Biomedical subjects

B Chaudhuri

Publications and source records attributed to B Chaudhuri.

At least 19 recordsLinked to original sources

PubMed2001

Human DNA replication initiation factors, ORC and MCM, associate with oriP of Epstein-Barr virus.

The 165-kb chromosome of Epstein-Barr virus (EBV) is replicated by cellular enzymes only once per cell cycle in human cells that are latently infected. Here, we report that the human origin recognition complex, ORC, can be detected in association with an EBV replication origin, oriP, in cells by using antibodies against three different subunits of human ORC to precipitate crosslinked chromatin. Mcm2, a subunit of the MCM replication licensing complex, was found to associate with oriP during G(1) and to dissociate from it during S phase. The detection of ORC and Mcm2 at oriP was shown to require the presence of the 120-bp replicator of oriP. Licensing and initiation of replication at oriP of EBV thus seem to be mediated by ORC. This is an example of a virus apparently using ORC and associated factors for the propagation of its genome.

Cell Cycle↗

PubMed2001

Replication from oriP of Epstein-Barr virus requires human ORC and is inhibited by geminin.

A hypomorphic mutation made in the ORC2 gene of a human cancer cell line through homologous recombination decreased Orc2 protein levels by 90%. The G1 phase of the cell cycle was prolonged, but there was no effect on the utilization of either the c-Myc or beta-globin cellular origins of replication. Cells carrying this mutation failed to support the replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV), and this defect was rescued by reintroduction of Orc2. Orc2 specifically associates with oriP in cells, most likely through its interaction with EBNA1. Geminin, an inhibitor of the mammalian replication initiation complex, inhibits replication from oriP. Therefore, ORC and the human replication initiation apparatus is required for replication from a viral origin of replication.

Alleles↗

PubMed2001

Chemical oxidation of methylene blue using a Fenton-like reaction.

Oxidation by Fenton-like reactions is proven and economically feasible process for destruction of a variety of hazardous pollutants in wastewater. We report herein the oxidation of methylene blue, a basic dye of thiazine series using a Fenton-like reaction at normal laboratory temperature and at atmospheric pressure. The effects of different parameters like the initial concentrations of dye, Fe2+, and H2O2, pH of the solution, reaction temperature, and added electrolytes on the oxidation of the dye present in dilute aqueous solution in the concentration range (3.13-9.39)x10(-5)mol dm(-3) (10-30 mg l(-1)) have been assessed. The results indicate that the dye can be most effectively oxidized in aqueous solution at dye:Fe(2+):H2O2 molar ratio of 1:1.15:14.1. More than 98% removal of the dye could be achieved in 1h in the pH range 2.2-2.6 at 299 K which corresponds to about 81% reduction of the initial COD. The results will be useful for designing the treatment systems of various dye-containing wastewaters.

Enzyme Inhibitors↗

PubMed2001

Selective in vivo and in vitro effects of a small molecule inhibitor of cyclin-dependent kinase 4.

BACKGROUND: Cyclin-dependent kinase 4 (Cdk4) represents a prime target for the treatment of cancer because most human cancers are characterized by overexpression of its activating partner cyclin D1, loss of the natural Cdk4-specific inhibitor p16, or mutation(s) in Cdk4's catalytic subunit. All of these can cause deregulated cell growth, resulting in tumor formation. We sought to identify a small molecule that could inhibit the kinase activity of Cdk4 in vitro and to then ascertain the effects of that inhibitor on cell growth and tumor volume in vivo. METHODS: A triaminopyrimidine derivative, CINK4 (a chemical inhibitor of Cdk4), was identified by screening for compounds that could inhibit Cdk4 enzyme activity in vitro. Kinase assays were performed on diverse human Cdks and on other kinases that were expressed in and purified from insect cells to determine the specificity of CINK4. Cell cycle effects of CINK4 on tumor and normal cells were studied by flow cytometry, and changes in phosphorylation of the retinoblastoma protein (pRb), a substrate of Cdk4, were determined by western blotting. The effect of the inhibitor on tumor growth in vivo was studied by use of tumors established through xenografts of HCT116 colon carcinoma cells in mice. Statistical tests were two-sided. RESULTS: CINK4 specifically inhibited Cdk4/cyclin D1 in vitro. It caused growth arrest in tumor cells and in normal cells and prevented pRb phosphorylation. CINK4 treatment resulted in statistically significantly (P: =.031) smaller mean tumor volumes in a mouse xenograft model. CONCLUSIONS: Like p16, the natural inhibitor of Cdk4, CINK4 inhibits Cdk4 activity in vitro and slows tumor growth in vivo. The specificity of CINK4 for Cdk4 raises the possibility that this small molecule or one with a similar structure could have therapeutic value.

