protozoa.

Cysteine proteinases have now been detected in most of the important species of parasitic protozoa. Characterization of the enzymes and sequence determinations have revealed that the enzymes are related to papain and the mammalian cathepsins. All of the protozoan enzymes analyzed to date are members of the cathepsin L/cathepsin H/papain branch of the papain superfamily and are more distantly related to cathepsin B. They thus share some characteristics with the cysteine proteinases of their hosts. Individual enzymes, however, are likely to have sufficient novel features to be potential targets for specific antiprotozoal drugs, and a number of proteinase inhibitors and substrates are currently being tested as possible chemotherapeutic agents.     

Food provision to nestlings in the Hoopoe Upupa epops: implications for the conservation of a small endangered population in the Swiss Alps

In an attempt to recognize the possible ecological causes of the decline of a population of Hoopoes Upupa epops in the Swiss Alps, we collected data on resource exploitation. The prey provisioned to nestlings by parents was investigated at four breeding sites using photographs (n = 4353, 80% of which enabled prey identification). Molecrickets Gryllotalpa gryllotalpa and Lepidoptera (larvae and pupae) were dominant in nestling diet (93% frequency; 97% biomass). Although Molecrickets were provisioned less frequently (26%) than Lepidoptera (67%), they represented 68% of the total biomass (vs 29% for Lepidoptera). There was an overall negative relationship between the proportion of Molecricket biomass in the diet and the parents' feeding rate, whereas a comparison between broods showed that a higher provisioning activity did not lead to an increase in the biomass supplied to the chicks. A diet based on Molecrickets therefore appears to be energetically advantageous. As Molecrickets are a traditional prey of Hoopoes in central Europe, this might be relevant to other populations. In the study area, Molecrickets occur only on the intensively cultivated plain, whereas the majority of Hoopoe pairs nest at various altitudes on the foothills adjacent to the plain as the latter provides at present almost no suitable nesting sites. Hoopoes breeding higher up on the foothills seem to experience greater provisioning costs and have, on average, lower breeding success. Providing nest sites on the plain is the main conservation measure proposed for the local Hoopoe population. Further attention should also be paid to Molecrickets as these may be crucial for Hoopoes.     

Exercise training in childhood-onset systemic lupus erythematosus: a controlled randomized trial

Introduction

Exercise training has emerged as a promising therapeutic strategy to counteract physical dysfunction in adult systemic lupus erythematosus. However, no longitudinal studies have evaluated the effects of an exercise training program in childhood-onset systemic lupus erythematosus (C-SLE) patients. The objective was to evaluate the safety and the efficacy of a supervised aerobic training program in improving the cardiorespiratory capacity in C-SLE patients.

Methods

Nineteen physically inactive C-SLE patients were randomly assigned into two groups: trained (TR, n = 10, supervised moderate-intensity aerobic exercise program) and non-trained (NT, n = 9). Gender-, body mass index (BMI)- and age-matched healthy children were recruited as controls (C, n = 10) for baseline (PRE) measurements only. C-SLE patients were assessed at PRE and after 12 weeks of training (POST). Main measurements included exercise tolerance and cardiorespiratory measurements in response to a maximal exercise (that is, peak VO₂, chronotropic reserve (CR), and the heart rate recovery (ΔHRR) (that is, the difference between HR at peak exercise and at both the first (ΔHRR1) and second (ΔHRR2) minutes of recovery after exercise).

Results

The C-SLE NT patients did not present changes in any of the cardiorespiratory parameters at POST (P > 0.05). In contrast, the exercise training program was effective in promoting significant increases in time-to-exhaustion (P = 0.01; ES = 1.07), peak speed (P = 0.01; ES = 1.08), peak VO₂ (P = 0.04; ES = 0.86), CR (P = 0.06; ES = 0.83), and in ΔHRR1 and ΔHRR2 (P = 0.003; ES = 1.29 and P = 0.0008; ES = 1.36, respectively) in the C-SLE TR when compared with the NT group. Moreover, cardiorespiratory parameters were comparable between C-SLE TR patients and C subjects after the exercise training intervention, as evidenced by the ANOVA analysis (P > 0.05, TR vs. C). SLEDAI-2K scores remained stable throughout the study.

Conclusion

A 3-month aerobic exercise training was safe and capable of ameliorating the cardiorespiratory capacity and the autonomic function in C-SLE patients.

Trial registration

NCT01515163.