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Biomedical subjects

A Srinivasan

Publications and source records attributed to A Srinivasan.

At least 19 recordsLinked to original sources
PubMed2000

Activation of caspase-3 in permanent and transient brain ischaemia in man.

Animal studies have shown brain ischaemia to cause oxidative damage to DNA and activation of caspase-3, leading to apoptosis. These changes may be exacerbated by reperfusion. To assess caspase-3 activation after transient and permanent brain ischaemia in man, we examined brain tissue from patients who had experienced a cardiac arrest with resuscitation or an atherothrombotic brain infarct, and died 12 h to 9 days later. Sections were immunostained for activated caspase-3 or the 89 kDa caspase-3-mediated cleavage product of poly(ADP-ribose) polymerase. Brain ischaemia caused activation of caspase-3 in macrophages/microglia. Some neurons showed delayed activation of caspase-3 after cardiac arrest, but very few in atherothrombotic infarcts. In man, activation of caspase-3 plays little part in neuronal death in atherothrombotic infarcts but may contribute to delayed death of neurons after cardiac arrest.

Adult↗
PubMed2000

Decreased apoptosis in proliferative and postmitotic regions of the Caspase 3-deficient embryonic central nervous system.

Caspase 3 (CPP32/Yama/apopain), a mammalian homolog of the Caenorhabditis elegans pro-cell death gene ced-3, is required for normal programmed cell death (PCD) in the nematode. Its prior deletion by homologous recombination in mice resulted in embryonic/early postnatal lethality associated with dramatic central nervous system (CNS) hyperplasia, yet a reported subtle decrease in cell death (Kuida et al. [1996] Nature 384:368-372). By comparison, the magnitude and distribution of dying cells identified using a DNA end-labeling technique, in situ end-labeling plus (ISEL+) (Blaschke et al. [1996] Development 122:1165-1174; Blaschke et al. [1998] J. Comp. Neurol. 396:39-50), supported an alternative explanation where the loss of caspase 3 function produces a more pervasive block in cell death, particularly among neuroblasts. To determine the relationship between loss of caspase 3 and dying cells identified by ISEL+, we analyzed caspase 3 +/+, +/-, and -/- embryos for normal caspase 3 expression and ISEL+ labeling. Both caspase 3 mRNA and active caspase 3 protein are present throughout the +/+ embryonic CNS, and both are absent from -/- embryonic cortices. Quantitation of dying cells identified by ISEL+ reveals a 30% reduction of labeled cells throughout the caspase 3 -/- embryonic cortices relative to +/+ littermates. Associated with this decrease is marked expansion of the total population of actively proliferating neuroblasts identified by 5-bromo-2;-deoxyuridine incorporation that nevertheless appears to maintain histological features of normal neurogenesis rather than dysregulated, neoplastic growth. These data indicate that caspase 3 deficiency results in a pervasive, albeit partial, decrease in embryonic neuroblast apoptosis that can account for the observed phenotypic hyperplasia in -/- embryos, and support the additional operation of caspase 3-independent PCD mechanisms during embryonic CNS development.

Animals↗
PubMed2000

Mechanical and physicochemical regulation of the action of insulin-like growth factor-I on articular cartilage.

