Ascorbic acid and copper in linoleate oxidation. II. Ascorbic acid and copper as oxidation catalysts

Both ascorbic acid and copper were strong prooxidants in the oxidation of linoleate in a buffered (pH 7.0) aqueous dispersion at 37 degrees C. Minimum concentrations at which catalytic activity was detected were 1.3 x 10(-7) m for copper and 1.8 x 10(-6) m for ascorbic acid. For concentrations up to 10(-3) m, the increase in rate of oxidation with increase in concentration of catalyst was greater for ascorbic acid than for copper. Ascorbic acid had maximum catalytic activity at 2.0 x 10(-3) m, but was still prooxidant at the highest concentration tested (5.0 x 10(-2) m). Dehydroascorbic acid was a weaker prooxidant than ascorbic acid. Further degradation products of ascorbic acid were not prooxidant. In early stages of the oxidation autocatalytic behavior was observed with copper, but not with ascorbic acid. Ascorbic acid functioned as a true catalyst, i.e., it accelerated the reaction but it was not oxidized simultaneously with the linoleate. It is proposed that the dehydroascorbic acid radical initiates the linoleate oxidation reaction.     

Depolarization-Dependent Protein Phosphorylation in Rat Cortical Synaptosomes: Characterization of Active Protein Kinases by Phosphopeptide Analysis of Substrates

Depolarization of synaptosomes is known to cause a calcium-dependent increase in the phosphorylation of a number of proteins. It was the aim of this study to determine which protein kinases are activated on depolarization by analyzing the incorporation of 32Pi into synaptosomal phosphoproteins and phosphopeptides. The following well-characterized phosphoproteins were chosen for study: phosphoprotein "87K," synapsin Ia and Ib, phosphoproteins IIIa and IIIb, the catalytic subunits of calmodulin kinase II, and the B-50 protein. Each was initially identified as a phosphoprotein in lysed synaptosomes after incubation with [gamma-32P]ATP. Mobility on two-dimensional polyacrylamide gels and phosphorylation by specific protein kinases were the primary criteria used for identification. A technique was developed that allowed simultaneous analysis of the phosphopeptides derived from all of these proteins. Phosphopeptides were characterized in lysed synaptosomes after activating cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases in the presence of [gamma-32P]ATP. Phosphoproteins labelled in intact synaptosomes after incubation with 32Pi were then compared with those seen after ATP-labelling of lysed synaptosomes. As expected from previous work, phosphoprotein "87K," and synapsin Ia and Ib were labelled, but for the first time, phosphoproteins IIIa, IIIb, and the B-50 protein were identified as being labelled in intact synaptosomes; the calmodulin kinase II subunits were hardly phosphorylated. From a comparison of the phosphopeptide profiles it was found that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases are all active in intact synaptosomes and their activity is dependent on extrasynaptosomal calcium. The activation of cyclic AMP-stimulated protein kinases in intact synaptosomes was confirmed by the addition of dibutyryl cyclic AMP and theophylline which specifically increased the labelling of phosphopeptides in synapsin Ia and Ib and in phosphoproteins IIIa and IIIb. On depolarization of intact synaptosomes, a number of phosphopeptides showed increased labelling and the pattern suggested that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases were all activated. No new peptides were phosphorylated, suggesting that depolarization simply increased the activity of already active protein kinases and that there was no depolarization-specific increase in protein phosphorylation.     

The heterochromatin of grasshoppers from the Caledia captiva species complex. I. Sequence evolution and conservation in a highly repeated DNA family

