Biomedical subjects

R Datta

Publications and source records attributed to R Datta.

At least 37 records · Page 2Linked to original sources

PubMed2000

XIAP regulates DNA damage-induced apoptosis downstream of caspase-9 cleavage.

The IAP (inhibitor of apoptosis) family of anti-apoptotic proteins regulates programmed cell death. Of the six known human IAP-related proteins, XIAP is the most potent inhibitor. To study the mechanistic effects of XIAP on DNA damage-induced apoptosis, we prepared U-937 cells that stably overexpress XIAP. The results demonstrate that XIAP inhibits apoptosis induced by 1-[beta-d-arabinofuranosyl]cytosine (ara-C) and other genotoxic agents. XIAP had no detectable effect on ara-C-induced release of mitochondrial cytochrome c and attenuated cleavage of procaspase-9. In addition, we show that ara-C induces the association of XIAP with the cleaved fragments of caspase-9 and thereby inhibition of caspase-9 activity. The results also demonstrate that ara-C induces cleavage of procaspase-3 by a caspase-8-dependent mechanism and that XIAP inhibits caspase-3 activity. These results demonstrate that XIAP functions downstream of procaspase-9 cleavage as an inhibitor of both proteolytically processed caspase-9 and -3 in the cellular response to genotoxic stress.

Apoptosis↗

PubMed2000

Requirement for caspase activation in monocytic differentiation of myeloid leukemia cells.

Human myeloid leukemia cells respond to 12-tetradecanoylphorbol-13-acetate (TPA) and other activators of protein kinase C (PKC) with the induction of terminal monocytic differentiation. The present studies demonstrate that TPA treatment of U-937 leukemia cells is associated with release of mitochondrial cytochrome c, activation of caspase-3 and induction of internucleosomal DNA fragmentation. By contrast, the TUR cell variant, which is deficient in PKCbeta, failed to respond to TPA with release of cytochrome c and induction of the caspase-3 cascade. Moreover, stable overexpression of PKCbeta in TUR cells reconstituted sensitivity to TPA-induced cytochrome c release and activation of caspase-3. The results also demonstrate that treatment of cells with the caspase inhibitor Z-VAD-fmk blocks both TPA-induced apoptosis and monocytic differentiation. Similar results were obtained in U-937 cells stably expressing the CrmA caspase inhibitor. These findings demonstrate that TPA induces cytochrome c release by a PKCbeta-dependent mechanism and that activation of caspase-mediated signaling is required for induction of the differentiated monocytic phenotype.

Amino Acid Chloromethyl Ketones↗

PubMed2000

Mitochondrial translocation of protein kinase C delta in phorbol ester-induced cytochrome c release and apoptosis.

Apoptosis is induced by the release of cytochrome c from mitochondria to the cytoplasm. The present studies demonstrate that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces translocation of protein kinase C (PKC) delta from the cytoplasm to mitochondria. The results also show that translocation of PKCdelta results in release of cytochrome c. The functional significance of this event is further supported by the demonstration that PKCdelta translocation is required for TPA-induced apoptosis. These findings demonstrate that translocation of PKCdelta to mitochondria is responsible, at least in part, for inducing cytochrome c release and apoptosis.

Apoptosis↗

PubMed2000

Proteolytic cleavage and activation of protein kinase C [micro] by caspase-3 in the apoptotic response of cells to 1-beta -D-arabinofuranosylcytosine and other genotoxic agents.

Protein kinase C (PKC) mu is a novel member of the PKC family that differs from the other isozymes in structural and biochemical properties. The precise function of PKCmu is not known. The present studies demonstrate that PKCmu is cleaved during apoptosis induced by 1-beta-d-arabinofuranosylcytosine (ara-C) and other genotoxic agents. PKCmu cleavage is blocked in cells that overexpress the anti-apoptotic Bcl-x(L) protein or the baculovirus p35 protein. Our results demonstrate that PKCmu is cleaved by caspase-3 at the CQND(378)S site. Cleavage of PKCmu is associated with release of the catalytic domain and activation of its kinase function. We also show that, unlike the cleaved fragments of PKCdelta and theta, overexpression of the PKCmu catalytic domain is not lethal. Cells stably expressing the catalytic fragment of PKCmu, however, are more sensitive to apoptosis induced by genotoxic stress. In addition, expression of the caspase-resistant PKCmu mutant partially inhibits DNA damage-induced apoptosis. These findings demonstrate that PKCmu is cleaved by caspase-3 and that expression of the catalytic domain sensitizes cells to the cytotoxic effects of ara-C and other anticancer agents.

