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R Posmantur

Publications and source records attributed to R Posmantur.

13 recordsLinked to original sources
PubMed2000

cAMP delays beta-amyloid (25-35) induced cell death in rat cortical neurons.

Beta-amyloid (A beta) accumulation is believed to contribute to neuronal cell death in Alzheimer's disease. To understand the role of cAMP in the regulation of A beta induced cell death, we used 8-chlorophenylthio-cAMP (8-CPT-cAMP, a cAMP analog) to raise intracellular cAMP levels. Exposure of rat cortical neurons to A beta(25-35) resulted in a gradual increase in lactate dehydrogenase (LDH) over 48 h, which was preceded by a transient elevation in caspase-3-like activity. In the presence of 8CPT-cAMP, both caspase-3 activity and LDH release was significantly reduced. These data suggest that elevation of intracellular cAMP levels attenuate A beta-induced neurotoxicity and may delay or prevent the onset of A beta-induced neurodegeneration.

Amyloid beta-Peptides↗
PubMed1998

Caspase-3-like activity is necessary for IL-2 release in activated Jurkat T-cells.

The caspase family of proteases has previously been implicated in the biochemical cascade leading to apoptotic cell death. Recently caspase-3 was reported to be cleaved into its catalytically active subunits (17 and 13 kDa) following phytohemagglutinin (PHA) activation of peripheral blood mononuclear cells (C. Miossec et al., J. Biol. Chem. 272, 13459-13462). More recently, J. M. Zapata and colleagues (J. Biol. Chem. 273, 6916-6920, 1998), however, proposed that caspase-3 activity detected during T-cell activation was due to a methodological artifact related to the composition of the cell lysis buffer. Here we show that in PHA-activated Jurkat T-cells using the recommended lysis buffer detailed by Zapata et al., a caspase-3-like protease is activated and is accompanied by cleavage of PARP and alpha-spectrin into cleavage products suggestive of caspase-3 proteolytic activation. LDH release did not increase following PHA stimulation in this paradigm. Two caspase inhibitors, carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-D-DCB) and acetyl-Asp-Glu-Val-Asp-CHO, blocked IL-2 release in a dose-dependent manner. Caspase-3-like protease-generated PARP and alpha-spectrin breakdown product formation was also reduced by Z-D-DCB. In addition, Jurkat T-cells costimulated with anti-CD3 plus anti-CD28 produced significant levels of IL-2 that were also blocked by these caspase inhibitors. Importantly, IL-2 was determined in cell culture supernatants, thus avoiding a cell lysis step that might have enabled activation of caspase-3 by granzyme B. Collectively, these data support the role of caspase-3-like protease activity in Jurkat T-cell activation and demonstrate that caspase-3 like activity is necessary for IL-2 release in PHA-activated and anti-CD3/anti-CD28 costimulated Jurkat T-cells.

Aspartic Acid↗
PubMed1998

Simultaneous degradation of alphaII- and betaII-spectrin by caspase 3 (CPP32) in apoptotic cells.

The degradation of alphaII- and betaII-spectrin during apoptosis in cultured human neuroblastoma SH-SY5Y cells was investigated. Immunofluorescent staining showed that the collapse of the cortical spectrin cytoskeleton is an early event following staurosporine challenge. This collapse correlated with the generation of a series of prominent spectrin breakdown products (BDPs) derived from both alphaII- and betaII-subunits. Major C-terminal alphaII-spectrin BDPs were detected at approximately 150, 145, and 120 kDa (alphaII-BDP150, alphaII-BDP145, and alphaII-BDP120, respectively); major C-terminal betaII-spectrin BDPs were at approximately 110 and 85 kDa (betaII-BDP110 and betaII-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that alphaII-BDP150 and alphaII-BDP120 were generated by cleavages at DETD1185*S1186 and DSLD1478*S1479, respectively. For betaII-spectrin, a major caspase site was detected at DEVD1457*S1458, and both betaII-BDP110 and betaII-BDP85 shared a common N-terminal sequence starting with Ser1458. An additional cleavage site near the C terminus, at ETVD2146*S2147, was found to account for betaII-BDP85. Studies using specific caspase or calpain inhibitors indicate that the pattern of spectrin breakdown during apoptosis differs from that during non-apoptotic cell death. We postulate that in concert with calpain, caspase rapidly targets critical sites in both alphaII- and betaII-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis.