The restriction enzyme TaqI digests 0.2% of the genomic DNA from the grasshopper Caledia captiva to a family of sequences 168 bp in length (length of consensus sequence). The sequence variation of this "Taq family" of repeat units was examined among four races from C. captiva to assay the pattern of evolution within this highly repeated DNA. The Taq-family repeats are located in C-banded heterochromatin on at least one member of each homologous pair of chromosomes; the locations range from centromeric to telomeric. Thirty-nine cloned repeats isolated from two population 1A individuals along with 11 clones from seven populations taken from three of the races demonstrated sequence variation at 72 positions. Pairwise comparisons of the cloned repeats, both within an individual and between different races, indicate that levels of intraspecific divergence, as measured by reproductive incompatibility, do not correlate with sequence divergence among the 168-bp repeats. A number of subsequences within the repeat remain unchanged among all 50 clones; the longest of these is 18 bp. That the same 18-bp subsequence is present in all clones examined is a finding that departs significantly (P less than 0.01) from what would be expected to occur at random. Two other cloned repeats, from a reproductively isolated race of C. captiva, have sequences that show 56% identity with this 18-bp conserved region. An analysis showed that the frequency of occurrence of an RsaI recognition site within the 168- bp repeat in the entire Taq family agreed with that found in the cloned sequences. These data, along with a partial sequence for the entire Taq family obtained by sequencing uncloned repeats, suggest that the consensus sequence from the cloned copies is representative of this highly repeated family and is not a biased sample resulting from the cloning procedure. The 18-bp conserved sequence is part of a 42-bp sequence that possesses dyad symmetry typical of protein-binding sites. We speculate that this may be significant in the evolution of the Taq family of sequences.      

Distribution of Calmodulin- and Cyclic AMP-Stimulated Protein Kinases in Synaptosomes

The subcellular location of calmodulin- and cyclic AMP stimulated protein kinases was assessed in synaptosomes which were prepared on Percoll density gradients. The distribution of the protein kinases between the outside and the inside and between the soluble and membrane fractions was determined by incubating intact and lysed synaptosomes, as well as supernatant and pellet fractions obtained from lysed synaptosomes, in the presence of [gamma-32P]ATP. Protein kinase activity was assessed by the labelling of endogenous proteins, or exogenous peptide substrates, under conditions optimized for either calmodulin- or cyclic AMP-stimulated protein phosphorylation. When assessed by calmodulin-stimulated autophosphorylation of the alpha subunit of calmodulin kinase II, 44% of this enzyme was on the outside of synaptosomes, and 41% was in the 100,000 g supernatant. Using an exogenous peptide substrate, the distribution of total calmodulin-stimulated kinase activity was 27% on the outside and 34% in the supernatant. The high proportion of calmodulin kinase II on the outside of synaptosomes is consistent with its known localization at postsynaptic densities. The proportion of calmodulin kinase II which was soluble depended on the ionic strength conditions used to prepare the supernatant, but the results suggest that a major proportion of this enzyme which is inside synaptosomes is soluble. When assessed by cyclic AMP-stimulated phosphorylation of endogenous substrates, no cyclic AMP-stimulated kinase activity was observed on the outside of synaptosomes, whereas 21% was found with an exogenous peptide substrate. This suggests that if endogenous substrates are present on the outside of synaptosomes, then the enzyme does not have access to them. The cyclic AMP-stimulated protein kinase present inside synaptosomes was largely bound to membranes and/or the cytoskeleton, with only 10% found in the supernatant when assessed by endogenous protein phosphorylation and 25% with an exogenous substrate. The markedly different distribution of the calmodulin- and cyclic AMP-stimulated protein kinases presumably reflects differences in the functions of these enzymes at synapses.     

Neurobiological consequences of acute footshock stress: effects on tyrosine hydroxylase phosphorylation and activation in the rat brain and adrenal medulla