The development and maintenance of healthy joints is a complex process involving many physical and biological stimuli. This study investigates the interaction between insulin-like growth factor-I (IGF-I) and static mechanical compression in the regulation of articular cartilage metabolism. Bovine cartilage explants were treated with concentrations of IGF-I from 0 to 300 ng/ml in the presence or absence of 0-50% static compression, and the transient and steady-state incorporation of [(3)H]proline and [(35)S]sulfate into matrix components were measured. In parallel studies, cartilage explants were treated with 0-300 ng/ml IGF-I at media pH ranging from 6.4 to 7.2 and the steady-state incorporation of [(3)H]proline and [(35)S]sulfate was measured. The effect of 50% static compression on IGF-I transport was determined by measuring the uptake of (125)I-labeled IGF-I into cartilage explants. Static compression decreased both [(3)H]proline and [(35)S]sulfate incorporation in a dose-dependent manner in the presence or absence of IGF-I. IGF-I increased [(3)H]proline and [(35)S]sulfate incorporation in a dose-dependent manner in the presence or absence of compression, but the anabolic effect of the growth factor was lessened when the tissue was compressed by 50%. The response of cartilage explants to IGF-I was similarly lessened in unstrained tissue cultured in media at pH 6.4, a condition which results in a similar intratissue pH to that when cartilage is compressed by 50%. The characteristic time constant (tau) for IGF-I stimulation of cartilage explants was approximately 24 h, while tau for inhibition of biosynthesis by static compression was approximately 2 h. Samples which were both compressed and treated with IGF-I demonstrated an initial decrease in biosynthetic activity at 2 h, followed by an increase at 24 h. Static compression did not alter tau for (125)I-labeled IGF-I transport into cartilage but decreased the concentration of (125)I-labeled IGF-I in the tissue at equilibrium.

Animals↗
PubMed2000

Dual role of caspase-11 in mediating activation of caspase-1 and caspase-3 under pathological conditions.

Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11-deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.

Animals↗
PubMed2000

Epitope-tagging approach to determine the stoichiometry of the structural and nonstructural proteins in the virus particles: amount of Vpr in relation to Gag in HIV-1.

We used an epitope-tagging approach to determine the ratio of Gag (structural) to Vpr (nonstructural) in the virus particles directed by human immunodeficiency virus type 1. For this purpose, chimeric Gag and Vpr expression plasmids were constructed with the Flag epitope (DYKDDDDK), and the sequences corresponding to the chimeric protein were introduced into human immunodeficiency virus type 1 proviral DNA (NL4-3) to determine the ratio in the virus particles when these proteins are expressed in cis. In addition, NL4-3 DNA was modified to disrupt Vpr synthesis to determine the extent of incorporation of Vpr-FL when it is expressed in trans through a heterologous promoter. The analysis of virus particles generated by transfection of proviral DNA into RD cells indicated that (1) the ratio of Gag to Vpr in virus particles, when Vpr-FL is expressed in cis (in the context of proviral DNA), is in the range of 150-200:1 (14-18 molecules of Vpr per virion) and (2) the expression of Vpr-FL in trans showed efficient incorporation with a Gag to Vpr ratio of 5-7:1 (392-550 molecules of Vpr). These results suggest that the presence of the same epitope on different viral proteins may provide an accurate comparison of these proteins in the virus particles.

Epitope Mapping↗
PubMed2000

Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson's disease.

Caspase-3 is an effector of apoptosis in experimental models of Parkinson's disease (PD). However, its potential role in the human pathology remains to be demonstrated. Using caspase-3 immunohistochemistry on the postmortem human brain, we observed a positive correlation between the degree of neuronal loss in dopaminergic (DA) cell groups affected in the mesencephalon of PD patients and the percentage of caspase-3-positive neurons in these cell groups in control subjects and a significant decrease of caspase-3-positive pigmented neurons in the substantia nigra pars compacta of PD patients compared with controls that also could be observed in an animal model of PD. This suggests that neurons expressing caspase-3 are more sensitive to the pathological process than those that do not express the protein. In addition, using an antibody raised against activated caspase-3, the percentage of active caspase-3-positive neurons among DA neurons was significantly higher in PD patients than in controls. Finally, electron microscopy analysis in the human brain and in vitro data suggest that caspase-3 activation precedes and is not a consequence of apoptotic cell death in PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2000

Extent of incorporation of HIV-1 Vpr into the virus particles is flexible and can be modulated by expression level in cells.