Amino Acid Sequence↗
PubMed1998

Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis.

Two cysteine protease families (caspase and calpain) participate in apoptosis. Here we report that the endogenous calpain inhibitor calpastatin is fragmented by caspase(s) to various extents during early apoptosis in two cell types. In anti-fas or staurosporine-treated Jurkat T-cells, the high-molecular-weight form (HMW) of calpastatin (apparent Mr 110 K) was extensively degraded to immunoreactive fragments of Mr 75 K and 30 K In apoptotic SH-SY5Y human neuroblastoma cells, HMW calpastatin was degraded to a major immunoreactive fragment of 75 K. In both cell types, fragmentation of HMW calpastatin was blocked by a caspase-specific inhibitor carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene. In vitro translated HMW calpastatin was sensitive to proteolysis by recombinant caspase-1, -3, and -7. By contrast, in vitro translated LMW calpastatin (which lacks domains L and I) was cleaved into multiple fragments only by caspase-1 and was relatively resistant to caspase-3, -7, and other caspases tested. Consistently with that, purified erythroid LMW calpastatin was also highly susceptible to caspase-1 digestion. Recombinant human calpastatin spanning domain I through III (CAST(DI-III)) was found cleaved by caspase-1 at at least three sites, located in either the A or the C helix of domains I and III (ALDD137*L, LSSD203*F and ALAD404*S), while only a single site (ALDD137*L) was cleaved by caspase-3. These findings suggest that both HMW and LMW calpastatins are more vulnerable to caspase-1 than to caspase-3. Surprisingly, both erythroid LMW calpastatin and recombinant CAST(DI-III) fragmented by caspase-1 suffered only a less than twofold reduction of inhibitory activity toward calpain. We propose that the proteolysis of calpastatin in early apoptosis might have yet unidentified effects on the cross-talk between the two protease systems.

Apoptosis↗
PubMed1998

Subcellular localization and duration of mu-calpain and m-calpain activity after traumatic brain injury in the rat: a casein zymography study.

Casein zymographic assays were performed to identify changes in mu-calpain and m-calpain activity in naive, sham-injured, and injured rat cortex at 15 minutes, 3 hours, 6 hours, and 24 hours after unilateral cortical impact brain injury. Cortical samples ipsilateral and contralateral to the site of injury were separated into cytosolic and total membrane fractions. Marked increases in mu-calpain activity in cytosolic fractions in the ipsilateral cortex occurred as early as 15 minutes, became maximal at 6 hours, and decreased at 24 hours to levels observed at 15 minutes after injury. A similar temporal profile of cytosolic mu-calpain activity in the contralateral cortex was observed, although the increases in the contralateral cortex were substantially lower than those in the ipsilateral cortex. Differences were also noted between cytosolic and total membrane fractions. The detection of a shift in mu-calpain activity to the total membrane fraction first occurred at 3 hours after traumatic brain injury and became maximal at 24 hours after traumatic brain injury. This shift in mu-calpain activity between the two fractions could be due to the redistribution of mu-calpain from the cytosol to the membrane. m-Calpain activity was detected only in cytosolic fractions. m-Calpain activity in cytosolic fractions did not differ significantly between ipsilateral and contralateral cortices, and increased in both cortices from 15 minutes to 6 hours after injury. Relative magnitudes of m-calpain versus mu-calpain activity in cytosolic fractions differed at different time points after injury. These studies suggest that traumatic brain injury can activate both calpain isoforms and that calpain activity is not restricted to sites of focal contusion and cell death at the site of impact injury but may represent a more global response to injury.

Animals↗
PubMed1997

A purine nucleoside phosphorylase (PNP) inhibitor induces apoptosis via caspase-3-like protease activity in MOLT-4 T cells.

