ATP Values Research Articles

Catalytic Site Modifications of TAP1 and TAP2 and Their Functional Consequences

The transporter associated with antigen processing (TAP), a member of the ATP binding cassette (ABC) family of transmembrane transporters, transports peptides across the endoplasmic reticulum membrane for assembly of major histocompatibility complex class I molecules. Two subunits, TAP1 and TAP2, are required for peptide transport, and ATP hydrolysis by TAP1.TAP2 complexes is important for transport activity. Two nucleotide binding sites are present in TAP1.TAP2 complexes. Compared with other ABC transporters, the first nucleotide binding site contains non-consensus catalytic site residues, including Asp(668) in the Walker B region of TAP1 (in place of a highly conserved glutamic acid), and Gln(701) in the switch region of TAP1 (in place of a highly conserved histidine). At the second nucleotide binding site, a glutamic acid (TAP2 Glu(632)) follows the Walker B motif, and the switch region contains a histidine (TAP2 His(661)). We found that alterations at Glu(632) and His(661) of TAP2 significantly reduced peptide translocation and/or TAP-induced major histocompatibility complex class I surface expression. Alterations of TAP1 Asp(668) alone or in combination with TAP1 Gln(701) had only small effects on TAP activity. Thus, the naturally occurring Asp(668) and Gln(701) alterations of TAP1 are likely to contribute to attenuated catalytic activity at the first nucleotide binding site (the TAP1 site) of TAP complexes. Due to its enhanced catalytic activity, the second nucleotide binding site (the TAP2 site) appears to be the main site driving peptide transport. A mechanistic model involving one main active site is likely to apply to other ABC transporters that have an asymmetric distribution of catalytic site residues within the two nucleotide binding sites.

Predicting Risk of Infection and Graft vs. Host Disease (GvHD), and Response to Immunosuppression (ISP) in Allogeneic Hematopoietic Stem Cell Transplant (HSCT) Patients (pts) Using the Cylex®ImmuKnow® Assay: A Pilot Study.

Background: GvHD, intensity of ISP and infection are major causes of toxicity post-HSCT. Yet, no method reliably predicts these complications. A functional assay for immunity, which predicts risk of GvHD and infection and allows tailoring ISP and infection prophylaxis, is needed. The ImmuKnow is an assay of global T-cell function that measures increased ATP production after stimulating CD4+ T-helper cells and has been shown useful for managing ISP therapies in solid-organ transplantation. We hypothesized that ImmuKnow could assist in predicting risk of GvHD and infection and response to ISP post-HSCT.Patients and Methods: Between January 2003 and May 2006, 34 adult allogeneic HSCT pts [mean age 47 years (range 22–71); 53% females; acute myelogenous leukemia, 8 pts; myeloma, 6 pts;] underwent blood sampling for determining ImmuKnow at various times post-HSCT (238 assays, mean of 7/pt). Results were correlated with clinical course (infection, stable, GvHD, response to ISP).Results: During the testing period, GvHD was documented in 16 pts [(acute, n=7 pts; chronic, n=9 pts] and 53 infections occurred [CMV viremia (n=20); bacteremia (n=20); others (n=13)]. The median immune function (ng/mL ATP) of GvHD pts was significantly higher than that of clinically stable pts (342 +/−105 vs. 167 ng/mL +/−136 respectively), p<0.001. Pts with infections had a median 89 ng/mL ATP (+/−119), also statistically different (p=0.002) from stable pts. A pt with ATP value ≥ 300 ng/mL was more likely (RR=4) to develop GvHD than one with lower immune response. Similarly, there was a higher risk (RR=5) for infection in pts with an ATP ≤ 80 ng/mL. Relative risk curves for infection and GvHD intersected in the low immune response zone (140 ng/mL ATP). Following therapy for GvHD in 10 pts, ATP values rapidly decreased (193 ng/mL ATP +/−122) over 17 days (range 3–35) and were associated with improvement, while stable or increasing values (50 ng/mL ATP +/−99) were observed among 6 pts who failed to respond to GvHD therapy.Conclusions: these data suggest that the FDA-approved ImmuKnow assay is an objective marker of risk for infection and GvHD, and of responsiveness to ISP therapy post-HSCT. Tailoring intensity and duration of ISP to risks of GvHD and infection in an individual pt may be possible and may improve outcomes. Validation of these preliminary data is ongoing.

