Temporal Association Between Pulmonary Inflammation and Antioxidant Induction Following Hyperoxic Exposure of the Preterm Guinea Pig

The time course and nature of the pulmonary inflammatory and antioxidant responses, both during and after hyperoxic-induced acute lung injury were studied in the preterm guinea pig. Three-day preterm (65 days gestation) guinea pigs were randomly exposed to either 21% O₂ (control) or 95% O₂ (hyperoxia) for 72 hours. All pups were then maintained in ambient conditions for up to a further 11 days, during which time lung damage was monitored. In animals exposed to hyperoxia, evidence of acute lung injury and inflammation was characterized by a marked increase in microvascular permeability and elevated numbers of neutrophils in bronchoalveolar lavage fluid. Protein concentration, elastase-like activity and elastase-inhibitory capacity in lavage fluid were at a maximum at the end of the 72 hours hyperoxic exposure. Four days later, all values had returned to control levels. In contrast, increased numbers of neutrophils, macrophages and lymphocytes were recovered in the lavage fluid during this early recovery period. Coinciding with the influx of inflammatory cells, there was a significant increase in glutathione peroxidase, manganese superoxide dismutase and catalase activities in immature lung. Lung copper/zinc superoxide dismutase activity remained unchanged during both experimental periods. The strong temporal relationship between the influx of inflammatory cells to the lung and the induction of pulmonary antioxidant enzyme defences suggests that a common mechanism underlies both responses. These findings have led us to regard inflammation in the hyperoxic-injured immature lung as a beneficial event and not, as previously suggested, as part of the injurious process.     

Thermoluminescence studies of zinc borate (ZnB2O4) phosphor doped with rare earths for dosimetric aspects

A series of ZnB₂O₄ phosphors doped with different concentrations of Eu and Dy (0.05 0.1, 0.2, 0.5, 1.0 mol%) and co-doped with Ce (1, 2, 5, 7, 10 mol%) respectively was prepared via the solid-state reaction technique and the thermoluminescence (TL) behaviour of gamma ray-irradiated samples was studied. The synthesized samples were irradiated with γ-rays for the dose range 0.03–1.20 kGy. The TL intensity variations with dose, dopant concentration, and the effect of co-doping were studied. The TL response curves for ZnB₂O₄:Eu³⁺ and ZnB₂O₄:Dy³⁺, ZnB₂O₄:Eu³,Ce³⁺ and ZnB₂O₄:Dy³⁺,Ce³⁺ phosphor were observed. It was revealed that ZnB₂O₄:Eu³⁺ showed a linear TL behaviour for the dose 0.03–1.20 kGy and ZnB₂O₄:Dy³⁺ showed linearity for the gamma dose range 0.03–0.10 kGy. Furthermore, fading for all the samples was observed to be less than 10% for a storage period of 30 days. In addition to this, the trapping parameters, especially activation energies were evaluated using the Ilich method and the initial rise method. The activation energy values obtained from both methods were in complete agreement with each other.     

Peptide-protein interaction markedly alters the functional properties of the catalytic subunit of aspartate transcarbamoylase.

Interaction of a 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites. The complex between the polypeptide (zinc domain) and wild-type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme. Moreover, the Scatchard plot for binding N-(phosphonacetyl)-L-aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state. Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu. As shown previously (Newell, J.O. & Schachman, H.K., 1990, Biophys. Chem. 37, 183-196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50-fold greater affinity for PALA than the free C subunit. Adding the zinc domain to the C trimer containing the Lys 164-->Glu substitution leads to a 50-fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme. A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508-8515) to be much less active as a holoenzyme than as the free C trimer. For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state. However, the addition of the zinc domain to the Gln 231-->Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme. This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein. This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change. The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.     

Overexpression of Slc30a7/ZnT7 increases the mitochondrial matrix levels of labile Zn2+ and modifies histone modification in hyperinsulinemic cardiomyoblasts