Antimetabolites, Antineoplastic↗

PubMed2000

Interaction between protein kinase C delta and the c-Abl tyrosine kinase in the cellular response to oxidative stress.

Protein kinase C (PKC) isoforms are phosphorylated on tyrosine in the response of cells to oxidative stress. The present studies demonstrate that treatment of cells with hydrogen peroxide (H(2)O(2)) induces binding of the PKCdelta isoform and the c-Abl protein-tyrosine kinase. The results show that c-Abl phosphorylates PKCdelta in the H(2)O(2) response. We also show that PKCdelta phosphorylates and activates c-Abl in vitro. In cells, induction of c-Abl activity by H(2)O(2) is attenuated by the PKCdelta inhibitor, rottlerin, and by overexpression of the regulatory domain of PKCdelta. These findings support a functional interaction between PKCdelta and c-Abl in the cellular response to oxidative stress.

Animals↗

PubMed1999

Increased phosphorylation of eukaryotic initiation factor 2alpha at the G2/M boundary in human osteosarcoma cells correlates with deglycosylation of p67 and a decreased rate of protein synthesis.

The rate of protein synthesis in higher eukaryotes is largely regulated at the level of eIF2alpha phosphorylation by its kinases. A cellular glycoprotein, p67, protects eIF2alpha from phosphorylation. An enzyme, p67-deglycosylase, when active, removes the carbohydrate moieties from p67 and inactivates it. Subsequently, protein synthesis is inhibited. During mitosis the overall rate of protein synthesis sharply declines. To understand the molecular mechanism underlying this inhibition of protein synthesis, we have examined the phosphorylation of eIF2alpha and the activity of p67. We find that the phosphorylation of eIF2alpha increases at the G2/M border of cycling U2-OS cells, and p67 is deglycosylated at the same period of the cell cycle. In addition, the level and the activity of p67-deglycosylase also increase at the G2/M boundary of cycling U2-OS cells. These results thus provide an important in vivo correlation between the increased phosphorylation of eIF2alpha and deglycosylation of p67 by p67-deglycosylase at the G2/M boundary of cycling U2-OS cells. This may explain in part the inhibition of protein synthesis in U2-OS cells approaching mitosis.

Acetylglucosaminidase↗

PubMed1999

U937 cells overexpressing bcl-xl are resistant to human immunodeficiency virus-1-induced apoptosis and human immunodeficiency virus-1 replication.

Many viruses, including human immunodeficiency virus type 1 (HIV-1), induce apoptosis and are affected by cellular expression of antiapoptotic genes. We sought to examine the effect of antiapoptotic gene expression on HIV replication by transfecting the promyelomonocytic cell line U937 with the bcl-xl gene to obtain clones of U937 cells that overexpressed bcl-xl (designated U937bcl-xl), a negative control U937 clone transfected with vector alone (designated U937neo) and a clone overexpressing bcl-2 (designated U937bcl-2). After infection with HIV-1, U937neo cells underwent apoptosis four times as frequently as the U937bcl-xl cells. Furthermore, U937bcl-xl cells produced 5-fold less HIV-1 protein than U937neo, whereas U937bcl-2 produced at least 2-fold more p24 than the U937neo control. Transient coexpression of bcl-2 or bcl-xl decreased HIV production and transcription from the HIV LTR. To define the mechanism by which bcl-xl, but not bcl-2, inhibits HIV expression, we examined bcl-2 and bcl-xl expression after HIV infection and CD4 cross-linking. Although HIV-1 infection or cross-linking CD4 led to a decrease in expression of bcl-2, it had no effect on bcl-xl expression. These results provide a mechanism for the resistance of U937bcl-xl transfectants, but not U937bcl-2 transfectants, to HIV-1 replication in monocytic cells in vitro. Therapies that up-regulate bcl-xl expression potentially provide a novel means to decrease the destructiveness of HIV-1.