Nuclear Ca++-influx, Ca++/Calmodulin-Dependent Protein Kinase IV Activity and CREB Protein Phosphorylation during Post-Hypoxic Reoxygenation in Neuronal Nuclei of Newborn Piglets: The Role of Nitric Oxide

The present study tests the hypothesis that post-hypoxic reoxygenation results in an nitric oxide (NO)-mediated increase in nuclear Ca(++)-influx, increased calmodulin kinase (CaM kinase) IV activity, and increased Ser(133) phosphorylation of cyclic AMP response element binding (CREB) protein in neuronal nuclei of the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx), hypoxic (Hx, FiO(2) = 0.07 for 1 h), hypoxic with 6 h reoxygenation (Hx + reox), and Hx + reox injected with 7-nitroindazole sodium salt (7-NINA), a nNOS inhibitor, immediately after hypoxia (Hx + 7-NINA). Cerebral tissue hypoxia was documented by ATP and phosphocreatine (PCr) levels. Nuclear Ca(++)-influx was determined using (45)Ca(++) and CaM kinase IV activity determined by (33)P-incorporation into syntide-2. Ser(133) phosphorylation of CREB protein was determined by Western blot analysis using a specific anti-phosphorylated Ser(133)-CREB protein antibody. ATP and PCr values in Hx, Hx + reox, and Hx + 7-NINA were significantly different from Nx (P < 0.05 versus Nx). Ca(++)-influx (pmoles/mg protein/min) was 3.79 +/- 0.91 in Nx; 11.81 +/- 2.54 in Hx (P < 0.05 versus Nx), 16.55 +/- 3.55 in Hx + reox (P < 0.05 versus Nx), and 12.40 +/- 2.93 in Hx + 7-NINA (P = NS versus Hx). CaM kinase IV activity (pmoles/mg protein/min) was 1,220 +/- 76 in Nx, 2,403 +/- 254 in Hx (P < 0.05 versus Nx), 1,971 +/- 147 in Hx + reox (P < 0.05 versus Hx), and 1,939 +/- 125 Hx + 7-NINA (P < 0.05 versus Hx). Ser(133) phosphorylated CREB protein expression (OD x mm(2)) was 87 +/- 2 in Nx, 203 +/- 24 in Hx (P < 0.05 versus Nx), 186 +/- 23 in Hx + reox (P < 0.05 Nx, P = NS versus Hx), and 128 +/- 10 in Hx + 7-NINA (P < 0.05 versus Hx and Hx + reox). The results show that post-Hx administration of 7-NINA prevents the increased nuclear Ca(++)-influx and CREB protein phosphorylation at Ser(133) during reox. We conclude that post-Hx increase in nuclear Ca(++)-influx leading to increased phosphorylation of CREB protein is mediated by NO derived from nNOS. However, hypoxia-induced increase in CaM Kinase IV activity decreased during the post-Hx reox. We propose that hypoxia-induced increase in CaM Kinase IV activity leads to increased phosphorylation of CREB protein and transcription of proapoptotic genes during post-Hx reox resulting in Hx neuronal death.

Structural Analysis of Protein Kinase A Mutants with Rho-kinase Inhibitor Specificity

Controlling aberrant kinase-mediated cellular signaling is a major strategy in cancer therapy; successful protein kinase inhibitors such as Tarceva and Gleevec verify this approach. Specificity of inhibitors for the targeted kinase(s), however, is a crucial factor for therapeutic success. Based on homology modeling, we previously identified four amino acids in the active site of Rho-kinase that likely determine inhibitor specificities observed for Rho-kinase relative to protein kinase A (PKA) (in PKA numbering: T183A, L49I, V123M, and E127D), and a fifth (Q181K) that played a surprising role in PKA-PKB hybrid proteins. We have systematically mutated these residues in PKA to their counterparts in Rho-kinase, individually and in combination. Using four Rho-kinase-specific, one PKA-specific, and one pan-kinase-specific inhibitor, we measured the inhibitor-binding properties of the mutated proteins and identify the roles of individual residues as specificity determinants. Two combined mutant proteins, containing the combination of mutations T183A and L49I, closely mimic Rho-kinase. Kinetic results corroborate the hypothesis that side-chain identities form the major determinants of selectivity. An unexpected result of the analysis is the consistent contribution of the individual mutations by simple factors. Crystal structures of the surrogate kinase inhibitor complexes provide a detailed basis for an understanding of these selectivity determinant residues. The ability to obtain kinetic and structural data from these PKA mutants, combined with their Rho-kinase-like selectivity profiles, make them valuable for use as surrogate kinases for structure-based inhibitor design.

