Increased nucleolar localization of SpiA3G in classically but not alternatively activated macrophages

Macrophages play a key role in innate immune response to pathogens and in tissue homeostasis, inflammation and repair. A serpin A3G (SpiA3G) is highly induced in classically activated macrophages. We show increased localization of SpiA3G in the nucleolus and co-localization with cathepsin L, upon classical, but not alternative activation of macrophages. Despite the increased expression of cathepsin L in the nuclei of classically activated macrophages, no cathepsin activity was detected. Since only pro-inflammatory, but not anti-inflammatory stimuli induce increased nucleolar localization of SpiA3G, we propose that SpiA3g translocation into the nucleolus is important in host defense against pathogens.

Structured summary

MINT-7714245: fibrillarin (uniprotkb:P35550) and SpiA3G(uniprotkb:Q5I2A0) co-localize (MI:0403) by fluorescence microscopy(MI:0416)MINT-7714241: SpiA3G (uniprotkb:Q5I2A0) and cathepsin L(uniprotkb:P06797) co-localize (MI:0403) by fluorescence microscopy (MI:0416)     

Rapid chemical reaction techniques developed for use in investigations of membrane-bound proteins (neurotransmitter receptors)

New techniques for investigating chemical reactions on cell surfaces in the microsecond-to-millisecond time region are described. Reactions mediated by membrane-bound neurotransmitter receptors that control signal transmission between 10¹² cells of the nervous system are taken as an example. Cells with receptors on their plasma membrane are equilibrated with photolabile, biologically inactive precursors of the neurotransmitters. Photolysis of these compounds releases free neurotransmitter that interacts with the receptors, leading to the transient opening of transmembrane receptor-formed channels that are permeant to small inorganic ions. The current thus induced can be measured. The technique can be used to measure the elementary steps of the receptor-mediated reactions. To illustrate the approach it was shown that an understanding of the mechanism of inhibition of the nicotinic acetylcholine receptor by the drug cocaine was obtained and led to the first proof that compounds exist that alleviate the inhibition.     

Importance of the +73/294 interaction in Escherichia coli RNase P RNA substrate complexes for cleavage and metal ion coordination

We have studied an interaction, the "73/294-interaction", between residues 294 in M1 RNA (the catalytic subunit of Escherichia coli RNase P) and +73 in the tRNA precursor substrate. The 73/294-interaction is part of the "RCCA-RNase P RNA interaction", which anchors the 3' R(+73)CCA-motif of the substrate to M1 RNA (interacting residues underlined). Considering that in a large fraction of tRNA precursors residue +73 is base-paired to nucleotide -1 immediately 5' of the cleavage site, formation of the 73/294-interaction results in exposure of the cleavage site. We show that the nature/orientation of the 73/294-interaction is important for cleavage site recognition and cleavage efficiency. Our data further suggest that this interaction is part of a metal ion-binding site and that specific chemical groups are likely to act as ligands in binding of Mg(2+) or other divalent cations important for function. We argue that this Mg(2+) is involved in metal ion cooperativity in M1 RNA-mediated cleavage. Moreover, we suggest that the 73/294-interaction operates in concert with displacement of residue -1 in the substrate to ensure efficient and correct cleavage. The possibility that the residue at -1 binds to a specific binding surface/pocket in M1 RNA is discussed. Our data finally rationalize why the preferred residue at position 294 in M1 RNA is U.     

Effect of bacitracin on erythroid differentiation of MEL cells

Bacitracin, an antibiotic widely utilized in clinical and veterinary use, was tested on murine erythroleukemia (MEL) cells. Tests were performed to evaluate the capacity of the drug to interfere with erythroid differentiation. Cells were exposed to a single treatment in S phase at sublethal doses of bacitracin. Two responses were found depending on the drug concentration. At higher concentrations (25 g/ml and 250ng/ml) a reduction in number of differentiating cells was observed but the kinetics of the process remained unchanged. At lower concentrations (from 2.5 ng/ml to 2.5 fglml) a dramatic alteration of the dynamic of differentiation was found. These two responses are related to different activities of the DNA repair mechanisms. Higher doses of bacitracin stimulate repair while lower concentrations are not able to activate repair, as demonstrated by tests with hydroxyurea. The bacitracin-induced damage can be considered a stable genetic andlor epigenetic alteration, as demonstrated by the high frequency of mutant clones isolatedfrom low-dose treated cells. The suitability of MEL cells system in evaluating genotoxicity of drugs for veterinary use is underlined.Abbreviations MEL murine erythroleukemia - HU hydroxyurea     

Transferrin binding capacity as a marker of differentiation and maturation of rat erythroid cells fractionated by counter current distribution in aqueous polymer two-phase systems

Rat bone marrow cell populations, containing different proportions of erythroid cells, have been fractionated by counter-current distribution in the non-charge-sensitive dextran/polyethyleneglycol two-phase systems on the basis of hydrophobic cell surface properties. Cell fractions with a low distribution coefficient, which contain non-erythroid cells and early erythoblasts, showed a low transferrin binding capacity and a low haemoglobin/cell ratio whereas cell fractions with a high distribution coefficient, which contain intermediate-late erythroblasts and mature red cells, showed an elevated transferrin binding capacity and the highest haemoglobin/cell ratio. These results support transferrin binding capacity as a good marker parameter for the erythroid bone marrow cell differentiation and maturation processes.     