Apoptosis↗

PubMed1999

Induction of apoptosis due to lowering the level of eukaryotic initiation factor 2-associated protein, p67, from mammalian cells by antisense approach.

p67, a cellular glycoprotein, protects eIF2alpha from phosphorylation by inhibitory kinases such as double-stranded RNA dependent eIF2 kinase, PKR, and heme-controlled repressor and thus promotes protein synthesis in mammalian cells. To investigate whether p67 is essential for the survival of mammalian cells, the basal level of p67 was lowered from rat tumor hepatoma cells using antisense approach. The antisense p67 RNA specifically lowered the levels of p67 message and the protein from these cells. As a result, the level of eIF2alpha phosphorylation increased significantly, the overall rate of protein synthesis decreased, and the rate of DNA synthesis also decreased in mammalian cells with low levels of p67 as compared to that seen in control cells. In addition, the majority of the cells with low levels of p67 are arrested at the G1 phase of the cell cycle and die with apoptosis. Taken together, these results suggest that appropriate levels of p67 is required for normal growth of mammalian cells.

Aminopeptidases↗

PubMed1999

Whey fermentation by anaerobiospirillum succiniciproducens for production of a succinate-based animal feed additive

Anaerobic fermentation processes for the production of a succinate-rich animal feed supplement from raw whey were investigated with batch, continuous, and variable-volume fed-batch cultures with Anaerobiospirillum succiniciproducens. The highest succinate yield, 90%, was obtained in a variable-volume fed-batch process in comparison to 80% yield in a batch cultivation mode. In continuous culture, succinate productivity was 3 g/liter/h, and the yield was 60%. Under conditions of excess CO2, more than 90% of the whey-lactose was consumed, with an end product ratio of 4 succinate to 1 acetate. Under conditions of limited CO2, lactose was only partially consumed and lactate was the major end product, with lower levels of ethanol, succinate, and acetate. When the succinic acid in this fermentation product was added to rumen fluid, it was completely consumed by a mixed rumen population and was 90% decarboxylated to propionate on a molar basis. The whey fermentation product formed under excess CO2, which contained mainly organic acids and cells, could potentially be used as an animal feed supplement.

Journal Article↗

PubMed1999

Role for caspase-mediated cleavage of Rad51 in induction of apoptosis by DNA damage.

We report here that the Rad51 recombinase is cleaved in mammalian cells during the induction of apoptosis by ionizing radiation (IR) exposure. The results demonstrate that IR induces Rad51 cleavage by a caspase-dependent mechanism. Further support for involvement of caspases is provided by the finding that IR-induced proteolysis of Rad51 is inhibited by Ac-DEVD-CHO. In vitro studies show that Rad51 is cleaved by caspase 3 at a DVLD/N site. Stable expression of a Rad51 mutant in which the aspartic acid residues were mutated to alanines (AVLA/N) confirmed that the DVLD/N site is responsible for the cleavage of Rad51 in IR-induced apoptosis. The functional significance of Rad51 proteolysis is supported by the finding that, unlike intact Rad51, the N- and C-terminal cleavage products fail to exhibit recombinase activity. In cells, overexpression of the Rad51(D-A) mutant had no effect on activation of caspase 3 but did abrogate in part the apoptotic response to IR exposure. We conclude that proteolytic inactivation of Rad51 by a caspase-mediated mechanism contributes to the cell death response induced by DNA damage.

Animals↗

PubMed1999

Current concepts in cardiopulmonary resuscitation in adults.

Cardiopulmonary resuscitation (CPR) provides artificial circulation and ventilation during cardiopulmonary arrest. CPR is further categorised as basic life support (BLS), advanced cardiac life support (ACLS) and postresuscitation support. BLS consists of provision of a patent upper airway, ventilation and circulation of blood by closed chest cardiac compressions. ACLS includes use of specialised equipment to maintain the airway, early defibrillation and pharmacologic therapy. Successful outcome from an arrest depends on the total duration of an arrest and early defibrillation, as ventricular fibrillation is the most common cardiac rhythm found in adult cardiac arrest. Initial drug therapy during CPR aims at correction of arterial hypoxaemia and restoring coronary and cerebral perfusion. Oxygen and epinephrine constitute the mainstay of drug therapy during CPR. In patients with ventricular tachycardia, lidocaine is the drug of choice, followed by bretylium. Magnesium has proved to be useful in both refractory pulseless ventricular tachycardia and fibrillation. Atropine has not been demonstrated to improve outcome from arrest but can be administered in bradyasystolic cardiac arrest. The routine administration of bicarbonate and calcium is no longer recommended but situations exist where they can be used appropriately. Administration of drugs during CPR should preferably be via a central route, but epinephrine, lidocaine and atropine can be administered via the endotracheal tube if intravenous access has not been established. Postresuscitation care includes mechanical ventilation if necessary to optimise oxygenation and ventilation and steps to maintain vital organ and optimal brain protection, which includes avoidance of hypertension, hypotension and hyperglycaemia.