Effect of nitric oxide synthase inhibition during post-hypoxic reoxygenation on Bax and Bcl-2 protein expression and DNA fragmentation in neuronal nuclei of newborn piglets

Previous studies have shown that cerebral tissue hypoxia results in increased generation of oxygen-free radicals including nitric oxide (NO), expression of the proapoptotic protein Bax and fragmentation of nuclear DNA. The present study tests the hypothesis that post-hypoxic reoxygenation for 6 h following hypoxia (FiO 2 = 0.06 for 1 h) results in continued hypoxia-induced, NO-mediated expression of the Bax protein and nuclear DNA fragmentation in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx), hypoxic (Hx, FiO 2 = 0.06 for 1 h), hypoxic with 6 h reoxygenation (Hx + reox) and hypoxic with 6 h reoxygenation injected with 7-nitroindazole sodium salt (7-NINA), a selective nNOS inhibitor, immediately after hypoxia (Hx + 7-NINA). Cerebral tissue hypoxia was documented by levels of ATP and phosphocreatine (PCr). Bax and Bcl-2 were analyzed by Western blot and DNA fragmentation was determined by agarose gel electrophoresis. ATP and PCr values in Hx, Hx + reox and Hx + 7-NINA were significantly different from Nx ( P < 0.05 vs. Nx). Bax protein (OD × mm 2) was 128.9 ± 38.7 in Nx; 223.6 ± 45.8 in Hx ( P < 0.05 vs. Nx); 340.5 ± 73.2 in Hx + reox ( P < 0.05 vs. Nx, Hx and Hx + 7-NINA); and 202.2 ± 34.8 in Hx + 7-NINA ( P = NS vs. Hx). Bcl-2 protein (OD × mm 2) was 14.9 ± 2.7 in Nx, 12.4 ± 2.1 in Hx, ( P < 0.05 vs. Nx), 15.7 ± 3.8 in Hx + reox, ( P < 0.05 vs. Hx) and 13.1 ± 2.2 in Hx + 7-NINA ( P = NS among groups). Nuclear DNA fragmentation (OD × mm 2) was 147 ± 15 in Nx; 797 ± 84 in Hx ( P < 0.05 vs. Nx); 1134 ± 127 in Hx + reox ( P < 0.05 vs. Nx, Hx and Hx + 7-NINA); and 778 ± 146 in Hx + 7-NINA ( P = NS vs. Hx, P < 0.05 vs. Hx + reox). The results show that post-hypoxic reoxygenation results in increased expression of Bax protein without affecting Bcl-2 protein and increased fragmentation of nuclear DNA, which are prevented by 7-NINA. We conclude that during post-hypoxic reoxygenation the increase in Bax protein expression and fragmentation of nuclear DNA are mediated by NO derived from nNOS. We propose that in addition to NO-mediated nuclear DNA damage, the hypoxia-induced increased ratio of Bax/Bcl-2 protein will lead to caspase-activated cascade of hypoxic neuronal death during post-hypoxic reoxygenation.

Epixylic biofilm and invertebrate colonization on submerged pine branches in a regulated lowland stream

Epixylic biofilm and invertebrate assemblages on pine branches (Pinus syl- vestris) experimentally submerged in October 2000 were investigated, after an initial colonization period of 3 months, by two-monthly sampling intervals for 13 months in a nutrient-rich sandy lowland stream subjected to flow disturbances caused by infre- quent impoundment openings. After 12 weeks of exposure an epixylic biofilm with 0.58 ± 0.25 µg/cm 2 chlorophyll-a (mean ± 1 SE) and 0.23 ± 0.04 µg/cm 2 ATP, as well as an abundant invertebrate community (14326 ± 2532 Ind/m 2 ; biomass: 974.6 ± 360.1 mg/m 2 ) became established on the branch surfaces. During the subsequent sam- pling dates invertebrate numbers decreased significantly during periods of high dis- charge and simultaneously chlorophyll-a values of epixylic biofilms increased, likely due to reduced feeding by invertebrate grazers. During periods with low discharge fluctuations and mainly low flow conditions, the epixylic biofilm, especially algal growth, was negatively correlated with invertebrate grazer and shredder numbers. After the initial growth period of 12 weeks, ATP values of the epixylic biofilm showed a strongly negative response to sand deposited on the wood surfaces. The present study displays the influence of seasonal discharge fluctuations and sand deposition on the wood surfaces on epixylic biofilms and invertebrate assembla- ges on experimentally submerged pine branches, but also indicate interactions between food sources (algae and fine particulate organic matter) and invertebrates (grazers and collector/gatherers).