To examine the factors that control the extent of incorporation of Vpr into the virus particles, we utilized an epitope-tagging approach with Flag (FL) as the epitope for quantitation. We generated expression plasmids containing Vpr-FL and Vpr E21,24P-FL and also HIV-1 proviral DNA containing Vpr-FL (NL-Vpr-FL). Immunoblot analysis using Flag antibodies revealed that virus particles derived from co-transfection of NL-Vpr-FL and Vpr-FL showed an enhanced level of Vpr-FL in comparison to NL-Vpr-FL derived virus. These results suggest that the amount of incorporation of Vpr into the virus particles is flexible and may be modulated by its expression level in cells.

Cell Line↗
PubMed2000

Three-dimensional structure of a presynaptic neurotoxic phospholipase A2 from Daboia russelli pulchella at 2.4 A resolution.

The phospholipase A(2 )from Daboia russelli pulchella (DPLA(2)) is the only known member of subclass II of group IIA. The three-dimensional structure of this presynaptic neurotoxic DPLA(2) enzyme has been determined at 2.4 A resolution. The structure was determined by the molecular replacement method using the model Crotalus atrox, and refined using X-PLOR to a final R-factor of 18.8 % for all data in the resolution range 20.0 A-2.4 A. The final refined model comprises 1888 atoms from two crystallographically independent protein molecules and 160 water oxygen atoms. The overall folding of DPLA(2), with three long helices and two short antiparallel beta-strands is grossly similar to those observed for other PLA(2)s. In the present structure, the calcium binding site is empty but the conformation of the calcium binding loop is similar to those observed in the calcium bound states. Two spatially adjacent regions of residues 55-61 (a typical beta-turn I) and 83-94 (a well defined loop) are remarkably different in conformation, electrostatic characteristics and inter-segmental interactions from those found in non-neurotoxic PLA(2)s. Yet another striking structural feature in DPLA(2 )pertains to the stretch of residues 53-77, which has a series of positively charged residues protruding outwardly. The above segment is presumed to be involved in the anticoagulant activity. A unique hydrophobic patch including residues Leu17, Ala18, Ile19, Pro20, Phe106 and Leu110 is found on the surface together with an equally emphatic region of -OH groups containing residues such as Ser21, Tyr22, Ser23, Ser24, Tyr25 and Tyr28. The interactions between two molecules of DPLA(2) in the asymmetric unit are remarkably different from those observed in the standard dimers and trimers of PLA(2)s, leaving the enzyme's active site fully exposed for enzyme-substrate reactions, it makes this structure one of the most favourable examples for structure-based drug design through soaking experiments.

Amino Acid Sequence↗
PubMed2000

Evidence that Wallerian degeneration and localized axon degeneration induced by local neurotrophin deprivation do not involve caspases.

The selective degeneration of an axon, without the death of the parent neuron, can occur in response to injury, in a variety of metabolic, toxic, and inflammatory disorders, and during normal development. Recent evidence suggests that some forms of axon degeneration involve an active and regulated program of self-destruction rather than a passive "wasting away" and in this respect and others resemble apoptosis. Here we investigate whether selective axon degeneration depends on some of the molecular machinery that mediates apoptosis, namely, the caspase family of cysteine proteases. We focus on two models of selective axon degeneration: Wallerian degeneration of transected axons and localized axon degeneration induced by local deprivation of neurotrophin. We show that caspase-3 is not activated in the axon during either form of degeneration, although it is activated in the dying cell body of the same neurons. Moreover, caspase inhibitors do not inhibit or retard either form of axon degeneration, although they inhibit apoptosis of the same neurons. Finally, we cannot detect cleaved substrates of caspase-3 and its close relatives immunocytochemically or caspase activity biochemically in axons undergoing Wallerian degeneration. Our results suggest that a neuron contains at least two molecularly distinct self-destruction programs, one for caspase-dependent apoptosis and another for selective axon degeneration.

Animals↗
PubMed2000

Atm and Bax cooperate in ionizing radiation-induced apoptosis in the central nervous system.