Children with congenital homozygous deficiency of purine nucleoside phosphorylase (PNP) have abnormalities in purine metabolism that result in T-cell selective immune deficiency. The mechanism of action for cell death has been attributed to intracellular accumulation of dGTP, a potent inhibitor of ribonucleotide reductase and subsequently DNA synthesis, in thymocytes and T-cells but not B-cells. However, the mode of cell death has not been determined to be either necrosis or apoptosis. To examine the involvement of apoptosis in T-cells following PNP inhibition, MOLT-4 cells, a human T cell leukemia cell line, were co-treated with the PNP inhibitor, CI-1000 (2-amino 3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d]-pyrimidin-4-one HCl), and 2'-deoxyguanosine (dGuo) which resulted in a concentration-dependent loss of cell viability (trypan blue) and inhibition of tritiated thymidine ([3H]-TdR) uptake. Staining of cells with the DNA dye Hoechst 33,258 showed nuclear morphology characteristic of apoptosis. Western blots (24 h lysates) were probed with antibodies against several proteins implicated in apoptosis. Anti-PARP revealed the presence of an 85 kD PARP breakdown product while, anti-alpha-spectrin revealed the accumulation a 120 kD breakdown product, both suggestive of CPP32 cleavage (caspase-3; an ICE-like cysteine protease). Western blots also detected the loss of the intact 32 kD caspase-3 isoform, a biochemical event associated with caspase-3 activation. Corresponding fluorometric activity assays detected a marked increase in caspase-3-like activity using the substrate Ac-DEVD-MCA. Lastly, a pan caspase inhibitor (Z-D-DCB) and 2'-deoxycytidine (dCyd), which is known to prevent dGTP accumulation following PNP inhibition, were able to prevent cell death and all indicators of caspase-3-like activity in MOLT-4 cells co-treated with dGuo and CI-1000. In summary, we provided several lines of evidence for the role of apoptosis and the contribution of caspase-3-like proteases in T-cell death following PNP inhibition.

Apoptosis↗
PubMed1997

A calpain inhibitor attenuates cortical cytoskeletal protein loss after experimental traumatic brain injury in the rat.

The capacity of a calpain inhibitor to reduce losses of neurofilament 200-, neurofilament 68- and calpain 1-mediated spectrin breakdown products was examined following traumatic brain injury in the rat. Twenty-four hours after unilateral cortical impact injury, western blot analyses detected neurofilament 200 losses of 65% (ipsilateral) and 36% (contralateral) of levels observed in naive, uninjured rat cortices. Neurofilament 68 protein levels decreased only in the ipsilateral cortex by 35% relative to naive protein levels. Calpain inhibitor 2, administered 10 min after injury via continuous arterial infusion into the right external carotid artery for 24 h, significantly reduced neurofilament 200 losses to 17% and 3% relative to naive neurofilament 200 protein levels in the ipsilateral and contralateral cortices, respectively. Calpain inhibitor administration abolished neurofilament 68 loss in the ipsilateral cortex and was accompanied by a reduction of putative calpain-mediated neurofilament 68 breakdown products. Spectrin breakdown products mediated by calpain 1 activation were detectable in both hemispheres 24 h after traumatic brain injury and were substantially reduced in animals treated with calpain inhibitor 2 both ipsilaterally and contralaterally to the site of injury. Qualitative immunofluorescence studies of neurofilament 200 and neurofilament 68 confirmed western blot data, demonstrating morphological protection of neuronal structure throughout cortical regions of the traumatically injured brain. Morphological protection included preservation of dendritic structure and reduction of axonal retraction balls. In addition, histopathological studies employing hematoxylin and eosin staining indicated reduced extent of contusion at the injury site. These data indicate that calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons after experimental traumatic brain injury in vivo.

Animals↗
PubMed1997

Characterization of CPP32-like protease activity following apoptotic challenge in SH-SY5Y neuroblastoma cells.

We characterized the activation of interleukin-1beta-converting enzyme (ICE)-like proteases (caspases) in human neuroblastoma cells (SH-SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti-poly(ADP-ribose) polymerase (anti-PARP) and anti-CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl-DEVD-7-amido-4-methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32-like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH-SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH-SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.

Antibody Specificity↗
PubMed1996

mu-calpain activation and calpain-mediated cytoskeletal proteolysis following traumatic brain injury.

