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PubMed1995

Glutamate, calcium, and free radicals as mediators of ischemic brain damage.

Calcium is considered a mediator of ischemic brain damage whether this is due to global or forebrain ischemia or to focal ischemia. Supporting evidence is the translocation of extracellular calcium into cells during ischemia, the precipitous rise in the free cytosolic calcium concentration, and the role of calcium in activating lipases, proteases, kinases, phosphatases, and endonucleases in potentially harmful metabolic cascades. In vitro and in vivo experiments suggest that the main route of entry is through channels gated by glutamate receptors. These experiments led to the excitotoxic hypothesis of cell death. The in vitro experiments further support the role of calcium as a mediator of cell death. Both cell calcium overload and acidosis enhance the production of partially reduced oxygen species, thus predisposing to free radical-related damage. In transient global or forebrain ischemia, free radicals formed during reperfusion may contribute to a perturbed membrane function, leading to a sustained alteration of cell calcium metabolism with ultimate mitochondrial calcium overload. In focal ischemia (stroke), free radicals may be important mediators of the infarction process. Infarction can be regarded as a form of secondary damage, which is probably caused by microvascular dysfunction. Very likely, such dysfunction is triggered by upregulation of adhesion molecules such as ICAM-1, microvascular "plugging," and an inflammatory response at the blood-endothelial cell interface. The involvement of free radicals in this type of secondary damage is supported by results showing that nitrones that act as free radical spin-traps ameliorate focal ischemic damage with a therapeutic window of many hours.

Animals↗

PubMed2002

Fibronectin accumulation in glomerulosclerotic lesions: self-assembly sites and the heparin II binding domain.

BACKGROUND: Glomerulosclerosis is a severe complication of many immunologically-mediated kidney diseases, eventually resulting in loss of renal function. In chronic graft-versus-host disease (GvHD) in mice, a model for human lupus nephritis, the end-stage sclerotic lesions were previously shown to contain large amounts of fibronectin (FN). This study investigated a domain-specific accumulation process of circulating plasma FN (pFN) in sclerotic lesions. METHODS: GvHD mice were injected with FITC-conjugated pFN or pFN-fragments, with or without heparin pre-incubation. pFN fragments were generated by digestion of pFN by cathepsin D, after which the fragments were separated on a heparin affinity column. Thus, two batches of fragments were obtained with either low or high affinity for heparin. RESULTS: FN accumulation was accompanied by an up-regulated expression of integrin alpha5beta1, the FN receptor, in the periphery of sclerotic lesions. pFN-FITC injected into GvHD mice was trapped in sclerotic glomeruli within 24 hours. Both heparin and non-anti-coagulant heparin blocked the accumulation of pFN-FITC, indicating that the protective effect of heparin in the trapping of FN is independent of its anticoagulant properties, and probably results from preventing direct binding of FN in the sclerotic lesions. To investigate whether FN binds in the glomerulus via the heparin-binding regions, pFN fragments were generated and injected into GvHD mice. Whereas the fraction with high affinity for heparin did not accumulate in the sclerotic glomeruli, the fraction with low affinity for heparin did. Partial sequencing of the isolated peptides showed that in the glomerulus fibronectin does not bind via the heparin II binding region. CONCLUSIONS: We hypothesize that the protective effect of heparin treatment may be the result of steric hindrance of the specific binding sites, that is, the I1-5 and/or III1 self-assembly sites of FN.

Animals↗

PubMed1996

Non-neural agrin codistributes with acetylcholine receptors during early differentiation of Torpedo electrocytes.