Ataxia-telangiectasia is a hereditary multisystemic disease resulting from mutations of ataxia telangiectasia, mutated (ATM) and is characterized by neurodegeneration, cancer, immune defects, and hypersensitivity to ionizing radiation. The molecular details of ATM function in the nervous system are unclear, although the neurological lesion in ataxia-telangiectasia becomes apparent early in life, suggesting a developmental origin. The central nervous system (CNS) of Atm-null mice shows a pronounced defect in apoptosis induced by genotoxic stress, suggesting ATM functions to eliminate neurons with excessive genomic damage. Here, we report that the death effector Bax is required for a large proportion of Atm-dependent apoptosis in the developing CNS after ionizing radiation (IR). Although many of the same regions of the CNS in both Bax-/- and Atm-/- mice were radioresistant, mice nullizygous for both Bax and Atm showed additional reduction in IR-induced apoptosis in the CNS. Therefore, although the major IR-induced apoptotic pathway in the CNS requires Atm and Bax, a p53-dependent collateral pathway exists that has both Atm- and Bax-independent branches. Further, Atm- and Bax-dependent apoptosis in the CNS also required caspase-3 activation. These data implicate Bax and caspase-3 as death effectors in neurodegenerative pathways.

Animals↗
PubMed2000

Hepatocytes sensitized to tumor necrosis factor-alpha cytotoxicity undergo apoptosis through caspase-dependent and caspase-independent pathways.

Hepatocytes can be sensitized to tumor necrosis factor (TNF)-alpha toxicity by repression of NF-kappaB activation or inhibition of RNA synthesis. To determine whether both forms of sensitization lead to TNF-alpha cytotoxicity by similar mechanisms, TNF-alpha-induced cell death in RALA255-10G hepatocytes was examined following infection with an adenovirus, Ad5IkappaB, that blocks NF-kappaB activation or following cotreatment with actinomycin D (ActD). TNF-alpha treatment of Ad5IkappaB-infected cells resulted in 44% cell death within 6 h. ActD/TNF-alpha induced no death within 6 h but did lead to 37% cell death by 24 h. In both instances, cell death occurred by apoptosis and was associated with caspase activation, although caspase activation in ActD-sensitized cells was delayed. CrmA and chemical caspase inhibitors blocked Ad5IkappaB/TNF-alpha-induced cell death but did not inhibit ActD/TNF-alpha-induced apoptosis. A Fas-associated protein with death domain (FADD) dominant negative decreased Ad5IkappaB/TNF-alpha- and ActD/TNF-alpha-induced cell death by 81 and 47%, respectively. However, downstream events differed, since Ad5IkappaB/TNF-alpha but not ActD/TNF-alpha treatment caused mitochondrial cytochrome c release. These results suggest that NF-kappaB inactivation and inhibition of RNA synthesis sensitize RALA255-10G hepatocytes to TNF-alpha toxicity through distinct cell death pathways that diverge below the level of FADD. ActD-induced hepatocyte sensitization to TNF-alpha cytotoxicity occurs through a FADD-dependent, caspase-independent pathway of apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed2000

BDNF blocks caspase-3 activation in neonatal hypoxia-ischemia.

Hypoxic-ischemic (H-I) injury to the brain in the perinatal period often leads to significant long-term neurological deficits. In a model of neonatal H-I injury in postnatal day 7 rats, our previous data have shown that cell death with features of apoptosis is prominent between 6 and 24 h after H-I and that neurotrophins, particularly BDNF, can markedly protect against tissue loss. During brain development, caspase-3 is required for normal levels of programmed cell death. Utilizing an antibody specific for the activated form of caspase-3, CM1, we now show that caspase-3 is specifically activated in neuronal cell bodies and their processes beginning at 6 h and peaking 24 h following unilateral carotid ligation and exposure to hypoxia in postnatal day 7 rats. Caspase-3 activation began to occur in cortex at 6 h and in striatum and hippocampus at 12-18 h. Caspase-3 activation was also observed in developing oligodendrocytes. Intracerebroventricular injection of BDNF prior to H-I injury almost completely abolished evidence of H-I-induced caspase-3 activation in vivo. Utilizing a specific molecular marker of an apoptotic pathway, these findings demonstrate that H-I injury to the developing brain is a strong apoptotic stimulus leading to caspase-3 activation, that BDNF can block this process in vivo, and that the ability of BDNF to inhibit caspase activation and subsequent apoptosis likely accounts in large part for its protection against neuronal injury in this model.

