Functional complexes of mitochondria with Ca,MgATPases of myofibrils and sarcoplasmic reticulum in muscle cells

Regulation of mitochondrial respiration in situ in the muscle cells was studied by using fully permeabilized muscle fibers and cardiomyocytes. The results show that the kinetics of regulation of mitochondrial respiration in situ by exogenous ADP are very different from the kinetics of its regulation by endogenous ADP. In cardiac and m. soleus fibers apparent K(m) for exogenous ADP in regulation of respiration was equal to 300-400 microM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 microM when about 70% of maximal rate of respiration was achieved. Respiration rate maintained by intracellular ATPases was suppressed about 20-30% during exogenous trapping of ADP with excess pyruvate kinase (PK, 20 IU/ml) and phosphoenolpyruvate (PEP, 5 mM). ADP flux via the external PK+PEP system was decreased by half by activation of mitochondrial oxidative phosphorylation. Creatine (20 mM) further activated the respiration in the presence of PK+PEP. It is concluded that in oxidative muscle cells mitochondria behave as if they were incorporated into functional complexes with adjacent ADP producing systems - with the MgATPases in myofibrils and Ca,MgATPases of sarcoplasmic reticulum.     

Endocrine regulation of cervical ripening in humans--potential roles for gonadal steroids and insulin-like growth factor-I

Cervical softening is crucial for a normal parturition and corresponds to remodeling of the dominating cervical extra cellular matrix (ECM). The onset of labor as well as cervical ripening is under hormonal control. To get further information about the endocrine regulation of term cervical ripening the following study was undertaken: cervical biopsies were obtained vaginally at elective caesareans, after normal vaginal delivery and after PGE2 or antiprogestin RU486. Biopsies from non-pregnant women served as controls. The concentrations of estrogen receptor (ER) and progesterone receptor (PR) protein were quantitated by EIA and the mRNA levels by solution hybridization. The ERalpha and beta were localized by immunohistochemistry, identified by RT-PCR and quantitated by solution hybridization. The co-localizations of CD45 (leukocyte antigen) and CD68 (macrophage antigen) were studied by immunohistochemistry. The cervical concentrations of ER and PR proteins decreased at term to 15 and 25%, respectively, compared to the non-pregnant levels. A further decrease was measured in the maximal ripened cervix at parturition. The mRNA levels were unchanged but IGF-I mRNA reached a maximum at term. ERalpha mRNA was significantly decreased until delivery, whereas ERbeta mRNA, like IGF-I; was maximum at term. By immunostaining ERbeta could be co-localized with CD45 leukocyte antigen and CD68 macrophage specific antigen. Oral administration of RU486 induced a significant increase in ER protein concentration, whereas PGE2 and spontaneous ripening did not. These findings indicate that cervical ripening is related to significant local hormonal changes.     

An immunohistochemical study on the regulation of estrogen receptor alpha by estradiol in the endometrium of the immature ewe

The effects of estradiol-17beta (E2) on the expression of estrogen receptor alpha (ERalpha) in stromal and epithelial cells of endometrium in prepubertal lambs were investigated. Twenty three-month-old lambs were treated or not treated with one, two or three i.m. injections of E2 (1 microg x kg(-1)) in corn oil at intervals of 24 h. Lambs were slaughtered 12 or 24 h after the last injection. An immunohistochemical technique was used to visualize ERalpha immunostaining which was then analyzed quantitatively by a computer imaging analysis system. Seven endometrial compartments defined by cell type and location were analyzed separately. Positive staining of ERalpha was seen in the nuclei of stromal and epithelial cells. Glandular epithelium located next to the myometrium was stained more intensely than that next to the luminal epithelium and this phenomenon was maintained during treatment. Significantly less immunostaining was found in stromal cells 12 and 24 h after the first injection compared to the control group. A similar pattern was found in the glandular epithelium, although the decrease was more pronounced and the restoration of ERalpha was faster. This study shows that E2 treatment down regulates ERalpha in the endometrium temporarily in both stromal and epithelial cells, but the characteristics of this effect seems to be cell type specific.     