Agrin, an extracellular matrix protein synthesized by nerves and muscles is known to promote the clustering of acetylcholine receptors and other synaptic proteins in cultured myotubes. This observation suggests that agrin may provide at least part of the signal for synaptic specialization in vivo. The extracellular matrix components agrin, laminin and merosin bind to alpha-dystroglycan, a heavily glycosylated peripheral protein part of the dystrophin-glycoprotein complex, previously characterized in the sarcolemma of skeletal and cardiac muscles and at the neuromuscular junction. In order to understand further the function of agrin and alpha DG in the genesis of the acetylcholine receptor-rich membrane domain, the settling of components of the dystrophin-glycoprotein complex and agrin was followed by immunofluorescence localization in developing Torpedo marmorata electrocytes. In 40-45 mm Torpedo embryos, a stage of development at which the electrocytes exhibit a definite structural polarity, dystrophin, alpha/beta-dystroglycan and agrin accumulated concomitantly with acetylcholine receptors at the ventral pole of the cells. Among these components, agrin appeared as the most intensely concentrated and sharply localized. The scarcity of the nerve-electrocyte synaptic contacts at this stage of development, monitored by antibodies against synaptic vesicles, further indicates that before innervation, the machinery for acetylcholine receptor clustering is provided by electrocyte-derived agrin rather than by neural agrin. These observations suggest a two-step process of acetylcholine receptor clustering involving: (i) an instructive role of electrocyte-derived agrin in the formation of a dystrophin-based membrane scaffold upon which acetylcholine receptor molecules would accumulate according to a diffusion trap model; and (ii) a maturation and/or stabilization step controlled by neural agrin. In the light of these data, the existence of more than one agrin receptor is postulated to account for the action of agrin variants at different stages of the differentiation of the postsynaptic membrane in Torpedo electrocytes.

Agrin↗

PubMed1994

Studies of the ultrastructure and permeability of the blood-brain barrier in the developing corpus callosum in postnatal rat brain using electron dense tracers.

The ultrastructure of the capillaries and their permeability to lanthanum ions and ferritin in the corpus callosum was examined in postnatal rat brain. In 1 and 7-d-old rats, numerous pinocytotic vesicles were observed in the endothelial cytoplasm of the callosal capillaries. Tight junctions were present between adjacent endothelial cells which were surrounded by an ill defined layer of basal lamina. The latter was almost devoid of astrocytic association in 1 d rats and partially covered by astrocytic end-feet in 7 d rats. Pericytes were a common feature. Amoeboid microglial cells were in direct contact with some parts of the vascular wall. Large extracellular spaces were present around the capillaries. In 14 d rats, the walls of the callosal capillaries became more well developed and were surrounded by a continuous sheath of astrocytic end-feet. The basal lamina became denser and well defined. The pericapillary spaces had diminished. Immunostaining for GFAP confirmed that, with age, the walls of the callosal capillaries became increasingly covered by the astrocytic end-feet. After perfusion with lanthanum, the tracer was deposited on the luminal surface but not in the abluminal side of the endothelial cells; the passage of the tracer was apparently obstructed in the intercellular space by the tight junctions in both the 1 and 14 d rats. When injected intravenously in 1-7 d rats, ferritin was transported across the endothelial cells by transcytosis and consequently taken up by the pericytes and amoeboid microglial cells contacting the basal lamina. In 14 d rats, the injected ferritin was only found in the endothelial cytoplasm. It was concluded that the difference in the capillary permeability to exogenous material in 1-7 and 14 d rats is due to the difference in the activities of the transendothelial transport. The pericytes and amoeboid microglial cells associated with the capillary probably play a role as phagocytes in maintaining the function of the blood-brain barrier by trapping any serum-derived foreign substances with astrocytes having a regulatory role in the formation of the barrier.

Animals↗

PubMed1989

D-xylulose-induced depletion of ATP and Pi in isolated rat hepatocytes.

