Bok Is Not Pro-Apoptotic But Suppresses Poly ADP-Ribose Polymerase-Dependent Cell Death Pathways and Protects against Excitotoxic and Seizure-Induced Neuronal Injury.

UNLABELLED Bok (Bcl-2-related ovarian killer) is a Bcl-2 family member that, because of its predicted structural homology to Bax and Bak, has been proposed to be a pro-apoptotic protein. In this study, we demonstrate that Bok is highly expressed in neurons of the mouse brain but thatbokwas not required for staurosporine-, proteasome inhibition-, or excitotoxicity-induced apoptosis of cultured cortical neurons. On the contrary, we found thatbok-deficient neurons were more sensitive to oxygen/glucose deprivation-induced injuryin vitroand seizure-induced neuronal injuryin vivo Deletion ofbokalso increased staurosporine-, excitotoxicity-, and oxygen/glucose deprivation-induced cell death inbax-deficient neurons. Single-cell imaging demonstrated thatbok-deficient neurons failed to maintain their neuronal Ca(2+)homeostasis in response to an excitotoxic stimulus; this was accompanied by a prolonged deregulation of mitochondrial bioenergetics.bokdeficiency led to a specific reduction in neuronal Mcl-1 protein levels, and deregulation of both mitochondrial bioenergetics and Ca(2+)homeostasis was rescued by Mcl-1 overexpression. Detailed analysis of cell death pathways demonstrated the activation of poly ADP-ribose polymerase-dependent cell death inbok-deficient neurons. Collectively, our data demonstrate that Bok acts as a neuroprotective factor rather than a pro-death effector during Ca(2+)- and seizure-induced neuronal injuryin vitroandin vivo SIGNIFICANCE STATEMENT Bcl-2 proteins are essential regulators of the mitochondrial apoptosis pathway. The Bcl-2 protein Bok is highly expressed in the CNS. Because of its sequence similarity to Bax and Bak, Bok has long been considered part of the pro-apoptotic Bax-like subfamily, but no studies have yet been performed in neurons to test this hypothesis. Our study provides important new insights into the functional role of Bok during neuronal apoptosis and specifically in the setting of Ca(2+)- and seizure-mediated neuronal injury. We show that Bok controls neuronal Ca(2+)homeostasis and bioenergetics and, contrary to previous assumptions, exerts neuroprotective activitiesin vitroandin vivo Our results demonstrate that Bok cannot be placed unambiguously into the Bax-like Bcl-2 subfamily of pro-apoptotic proteins.

Tachykinins Processing is Significantly Impaired in PC1 and PC2 Mutant Mouse Spinal Cord S9 Fractions

Substance P (SP) play a central role in nociceptive transmission and it is an agonist of the Neurokinin-1 receptor located in the lamina I of the spinal cord. SP is a major proteolytic product of the protachykinin-1 primarily synthesized in neurons. Proprotein convertases (PCs) are extensively expressed in the central nervous system (CNS) and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous protachykinins has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis surrogate protachykinins (i.e. Tachykinin 20-68 and Tachykinin 58-78) using cellular fractions of spinal cords from wild type (WT), PC1-/+ and PC2-/+ animals and mass spectrometry. Full-length Tachykinin 20-68 and Tachykinin 58-78 was incubated for 30 minutes in WT, PC1-/+ and PC2-/+ mouse spinal cord S9 fractions and specific C-terminal peptide fragments were identified and quantified by mass spectrometry. The results clearly demonstrate that both PC1 and PC2 mediate the formation of SP and Tachykinin 58-71, an important SP precursor, with over 50% reduction of the rate of formation in mutant PC 1 and PC2 mouse S9 spinal cord fractions. The results obtained revealed that PC1 and PC2 are involved in the C-terminal processing of protachykinin peptides and suggest a major role in the maturation of the protachykinin-1 protein.

Aerobic cultivation of Streptococcus zooepidemicus and the role of NADH oxidase

A metabolic flux model was developed for Streptococcus zooepidemicus to compare the metabolism of glucose and maltose during aerobic batch cultivation. Lactic acid was the main product of glucose metabolism whereas acetic acid was the main product of maltose metabolism. This difference was chiefly attributed to the two-fold higher flux through NADH oxidase in maltose-grown cells that enabled the ATP generation rate to remain high despite a slower maltose consumption rate. The two-fold higher flux was matched by a two-fold increase in NADH oxidase activity, 2.53 +/- 0.1 mumol NADH min(-1) mg(-1) protein on maltose versus 1.07 +/- 0.04 Rmol NADH min(-1) mg(-1) protein on glucose, indicating that NADH oxidase activity is regulated by the energy status of the cell. Surprisingly, the energy status of the cell had little impact on hyaluronic acid (HA) yield and molecular weight. (C) 2003 Elsevier Science B.V. All rights reserved.