Animals↗

PubMed2001

DNA binding properties of the marine sponge pigment fascaplysin.

Association of fascaplysin with double-stranded calf thymus DNA was investigated by means of isothermal titration calorimetry, absorption spectroscopy, and circular dichroism. The UV spectroscopic data could be well interpreted in terms of a two-site model for the binding of fascaplysin to DNA revealing affinity constants of K1 = 2.5 x 10(6) M(-1) and K2 = 7.5 x 10(4) M(-1) (base pairs of DNA). Based on the typical change observed in the absorption and circular dichroism spectra, intercalation of fascaplysin is regarded as the major binding mode. The calorimetric titration curves showed an exothermic reaction which was exhausted at a 2:1 base pair/drug; ratio. This finding is in agreement with an intercalation model comprising nearest neighbor exclusion. In addition, significantly weaker non-intercalative DNA interactions can be observed at high drug concentration. By comparison of all these data with the binding behavior of known intercalating agents, it is concluded that fascaplysin intercalates into DNA.

Alkaloids↗

PubMed2001

Cell cycle arrest mediated by a pyridopyrimidine is not abrogated by over-expression of Bcl-2 and cyclin D1.

Inhibition of cyclin dependent kinases (Cdks) is of pivotal importance in tumor cell biology as these kinases are the drivers of cell proliferation. This inhibition can be achieved either by naturally occurring biological proteins or by small molecule compounds. They cause cell cycle arrest and/or apoptosis depending upon the specificity and efficacy of the inhibitor in question. We have reported earlier that specific pyridopyrimidines (novel Cdk inhibitors) cause cell cycle arrest in mink lung epithelial cells and the arrest is abrogated by over-expression of Cdk4. In contrast, we show here that one of these inhibitors effectively maintains cell cycle arrest in a leukemic or a breast cancer cell line even after the respective cells over-express an oncogene, either Bcl-2 or cyclin D1. However, in the leukemic cells, Bcl-2 over-expression suppresses apoptosis induced by the pyridopyrimidine. Thus, novel Cdk inhibitors can prove to be useful chemical genetics tools for understanding the underlying mechanisms of growth arrest and/or apoptosis in normal versus tumor cells. This could also lead to the development of improved inhibitors of cell proliferation.

Apoptosis↗

PubMed2000

Inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin, a marine natural product.

Small chemical molecules that interfere with biological proteins could be useful for gaining insight into the complex biochemical processes in mammalian cells. Cdk4 is a key protein whose activity is required not only for emergence of cells from quiescence but also at the G1/S transition in the cell cycle and which is misregulated in 60-70% of human cancers. We set out to identify chemical inhibitors of Cdk4 and discovered that, in vitro, fascaplysin specifically inhibited Cdk4. Molecular modelling based on the crystal structure of Cdk2 suggests that fascaplysin inhibits Cdk4 by binding to the ATP pocket of the kinase. Treatment of tumour (p16(-), pRb(+)) and normal (p16(+), pRb(+)) cell lines with fascaplysin caused G1 arrest and prevented pRb phosphorylation at sites implicated as being specific for Cdk4 kinase. Fascaplysin will therefore prove to be a useful tool in studying the consequence of Cdk4 inhibition, especially in cells containing inactivated p16.

