A rapid Percoll gradient procedure for preparation of synaptosomes

Homogenization of fresh brain tissue in isotonic medium shears plasma membranes causing nerve terminals to become separated from their axons and postsynaptic connections. The nerve terminal membranes then reseal to form synaptosomes. The discontinuous Percoll gradient procedure described here is designed to isolate synaptosomes from brain homogenates in the minimum time to allow functional experiments to be performed. Synaptosomes are isolated using a medium-speed centrifuge, while maintaining isotonic conditions and minimizing mechanically damaging resuspension steps. This protocol has advantages over other procedures in terms of speed and by producing relatively homogeneous synaptosomes, minimizing the presence of synaptic and glial plasma membranes and extrasynaptosomal mitochondria. The purified synaptosomes are viable and take up and release neurotransmitters very efficiently. A typical yield of synaptosomes is between 2.5 and 4 mg of synaptosomal protein per gram rat brain. The procedure takes approximately 1 h from homogenization of the brain until collection of the synaptosomal suspension from the Percoll gradient.     

Mucosal immunology down under: Special Interest Group in Mucosal Immunology workshop, Australasian Society for Immunology, Sydney, Australia, 2 December 2007

The Mucosal Immunology Special Interest Group (SIG-MI) of the Australasian Society of Immunology was formed 14 years ago and has run regular symposia and workshops in conjunction with the Australasian Society of Immunology since that time. In December 2007 the Mucosal Immunology Special Interest Group held a 1-day satellite workshop in conjunction with the annual Australasian Society of Immunology scientific meeting in Sydney to celebrate the decade since hosting the 9th International Congress of Mucosal Immunology (9-ICMI) in 1997, which was also held in Sydney. The meeting that was attended by 65 delegates focussed on 4 session themes: reproductive immunology, respiratory immunology, mucosal immunology of the gastrointestinal tract and mucosal vaccines.     

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells: the role of MAPKs after angiotensin II stimulation

Angiotensin II (AII, 100 nM) stimulation of bovine adrenal chromaffin cells (BACCs) produced angiotensin II receptor subtype 1 (AT1)-mediated increases in extracellular regulated protein kinase 1/2 (ERK1/2) and stress-activated p38MAPK (p38 kinase) phosphorylation over a period of 10 min. ERK1/2 and p38 kinase phosphorylation preceded Ser31 phosphorylation on tyrosine hydroxylase (TOH). The inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) activation, PD98059 (0.1-50 microM) and UO126 (0.1-10 microM), dose-dependently inhibited both ERK2 and Ser31 phosphorylation on TOH in response to AII, suggesting MEK1/2 involvement. The p38 kinase inhibitor SB203580 (20 microM, 30 min) abolished Ser31 and Ser19 phosphorylation on TOH and partially inhibited ERK2 phosphorylation produced by AII. In contrast, 1 microM SB203580 did not affect AII-stimulated TOH phosphorylation, but fully inhibited heat shock protein 27 (HSP27) phosphorylation produced by AII. Also, 1 microM SB203580 fully inhibited Ser19 phosphorylation on TOH and HSP27 phosphorylation in response to anisomycin (30 min, 10 microg/mL). The results suggest that ERKs mediate Ser31 phosphorylation on TOH in response to AII, but p38 kinase is not involved. Previous studies suggesting a role for p38 kinase in the phosphorylation of Ser31 are explained by the non-specific effects of 20 microM SB203580 in BACCs. The p38 kinase pathway is able to phosphorylate Ser19 on TOH in response to anisomycin, but does not do so in response to AII.     