Reduced ATP release from erythrocytes of humans with type 2 diabetes is increased by the phosphodiesterase 3 inhibitor cilostazol

The G protein, Gi, and adenylyl cyclase (AC) are components of a pathway for ATP release from erythrocytes (RBCs), a stimulus for vascular NO synthesis. Exposure to reduced oxygen tension (PO2) or Gi activation with mastoparan 7 (MAS7) results in ATP release from healthy human (HH) RBCs. Here, we investigate the hypothesis that ATP release from and cAMP accumulation in RBCs of humans with diabetes type 2 (DM2) are decreased and that a phosphodiesterase 3 (PDE3) inhibitor, cilostazol (CZ), attenuates these defects. ATP values are normalized to 4x105RBC/mm3 and cAMP values to 5x108RBC/mm3. In response to reduced PO2 (24±1 mm Hg), ATP release from DM2 RBCs (n=3) and HH RBCs (n=7) was 1±1 μM and 12±4 μM, respectively (P<0.01). MAS7-induced ATP release from DM2 RBCs (10 μM, n=8) was 0.3±0.1 μM compared to 0.9±0.2 μM from HH RBCs (n=8) (P<0.01). Pretreatment of DM2 RBCs with CZ (10 μM) increased MAS7-induced cAMP from 2.1 ±0.1 (n=9) to 2.9±0.9 pmole (n=7) (P<0.01) and ATP release from 0.3±1 to 0.5±1 μM (n=4) (P<0.05). Thus, both hypoxia- and MAS7-induced increases in ATP release are reduced in DM2 RBCs. Importantly, MAS7-induced ATP release and cAMP accumulation in DM2 RBCs were increased by CZ, a PDE3 inhibitor approved for human use in peripheral vascular disease. These findings are consistent with the hypothesis that decreased release of ATP from RBCs may contribute to reduced vascular reactivity in DM2 and that PDE3 inhibitors could improve this defect via a heretofore unrecognized effect on RBCs. Supported by NIH grants HL-64180 and HL-51298 and an ADA Innovation Award.

Ameliorating Injury during Preservation and Isolation of Human Islets Using the Two-Layer Method with Perfluorocarbon and UW Solution

This study assessed the effects of a two-layer method (TLM), using perfluorocarbon and UW solution, on the quality of human pancreata following storage and islet yield/function after isolation. In part A, TLM was applied immediately after procurement and the energetic profile was compared to a group treated with UW solution only (control) throughout 24-h storage. In part B, cadaveric human pancreata were procured and subjected to a TLM after cold storage in UW solution (TLM group) or UW solution (control group). Energetics, lipid peroxidation, and islet recovery/function were assessed after preservation at 4 degrees C. In part A, after 9-h storage, the energetic profile (ATP, ATP/ADP, energy charge) for the TLM group was superior to controls. In part B, TLM treatment resulted in consistently greater ATP, ATP/ADP, and energy charge values than with storage in UW solution alone (p < 0.05). UW treatment resulted in 40% greater peroxidative damage than in the TLM group (p < 0.05). Islet recovery and functional viability were 30-40% higher following TLM treatment (p < 0.05). These data support the hypothesis that islet viability and yields can be significantly improved using a brief period of TLM treatment following conventional UW storage; reduced energetic and oxidative stress are implicated as potential mechanisms.

Protective Effect of Diltiazem on Hepatic Ischemia-Reperfusion Injury in Rats by Improving Liver Tissue Blood Flow

Background Cytosolic calcium ions are known to play an important role in ischemia-reperfusion (IR) injury. However, the protective effect of calcium channel blockers remains controversial in liver IR injury. Moreover, calcium channel blockers improve hepatic IR injury not due to blocking an increase in hepatic calcium concentration. Therefore, we hypothesized that calcium antagonists protected a liver from IR injury by a vasodilatory action rather than by the inhibition of an increase in Ca 2+ within parenchymal cells. This study evaluated the effects of diltiazem, a calcium channel blocker, on liver energy metabolism and blood flow after IR injury. Methods Twenty-seven rats underwent hepatic ischemia for 30 minutes followed by 60 minutes of reperfusion. The animals were allocated into group C (without drug); group D5 (diltiazem, 5 μg/kg per min); or group D10 (diltiazem, 10 μg/kg per min). Diltiazem was infused before laparotomy and then throughout the experiment. Results After 60 minutes of reperfusion, liver tissue blood flow and ATP concentrations were significantly higher in group D10 than the other animals (both, P < .05). Changes in ATP values strongly correlated those observed in blood flow (R = 0.80, P < .001). Conclusion Diltiazem improved ATP-generating capacity during reperfusion by improving liver tissue blood flow. An improvement in hepatic tissue perfusion may be a therapeutic strategy for liver IR injury.