Uracil-DNA glycosylases SMUG1 and UNG2 coordinate the initial steps of base excision repair by distinct mechanisms

DNA glycosylases UNG and SMUG1 excise uracil from DNA and belong to the same protein superfamily. Vertebrates contain both SMUG1 and UNG, but their distinct roles in base excision repair (BER) of deaminated cytosine (U:G) are still not fully defined. Here we have examined the ability of human SMUG1 and UNG2 (nuclear UNG) to initiate and coordinate repair of U:G mismatches. When expressed in Escherichia coli cells, human UNG2 initiates complete repair of deaminated cytosine, while SMUG1 inhibits cell proliferation. In vitro, we show that SMUG1 binds tightly to AP-sites and inhibits AP-site cleavage by AP-endonucleases. Furthermore, a specific motif important for the AP-site product binding has been identified. Mutations in this motif increase catalytic turnover due to reduced product binding. In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER. In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms. UNG2 is apparently adapted to rapid and highly coordinated repair of uracil (U:G and U:A) in replicating DNA, while the less efficient SMUG1 may be more important in repair of deaminated cytosine (U:G) in non-replicating chromatin.     

Regulation of thioredoxin mRNA in the rat uterus by gonadal steroids

Estradiol has been shown to increase the level of thioredoxin mRNA in the uterus of the ovariectomized (ovx) rat. In this study the influence of progesterone, androgens, the anti-estrogen ICI 182780 and the anti-androgen Flutamid on thioredoxin expression, has been studied in the rat uterus. Thioredoxin mRNA concentrations were determined by solution hybridization. Ovx rats treated with progesterone alone showed no effect on thioredoxin expression. Combined treatment of ICI 182780 and estradiol attenuated the estradiol-induced increase in thioredoxin mRNA. When ovx rats were treated with a testosterone depot, the amount of thioredoxin mRNA was increased five-fold after 48 h and remained at that level during the rest of the 168 h monitored. A similar increase in thioredoxin mRNA could be seen after 5-dihydrotestosterone treatment, indicating a true androgenic effect. In addition, the anti-androgen Flutamid attenuated the thioredoxin mRNA increase seen after 5-dihydrotestosterone treatment alone.

It is concluded that thioredoxin mRNA is regulated by growth promoting gonadal steroids in the rat uterus. The attenuation of the estrogen and androgen-induced increases of the thioredoxin mRNA with ICI 182780 and Flutamid, indicate that the effect is mediated via the estrogen receptor and androgen receptor respectively. None of these hormones affected the hepatic thioredoxin mRNA level in the same animals.     

A model of wild bee populations accounting for spatial heterogeneity and climate‐induced temporal variability of food resources at the landscape level

The viability of wild bee populations and the pollination services that they provide are driven by the availability of food resources during their activity period and within the surroundings of their nesting sites. Changes in climate and land use influence the availability of these resources and are major threats to declining bee populations. Because wild bees may be vulnerable to interactions between these threats, spatially explicit models of population dynamics that capture how bee populations jointly respond to land use at a landscape scale and weather are needed. Here, we developed a spatially and temporally explicit theoretical model of wild bee populations aiming for a middle ground between the existing mapping of visitation rates using foraging equations and more refined agent‐based modeling. The model is developed for Bombus sp. and captures within‐season colony dynamics. The model describes mechanistically foraging at the colony level and temporal population dynamics for an average colony at the landscape level. Stages in population dynamics are temperature‐dependent triggered by a theoretical generalized seasonal progression, which can be informed by growing degree days. The purpose of the LandscapePhenoBee model is to evaluate the impact of system changes and within‐season variability in resources on bee population sizes and crop visitation rates. In a simulation study, we used the model to evaluate the impact of the shortage of food resources in the landscape arising from extreme drought events in different types of landscapes (ranging from different proportions of semi‐natural habitats and early and late flowering crops) on bumblebee populations.     

Reduced forms of the iron-containing small subunit of ribonucleotide reductase from Escherichia coli

The B2 subunit of ribonucleotide reductase from Escherichia coli contains a stable tyrosyl free radical and an antiferromagnetically coupled dimeric iron center with high-spin ferric ions. The tyrosyl radical is an oxidized form of tyrosine-122. This study shows that the B2 protein has a fully reduced state, denoted reduced B2, characterized by a normal nonradical tyrosine-122 residue and a dimeric ferrous iron center. Reduced B2 can be formed either from active B2 by a three-electron reduction in the presence of suitable mediators or from apoB2 by addition of two equimolar amounts of ferrous ions in the absence of oxygen. The oxidized tyrosyl radical and the ferric iron center can be generated from reduced B2 by the admission of air. The tyrosyl radical can be selectively reduced by one-electron reduction in the presence of a suitable mediator, yielding metB2, a form that seems identical with the form resulting from treatment of active B2 with hydroxyurea. 1H NMR was used to characterize the paramagnetically shifted resonances associated with the reduced iron center. Prominent resonances were observed around 45 ppm (nonexchangeable with solvent) and 57 ppm (exchangeable with solvent) at 37 degrees C. From the temperature dependence of the chemical shifts of these resonances it was concluded that the ferrous ions in reduced B2 are only weakly, if at all, antiferromagnetically coupled. By comparison with data on the similar iron center of deoxyhemerythrin it is suggested that the 57 ppm resonance should be assigned to protons in histidine ligands of the iron center.     