Animals↗
PubMed2000

Caspases: decoders of apoptotic signals during mammary involution. Caspase activation during involution.

At weaning most of the alveolar epithelial cells in the mammary gland die by apoptosis and are removed by phagocytosis. Caspases are a family of aspartate specific cysteine proteases. Activation of caspases is generally thought to represent a major and irreversible event in the apoptotic process. We analyzed caspase expression and activation during mammary gland involution. A quantitative RT-PCR based approach revealed that levels of mRNA expression of several caspases are induced during involution. Using an antibody that specifically recognizes activated caspases we measured a transient induction of caspase activity in situ and found a maximal activation at days two and three of involution. These data were corroborated by monitoring caspase-3 like activity in mammary extracts with a synthetic DEVD-afc peptide as caspase-3 substrate. Using Fas-deficient mice we present evidence that the Fas signaling pathway is not essential for caspase activation and apoptosis during mammary gland involution. In summary, signaling pathways during involution seem to involve activation of caspases as intraepithelial triggers of mammary epithelial cell apoptosis.

Animals↗
PubMed2000

In vivo study of motoneuron death induced by nerve injury in mice deficient in the caspase 1/ interleukin-1 beta-converting enzyme.

The apoptotic cell death program is orchestrated by members of the caspase family. Among these caspases, several in vitro and in vivo reports indicate that the interleukin-1 beta-converting enzyme (or caspase 1) may be involved in neurodegenerative processes. In view of these findings, and in order to characterize the role of the interleukin-1beta-converting enzyme in mediating or modulating cell death processes in vivo, we have investigated the effects of its deletion on motoneuron survival after a facial nerve transection in newborn and adult interleukin-1 beta-converting enzyme knock-out mice. During the postnatal period of development, when facial motoneurons are highly vulnerable to axotomy, we did not observe any significant effect of the interleukin-1 beta-converting enzyme-deletion on the percentage of cell death in the lesioned nuclei. In addition, the spontaneous cell death characteristic of the postnatal period was not altered in knock-out mice. In contrast, in adult knock-out mice, a significant reduction (16%) in the number of surviving facial motoneurons was observed six weeks after axotomy. We therefore conclude that the interleukin-1 beta-converting enzyme does not appear to be critical for cell death during the postnatal period but may favor motoneuron survival during adulthood. Given the key role of caspase 3 in neuronal apoptosis during embryonic development of the central nervous system, we also investigated the role of this caspase in cell death following axotomy. Combined immunofluorescence revealed that, at least during the postnatal period, axotomized motoneurons that have apoptotic nuclear morphologies were immunopositive for the active form of caspase 3. Double-stained cells could be also observed on the unlesioned side. These results strongly suggest that caspase 3 may be involved in both the postnatal spontaneous- and axotomy-induced facial motoneuron death processes. Similar results were obtained in interleukin-1 beta-converting enzyme-deficient and wild-type mice, indicating that the interleukin-1 beta-converting enzyme may not be required for caspase 3 activation.

Age Factors↗
PubMed2000

Radioiodination and astatination of octreotide by conjugation labeling.