Determination of phosphorylation levels of tyrosine hydroxylase by electrospray mass spectrometry

A novel approach has been developed to quantify the extent of phosphorylation of tyrosine hydroxylase (TH). The strategy consists of a chemical cleavage and characterization of the products using electrospray mass spectrometry (ESMS). The chemical cleavage involves selective hydrolysis of the aspartyl-peptide bond. Of the peptides formed, an 8-kDa NH2-terminus fragment is found to accurately duplicate the phosphorylation of TH using standard mixtures of TH-P/TH. The calibration yields a straight line with an R2 of 0.996, which is valid within the 10-90% range. The ESMS protocol has been used to determine the extent of phosphorylation of TH in the presence of CaM-PKII. The experimental conditions were designed to produce low levels of phosphorylation. Nevertheless, the ESMS analysis yielded single, double, and nonphosphorylation forms of TH. With respect to in vivo measurements, this ESMS protocol may be a generic procedure for determining the extent of phosphorylation of proteins.     

Circulating T-cell response to Helicobacter pylori infection in chronic gastritis

Background. Helicobacter pylori elicits a specific humoral and cellular immune response. There is increasing evidence that the type of T-cell response contributes to clinical outcome in H. pylori infection.
Materials and Methods. The host response to H. pylori infection in 34 subjects with chronic gastritis was examined in terms of T-cell proliferation and cytokine production in whole-blood cultures stimulated or unstimulated with H. pylori acid-glycine extract antigens (AGE).
Results. The proliferative response in whole-blood cultures was similar for both H. pylori –positive and –negative subjects stimulated with H. pylori AGE. While an increase in interferon-γ (IFN-γ) production was observed from both H. pylori –positive and –negative subjects with gastritis, significantly higher levels of IFN-γ were detected in the former when stimulated with H. pylori AGE. In contrast, interleukin 4 (IL-4) was undetectable regardless of antigen stimulation. However, if an in situ IL-4 antibody capture assay was used, antigen-independent production of IL-4 was detected, but there was no difference between H. pylori –positive and –negative subjects with gastritis. After eradication of H. pylori , antigen-induced production of IL-4 was increased, with no decrease in the levels of secretion of IFN-γ. IL-4 production was dependent on CD4+ T cells, as addition of anti-CD4 but not anti-CD8 mouse monoclonal antibody or matched IgG isotype to the whole-blood culture inhibited the production of IL-4.
Conclusion. The results suggest that a shift toward a balanced Th1-Th2 response due to an increase in antigen-induced IL-4 production from CD4+ T cells follows eradication. We suggest that the downregulation of mucosal inflammation consequent on reduction in antigen levels or removal of downregulation after eradication of H. pylori contributes to this shift in cytokine balance.     

Angiotensin II regulates tyrosine hydroxylase activity and mRNA expression in rat mediobasal hypothalamic cultures: the role of specific protein kinases

Dopamine secreted by hypothalamic neurons is crucial in regulating prolactin secretion from the pituitary. We have examined the ability of angiotensin II (AngII) to regulate the activity of these dopaminergic neurons and thus act as a potential physiological regulator of prolactin secretion. Using a hypothalamic cell culture preparation we determined the effect of AngII on tyrosine hydroxylase activity and expression (TOH). This is important because TOH is the rate-limiting enzyme in dopamine biosynthesis. AngII stimulated a time- and concentration-dependent increase in TOH activity which was suppressed by inhibitors able to act on protein kinase A (PKA), protein kinase C (PKC) and Ca(2+)/calmodulin-dependent protein kinase II (CaMPKII). An inhibitor of the mitogen-activated protein kinase (MAPK) pathway, PD 98059, reduced basal TOH activity but the AngII response was still detectable. AngII stimulation enhanced the phosphorylation of TOH at Ser19, Ser31 and Ser40. AngII also induced a time-dependent increase in TOH mRNA expression which was unaffected by inhibitors able to act on PKA and CaMPKII, but was abolished by inhibitors able to act on ERK and PKC. AngII responses were very much larger in cultures prepared from female when compared to male rat pups. Data from adult hypothalamic slices confirmed this sexual dimorphism and supported the role of the protein kinases noted above. Therefore AngII can regulate both the activity and expression of TOH in hypothalamic neurons employing multiple, but only partially overlapping, signaling pathways.     