Increased concentrations of P(i) and lactic acid reduce creatine-stimulated respiration in muscle fibers.

We tested the hypothesis that the respiratory function of skeletal muscle mitochondria is impaired by lactic acidosis and elevated concentrations of P(i). The rate of respiration of chemically skinned fiber bundles from rat soleus muscle was measured at [P(i)] (brackets denote concentration) and pH values similar to those at rest (3 mM P(i), pH 7.0) and high-intensity exercise (20 mM P(i), pH 6.6). Respiration was measured in the absence of ADP and after sequential additions of 0.1 mM ADP, 20 mM creatine (Cr; V(Cr)), and 4 mM ADP. Respiration at 0.1 mM ADP increased after addition of Cr. However, V(Cr) was 23% lower (P < 0.05) during high-intensity conditions than during resting conditions. V(Cr) was also reduced when P(i) or H(+) was increased separately (P < 0.05). Respiration in the absence of ADP and after additions of 0.1 mM ADP and 4 mM ADP was not affected by changes in [P(i)] or [H(+)]. The response was similar, irrespective of when acidosis was induced (i.e., quiescent or actively respiring mitochondria). In conclusion, Cr-stimulated respiration is impaired by increases in [H(+)] and [P(i)] corresponding to those in exercising muscle. Although the reduced Cr-stimulated respiration could be compensated for by increased [ADP], this might have implications for intracellular homeostasis.     

UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation

Cytotoxicity of 5-fluorouracil (FU) and 5-fluoro-2′-deoxyuridine (FdUrd) due to DNA fragmentation during DNA repair has been proposed as an alternative to effects from thymidylate synthase (TS) inhibition or RNA incorporation. The goal of the present study was to investigate the relative contribution of the proposed mechanisms for cytotoxicity of 5-fluoropyrimidines. We demonstrate that in human cancer cells, base excision repair (BER) initiated by the uracil–DNA glycosylase UNG is the major route for FU–DNA repair in vitro and in vivo. SMUG1, TDG and MBD4 contributed modestly in vitro and not detectably in vivo. Contribution from mismatch repair was limited to FU:G contexts at best. Surprisingly, knockdown of individual uracil–DNA glycosylases or MSH2 did not affect sensitivity to FU or FdUrd. Inhibitors of common steps of BER or DNA damage signalling affected sensitivity to FdUrd and HmdUrd, but not to FU. In support of predominantly RNA-mediated cytotoxicity, FU-treated cells accumulated ~3000- to 15 000-fold more FU in RNA than in DNA. Moreover, FU-cytotoxicity was partially reversed by ribonucleosides, but not deoxyribonucleosides and FU displayed modest TS-inhibition compared to FdUrd. In conclusion, UNG-initiated BER is the major route for FU–DNA repair, but cytotoxicity of FU is predominantly RNA-mediated, while DNA-mediated effects are limited to FdUrd.     

Dietary inorganic nitrate improves mitochondrial efficiency in humans

Nitrate, an inorganic anion abundant in vegetables, is converted in vivo to bioactive nitrogen oxides including NO. We recently demonstrated that dietary nitrate reduces oxygen cost during physical exercise, but the mechanism remains unknown. In a double-blind crossover trial we studied the effects of a dietary intervention with inorganic nitrate on basal mitochondrial function and whole-body oxygen consumption in healthy volunteers. Skeletal muscle mitochondria harvested after nitrate supplementation displayed an improvement in oxidative phosphorylation efficiency (P/O ratio) and a decrease in state 4 respiration with and without atractyloside and respiration without adenylates. The improved mitochondrial P/O ratio correlated to the reduction in oxygen cost during exercise. Mechanistically, nitrate reduced the expression of ATP/ADP translocase, a protein involved in proton conductance. We conclude that dietary nitrate has profound effects on basal mitochondrial function. These findings may have implications for exercise physiology- and lifestyle-related disorders that involve dysfunctional mitochondria.