ATP Bioluminescence Assay for Estimation of Microbial Populations of Fresh-Cut Melon

Estimation of microbial numbers in foods by conventional microbiological techniques takes days, so there is a need for faster methods that can give results in minutes. Research was undertaken to investigate the use of bioluminescent ATP determination and a firefly luciferase assay to estimate the initial population of aerobic mesophilic bacteria on fresh-cut melons immediately after preparation and during storage at 5 or 15°C for up to 12 days. Populations of aerobic mesophilic bacteria on fresh-cut cantaloupe prepared immediately from unsanitized whole melons averaged 3.42 log CFU/g, corresponding to an ATP value of 5.40 log fg/g. Populations for fresh-cut honeydew prepared from unsanitized whole melon averaged 1.97 log CFU/g, corresponding an ATP value of 3.94 log fg/g. Fresh-cut pieces prepared from cantaloupe or honeydew melons sanitized with either chlorine (200 ppm free chlorine) or hydrogen peroxide (2.5%) had similar ATP values: 3.1 log fg/g (corresponding to bacterial counts 1.7 log CFU/g) for cantaloupes and 2.6 log fg/g (corresponding to bacterial counts of 0.48 CFU/g) for fresh-cut honeydew. Positive linear correlations for ATP concentrations and microbial populations were found for fresh-cut cantaloupe (R2 = 0.99) and honeydew R2 = 0.95) during storage at 5°C for up to 12 days. ATP values in fresh-cut melons inoculated with either aerobic mesophilic bacteria or yeast and mold were significantly higher (P < 0.05) than control values and parallel total plate counts on plate count agar. Results of this study indicate that the bioluminescent ATP assay can be used to monitor total microbial populations on fresh-cut melon after preparation and during storage for quality control purposes to establish specific sell-by or consume-by dates.

THE ACTIVITY AND KINETICS OF PYRUVATE KINASE IN HYPOXIC NEWBORNS

Pyruvate kinase (PK) plays a key role in erythrocytes, which obtain most of their energy from glycolysis. This study investigated erythrocyte energy metabolism in hypoxic newborns, measuring pyruvate kinase activity, kinetic, and ATP levels in hypoxia. Forty-nine babies who had cord pH value lower than 7.2 and Apgar scores lower than 7 in the first minute were accepted as the hypoxic group, and 48 babies who had cord pH value higher than 7.2 and an Apgar score higher than 7 in the first minute were taken as controls. The erythrocyte mean PK activity was found to be lower (16.9 ± 8.5 [5.8−47.9] EU/gHb) in the hypoxic group than the control group (21.3 ± 10.9 [3.9−44.3] EU/gHb) (p <. 05). The mean ATP value of hypoxic group was higher (19.2 ± 11.3 [3.9−37.6] mM) compared to control group (13.8 ± 7.16 [3.9−28.7] mM). In the kinetic study, with different ADP concentrations in the control group, the substrate amount (Km) that is needed to reach the half-maximum of enzyme activity (Vmax = 27.7 Eu/gHb) was found to be 2.70 mM, but it was 1.47 mM to reach Vmax (22.7 Eu/gHb) in the hypoxic group. Vmax was 41.67 Eu/gHb and Km was 8.33 mM in the control group at different PEP concentrations, whereas Vmax was 21.7 Eu/gHb and Km was 0.89 mM in the hypoxic group. Increase in the ATP level while ePK activity decreases, suggesting that glycolysis increases in hypoxia. In the kinetic study, the substrate amount needed for reaching the half-maximum of enzyme activity was less in the hypoxic group, probably suggests that pyruvate kinase increases glycolysis by increasing its affinity to the substrates. In this way, erythrocytes may gain the energy required for oxygen delivery to tissues and maintaining ion gradient. This arrangement possibly proceeds from sygmoidal structure of pyruvate kinase.