Lectin-induced haemocyte inactivation in insects

Most multimeric lectins are adhesion molecules, promoting attachment and spreading on surface glycodeterminants. In addition, some lectins have counter-adhesion properties, detaching already spread cells which then acquire round or spindle-formed cell shapes. Since lectin-mediated adhesion and detachment is observed in haemocyte-like Drosophila cells, which have haemomucin as the major lectin-binding glycoprotein, the two opposite cell behaviours may be the result of lectin-mediated receptor rearrangements on the cell surface. To investigate oligomeric lectins as a possible extracellular driving force affecting cell shape changes, we examined lectin-mediated reactions in lepidopteran haemocytes after cytochalasin D-treatment and observed that while cell-spreading was dependent on F-actin, lectin-uptake was less dependent on F-actin. We propose a model of cell shape changes involving a dynamic balance between adhesion and uptake reactions. (C) 2004 Elsevier Ltd. All rights reserved.

Freshwater to seawater acclimation of juvenile bull sharks ( Carcharhinus leucas): plasma osmolytes and Na +/K +-ATPase activity in gill, rectal gland, kidney and intestine

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater ( SW). Juvenile C. leucas captured in FW ( 3 mOsm l(-1) kg(-1)) were acclimated to SW ( 980 - 1,000 mOsm l(-1) kg(-1)) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l(-1) kg(-1). In SW, bull sharks had significantly higher plasma osmolarities ( 940 mOsm l(-1) kg(-1)) than FW-acclimated animals and were slightly hypoosmotic to the environment. Plasma Na+, Cl-, K+, Mg2+, Ca2+, urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na+/ K+-ATPase activity. Na+/ K+-ATPase activity in the gills and intestine was less than 1 mmol Pi mg(-1) protein h(-1) and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na+/ K+-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na+/ K+-ATPase activity was 5.6 +/- 0.8 and 9.2 +/- 0.6 mmol Pi mg(-1) protein h(-1), respectively. Na+/ K+-ATPase activity in the kidney of FW and SW acclimated animals was 8.4 +/- 1.1 and 3.3 +/- 1.1 Pi mg(-1) protein h(-1), respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.

Uptake and metabolism of ginsenoside Rh2 and its aglycon protopanaxadiol by Caco-2 cells

The uptake and metabolism profiles of ginsenoside Rh2 and its aglycon protopanaxadiol (ppd) were studied in the human epithelial Caco-2 cell line. High-performance liquid chromatography-mass spectrometry was applied to determine Rh2 and its aglycon ppd concentration in the cells at different pH, temperature, concentration levels and in the presence or absence of inhibitors. Rh2 uptake was time and concentration dependent, and its uptake rates were reduced by metabolic inhibitors and influenced by low temperature, thus indicating that the absorption process was energy-dependent. Drug uptake was maximal when the extracellular pH was 7.0 for Rh2 and 8.0 for ppd. Rh2 kinetic analysis showed that a non-saturable component (K-d 0.17 nmol (.) h(-1) (.) mg(-1) protein) and an active transport system with a K-m of 3.95 mumol (.) l(-1) and a V-max of 4.78 nmol(.)h(-1) (.)mg(-1) protein were responsible for the drug uptake. Kinetic analysis of ppd showed a non-saturable component (K-d 0.78 nmol (.) h(-1) (.) mg(-1) protein). It was suggested that active extrusion of P-glycoprotein and drug degradation in the intestine may influence Rh2 bioavailability.

The PCNA-associated factor KIAA0101/p15(PAF) binds the potential tumor suppressor product p33ING1b

The KIAA0101/p15(PAF)/OEATC-1 protein was initially isolated in a yeast two-hybrid screen for proliferating cell nuclear antigen (PCNA) binding partners, and was shown to bind PCNA competitively with the cell cycle regulator p21(WAF). PCNA is involved in DNA replication and damage repair. Using polyclonal antisera raised against a p15(PAF) fusion protein, we have shown that in a range of mammalian tumor and non-tumor cell lines the endogenous p15(PAF) protein localises to the nucleus and the mitochondria. Under normal conditions no co-localisation with PCNA could be detected, however following exposure to UV it was possible to co-immunoprecipitate p15(PAF) and PCNA from a number of cell lines, suggesting a UV-enhanced association of the two proteins. Overexpression of p15(PAF) in mammalian cells was also found to protect cells from UV-induced cell death. Based on similarities between the behaviour of p15(PAF) and the potential tumor suppressor product p33ING1b, we have further shown that these two proteins interact in the same complex in cell cultures. This suggests that p15(PAF) forms part of a larger protein complex potentially involved in the regulation of DNA repair, apoptosis and cell cycle progression. (c) 2005 Elsevier Inc. All rights reserved.