3T3 Cells↗

PubMed2000

Novel Cdk inhibitors restore TGF-beta sensitivity in cdk4 overexpressing epithelial cells.

Transforming growth factor-beta (TGF-beta) is a potent mitogen that effects a wide variety of cells by blocking cell growth. TGF-beta acts by interacting with components of cell cycle machinery to cause G1 arrest and in mink lung epithelial cells (Mv1Lu) it does so by inhibiting Cdk4 synthesis. Overexpression of Cdk4 in these cells (B7) renders them resistant to the effects of TGF-beta. Here we report that two novel Cdk inhibitors (pyridopyrimidines) that not only inhibit Cdk4 and Cdk2 in an in vitro kinase assay but also, in the absence of TGF-beta, block growth of Mv1Lu cells in G1 more efficiently than their B7 (overexpressing Cdk4) counterparts. Interestingly, these inhibitors restored sensitivity of B7 cells towards TGF-beta. This may have implications for the treatment of tumors that have lost TGF-beta responsiveness due to deregulated cellular growth in vivo. These Cdk inhibitors could therefore be used in conjunction with TGF-beta to understand the mechanism of growth arrest in normal versus tumour cells.

Animals↗

PubMed2000

Measurement of cdk4 kinase activity using an affinity peptide-tagging technology.

Cyclin-dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus. This affinity peptide-tagged protein (GST-Rb152-StrepTag), which contains the two known phosphorylation sites of Rb, specifically phosphorylated by Cdk4 in vivo, was used as a substrate in the current in vitro kinase assay. After phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was brought in close proximity to the SPA scintillant beads through the interaction between StrepTag and streptavidin, resulting in the emission of light from beads. By applying the affinity peptide-tagging technology, we have eliminated the separation and wash steps which are normally required in a radioactive filtration assay. Therefore, this homogeneous method is simple, robust, and highly amenable to high-throughput screening of Cdk4-specific inhibitors. Furthermore, the affinity peptide tagging technique reported here is a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.

Affinity Labels↗

PubMed1999

A selection system for human apoptosis inhibitors using yeast.

Apoptosis is a regulated series of events which leads to the death and elimination of mammalian cells during development or in disease control. It is regulated in part by members of the Bcl-2 family of genes. Some of these stimulate cell death, while others prevent it. Expression of one of these death inducers, Bax-alpha (Bax), in the yeast Saccharomyces cerevisiae induces growth arrest and subsequently can cause cell death. Proteins of the Bcl-2 family that are known to inhibit apoptosis in mammalian cells overcome Bax-induced growth arrest in yeast. We describe here a system for isolation of human genes that are able to overcome Bax sensitivity in yeast. Two novel proteins, identified with this system, have been named 'Bax antagonists selected in saccharomyces' (BASS). These proteins not only overcome toxicity of Bax in yeast but also protect mammalian cells from apoptosis that is induced by staurosporine or Bax overexpression. We find that BASS2 is the more effective of the two genes.

Adult↗

PubMed1999

Comparison of the effects of bax-expression in yeast under fermentative and respiratory conditions: investigation of the role of adenine nucleotides carrier and cytochrome c.

A new system for bax-expression in yeast has been devised to investigate bax's effect under fermentative and respiro-fermentative conditions. This has allowed us to show unambiguously that the ability of bax to kill yeast is higher under respiratory conditions than under purely fermentative conditions. The extent of killing under respiro-fermentative conditions (non-repressive sugars) is intermediate. It has been proposed that the two proteins adenine nucleotides carrier (ANC) and cytochrome c play a crucial role in bax-induced cell death. We have investigated the effects of deletion of the genes encoding the two proteins on the toxicity induced by bax, using this new system. The absence of ANC did not modify bax-induced lethality in any way. Moreover, the absence of cytochrome c also did not prevent bax-induced death. Only the kinetics of lethality were altered. All these effects are prevented by co-expression of bcl-xL.

Apoptosis↗

PubMed1999

DNA binding protein dbpA binds Cdk5 and inhibits its activity.