Xylitol is known to cause hepatic ATP catabolism by inducing the trapping of Pi in the form of glycerol 3-P as a consequence of an increase in the NADH:NAD+ ratio, resulting from the oxidation of xylitol to D-xylulose. The question was therefore raised whether D-xylulose also depletes hepatic ATP. In isolated rat hepatocytes, 5 mM D-xylulose decreased ATP by 80% within 5 min compared to 40% with 5 mM xylitol. Intracellular Pi decreased by 70% within the same time interval with both compounds, but was restored three-fold faster with D-xylulose. The rate of utilization of D-xylulose reached 5 mumol.min-1.g-1 of cells, as compared with 1.5 for xylitol, indicating that reduction of xylitol into D-xylulose is a rate-limiting step in the metabolism of the polyol. D-Xylulose barely modified the concentration of glycerol 3-P but increased xylulose 5-P from 0.02 to 0.5 mumol/g within 5 min. The main cause of the ATP- and Pi-depleting effects of D-xylulose was found to be an accumulation of sedoheptulose 7-P from a basal value of 0.1 to 5 mumol/g of cells after 10 min. Ribose 5-P increased from 0.03 to 0.5 mumol/g at 5 min. Ribose 1-P also accumulated, albeit outside of the cells. This extracellular accumulation can be explained by the release of intracellular purine nucleoside phosphorylase from damaged hepatocytes acting on inosine that had diffused out of the cells. Smaller increases in the concentrations of sedoheptulose 7-P and pentose phosphates were recorded after incubations of the cells with xylitol.

Adenosine Triphosphate↗

PubMed1994

New approaches to tissue regeneration and repair.

Several Heparin Binding Growth Factors (HBGFs) are thought to play a key role in the natural processes of tissue regeneration or repair after being released by neighbouring, inflammatory or circulating cells as well as from extracellular matrix associated heparan sulfate proteoglycosaminoglycans. In order to better understand how the bioavailability of these HBGFs can take part in the regulation of the wound healing processes, we have studied the healing effect of various chemically substituted dextrans (CMDBS) selected for their affinity for HBGFs, alone and in association with HBGFs. The CMDBS was obtained by substitution of methylcarboxylic (CM), benzylamide (B) and benzylamine sulfonate (S) groups in proportion of 83%, 23% and 13% respectively for CMDBS K that we have further used (Mauzac et al., 1985 Biomaterials. 6: 61-63). CMDBS K could 1: potentiate the biological activity of 1 or 2 FGFs, 2: protect 1 and 2 FGFs against thermal or pH inactivation, 3: protect a and b FGFs against proteolytic degradation (Tardieu et al., 1992 J. Cell. Physiol. 150: 194-203). CMDBS K was tested alone in cutaneous and bone wound healing models and for its ability to stabilize FGFs. Rats were punched and skin regeneration was studied by morphometric and histological analysis. The wounds (6 mm diameter) were filled with collagen plaster alone or soaked with CMDBS. CMDBS K in collagen plaster was able to induce a remarkable effect both on the kinetics and on the quality of the restored skin. These results suggest that endogenous growth factors naturally released during the regeneration process could be trapped, protected and released by CMDBS.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2006

(+/-)-2-Chloropropionic acid elevates reactive oxygen species formation in human neutrophil granulocytes.

(+/-)-2-Chloropropionic acid (2-CPA) is a neurotoxic compound which kills cerebellar granule cells in vivo, and makes cerebellar granule cells in vitro produce reactive oxygen species (ROS). We have studied the effect of 2-CPA on ROS formation in human neutrophil granulocytes in vitro. We found an increased formation of ROS after 2-CPA exposure using three different methods; the fluorescent probe DCFH-DA and the chemiluminescent probes lucigenin and luminol. Four different inhibitors of ROS formation were tested on the cells in combination with 2-CPA to characterize the signalling pathways. The spin-trap s-PBN, the ERK1/2 inhibitor U0126 and the antioxidant Vitamin E inhibited the 2-CPA-induced ROS formation completely, while the mitochondrial transition permeability pore blocker cyclosporine A inhibited the ROS formation partly. We also found that 2-CPA induced an increased nitric oxide production in the cells by using the Griess reagent. The level of reduced glutathione, measured with the DTNB assay, was decreased after exposure to high concentrations of 2-CPA. Western blotting analysis showed that 2-CPA exposure led to an elevated phosphorylation of ERK MAP kinase. This phosphorylation was inhibited by U0126. Based on these experiments it seems like the mechanisms for 2-CPA induced toxicity involves ROS formation and is similar in neutrophil granulocytes as earlier shown in cerebellar granule cells. This also implies that 2-CPA may be immunotoxic.

Acridines↗

PubMed2000

The role of the hinge region of the luteinizing hormone receptor in hormone interaction and signal generation.