Role of protein phosphatase 2C from bovine adrenal chromaffin cells in the dephosphorylation of phospho-serine 40 tyrosine hydroxylase

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. It is dephosphorylated by protein phosphatase (PP) 2A and PP2C. In this study we used a fixed amount of bacterially expressed rat TH (5 microM), phosphorylated only at serine 40 (pSer40TH), to determine the PP activities against this site that are present in extracts from the bovine adrenal cortex, adrenal medulla, adrenal chromaffin cells and rat striatum. We found that PP2C was the main TH phosphatase activity in extracts from the adrenal medulla and adrenal chromaffin cells. In adrenal cortex extracts PP2C and PP2A activities toward pSer40TH did not differ significantly. PP2A was the main TH phosphatase activity in extracts from rat striatum. Kinetic studies with extracts from adrenal chromaffin cells showed that when higher concentrations of pSer40TH (> 5 microM) were used the activity of PP2C increased more than the activity of PP2A. PP2C was maximally activated by 1.25 mM Mn2+ and by 5 mM Mg2+ but was inhibited by calcium. Our data suggest a more important role for PP2C than was previously suggested in the dephosphorylation of serine 40 on TH.     

Localization of organelle proteins by isotope tagging (LOPIT)

We describe a proteomics method for determining the subcellular localization of membrane proteins. Organelles are partially separated using centrifugation through self-generating density gradients. Proteins from each organelle co-fractionate and therefore exhibit similar distributions in the gradient. Protein distributions can be determined through a series of pair-wise comparisons of gradient fractions, using cleavable ICAT to enable relative quantitation of protein levels by MS. The localization of novel proteins is determined using multivariate data analysis techniques to match their distributions to those of proteins that are known to reside in specific organelles. Using this approach, we have simultaneously demonstrated the localization of membrane proteins in both the endoplasmic reticulum and the Golgi apparatus in Arabidopsis. Localization of organelle proteins by isotope tagging is a new tool for high-throughput protein localization, which is applicable to a wide range of research areas such as the study of organelle function and protein trafficking.     

Depolarisation-Dependent Protein Phosphorylation and Dephosphorylation in Rat Cortical Synaptosomes Is Modulated by Calcium

The effect of calcium on protein phosphorylation was investigated using intact synaptosomes isolated from rat cerebral cortex and prelabelled with 32Pi. For nondepolarised synaptosomes a group of calcium-sensitive phosphoproteins were maximally labelled in the presence of 0.1 mM calcium. The phosphorylation of these proteins was slightly decreased in the presence of strontium and absent in the presence of barium, consistent with the decreased ability of these cations to activate calcium-stimulated protein kinases. Addition of calcium alone to synaptosomes prelabelled in its absence increased phosphorylation of a number of proteins. On depolarisation in the presence of calcium certain of the calcium-sensitive phosphoproteins were further increased in labelling above nondepolarised levels. These increases were maximal and most sustained after prelabelling at 0.1 mM calcium. On prolonged depolarisation at this calcium concentration a slow decrease in labelling was observed for most phosphoproteins, whereas a greater rate and extent of decrease occurred at higher calcium concentrations. At 2.5 mM calcium a rapid and then a subsequent slow dephosphorylation was observed, indicating two distinct phases of dephosphorylation. Of all the phosphoproteins normally stimulated by depolarisation, only phosphoprotein 59 did not exhibit the rapid phase of dephosphorylation at high calcium concentrations. Replacing calcium with strontium markedly decreased the extent of change observed on depolarisation whereas barium decreased phosphorylation changes even further. Taken together these data suggest that an influx of calcium into synaptosomes initially activates protein phosphorylation, but as the levels of intrasynaptosomal calcium rise protein dephosphorylation predominates. Other phosphoproteins were dephosphorylated immediately on depolarisation in the presence of calcium. The fine control of protein phosphorylation levels exerted by calcium supports the idea that the synaptosomal phosphoproteins could play a role in modulating events such as neurotransmitter release in the nerve terminal.