Progress in the cell cycle is governed by the activity of cyclin dependent kinases (Cdks). Unlike other Cdks, the Cdk5 catalytic subunit is found mostly in differentiated neurons. Interestingly, the only known protein that activates Cdk5 (i.e. p35) is expressed solely in the brain. It has been suggested that, besides its requirement in neuronal differentiation, Cdk5 activity is induced during myogenesis. However, it is not clear how this activity is regulated in the pathway that leads proliferative cells to differentiation. In order to find if there exists any Cdk5-interacting protein, the yeast two-hybrid system was used to screen a HeLa cDNA library. We have determined that a C-terminal 172 amino acid domain of the DNA binding protein, dbpA, binds to Cdk5. Biochemical analyses reveal that this fragment (dbpA(Cdelta)) strongly inhibits p35-activated Cdk5 kinase. The protein also interacts with Cdk4 and inhibits the Cdk4/cyclin D1 enzyme. Surprisingly, dbpA(Cdelta) does not bind Cdk2 in the two-hybrid assay nor does it inhibit Cdk2 activated by cyclin A. It could be that dbpA's ability to inhibit Cdk5 and Cdk4 reflects an apparent cross-talk between distinct signal transduction pathways controlled by dbpA on the one hand and Cdk5 or Cdk4 on the other.

Animals↗

PubMed1999

Identification of ribosomal protein L34 as a novel Cdk5 inhibitor.

The cell cycle is regulated by sequential activation, inactivation of cyclin dependent kinases (Cdk-s). Like all other Cdk-s, the catalytic subunit of Cdk5 is present in cycling cells. However, its highest concentration is found in differentiated neurons, and the only known protein that activates Cdk5 (i.e., p35) is expressed solely in the brain. Active Cdk5 is thought to be involved in the in vivo phosphorylation of the neurofilament proteins and tau which are hyperphosphorylated in neurodegenerative diseases. Recent reports suggest that Cdk5 may also contribute to cellular differentiation. Therefore, it would not be unusual to surmise that there exist specific proteins that regulate Cdk5 activity in cycling cells. In order to find if this was true, a cDNA library prepared from HeLa cells was screened using the yeast-two-hybrid system. The 60S ribosomal protein, L34, was identified as a Cdk5-interacting protein. Biochemical analyses reveal that L34 cannot activate Cdk5 but potently inhibits the p35-activated kinase. L34 also interacts with Cdk4 and, in parallel, inhibits the Cdk4/cyclin D1 activity. Interestingly, L34 does not interact with Cdk2 in the two-hybrid assay nor does it inhibit the Cdk2/cyclin A enzyme. The fact that a ribosomal protein inhibits Cdk5 and Cdk4 may suggest that these two kinases have a cellular role in translational regulation.

Cell Cycle↗

PubMed1999

Role of the C-terminal domain of Bax and Bcl-XL in their localization and function in yeast cells.

It has been suggested that the C-terminal domain of Bcl-2 family members may contain a signal anchor sequence that targets these proteins to the mitochondrial outer membrane. We have investigated the consequence of deleting this domain upon cytochrome c release in yeast strains that coexpress truncated forms of Bax (i.e. BaxA) and Bcl-X(L) (i.e. Bcl-X(L)delta). We find that (i) Bax(delta) is as efficient as full-length Bax in promoting cytochrome c release, but Bcl-x(L)delta has remarkably reduced rescuing ability compared to full-length Bcl-x(L); (ii) full-length Bcl-X(L) protein acts by relocalizing Bax from the mitochondrial fraction to the soluble cytosolic fraction; (iii) Bax undergoes N-terminal cleavage when expressed in yeast, which is prevented by coexpression of Bcl-X(L), suggesting that Bcl-x(L) may mask the cleavage site of Bax through a direct physical interaction of the two proteins.

Cytochrome c Group↗

PubMed1999

Estimation of .OH radical reaction rate constants for phenol and chlorinated phenols using UV/H2O2 photo-oxidation.