Luteinizing hormone receptor, a G protein-coupled receptor, consists of two halves, the N-terminal extracellular hormone binding domain (exodomain) and the C-terminal membrane-associated, signal-generating domain (endodomain). The exodomain has seven to nine Leu-rich repeats, which are generally thought to form a 1/3 donut-like structure and interact with human choriogonadotropin (hCG). The resulting hCG-exodomain complex adjusts the structure and its association with the endodomain, which results in signal generation in the endodomain. It is unclear whether the rigid 1/3 donut structure could provide the agility and versatility of this dynamic action. In addition, there is no clue as to where the endodomain contact point (the signal modulator) in the exodomain is. To address these issues, the exodomain was examined by Ala scan and multiple substitutions, while receptor peptides were used for photoaffinity labeling and affinity cross-linking. Our results show that the C-flanking sequence (hinge region), Thr(250)-Gln(268), of the Leu-rich repeats (LRRs) specifically interacts with hCG, preferentially hCGalpha. This interaction is inhibited by exoloop 2 of the endodomain but not by exoloops 1 and 3, suggesting an intimate relationship between Thr(250)-Gln(268), exoloop 2, and hCG. Taken together, our observations in this article suggest a new paradigm that the LRRs contact the front of hCG, while both flanking regions of the LRRs interact with the sides of hCG. This would trap hCG in the 1/3 donut structure of the LRRs and enhance the binding affinity. In addition, mutations of conserved Ser(255) in the sequence can constitutively activate the receptor. This provides a clue for the signal modulator in the exodomain. In contrast, a phenyl or phenolic group is necessary at conserved Tyr(253) for targeting the receptor to the surface.

Amino Acid Motifs↗

PubMed2004

Understanding experimental biology of skin equivalent: from laboratory to clinical use in patients with burns and chronic wounds.

A major breakthrough in burn wound care was the early excision of the burn and its immediate coverage with a skin autograft. A search for a skin-graft substitute began to reduce the autografting-related trauma at the donor site. One entry was skin equivalence, which contains 3 components: (1) living fibroblasts, suspended in (2) a native collagen matrix, the surface of which is covered with (3) viable keratinocytes. The tissue-cultured dermal fibroblasts are derived from human foreskin. The fibroblasts are grown in cell culture dishes as a monolayer and are retrieved by limited trypsin digestion. The fibroblast suspension is mixed with serum-supplemented culture medium and native acid-soluble collagen. The entire mixture, called a dermal equivalent, is placed in a bacteriological Petri dish before transfer to a 37 degrees C incubator. The collagen rapidly polymerizes, trapping cells in the dermal equivalent. During the initial 4 hours, fibroblasts elongate and spread, causing a decrease in the thickness of the dermal equivalent. After 6 hours, the dermal equivalent undergoes a decrease in diameter as a consequence of the reorganization of the collagen. A freshly isolated suspension of human skin-derived keratinocytes is seeded on the surface of a several-day-old floating dermal equivalent. The keratinocytes proliferate, covering the surface of the dermal equivalent. The keratinocytes deposit basement membranes beneath them and undergo epidermal cell differentiation, leading to the formation of a basal layer beneath differentiated cell layers. Both cell populations retain viability and release cell factors that have a positive effect on wound closure. The placement of skin equivalence within a chronic wound may share structural attributes with a skin graft, but its function is to accelerate closure.

Animals↗

PubMed1994

The folding of bovine pancreatic trypsin inhibitor in the Escherichia coli periplasm.