Octreotide was coupled to 3-iodobenzoyl and 3-iodonicotinoyl moieties to obtain [N-(3-iodobenzoyl)-D-Phe(1)]octreotide (IBO) and [N-(3-iodonicotinoyl)-D-Phe(1)]octreotide (INO), respectively. The IC(50) values for the binding of IBO and INO to CA20948 rat pancreatic tumor membranes were 0.90 and 0.13 nM, respectively, compared with 0.35 nM for octreotide itself. Starting from N-succinimidyl 3-[(131)I]iodobenzoate and N-succinimidyl 5-[(131)I]iodopyridine-3- carboxylate, [(131)I]IBO and [(131)I]INO were prepared in overall radiochemical yields of 35%-50%. Likewise, ¿N-(3-[(211)At]astatobenzoyl)-D-Phe(1)¿octreotide ([(211)At]ABO) was prepared in similar yield from N-succinimidyl 3-[(211)At]astatobenzoate. In vitro assays with AR42J rat pancreatic tumor cells demonstrated a higher retention of cell-internalized radioiodine activity for [(131)I]INO compared with [(125)I]IBO. Tissue distribution studies with both conjugates revealed low levels of activity in the thyroid suggesting that dehalogenation of these peptides was minimal.

Animals↗
PubMed2000

Temporal profile and cell subtype distribution of activated caspase-3 following experimental traumatic brain injury.

This study investigated the temporal expression and cell subtype distribution of activated caspase-3 following cortical impact-induced traumatic brain injury in rats. The animals were killed and examined for protein expression of the proteolytically active subunit of caspase-3, p18, at intervals from 6 h to 14 days after injury. In addition, we also investigated the effect of caspase-3 activation on proteolysis of the cytoskeletal protein alpha-spectrin. Increased protein levels of p18 and the caspase-3-specific 120-kDa breakdown product to alpha-spectrin were seen in the cortex ipsilateral to the injury site from 6 to 72 h after the trauma. Immunohistological examinations revealed increased expression of p18 in neurons, astrocytes, and oligodendrocytes from 6 to 72 h following impact injury. In contrast, no evidence of caspase-3 activation was seen in microglia at all time points investigated. Quantitative analysis of caspase-3-positive cells revealed that the number of caspase-3-positive neurons exceeded the number of caspase-3-positive glia cells from 6 to 72 h after injury. Moreover, concurrent assessment of nuclear histopathology using hematoxylin identified p18-immunopositive cells exhibiting apoptotic-like morphological profiles in the cortex ipsilateral to the injury site. In contrast, no evidence of increased p18 expression or alpha-spectrin proteolysis was seen in the ipsilateral hippocampus, contralateral cortex, or hippocampus up to 14 days after the impact. Our results are the first to demonstrate the concurrent expression of activated caspase-3 in different CNS cells after traumatic brain injury in the rat. Our findings also suggest a contributory role of activated caspase-3 in neuronal and glial apoptotic degeneration after experimental TBI in vivo.

Animals↗
PubMed2000

Antiviral agent based on the non-structural protein targeting the maturation process of HIV-1: expression and susceptibility of chimeric Vpr as a substrate for cleavage by HIV-1 protease.

The processing of precursor proteins (Gag and Gag-pol) by the viral protease is absolutely required in order to generate infectious particles. This prompted us to consider novel strategies that target viral maturation. Towards this end, we have engineered an HIV-1 virion associated protein, Vpr, to contain protease cleavage signal sequences from Gag and Gag-pol precursor proteins. We previously reported that virus particles derived from HIV-1 proviral DNA, encoding chimeric Vpr, showed a lack of infectivity, depending on the fusion partner. As an extension of that work, the potential of chimeric Vpr as a substrate for HIV-1 protease was tested utilizing an epitope-based assay. Chimeric Vpr molecules were modified such that the Flag epitope is removed following cleavage, thus allowing us to determine the efficiency of protease cleavage. Following incubation with the protease, the resultant products were analyzed by radioimmunoprecipitation using antibodies directed against the Flag epitope. Densitometric analysis of the autoradiograms showed processing to be both rapid and specific. Further, the analysis of virus particles containing chimeric Vpr by immunoblot showed reactivities to antibodies against the Flag epitope similar to the data observed in vitro. These results suggest that the pseudosubstrate approach may provide another avenue for developing antiviral agents.

Aminobiphenyl Compounds↗