Adult↗

PubMed1998

Understanding Nonidealities of the Osmotic Pressure of Concentrated Bovine Serum Albumin.

Previously Vilker et al. (J. Colloid Interface Sci. 79(2), (1981)) reported the osmotic pressure of concentrated bovine serum albumin (BSA) up to 475 g/L in 0.15 M sodium chloride at pH 4.5, 5.4, and 7.4. The authors used a semiempirical model based on Donnan theory to predict the osmotic pressure with good agreement. However, the formal application of a three-term virial expansion with the coefficients determined from the potential energy of interaction between BSA molecules resulted in poor agreement with their data. In this study, modeling of the osmotic pressure was reexamined using a free-solvent model that considered average solute-solvent and microion-solute interactions in a mole fraction concentration variable. The resulting fits were excellent for all three pH. The model is designed with no fitted parameters; however, the model results were highly sensitive to the selected hydration and microion binding. Therefore the hydration was further regressed from its initial estimate of 1 g H2O/g BSA (based on water-17O magnetic resonance studies of other globular proteins) to minimize the least-squares error between the predicted values and data. The resulting average hydration was determined to be 1.14 +/- 0.03 g H2O/g BSA for all pH values. However, the standard error in hydration for each pH was no greater than +/-0.0063 g H2O/g BSA. These results demonstrate that solvent-solute interaction and the concentration variable may be critical factors when evaluating osmotic pressure data of concentrated protein solutions. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

Expression and activity of p67 are induced during heat shock.

p67, a cellular glycoprotein, protects eIF2 alpha from phosphorylation by inhibitory kinases such as PKR and HCR. p67 promoter contains heat shock element (HSE). To investigate whether this HSE of p67 has any role during heat-shock, rat tumor hepatoma cells were transiently transfected with CAT reporters linked to p67 promoter with HSE and without HSE. Heat shock induced CAT activity when p67 promoter contained HSE and this induction was not observed when HSE was deleted from the p67 promoter. In response to heat-shock, the endogenous p67 mRNA was also induced to more than 36-fold, and much of it translated into protein which was modified by GlcNAc moieties. The time of induced glycosyl modification at the later stages of the heat-shock correlates with the reduced level of eIF2 alpha phosphorylation. During later stages of the heat shock of animal cells, there is a preferential translation of a small class of messages encoding heat shock proteins. Our results suggest that the expression and activity of p67 are induced at the later stages of the heat-shock, and may be involved in the preferential translation of the heat-shock messages.

Aminopeptidases↗

PubMed1998

Abrogation of mitochondrial cytochrome c release and caspase-3 activation in acquired multidrug resistance.

Acquired multidrug resistance to anti-cancer agents has been associated with overexpression of the P-glycoprotein and other members of the ATP-binding cassette superfamily. The present studies demonstrate that SCC-25 cells selected for resistance to the alkylating agent cisplatin (CDDP) overexpress the anti-apoptotic Bcl-xL protein. In contrast to parental cells, the SCC-25/CDDP-resistant variant failed to exhibit activation of caspase-3, cleavage of protein kinase C delta, and other characteristics of apoptosis in response to CDDP. Similar results were obtained when SCC-25/CDDP cells were exposed to the structurally and functionally unrelated antimetabolite 1-beta-D-arabinofuranosyl-cytosine (ara-C). Other cells selected for resistance to doxorubicin or vincristine also exhibited overexpression of Bcl-xL and failed to respond to CDDP and ara-C with activation of caspase-3. The results further demonstrate that multidrug-resistant cells exhibit a block in the release of mitochondrial cytochrome c into the cytosol and that this effect is dependent on overexpression of Bcl-xL. The demonstration that lysates from the resistant cells respond to the addition of cytochrome c with activation of caspase-3 confirms that the block in apoptosis is because of inhibition of mitochondrial cytochrome c release. These findings demonstrate that cells respond to diverse classes of anti-cancer drugs with overexpression of Bcl-xL and that this response represents another mechanism of acquired multidrug resistance.