PreOmpA-bovine pancreatic trypsin inhibitor (BPTI) (Goldenberg, D. P. (1988) Biochemistry 27, 2481-2489) was expressed in Escherichia coli, and the folding pathway of the mature protein in the periplasmic space was analyzed by pulse-chase experiments. Folding intermediates were trapped with iodoacetamide, immunoprecipitated with antisera specific for either the reduced or the native protein, and resolved by electrophoresis. In vivo, native BPTI formed with a half-life of 7 min which is 3-fold faster than the optimal in vitro folding rate in growth media supplemented with low molecular weight disulfides. The measured in vivo half-life includes the time required for translocation and processing by leader peptidase and therefore represents the lower limit for the actual folding rate in the cell. In addition to the native species, two-disulfide intermediates accumulated in the cell at appreciable levels and did not chase to the native species for at least 20 min. We found that the folding of BPTI in E. coli was absolutely dependent on DsbA, a protein which accelerates the formation of disulfide bonds in the periplasm. In a dsbA mutant strain, trace amounts of oxidized BPTI could be detected only in cultures grown under strongly oxidizing conditions. In wild-type cells, the addition of different concentrations of GSH/GSSG or oxidized dithiothreitol did not affect the kinetics of BPTI oxidation by DsbA. Finally, even though the folding of BPTI in vitro decreases by almost 10-fold/unit pH decrease, folding in cells grown at pH 6.0 was only marginally slower than folding in cells grown at neutral pH, despite the fact that the pH of the periplasmic space varies in response to the extracellular fluid.

Animals↗

PubMed2000

The protective effects of hypoxia-induced hypometabolism in the Nautilus.

Specimens of Nautilus pompilius were trapped at depths of 225-300 m off the sunken barrier reef southeast of Port Moresby, Papua New Guinea. Animals transported to the Motupore Island laboratory were acclimated to normal habitat temperatures of 18 degrees C and then cannulated for arterial and venous blood sampling. When animals were forced to undergo a period of progressive hypoxia eventually to encounter ambient partial pressure of oxygen (PO2) levels of approximately 10 mmHg (and corresponding arterial PO2's of approximately 5 mmHg), they responded by lowering their aerobic metabolic rates to 5-10% of those seen in resting normoxic animals. Coincident with this profound metabolic suppression was an overall decrease in activity, with brief periods of jet propulsion punctuating long periods of rest. Below ambient PO2 levels of 30-40 mmHg, ventilatory movements became highly periodic and at the lowest PO2 levels encountered, ventilation occasionally ceased altogether. Cardiac output estimated by the Fick equation decreased during progressive hypoxia by as much as 75 80%, and in the deepest hypometabolic states heart rates slowed to one to two cycles of very low amplitude per minute. By the end of 500 min exposure to ambient PO2 levels of 10 mmHg or less, the anaerobic end products octopine and succinate had increased significantly in adductor muscle and heart, respectively. Increased concentrations of octopine in adductor muscle apparently contributed to a small intracellular acidosis and to the development of a combined respiratory and metabolic acidosis in the extracellular compartment. On the other hand, increases in succinate in heart muscle occurred in the absence of any change in cardiac pHi. Taken together, we estimate that these anaerobic end products would make up less than 2% of the energy deficit arising from the decrease in aerobic metabolism. Thus, metabolic suppression is combined with a massive downregulation of systemic O2 delivery to match metabolic supply to demand.

Acid-Base Equilibrium↗

PubMed1997

Biosensor for neurotransmitter L-glutamic acid designed for efficient use of L-glutamate oxidase and effective rejection of interference.

An amperometric biosensor for L-glutamic acid (Glu) was constructed by the adsorption and dip coating of L-glutamate oxidase (GluOx, 200 U ml-1 phosphate buffer, pH 7.4) onto 60-micron radius Teflon-coated Pt wire (1 mm exposed length). The enzyme was then trapped on the surface by electropolymerisation of o-phenylenediamine that also served to block electroactive interference. This procedure afforded electrodes with similar substrate sensitivity compared with the classical approach of immobilising enzyme from a solution of monomer, and represents an approximately 10,000-fold increase in the yield of biosensors from a batch of enzyme. A number of strategies were examined to enhance the sensitivity and selectivity of the Pt/PPD/GluOx sensors operating at 0.7 V versus SCE. Pre-coating the Pt with lipid and incorporation of the protein bovine serum albumin into the polymer matrix were found to improve the performance of the electrode. The sensors had a fast response time, high sensitivity to Glu, with an LOD of about 0.3 mumol l-1, and possessed selectivity characteristics suggesting that monitoring Glu in biological tissues in vivo may be feasible.

Amino Acid Oxidoreductases↗

PubMed1994

Human recombinant extracellular-superoxide dismutase type C improves cardioplegic protection against ischemia/reperfusion injury in isolated rat heart.