Increasing evidence suggests that excessive activation of the calcium-activated neutral protease mu-calpain could play a major role in calcium-mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of mu-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of mu-calpain to its 80-kDa precursor was measured by western blotting. This mu-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24-48 h after injury. We also investigated the effect of mu-calpain activation on proteolysis of the neuronal cytoskeletal protein alpha-spectrin. Immunoreactivity for alpha-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of alpha-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of alpha-spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of mu-calpain autolysis, calpain-mediated alpha-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although mu-calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain-mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent mu-calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.

Analysis of Variance↗
PubMed1996

Calpain inhibitors protect against depolarization-induced neurofilament protein loss of septo-hippocampal neurons in culture.

We examined the effect of a 6 min depolarization with 60 mM KCl and 1.8, 2.8 or 5.8 mM extracellular CaCl2 on neurofilament proteins of high (NF-H), medium (NF-M) and low (NF-L) molecular weight in primary septohippocampal cultures. One day after depolarization, Western blot analyses revealed losses of all three neurofilament proteins. Increasing the extracellular calcium concentration from 1.8 to 5.8 mM CaCl2 in the presence of 60 mM KCl produced increased losses of all three neurofilament proteins to approximately 80% of control values in the absence of cell death. Calcium-dependent losses of the neurofilament proteins correlated with calcium-dependent increases in calpain 1-mediated breakdown products of alpha-spectrin. Calpain inhibitors 1 and 2, applied immediately after depolarization and made available to cultures for 24 h, reduced losses of all three neurofilament proteins to approximately 14% of control values. The protective effects of calpain inhibitors 1 and 2 were influenced by different levels of extracellular calcium. Qualitative immunohistochemical evaluations confirmed semiquantitative Western blot data on neurofilament loss and protection by calpain inhibitors 1 and 2. We propose that brief depolarization causes loss of neurofilament proteins, possibly due to calpain activation. Thus, calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons.

Animals↗
PubMed1995

Calpain inhibitors reduce depolarization induced loss of tau protein in primary septo-hippocampal cultures.

We studied the effects of a 6-min potassium depolarization injury produced by 60 mM KCl and 1.8 mM or 5.8 mM extracellular CaCl2 on tau protein levels in primary rat septo-hippocampal cultures. One day after injury, Western blot analyses revealed a calcium dependent loss of tau protein of approximately 50% of control values. Loss of tau protein was associated with calpain 1 mediated breakdown products to alpha-spectrin. Calpain inhibitors 1 and 2, applied immediately after depolarization injury and available to cultures for 24 h reduced depolarization induced degradation of tau protein to approximately 35% or 25% of control values, respectively. We propose that brief potassium depolarization causes degradation of tau protein, possibly due to calpain activation. Thus, calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons.

Animals↗
PubMed1994

Neurofilament 68 and neurofilament 200 protein levels decrease after traumatic brain injury.

We have examined the effect of lateral cortical impact injury on the levels of axonal cytoskeletal proteins in adult rats. Traumatic brain injury (TBI) causes a significant decrease in the protein levels of two prominent neurofilament (NF) proteins, NF68 and NF200. We employed quantitative immunoreactivity measurements on Western blots to examine NF68 and NF200 levels in homogenates of hippocampal and cortical tissue taken at several intervals postinjury. Sham injury had no effect on NF protein levels. However, injury was associated with a significant loss of NF68, restricted to the cortex ipsilateral to the injury site. NF68 loss was detectable as early as 3 h and lasted at least 2 weeks postinjury. Similarly, TBI induced a decrease in NF200 protein, although losses were observed both ipsilateral and contralateral to the injury site. No loss of NF68 or NF200 protein was detected in hippocampal samples obtained from the same injured animals. An increase in the presence of lower molecular weight (MW) NF68 immunopositive bands was associated with the decrease of NF68 in the ipsilateral cortex. This NF68 antigenicity pattern suggests the production of NF68 breakdown products caused by the pathologic activation of neuronal proteases, such as calpain. Putative NF68 breakdown products increase significantly until 1 day postinjury, suggesting that NF degradation may be ongoing until that time and indicating that a potential therapeutic window may exist within the first 24 h postinjury. In summary, these data identify specific biochemical alterations of the neuronal cytoskeleton following TBI and lay a foundation for further investigation of postinjury cytoskeletal changes in neuronal processes.

Animals↗