Characterization and effects on cAMP accumulation of adrenomedullin and calcitonin gene-related peptide (CGRP) receptors in dissociated rat spinal cord cell culture

1 Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) have structural similarities, interact with each others receptors (calcitonin receptor-like receptor (CLR)/receptor-activity-modifying proteins (RAMPs)) and show overlapping biological activities. AM and CGRP receptors are chiefly coupled to cAMP production. In this study, a method of primary dissociated cell culture was used to investigate the presence of AM and CGRP receptors and their effects on cAMP production in embryonic spinal cord cells. 2 Both neuronal and non-neuronal CLR immunopositive cells were present in our model. 3 High affinity, specific [ 125I]-AM binding sites (K(d) 79±9 pM and B(max) 571±34 fmol mg -1 protein) were more abundant than specific [ 125I]-CGRP binding sites (K(d) 12±0.7 pM and B(max) 32±2 fmol mg -1 protein) in embryonic spinal cord cells. 4 Specific [ 125I]-AM binding was competed by related molecules with a ligand selectivity profile of rAM>hAM(22-52)>rCGRPα>CGRP(8-37) ≫[r-(r*,s*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl] carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1, 4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-piperidinecarboxamide (BIBN4096BS). 5 Specific [ 125I]-CGRP binding was competed by rCGRPα>rAM≥ CGRP(8-37)≥BIBN4096BS>hAM(22-52). 6 Cellular levels of cAMP were increased by AM (pEC"5"0 10.2±0.2) and less potently by rCGRPα (pEC"5"0 8.9±0.4). rCGRPα-induced cAMP accumulation was effectively inhibited by CGRP(8-37) (pA"2 7.63±0.44) and hAM(22-52) (pA"2 6.18±0.21) while AM-stimulation of cAMP levels was inhibited by CGRP(8-37) (pA"2 7.41±0.15) and AM(22-52) (pA"2 7.26±0.18). BIBN4096BS only antagonized the effects of CGRP (pA"2 8.40±0.30) on cAMP accumulation. 7 These pharmacological profiles suggest that effects of CGRP are mediated by the CGRP"1 (CLR/RAMP1) receptor in our model while those of AM are related to the activation of the AM"1 (CLR/RAMP2) receptor subtype. © 2006 Nature Publishing Group All rights reserved.

Fibronectin-tissue transglutaminase matrix rescues RGD-impaired celladhesion through syndecan-4 and β integrin co-signaling

Heterotropic association of tissue transglutaminase (TG2) with extracellular matrix-associated fibronectin (FN) can restore the adhesion of fibroblasts when the integrin-mediated direct binding to FN is impaired using RGD-containing peptide. We demonstrate that the compensatory effect of the TG-FN complex in the presence of RGD-containing peptides is mediated by TG2 binding to the heparan sulfate chains of the syndecan-4 cell surface receptor. This binding mediates activation of protein kinase Ca (PKCa) and its subsequent interaction with ß1 integrin since disruption of PKCa binding to ß1 integrins with a cell-permeant competitive peptide inhibits cell adhesion and the associated actin stress fiber formation. Cell signaling by this process leads to the activation of focal adhesion kinase and ERK1/2 mitogen-activated protein kinases. Fibroblasts deficient in Raf-1 do not respond fully to the TG-FN complex unless either the full-length kinase competent Raf-1 or the kinase-inactive domain of Raf-1 is reintroduced, indicating the involvement of the Raf-1 protein in the signaling mechanism. We propose a model for a novel RGD-independent cell adhesion process that could be important during tissue injury and/or remodeling whereby TG-FN binding to syndecan-4 activates PKCa leading to its association with ß1 integrin, reinforcement of actin-stress fiber organization, and MAPK pathway activation.