The cardioprotective effects of human recombinant extracellular-superoxide dismutase type C (hr-EC-SOD C) were compared with those of bovine Cu,Zn-SOD in isolated working rat heart subjected to 35-min global normothermic ischemia followed by 55-min reperfusion. hr-EC-SOD C or bovine Cu,Zn-SOD (3 x 10(4) and 6 x 10(4) IU/L, respectively) was added to St. Thomas' Hospital (STH) cardioplegic solution infused 5 min before and 10 min after the ischemic period. Control hearts were treated with STH cardioplegic solution without SOD. By the end of reperfusion, hr-EC-SOD C-treated hearts recovered left ventricular systolic pressure (LVSP), aortic flow (AF) and cardiac output (CO) to 95 +/- 4, 60 +/- 4, 69 +/- 6% of preischemic value, respectively, as compared with 86 +/- 3, 44 +/- 5, and 52 +/- 6% in the control (p < 0.05). Cardioplegia with hr-EC-SOD C significantly reduced lactate dehydrogenase (LDH) release into myocardial effluent during reperfusion (p < 0.05) and increased ATP, AMP, and total creatine (Cr) tissue contents in reperfused hearts (by 21 +/- 3, 42 +/- 4, and 34 +/- 3%, respectively, as compared with control hearts, p < 0.05). The effects of bovine SOD on functional and biochemical indexes were similar but not statistically significant as compared with control. Treatment with hr-EC-SOD C, but not with bovine SOD, resulted in reduction in hydroxyl radical formation assessed by 5-5-dimethy-1-pyrroline-N-oxide spin trap (DMPO) in coronary effluent at early reperfusion with electron spin resonance (ESR) technique. The results suggest that enhanced myocardial protection against ischemia/reperfusion injury afforded by hr-EC-SOD C is related to scavenging of oxygen-derived free radicals.

Animals↗

PubMed1988

Perfusion distribution and lung thermal volume in canine hydrochloric acid aspiration.

We investigated the effects of a brief period of positive end-expiratory pressure (PEEP) ventilation or nitroglycerin (NTG) infusion on the distribution of pulmonary blood flow and extravascular thermal volume (ETV) in anesthetized dogs with unilateral HCl lung injury. ETV was determined by the thermal dye technique by use of a monoexponential extrapolation to exclude recirculating indicator, and regional blood flow was determined by a particle distribution technique (radiolabeled plastic microspheres). The lungs were weighted after the animals were killed, and extravascular lung mass (ELM) was determined with the use of hemoglobin to correct for trapped lung blood. Measurements were obtained before instillation of HCl into the right lung and repeated 3 h later before, during, and after PEEP ventilation or NTG infusion. Fractional perfusion of the severely injured portion of the right lung (Qinj/QT) fell from 44.3 +/- 11.1% at base line to 27.8 +/- 15.4% after the onset of lung injury. PEEP produced an acute reversible increase in ETV (63 +/- 37% over average of pre- and post-PEEP values), and the changes in ETV were closely correlated with changes in Qinj/QT (r = 0.91). NTG infusion produced insignificant increases in ETV (14 +/- 10% over average of pre- and postinfusion values) and Qinj/QT (59 +/- 35%), but the changes in ETV and Qinj/QT were strongly correlated (r = 0.92). The fraction of extravascular lung mass detected by the thermodilution measurement averaged 0.44 (range 0.24-0.77).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2001

Phosphotyrosines 627 and 659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) conferring binding and activation of SHP2.