Analysis of the catalytic mechanism of pyruvate dehydrogenase kinase

It has been proposed that “Glu238” within the N-box of pyruvate dehydrogenase kinase (PDK) is a base catalyst. The pH dependence of k cat of Arabidopsis thaliana PDK indicates that ionizable groups with p K values of 6.2 and 8.4 are necessary for catalysis, and the temperature dependence of these values suggests that the acidic p K is due to a carboxyl- or imidazole-group. The E238 and K241 mutants had elevated K m, ATP values. The acidic p K value of the E238A mutant was shifted to 5.5. The H233A, L234H, and L234A mutants had the same p K values as wild-type AtPDK, contrary to the previous proposal of a “Glu-polarizing” His. Instead, we suggest that the conserved Glu, Lys, and Asn residues of the N-box contribute to coordinating Mg 2+ in a position critical for formation of the PDK-MgATP-substrate ternary complex.

Effect of hypoxia on protein tyrosine kinase activity in cortical membranes of newborn piglets—the role of nitric oxide

Previous studies have shown that cerebral hypoxia results in increased tyrosine phosphorylation of cerebral cortical cell membrane proteins as well as nuclear membrane anti-apoptotic protein, Bcl-2. The present study tests the hypothesis that hypoxia results in increased protein tyrosine kinase activity in cortical cell membranes of newborn piglets and that the inhibition of neuronal NOS by administration of 7-nitroindazole sodium salt (7-NINA), a selective inhibitor of nitric oxide synthase (NOS), will prevent the hypoxia-induced increase in protein tyrosine kinase activity. To test this hypothesis, protein tyrosine kinase activity was determined in cerebral cortical membranes of 2- to 4-day-old newborn piglets divided into normoxic ( n = 6), hypoxic ( n = 5) and 7-NINA-treated hypoxic ( n = 5) (7-NINA, 1 mg/kg, i.p., prior to hypoxia) groups. Tissue hypoxia was achieved by exposing the animals to an FiO 2 of 0.07 for 60 min and was documented biochemically by determining tissue ATP and phosphocreatine (PCr) levels. Cortical P 2 membranes were isolated and protein tyrosine kinase activity determined by 33P incorporation into a specific peptide substrate for 15 min at 37 °C in a medium containing 100 mM HEPES, pH 7.0, 1 mM EDTA, 125 mM MgCl 2, 25 mM MnCl 2, 2 mM DTT, 0.2 mM sodium orthovanadate, 2 mM EGTA, 150 μM tyrosine kinase peptide substrate [Lys 19] cdc2(6–20)-NH 2, 33P-ATP, and 10 μg of membrane protein. Protein tyrosine kinase activity was determined by the difference between 33P incorporation in the presence and absence of specific peptide substrate and expressed as pmol/mg protein/h. The ATP values in the normoxic, hypoxic and 7-NINA-treated hypoxic animals were ATP: 4.57 ± 0.45 μmol/g, 1.29 ± 0.23 μmol/g ( p < 0.05 versus normoxic) and 1.50 ± 0.14 μmol/g brain ( p < 0.05 versus normoxic), respectively. The PCr values in the normoxic, hypoxic and 7-NINA-treated hypoxic animals were: 3.77 ± 0.36 μmol/g, 0.77 ± 0.13 μmol/g ( p < 0.05 versus normoxic) and 1.02 ± 0.24 μmol/g brain ( p < 0.05 versus normoxic), respectively. Protein tyrosine kinase activity in the normoxic, hypoxic and the 7-NINA-treated groups was 378 ± 77 pmol/mg protein/h, 854 ± 169 pmol/mg protein/h ( p < 0.05 versus normoxic) and 464 ± 129 pmol/mg protein/h ( p < 0.05 versus hypoxic), respectively. The data show that cerebral tissue hypoxia results in increased protein tyrosin kinase activity in cortical membranes of newborn piglets and pretreatment with 7-NINA prevents the hypoxia-induced increase in protein tyrosine kinase activity. We conclude that the hypoxia-induced increase in protein tyrosine kinase activity is NO-mediated. We propose that the hypoxia-induced increase in protein tyrosine kinase activity leading to increased phosphorylation of Bcl-2 is a critical link to hypoxic neuronal injury pathway.

Ablation of PLB exacerbates ischemic injury to a lesser extent in female than male mice: protective role of NO.

Recent studies suggest a role for phospholamban phosphorylation during ischemia and reperfusion. The role of phospholamban in ischemia was studied by subjecting hearts from male and female wild-type (MWT/FWT) and phospholamban-knockout (MKO/FKO) mice to 20 min of ischemia-40 min of reperfusion while (31)P NMR spectra were acquired. ATP and pH values fell lower during ischemia, and postischemic contractility was less, in MKO and FKO versus WT hearts. After shorter ischemia (15 min), recoveries of contraction, ATP, and pH were greater in FKO than MKO hearts. To examine the role of nitric oxide (NO) synthases (NOS) in the protection in FKO versus MKO hearts, we utilized 1 microM l-NAME, a NOS inhibitor, or 100 microM S-nitroso-N-acetylpenicillamine (SNAP), an NO donor. Recoveries of function, ATP, and pH were less in l-NAME-treated FKO than untreated FKO hearts and greater in SNAP-treated MKO than untreated MKO hearts. In conclusion, phospholamban ablation increased ischemic injury in both males and females; however, female hearts were less susceptible than male hearts. Protection in females was decreased by a NOS inhibitor and mimicked in males by an NO donor, implying that protection was NOS mediated.