Stress activates selected neuronal systems in the brain and this leads to activation of a range of effector systems. Our aim was to investigate some of the relationships between these systems under basal conditions and over a 40‐min period in response to footshock stress. Specifically, we investigated catecholaminergic neurons in the locus coeruleus (LC), ventral tegmental area and medial prefrontal cortex (mPFC) in the brain, by measuring tyrosine hydroxylase (TH) protein, TH phosphorylation and TH activation. We also measured the effector responses by measuring plasma adrenocorticotrophic hormone, corticosterone, glucose and body temperature as well as activation of adrenal medulla protein kinases, TH protein, TH phosphorylation and TH activation. The LC, ventral tegmental area and adrenal medulla all had higher basal levels of Ser19 phosphorylation and lower basal levels of Ser31 phosphorylation than the mPFC, presumably because of their cell body versus nerve terminal location, while the adrenal medulla had the highest basal levels of Ser40 phosphorylation. Ser31 phosphorylation was increased in the LC at 20 and 40 min and in the mPFC at 40 min; TH activity was increased at 40 min in both tissues. There were significant increases in body temperature between 10 and 40 min, as well as increases in plasma adrenocorticotropic hormone at 20 min and corticosterone and glucose at 20 and 40 min. The adrenal medulla extracellular signal‐regulated kinase 2 was increased between 10 and 40 min and Ser31 phosphorylation was increased at 20 min and 40 min. Protein kinase A and Ser40 phosphorylation were increased only at 40 min. TH activity was increased between 20 and 40 min. TH protein and Ser19 phosphorylation levels were not altered in any of the brain regions or adrenal medulla over the first 40 min. These findings indicate that acute footshock stress leads to activation of TH in the LC, pre‐synaptic terminals in the mPFC and adrenal medullary chromaffin cells, as well as changes in activity of the hypothalamic‐pituitary‐adrenal axis.

     

Microhabitats of sharknose goby (Elacatinus evelynae) cleaning stations and their links with cleaning behaviour

Coral Reefs - Coral reefs are renowned for the complexity of their habitat structures and their resulting ability to host more species per unit area than any another marine ecosystem. Dedicated...     

Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease

A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12-15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.     

Growth and genetic responses of Salmonella Typhimurium to pH-shifts in an anaerobic continuous culture

Salmonella infection of chickens that leads to potential human foodborne salmonellosis continues to be a concern. Changes in the pH of poultry gastrointestinal tract could influence Salmonella growth and virulence response. In the current study, growth responses of a chicken isolate Salmonella enterica serovar Typhimurium (ST) to three incremental pH-shifts (6.17-7.35) in continuous cultures (CC) were evaluated. The expression of rpoS and hilA was determined by real time-polymerase chain reaction (RT-PCR) as well. Increases in pH resulted in higher cell protein concentrations, glucose disappearance, and glucose and ATP yields. Although with some inconsistency between the two trials, the data indicated that the ammonia release into media was favored by low pH. The pH shifts did not significantly affect acetate biosynthesis. No consistent trends of pH influence on propionate and butyrate production could be detected. In all three pH shifts, relative expression of hilA was dominant at 0h which represented CC steady state. In pH shift 7.35-6.86 (Trial 1), the relative expression of rpoS at time 0 and 1h were over five-fold higher than after 3 and 6h of growth. Overall, the results suggest that ST physiology is altered by changes in pH, which could be determinant factors for ST survival in the poultry gastrointestinal ecosystems.     

Angiotensin II promotes the phosphorylation of cyclic AMP‐responsive element binding protein (CREB) at Ser133 through an ERK1/2-dependent mechanism

In cells from the adrenal medulla, angiotensin II (AII) regulates both the activity and mRNA levels of catecholamine biosynthetic enzymes whose expression is thought to be under the control of cAMP-responsive element (CRE) binding protein (CREB). In this study, we evaluated the effect of AII stimulation on CREB phosphorylation at Ser133 (pCREB) in bovine adrenal chromaffin cells (BACC). We found that AII produces a rapid and AII type-1 receptor (AT1)-dependent increase in pCREB levels, which is blocked by the MEK1/2 inhibitor U0126 but not by H-89, SB203580 or KN-93, suggesting that it is mediated by the extracellular-regulated protein kinases 1 and 2 (ERK1/2) and not by cAMP-dependent protein kinase (PKA), p38 mitogen-activated protein kinase (p38MAPK) or Ca(2+)/calmodulin-dependent protein kinases (CaMKs) dependent pathways. Gel-shift experiments showed that the increase in pCREB levels is accompanied by an ERK1/2-dependent upregulation of CRE-binding activity. We also found that AII promotes a rapid and reversible increase in the activity of the non-receptor tyrosine kinase Src and that the inhibition of this enzyme completely blocks the AII-induced phosphorylation of ERK1/2, the CREB kinase (p90)RSK and CREB. Our data support the hypothesis that in BACC, AII upregulates CREB functionality through a mechanism that requires Src-mediated activation of ERK 1/2 and (p90)RSK.