BackgroundCellular free Zn²⁺ concentrations ([Zn²⁺]) are primarily coordinated by Zn²⁺-transporters, although their roles are not well established in cardiomyocytes. Since we previously showed the important contribution of a Zn²⁺-transporter ZnT7 to [Zn²⁺]_i regulation in hyperglycemic cardiomyocytes, here, we aimed to examine a possible regulatory role of ZnT7 not only on [Zn²⁺]_i but also both the mitochondrial-free Zn²⁺ and/or Ca²⁺ in cardiomyocytes, focusing on the contribution of its overexpression to the mitochondrial function.MethodsWe mimicked either hyperinsulinemia (by 50-μM palmitic acid, PA-cells, for 24-h) or overexpressed ZnT7 (ZnT7OE-cells) in H9c2 cardiomyoblasts.ResultsOpposite to PA-cells, the [Zn²⁺]_i in ZnT7OE-cells was not different from untreated H9c2-cells. An investigation of immunofluorescence imaging by confocal microscopy demonstrated a ZnT7 localization on the mitochondrial matrix. We demonstrated the ZnT7 localization on the mitochondrial matrix by using immunofluorescence imaging. Later, we determined the mitochondrial levels of [Zn²⁺]_Mit and [Ca²⁺]_Mit by using the Zn²⁺ and Ca²⁺ sensitive FRET probe and a Ca²⁺-sensitive dye Fluo4, respectively. The [Zn²⁺]_Mit was found to increase significantly in ZnT7OE-cells, similar to the PA-cells while no significant changes in the [Ca²⁺]_Mit in these cells. To examine the contribution of ZnT7 overexpression on the mitochondria function, we determined the level of reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) in these cells in comparison to the PA-cells. There were significantly increased production of ROS and depolarization in MMP and increases in marker proteins of mitochondria-associated apoptosis and autophagy in ZnT7-OE cells, similar to the PA-cells, parallel to increases in K-acetylation. Moreover, we determined significant increases in trimethylation of histone H3 lysine27, H3K27me3, and the mono-methylation of histone H3 lysine36, H3K36 in the ZnT7OE-cells, demonstrating the role of [Zn²⁺]_Mit in epigenetic regulation of cardiomyocytes under hyperinsulinemia through histone modification.ConclusionsOverall, our data have shown an important contribution of high expression of ZnT7-OE, through its buffering and muffling capacity in cardiomyocytes, on the regulation of not only [Zn²⁺_]i but also both [Zn²⁺]_Mit and [Ca²⁺]_Mit affecting mitochondria function, in part, via histone modification.     

The relationship between beta cell activation and SLC30A8/ZnT8 levels of the endocrine pancreas and maternal zinc deficiency in rats

ObjectivesZinc, which is found in high concentrations in the β-cells of the pancreas, is also a critical component for the endocrine functions of the pancreas. SLC30A8/ZnT8 is the carrier protein responsible for the transport of zinc from the cytoplasm to the insulin granules. The aim of this study was to investigate how dietary zinc status affects pancreatic beta cell activation and ZnT8 levels in infant male rats born to zinc-deficient mothers.MethodsThe study was performed on male pups born to mothers fed a zinc-deficient diet. A total of 40 male rats were divided into 4 equal groups. Group 1: In addition to maternal zinc deficiency, this group was fed a zinc-deficient diet. Group 2: In addition to maternal zinc deficiency, this group was fed a standard diet. Group 3: In addition to maternal zinc deficiency, this group was fed a standard diet and received additional zinc supplementation. Group 4: Control group. Pancreas ZnT8 levels were determined by ELISA method and insulin-positive cell ratios in β-cells by immunohistochemistry.ResultsThe highest pancreatic ZnT8 levels and anti-insulin positive cell ratios in the current study were obtained in Group 3 and Group 4. In our study, the lowest pancreatic ZnT8 levels were obtained in Group 1 and Group 2, and the lowest pancreatic anti-insulin positive cell ratios were obtained in Group 1.ConclusionThe results of the present study; in rats fed a zinc-deficient diet after maternal zinc deficiency has been established shows that ZnT8 levels and anti-insulin positive cell ratios in pancreatic tissue, which is significantly suppressed, reach control values with intraperitoneal zinc supplementation.     

GROWTH OF HETEROSIGMA CARTERAE (RAPHIDOPHYCEAE) ON NITRATE AND AMMONIUM AT THREE PHOTON FLUX DENSITIES: EVIDENCE FOR N STRESS IN NITRATE-GROWING CELLS

The marine alga Heterosigma carterae Hulburt (Raphidophyta) was grown in N-limiting batch cultures using either nitrate or ammonium as the N source, at photon flux densities (PFDs) of 50, 200, and 350 μmol·m^-2·s^-1 in a 12:12 h LD cycle. Carbon content could be estimated from biovolume (μg C = 0.278 × nL; R = 0.98) but not reliably from pigment content. During exponential growth, ammonium-grown cells (in comparison with nitrate-grown cells at the same PFD) attained higher growth rates by at least 20%, contained more N, and had a lower C:N ratio, higher concentrations of intracellular free amino acids, and higher ratios of glutamine: glutamate (Gln: Glu) and asparagine: aspartate (Asn:Asp). Growth was nearly light-saturated on ammonium at 200 μmol·m^-2·s^-1 (cell-specific growth rate of 1.2 d^-1) but probably not saturated in nitrate-grown cells at 350 μmol·m^-2·s^-1. PFD did not affect Gln: Glu or Asn: Asp for a given N source. These results indicate that the nitrate-growing cells were more N-stressed than those using ammonium (which in contrast were relatively C-stressed) and that this organism would show an enhanced competitive advantage against other species when supplied with a transient supply of ammonium rather than nitrate .     