Studies related to the head-maturation pathway of bacteriophages T4 and T2: II. Nuclear disruption,protein synthesis and particle formation with the mutant 43−·30−·46−

We describe the aberrant phage multiplication of the triple conditional lethal mutant 43^?(polymerase)· 30^?(ligase)·46^?(exonuclease) of bacteriophage T4D in which phage DNA replication is arrested but some late protein synthesis occurs (33). The nuclear disruption is indistinguishable from wild type. Forty-five empty small and empty large particles are assembled per cell when the multiplicity of infection (m.o.i.) is 100. This number corresponds closely to the 38 phage equivalents of cleaved major head protein determined biochemically. By reducing the m.o.i. the number of observable particles decreases, reaching 1–5 per cell at an m.o.i. of 5(+5). The total synthesis of phage related proteins is not significantly dependant on the m.o.i. The synthesis of late proteins is about 10% of that of wild type at high m.o.i. and decreases with the m.o.i. The different early and late proteins do not show the same relative proportions as in wild type and respond differently to an increased m.o.i. These and other results are discussed with respect to the role of phage DNA in prehead assembly and head maturation.     

Phenylalanine ammonia-lyase activity and capsaicin-precursor compounds in p-fluorophenylalanine-resistant and -sensitive variant cells of chili pepper (Capsicum annuum)

Cell suspension cultures of chili pepper ( Capsicum annuum L. cv. Tampiqueño 74) displaying differences in their resistance to p -fluorophenylalanine (PFP) and in their contents of capsaicin (the compound which is responsible for the hot taste of chili pepper fruits) were characterized in relation to the activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the levels of free l -phenylalanine, phenolics and the phenylpropanoid acids involved in capsaicin biosynthesis. A nonselected cell line, a sensitive line (CA-02), a moderately resistant cell line (CA-29) and two resistant cell lines (CA-04 and CA-16) were studied. Higher PAL activities and higher levels of phenylalanine and phenolics were found in the PFP-resistant cells even after a minimum of 9 subcultures (15 days each) in the absence of the analog, indicating that the selected trait was stable. PFP-resistant chili pepper cells accumulated higher amounts of capsaicin precursors (cinnamic, caffeic and ferulic acids) than either the nonselected cells or the sensitive cell line. p -Coumaric acid was not detected at significant levels in any of the cell cultures. Overall, accumulation of free phenyl-alanine correlated well with PAL activity, phenolics, phenylpropanoids and capsaicin levels, suggesting an active flow through the phenylpropanoid pathway in PFP-resistant cells of chili pepper.     

PGC‐1α protects from myocardial ischaemia‐reperfusion injury by regulating mitonuclear communication

The recovery of blood supply after a period of myocardial ischaemia does not restore the heart function and instead results in a serious dysfunction called myocardial ischaemia‐reperfusion injury (IRI), which involves several complex pathophysiological processes. Mitochondria have a wide range of functions in maintaining the cellular energy supply, cell signalling and programmed cell death. When mitochondrial function is insufficient or disordered, it may have adverse effects on myocardial ischaemia‐reperfusion and therefore mitochondrial dysfunction caused by oxidative stress a core molecular mechanism of IRI. Peroxisome proliferator‐activated receptor gamma co‐activator 1α (PGC‐1α) is an important antioxidant molecule found in mitochondria. However, its role in IRI has not yet been systematically summarized. In this review, we speculate the role of PGC‐1α as a key regulator of mitonuclear communication, which may interacts with nuclear factor, erythroid 2 like ‐1 and ‐2 (NRF‐1/2) to inhibit mitochondrial oxidative stress, promote the clearance of damaged mitochondria, enhance mitochondrial biogenesis, and reduce the burden of IRI.     

Similar protective effects of BQ-123 and erythropoietin on survival of neural cells and generation of neurons upon hypoxic injury

Our recent study [Danielyan et al., 2005. Eur. J. Cell Biol. 84, 567-579] showed an additive protective effect of endothelin (ET) receptor A (ETA-R) blockade and erythropoietin (EPO) on the survival and rejuvenation of rat astroglial cells exposed to hypoxia. Whether the effects observed with rodent astroglial cells can be reproduced in human astrocytes and whether these effects of ETA-R blockade and EPO on astrocytes are associated with neuronal survival remained open. Therefore, in the present study, the effects of the ETA-R antagonist BQ-123 and EPO on the maintenance of the neuronal population and survival of the human fetal astroglial cell line (SV-FHAS) under normoxic and hypoxic conditions (NC and HC, respectively) were investigated. Rat brain primary cultures exposed to BQ-123 and/or EPO revealed an increase in the number of beta-III tubulin-positive neurons under NC. The hypoxia-caused loss of neurons was abolished by administration of BQ-123 or EPO. Simultaneous application of EPO and BQ-123 led to an additive protective effect on the generation of neurons under NC only. By contrast, BQ-788, the selective ETB-R antagonist, diminished the neuronal population both in NC and HC. Both under NC and HC the number of non-differentiated nestin+/GFAP- neural cells increased upon application of EPO or BQ-123. SV-FHAS responded to BQ-123 or EPO by a decrease in LDH activity in the culture medium under NC (35%) and HC (26% LDH decrease). Concomitant effects of EPO and BQ-123 were illustrated in an additional increase in the survival of human astrocytes (33% under NC and 17% under HC). These data hint at a neuroprotective therapeutic potency of ETA-R blockade, which either alone or in combination with EPO may improve the survival of astroglial and neuronal cells upon hypoxic injury.