THE PROTECTIVE POTENCY OF VITAMINS E AND C IN METHANOL-INDUCED OXIDATIVE STRESS AND RETINOTOXICITY

The protective effects of vitamins E and C against methanol-induced free radical production with its subsequent tissue injury in liver and retina of male albino rats were assessed. The rats were divided into four groups: (1) control, (2) antioxidant (Ao) control group receiving 5 mg of each vitamin, E and C, (3) daily ip-injected methanol (2 mL/kg b.wt.) group, killed after three and six doses, and (4) Ao/methanol group administered the vitamins 3 weeks prior to and along with methanol injection and killed as the latter group [Sharpe et al. Methanol Optic Neuropathy: A Histopathological Study. Neurology 1982, 32 (10), 1093–1100].Methyl alcohol drastically altered the Ao defense system and energy status of rat liver, where highly significant depletion of glutathione levels and inhibition of superoxide dismutase activity, concurrent with significant increase in thiobarbituric acid reactive substances indicating marked elevation in lipid peroxidation. These effects were reversed when the vitamins were administered denoting their role in promoting the Ao defense system. Furthermore, they also increased the methanol-induced depletion in adenylate energy charge, phosphate potential, and ATP values. The amelioration in the energy status as a result of vitamins E and C supplementation suggests that their role as Aos is effective in relieving the impaired oxidative phosphorylation in order to increase the energy demand under physiologically stressful conditions.Histopathological and ultrastructural results of rat retina confirmed the protective effect of Ao vitamins. As compared to the methanol-intoxicated group, the protected group showed preservation of the membranous structures of the retinal cells, especially mitochondria that assumed their normal shape. This may be attributed to the inhibition of free radical production and lipid peroxidation and subsequently minimum degree of tissue damage. Amelioration of mitochondrial structure reflected the improvement of impaired oxidative phosphorylation of intoxicated animals with an approximately normal level of energy demand. This suggests that Ao vitamins may alter the retinotoxic effects of methanol by promoting the internal Ao defense system and preserving the energy status of the animal.

Heavy metal concentrations and enzyme activities in soil from a contaminated Korean shooting range

Soil enzyme activities and heavy metal concentrations (Cd, Zn, Cu and Pb) were measured at a general shooting range in South Korea. Total heavy metal concentrations varied with pH, and were negatively correlated with moisture content and organic matter. Most enzyme activities (dehydrogenase, acid phosphatase and β-glucosidase) and ATP values (measure of biomass) were lower in polluted soils, especially in bald spots contaminated by heavy metals and TNT (2,4,6-trinitrotoluene). These data indicate that concentrated shooting activity inhibits enzyme activity and microbial growth. Soil enzyme activities in richly vegetated spots around the general shooting range were higher (2- to 3-fold) than in bald spots, but lower in areas of heavy metal contamination. Our results demonstrate that the dominant plant species in richly vegetated spots accumulated heavy metals from soils, and suggest certain plants may be useful for decontaminating polluted soil.

Estimation of ATP in soil: extraction methods and calculation of extraction efficiency