A major Grb2-associated binder-1 (Gab1) binding partner in epidermal growth factor (EGF)-stimulated cells is protein-tyrosine phosphatase (PTPase) SHP2, which contains tandem SH2 domains. The SHP2 PTPase activity is required for activation of the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinase by EGF. To investigate the mechanism by which Gab1 and SHP2 mediate ERK activation, we characterized the Gab1-SHP2 interaction. We found that both Tyr-627 and Tyr-659 of Gab1 were required for SHP2 binding to Gab1 and for ERK2 activation by EGF. Far Western blot analysis suggested that the tandem SH2 domains of SHP2 bind to Gab1 in a specific orientation, in which the N-SH2 domain binds to phosphotyrosine (Tyr(P))-627 and the C-SH2 domain binds to Tyr(P)-659. When assayed with peptide substrates, SHP2 PTPase was activated by a bisphosphopeptide containing both Tyr(P)-627 and Tyr(P)-659, but not by monophosphopeptides containing Tyr(P)-627 or Tyr(P)-659 or a mixture of these monophosphopeptides. These results suggest that Tyr(P)-627 and Tyr(P)-659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) that binds and activates SHP2. Remarkably, while a constitutively active SHP2 (SHP2DeltaN) could not rescue the defect of a SHP2-binding defective Gab1 (Gab1FF) in ERK2 activation, expression of a Gab1FF-SHP2DeltaN chimera resulted in constitutive activation of ERK2 in transfected cells. Thus, physical association of activated SHP2 with Gab1 is necessary and sufficient to mediate the ERK mitogen-activated protein kinase activation. Phosphopeptides derived from Gab1 were dephosphorylated by active SHP2 in vitro. Consistently, substrate-trapping experiments with a SHP2 catalytic inactive mutant suggested that Gab1 was a SHP2 PTPase substrate in the cells. Therefore, Gab1 not only is a SHP2 activator but also is a target of its PTPase.

Amino Acid Motifs↗

PubMed1999

Tartrate-resistant purple acid phosphatase is synthesized as a latent proenzyme and activated by cysteine proteinases.

Purple acid phosphatases (PAPs) are binuclear acid metallohydrolases also referred to as tartrate-resistant acid phosphatases (TRAPs) or type 5 acid phosphatases. The cDNA sequences of TRAP/PAP enzymes from different species and organs indicate that these enzymes are translated as monomeric polypeptides of approx. 35 kDa, contrasting with the predominantly two-subunit structure observed in purified enzyme preparations. In the present study we have compared certain structural and enzyme-kinetic properties of recombinant rat PAP (monomeric) with those of the native rat bone TRAP/PAP enzyme (two-subunit), and examined effects on these parameters by cleaving the monomeric recombinant PAP with the serine proteinase trypsin or the cysteine proteinases papain or cathepsin B. Cleavage with trypsin resulted in a moderate activation of the recombinant enzyme and shifted the pH optimum to a slightly more basic value (5.0-5.5). Cleavage with papain resulted in complete activation and conferred similar properties to those of the bone PAP variant with regard to pH optimum (5.5-6.0) and sensitivity to reducing agents, as well as in the sizes of the subunits. Substrate specificity studies showed that the two-subunit bone PAP was considerably more active than the monomeric recombinant rat PAP towards a variety of serine-, threonine- and tyrosine-phosphorylated substrates. Of these substrates, bovine milk osteopontin seemed to be the most readily dephosphorylated substrate. In conclusion, the results suggest that the monomeric form of PAP represent a latent proenzyme with low enzymic activity towards both tyrosine- and serine/threonine-containing phosphorylated substrates. Besides being implicated in the catabolism of the extracellular matrix, members of the cysteine proteinase family might also exert a regulatory role in degradative processes involving the PAP enzymes by converting the newly synthesized PAPs to enzymically active and microenvironmentally regulated species.

Acid Phosphatase↗

PubMed1994

Membrane-permeant derivatives of cyclic AMP optimized for high potency, prolonged activity, or rapid reversibility.