Description of two-metal biosorption equilibria by Langmuir-type models

A biosorbent prepared from Ascophyllum nodosum seaweed biomass, FCAN2, was examined for its sorption capacity. Equilibrium batch sorption studies were performed using two-matal systems containing either (Cu + Zn), (Cu + Cd), or (Zn + Cd). In the evaluation of the two-metal sorption system performance, simple isotherm curves had to be replaced by three-dimensional sorption isotherm surfaces. In order to describe the isotherm surfaces mathematically, three Langmuir-type models were evaluated. The apparent one-parameter Langmuir constant (b) was used to quantify FCAN2 "affinity" for one metal in the presence of another one. The uptake of Zn decreased drastically when Cu or Cd were present. The uptake of Cd wasmuch more sensitive to the presence of Cu than to that of Zn. The presence of Cd and Zn alter the "affinity" of FCAN2 for Cu the least at high Cu equilibrium concentrations. The mathematical model of the two-metal sorption system enabled quantitative estimation of one-metal (bio)sorption inhibition due to the influence of a second metal. (c) 1995 John Wiley & Sons Inc.     

Intracellular flux analysis in hybridomas using mass balances and in vitro (13)C nmr

Intracellular fluxes are important in defining cellular physiology and its changes in response to environmental variations. Stoichiometric balances combined with extra cellular metabolite measurements were applied to the estimation of intracellular fluxes and the study of energy metabolism in the hybridoma cell line ATCC CRL 1606. Redundant measurements allowed the evaluation of the consistency of the stoichiometry, measurements, and pseudo-steady-state assumption leading to refinement of the assumed biochemistry and identification of measurement errors. To validate the flux estimates, two batch experiments were performed with glucose labeled in the 1 position with (13)C. The distribution of (13)C in secreted lactate was measured via nuclear magnetic resonance spectroscopy (NMR) and compared to that predicted from the estimated intracellular fluxes. There was good agreement between the measured and estimated isotope distributions, demonstrating the validity of the flux estimates obtained from stoichiometric balances. (c) 1995 John Wiley & Sons, Inc.     

Liver fatty acid binding protein enhances sterol transfer by membrane interaction

Among the large family of fatty acid binding proteins, the liver L-FABP is unique in that it not only binds fatty acids but also interacts with sterols to enhance sterol transfer between membranes. Nevertheless, the mechanism whereby L-FABP potentiates intermembrane sterol transfer is unknown. Both fluorescence and dialysis data indicate L-FABP mediated sterol transfer between L-cell fibroblast plasma membranes occurs by a direct membrane effect: First, dansylated-L-FABP (DNS-L-FABP) is bound to L-cell fibroblast plasma membranes as indicated by increased DNS-L-FABP steady state polarization and phase resolved limiting anisotropy. Second, coumarin-L-FABP (CPM-L-FABP) fluorescence lifetimes were significantly increased upon interaction with plasma membranes. Third, dialysis studies with³H-cholesterol loaded plasma membranes showed that L-FABP added to the donor compartment of the dialysis cell stimulated³H-cholesterol transfer whether or not the dialysis membrane was permeable to L-FABP. However, L-FABP mediated intermembrane sterol transfer did require a sterol binding site on L-FABP. Chemically blocking the ligand binding site also inhibited L-FABP activity in intermembrane sterol transfer. Finally, L-FABP did not act either as an aqueous carrier or in membrane fusion. The fact that L-FABP interacted with plasma membrane vesicles and required a sterol binding site was consistent with a mode of action whereby L-FABP binds to the membrane prior to releasing sterol from the bilayer.Abbreviations ³H-CHO [1,2-³H(N)]-cholesterol - ANTS 8-aminonaphthalene-1,3,6-trisulfonic acid - CF carboxyfluorescein - CHO cholesterol - CPM (coumarin maleimide) 7-diethylamino-3-(4-maleimidylphenyl)-4-methylcoumarin - cPNA cisparinaric acid - DHE (dehydroergosterol) ^5,7,9(11),22-ergostatetraen-3-ol - DMF dimethyl formamide - DMPOPOP 1,4-bis[4-methyl-5-phenyl-2-oxazolyl]benzene - DNS (dansyl chloride) 5-dimethylaminonaphthalene-1-sulfonylchloride - DPX p-xylene-bis-pyridinium bromide - FBS fetal bovine serum - fluorescamine 4-phenylspiro[furan-2(3H), 1 phthalan]-3,3-dione - L-FABP liver fatty acid binding protein - NPG p-nitrophenylglyoxal - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine - SUV small unilamellar vesicle(s) - TNM tetranitromethane This work was supported in part by the National Institutes of Health United States Public Health Service (GM31651 and DK41402) and the American Heart Association (Postdoctoral Fellowship to JKW). The helpful assistance of Dr. Scott M. Colles and Mr. Daniel R. Prows in isolating L-FABP was much appreciated.