REST mediates resolution of HIF-dependent gene expression in prolonged hypoxia

The hypoxia-inducible factor (HIF) is a key regulator of the cellular response to hypoxia which promotes oxygen delivery and metabolic adaptation to oxygen deprivation. However, the degree and duration of HIF-1α expression in hypoxia must be carefully balanced within cells in order to avoid unwanted side effects associated with excessive activity. The expression of HIF-1α mRNA is suppressed in prolonged hypoxia, suggesting that the control of HIF1A gene transcription is tightly regulated by negative feedback mechanisms. Little is known about the resolution of the HIF-1α protein response and the suppression of HIF-1α mRNA in prolonged hypoxia. Here, we demonstrate that the Repressor Element 1-Silencing Transcription factor (REST) binds to the HIF-1α promoter in a hypoxia-dependent manner. Knockdown of REST using RNAi increases the expression of HIF-1α mRNA, protein and transcriptional activity. Furthermore REST knockdown increases glucose consumption and lactate production in a HIF-1α- (but not HIF-2α-) dependent manner. Finally, REST promotes the resolution of HIF-1α protein expression in prolonged hypoxia. In conclusion, we hypothesize that REST represses transcription of HIF-1α in prolonged hypoxia, thus contributing to the resolution of the HIF-1α response.

Monitoring changes in thioredoxin and over-oxidised peroxiredoxin in response to exercise in humans

Introduction. Peroxiredoxin (PRDX) and thioredoxin (TRX) are antioxidant proteins that control cellular signalling and redox balance, although their response to exercise is unknown. This study aimed to assess key aspects of the PRDX-TRX redox cycle in response to three different modes of exercise. Methods. Healthy males (n = 10, mean ± SD: 22 ± 3 yrs) undertook three exercise trials on separate days: two steady-state cycling trials at moderate (60% VO2MAX; 27 min, MOD) and high (80% VO2MAX; 20 min, HIGH) intensities, and a low-volume high-intensity interval training trial (10 × 1 min 90% VO2MAX, LV-HIIT). Peripheral blood mononuclear cells were assessed for TRX-1 and over-oxidised PRDX (isoforms I-IV) protein expression before, during, and 30 min following exercise (post + 30). The activities of TRX reductase (TRX-R) and the nuclear factor kappa B (NF-κB) p65 subunit were also assessed. Results. TRX-1 increased during exercise in all trials (MOD, + 84.5%; HIGH, + 64.1%; LV-HIIT, + 205.7%; p < 05), whereas over-oxidised PRDX increased during HIGH only (MOD, - 28.7%; HIGH, + 202.9%; LV-HIIT, - 22.7%; p < .05). TRX-R and NF-κB p65 activity increased during exercise in all trials, with the greatest response in TRX-R activity seen in HIGH (p < 0.05). Discussion. All trials stimulated a transient increase in TRX-1 protein expression during exercise. Only HIGH induced a transient over-oxidation of PRDX, alongside the greatest change in TRX-R activity. Future studies are needed to clarify the significance of heightened peroxide exposure during continuous high-intensity exercise and the mechanisms of PRDX-regulatory control.

A deimmunised form of the ribotoxin, α-sarcin, lacking CD4+ T cell epitopes and its use as an immunotoxin warhead

Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus that functions by catalytically cleaving a single phosphodiester bond in the sarcin–ricin loop of the large ribosomal subunit, thus making the ribosome unrecognisable to elongation factors and leading to inhibition of protein synthesis. Peptide mapping using an ex vivo human T cell assay determined that α-sarcin contained two T cell epitopes; one in the N-terminal 20 amino acids and the other in the C-terminal 20 amino acids. Various mutations were tested individually within each epitope and then in combination to isolate deimmunised α-sarcin variants that had the desired properties of silencing T cell epitopes and retention of the ability to inhibit protein synthesis (equivalent to wild-type, WT α-sarcin). A deimmunised variant (D9T/Q142T) demonstrated a complete lack of T cell activation in in vitro whole protein human T cell assays using peripheral blood mononuclear cells from donors with diverse HLA allotypes. Generation of an immunotoxin by fusion of the D9T/Q142T variant to a single-chain Fv targeting Her2 demonstrated potent cell killing equivalent to a fusion protein comprising the WT α-sarcin. These results represent the first fungal ribotoxin to be deimmunised with the potential to construct a new generation of deimmunised immunotoxin therapeutics.

In vitro expansion of U87-MG human glioblastoma cells under hypoxic conditions affects glucose metabolism and subsequent in vivo growth

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A Putative Otu Domain-containing Protein 1 Deubiquitinating Enzyme Is Differentially Expressed In Thyroid Cancer And Identifies Less-aggressive Tumours.

This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.