Methodological problems still exist when soil ATP is estimated using a specific extractant in different laboratories or when different extractants are compared in one laboratory. I hypothesised that this may possibly be caused by uncertainties inherent in addition of an ATP internal standard. Therefore, the reliability of this method was tested using 14 agricultural soils. I chose the TCA/HPO42−/paraquat extractant with sonification (Jenkinson and Oades, 1979) to test whether the recovery of an ATP standard added to the extractant represents the true loss of ATP during extraction process. For this purpose soils were repeatedly extracted with fresh extractant, until the amount of ATP reached the limit of detection in the luciferin–luciferase system. This was reached at the latest after six extraction steps. The summed ATP contents obtained in the six extracts were compared with the amount of ATP as estimated by the conventional way of a single extraction and correction for recovery of an added ATP standard. It was found that the latter procedure considerably underestimated the amount of soil ATP, because the recovery of standards underestimated losses. The ATP contents in the extracts 2–6 during the six-fold extraction procedure were by a factor of 2–9 higher than indicated by losses of added ATP standards after a single extraction. The amount of standard added to the extraxtant did not influence its recovery. It was shown that the application of fresh extractant is the decisive step for obtaining higher ATP values by multiple extraction and not a prolonged sonification. Since a repeated extraction of a soil sample is too time- and labour-consuming for practical application, a more convenient and efficient technique was required. Extraction with dimethylsufoxide (DMSO), Na3PO4 (pH 11.7) and NRB® as proposed by Bai et al. (1988) was found to be rapid and efficient. This extractant gave nearly identical ATP contents of soils as did the repeated extraction with the TCA/HPO42−/paraquat extractant. A useful indicator of ATP extraction efficiency is the biomass C–ATP ratio which was 208–217 for all soils and both methods. This good agreement between both methods, and the use of non toxic chemicals encourage me to propose the DMSO based extraction procedure as a suitable technique to estimate soil ATP in agricultural soils.

Rapid Assessment of the Bacteriological Quality of Raw Milk Using ATP Bioluminescence

Research was conducted to assess the practical use of an ATP bioluminescence assay to evaluate the bacteriological quality of raw milk. Filtration was used to precondition samples before ATP determination, which was measured in relative light units (RLUs). The Lumac ATP bioluminescence assay results were compared with standard plate counts (SPCs) of samples to estimate the microbial load for 246 raw milk samples that were split and either tested immediately or subjected to two preliminary incubation temperatures, 12.8 and 15.6°C, for 18 h. Linear regression analysis procedures were used to analyze the data. Preincubation treatments were analyzed separately. For all treatments, linear regression coefficients were significantly different from zero (P < 0.01). The R2 values calculated using log10-transformed SPC and log10-transformed RLUs for samples tested immediately and samples preliminarily incubated at 12.8 and 15.6°C were 0.58, 0.78, and 0.80, respectively. The R2 for all samples combined was 0.78. Differences in regressions among treatments were tested using a multiple slope and intercept model. Treatment intercepts and slopes were significantly different (P < 0.01). A linear regression equation was used to predict SPC from ATP values. Comparison of predicted values with actual SPCs indicated that ATP could be useful in predicting SPC in raw milk.

Measurement of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis

In this study the relative levels of ADP and ATP have been measured in cells undergoing apoptosis. Using HL60, CEM7, Jurkat and U937 cell lines and cytotoxic agents known to induce apoptosis, there was a significant correlation ( P<0.01 for all models) between the ADP:ATP ratio and the degree of apoptosis measured by TUNEL and estimation of the sub G 0 fraction by propidium iodide staining and flow cytometry. The ratio measured in viable proliferating cells was found to be less than 0.11 compared with ratios between 0.11 and 1.0 seen in cells undergoing apoptosis. The higher the percentage of hypodiploidy the greater the ratio. Necrosis induced by heat shock resulted in ADP:ATP ratios in excess of 15.0. When primary cultures of AML blast cells were used, there was again a significant correlation between the ADP:ATP ratio and the degree of hypodiploidy. Recent evidence suggests that apoptosis is accompanied by opening of the mitochondrial permeability pores, leading to disruption of the mitochondrial transmembrane potential (Δ Ψ m). This results in caspase activation due to the release of cytochrome c and apoptogenic factors into the cytosol. In five experiments using CEM7 and dexamethasone the mitochondrial transmembrane potential was assessed using the fluorescent cyanine dye JC-1 and flow cytometry. Functioning mitochondria concentrate the JC-1 to produce red fluorescence. Loss of mitochondrial transmembrane potential results in green fluorescence only. The percentage of cells exhibiting red fluorescence correlated positively with the ATP values and negatively with the ADP:ATP ratio.

Metabolism of Lactococcus lactis subsp. cremoris MG 1363 in acid stress conditions.

The metabolism of glucose by Lactococcus lactis subsp. cremoris MG 1363 remains homolactic whatever the pH of the culture medium. The growth rate decreased with the acidification of the medium until a limit pH value of 4.0 for which no growth was observed. In contrast, the specific rate of glucose consumption decreased only for very low pH values, i.e., below 4.5. The efficiency of biomass synthesis relative to the energy supply decreased when the medium pH diminished, as illustrated by Y ATP values. This observation was related to the increase in both components of the proton-motive force when the pH decreased. The growth stopped when the internal pH reached a limit value of 5.4 due to organic acid accumulation.