Antineoplastic Agents↗

PubMed1998

Free-Solvent Model of Osmotic Pressure Revisited: Application to Concentrated IgG Solution under Physiological Conditions

The osmotic pressure measurements of bovine immuno-gamma globulin in phosphate-buffered solution at pH 7.4 and 0.13 M total salt concentration were extended to near saturation concentrations for ambient temperature. The osmotic pressure at the highest measured concentration of 424 g/L was 4.18 psi (28.3 kPa). A free-solvent model, considering solute-solvent interaction in the concentration variable, provided an excellent fit to observed osmotic pressure nonideality at even the highest protein concentration. The calculated mass of hydrated solvent compared with amounts determined from water-17O magnetic resonance for other globular proteins. This model provides an improved correlation to the data over virial equations (truncated to the third term) when only solute-solute interactions are considered. The use of mole fraction as the composition variable was critical in obtaining the excellent fit of the free-solvent model. A combination of the free-solvent correction for the concentration variable coupled with models incorporating solute-solute interaction, such as a virial expansion, will be necessary to generally describe the osmotic pressure of protein solutions for all concentrations. Copyright 1998 Academic Press. Copyright 1998Academic Press

Journal Article↗

PubMed1998

Amylases synthesis in scutellum and aleurone layer of maize seeds.

The endosperm of germinating maize seeds contains four isozymes of alpha-amylases (alpha-amylase-1 to -4) and one isozyme of beta-amylase. The alpha-amylases were purified by affinity chromatography on amylose and separated by DEAE-cellulose chromatography, into two groups, namely alpha-amylases-1,2 and alpha-amylases-3,4; and beta-amylase was purified by precipitation as a glycogen-enzyme complex. The molecular weight of alpha-amylases-1 and -2 was 46 kD, alpha-amylases-3 was 44.5 kD and of alpha-amylases-4 was 47.5 kD. The molecular weight of beta-amylase was 56 kD. During seed germination increase in amylolytic activity in endosperm was mainly contributed by secretion of alpha-amylases from adjoining aleurone layer and scutellum. The synthesis and secretion of alpha-amylases was first initiated in the scutellum followed by aleurone layers. Exogeneous Ca2+ stimulated synthesis of alpha-amylases in both eleurone layer and scutellum. In contrast, though scutellum and aleurone layer synthesized beta-amylase but it was not secreted to the medium. These results suggest that during the early germination period, alpha-amylases secreted from scutellum mobilizes starch.

Amylases↗

PubMed1998

Containment of herbicide resistance through genetic engineering of the chloroplast genome.

Glyphosate is a potent herbicide. It works by competitive inhibition of the enzyme 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS), which catalyzes an essential step in the aromatic amino acid biosynthetic pathway. We report the genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector. Southern blot analysis confirms stable integration of the EPSPS gene into all of the chloroplast genomes (5000-10,000 copies per cell) of transgenic plants. Seeds obtained after the first self-cross of transgenic plants germinated and grew normally in the presence of the selectable marker, whereas the control seedlings were bleached. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations of glyphosate. Chloroplast transformation provides containment of foreign genes because plastid transgenes are not transmitted by pollen. The escape of foreign genes via pollen is a serious environmental concern in nuclear transgenic plants because of the high rates of gene flow from crops to wild weedy relatives.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1997

Caspase-3-mediated cleavage of protein kinase C theta in induction of apoptosis.

Protein kinase C theta (PKCtheta) is a member of the novel or nPKC family. A functional role for PKCtheta is unknown. The present studies demonstrate that PKCtheta is cleaved in the third variable region (V3) in apoptosis induced by diverse agents. PKCtheta cleavage is blocked in cells that overexpress the anti-apoptotic Bcl-xL or the baculovirus p35 protein. PKCtheta is cleaved by Caspase-3 and by apoptotic cell lysates at a DEVD354/K site. We also show that overexpression of the cleaved kinase-active PKCtheta fragment, but not full-length PKCtheta or a kinase-inactive fragment, results in induction of sub-G1 phase DNA, nuclear fragmentation, and lethality. These findings indicate that proteolytic cleavage of PKCtheta by Caspase-3 induces events characteristic of apoptosis.

Apoptosis↗