A novel membrane-permeant derivative of cAMP, cAMP acetoxymethyl ester (cAMP/AM), was synthesized via silylated intermediates. Its ability to induce Cl- secretion by T84 cells, a human colon cancer cell line, was compared with that of two other membrane-permeant cAMP derivatives that were recently introduced, N6,O2'-dibutyryl-cAMP acetoxymethyl ester (bt2cAMP/AM) and Sp-5,6-dichlorobenzimidazole-1-beta-D-ribofuranoside 3',5'-cyclic phosphorothioate (Sp-5,6-DCl-cBIMPS). All of these compounds are powerful activators of Cl- secretion when applied extracellularly, with EC50 values of 60 microM, 0.7 microM, and 3 microM, respectively. However, cAMP/AM was expected to be readily degraded inside cells, in contrast to the cyclophosphodiesterase-resistant Sp-5,6-DCI-cBIMPS or the only slowly metabolizable N6-butyryl-cAMP derived from bt2cAMP/AM. Reversibility of cAMP/AM action was demonstrated by wash-out experiments; Cl- secretion induced by high doses of cAMP/AM (100 microM) could be quickly abolished by rinsing of the cells, whereas similar experiments with bt2cAMP/AM and Sp-5,6-DCI-cBIMPS showed much slower decreases. Even more sensitive to residual cAMP derivatives was the synergistic effect of carbachol, which was applied after the incubation with membrane-permeant derivatives and their subsequent wash-out. Although doses of cAMP derivatives that barely activated Cl- secretion were readily capable of inducing a synergistic response with carbachol, cells incubated with high doses of cAMP/AM (100 microM) and subsequently washed showed only a nonsynergistic carbachol response, in contrast to cells incubated with bt2cAMP/AM or Sp-5,6-DCI-cBIMPS. We therefore characterize cAMP/AM as a membrane-permeant derivative of cAMP that is easily metabolizable inside cells and hence is most useful for applications where a transient intracellular cAMP signal is desired. In contrast, completely nonmetabolizable Sp-5,6-DCI-cBIMPS seems to be more useful in longer incubations that require steady levels of cAMP-dependent protein kinase activation. bt2cAMP/AM combines the advantages of intracellular trapping by ester hydrolysis and reduced cyclophosphodiesterase sensitivity of its active intracellular product, which probably lead to its particularly high potency.

Cell Membrane↗

PubMed1998

Glycoxidation in aortic collagen from STZ-induced diabetic rats and its relevance to vascular damage.

Glycoxidation reactions lead to the formation of permanent, irreversible chemical modifications and cross-links in protein, such as the glycoxidation products carboxymethyllysine (CML) and pentosidine. It has been implicated that CML as well as Amadori products play a role in the formation of superoxidative products, such as H2O2 and advanced glycosylation endproducts in trapping LDL. Therefore, a possible relationship between glycoxidation and lipoperoxidation might exist because oxidized lipoprotein, which has been directly linked to atheroma formation, could be produced by the superoxidative products released from the pathway of CML formation. Using a CML-specific monoclonal antibody (6D12) and a specific antiserum against hexitol-lysine (HL), an Amadori product, we studied the relationship between glycoxidation and lipoperoxidation by determining the aortic CML contents with ELISA and the fluorescence levels of lipoperoxidation side products, malondialdehyde (MDA) and hydroxynonenal (HNE) from STZ-induced diabetic rats and age-matched control rats. The immunohistochemical and ultrastructural changes relevant to glycoxidation and lipoperoxidation were also studied. The CML content measured by ELISA in DM rats was significantly higher than that in the control rats at 28 weeks (n = 11, P < 0.01). The levels of MDA-linked and HNE-linked fluorescence in the DM rats increased in a similar way and were significantly higher than the levels in control rats at 28 weeks (n = 11, both P < 0.01 at 28 weeks). The CML contents correlated with the fluorescence levels of both MDA-linked (n = 19, r = 0.638, P < 0.01) and HNE-linked fluorescence (n = 19, r = 0.629, P < 0.01) only in the DM rats, but not in the control rats. Our immunohistochemical study thus demonstrated that CML was initially formed in the aortic media of diabetic rats in the 16th week of diabetes, localized primarily in the extracellular matrix surrounding the aortic smooth muscle cells after HL occurred early in the 2nd week of diabetes. Consequently, a significant increase in the extracellular matrix and decrease in the area of the SMCs were observed in the aortic media in the DM rats by a morphometrical study. The in vivo results of this study provided the first evidence that CML correlated with fluorescence levels of MDA and HNE, and thus suggested the existence of a close relationship between glycoxidation and lipoperoxidation in vivo. This information is thus considered to shed some new light on the etiology of atherogenesis in diabetes.

Aldehydes↗