A detailed investigation on UV/H2O2 photooxidation has been carried out in order to determine the kinetics of the oxidative degradation of phenol and 2- and 4-chlorophenols in dilute aqueous solutions. Effects of different process parameters, such as initial substrate and H2O2 concentrations, substrate to H2O2 ratio on the degradation kinetics of the phenolic substrates, have been studied. Degradation rates of phenol and chlorophenols are insignificantly small with ultraviolet radiation only and also with hydrogen peroxide (in the absence of UV radiation), but the synergistic effect of UV/H2O2 results in a marked enhancement of the rates of degradation. A mechanistic model for UV/H2O2 photooxidation has been developed. Room temperature (27 degrees C) rate constants for the reaction of .OH radical (formed by absorption of photons) with the substrates have been estimated by using the model equation. The calculated rate constants are of the same order of magnitude as reported for other similar aromatic compounds.

Absorption↗

PubMed1999

Investigation of bax-induced release of cytochrome c from yeast mitochondria permeability of mitochondrial membranes, role of VDAC and ATP requirement.

Recent studies that attempt to explore the action of pro- and anti-apoptotic proteins of the bcl2 family demonstrate the crucial role of relocalization of cytochrome c from the mitochondrial intermembrane space to the cytosol. This early event of apoptosis can be mimicked in the yeast Saccharomyces cerevisiae following expression of bax. In mammalian mitochondria, the mechanism of relocalization is thought to involve the opening of the so-called permeability transition pore. We show in this paper: (a) that bax-induced release of cytochrome c in yeast does not involve any permeability transition of the inner mitochondrial membrane but involves a general alteration of the permeability of the outer mitochondrial membrane to macromolecules. This suggests that a permeability transition of the inner mitochondrial membrane is not an event required for the relocalization of cytochrome c in yeast. (b) The outer-membrane voltage-dependent anion channel (VDAC), a putative component of the permeability transition pore, is not involved in bax-induced release of cytochrome c or in the prevention of this release by bcl-xL. (c) Bax devoid of its C-terminal putative hydrophobic alpha-helix is as efficient as full-length bax to allow the relocalization of cytochrome c, demonstrating this segment of the protein is not required for membrane-targeting. (d) We finally observe that the action of bax on the outer mitochondrial membrane requires the presence of ATP both in vitro and in vivo, and it is shown that ATP directly increases the amount of bax inserted to mitochondria.

Adenosine Triphosphate↗

PubMed1998

Identification of a human cDNA encoding a kinase-defective cdk5 isoform.

The cyclin-dependent kinase 5 (Cdk5) catalytic subunit is expressed in both cycling and noncycling cells and is present in many tissues. Neuronal and muscle cells contain the highest amount of this protein. The p35 protein, which is expressed solely in the brain, activates Cdk5. Cdk5 activity is involved in terminal differentiation of neurons and muscle cells. We attempted to clone cdk5 by PCR from a human fetal brain cDNA library. Surprisingly, we amplified two forms of the cdk5 gene, the wild type and a cdk5 variant that lacks the complete kinase domain VI. The variant is also found in SH-SY-5Y neuroblastoma cells but not in T-cells, HeLa cells, the thymus, and placental tissue. The protein encoded by the cdk5 variant, the Cdk5 isoform (Cdk5i), purifies with p35 when coexpressed in insect cells. The activity associated with the heterodimer Cdk5i/p35 is found to be appreciably weaker than the wild-type Cdk5/p35 kinase. Moreover, Cdk5i/p35 cannot autophosphorylate its two subunits as with Cdk5/p35. Interestingly, kinase-defective Cdk5i can abolish the activity of wild-type Cdk5 when both are coexpressed with p35 in insect cells, suggesting that Cdk5i may have a function in regulating Cdk5 activity in human cells too.

Amino Acid Sequence↗

PubMed1998

Under respiratory growth conditions, Bcl-x(L) and Bcl-2 are unable to overcome yeast cell death triggered by a mutant Bax protein lacking the membrane anchor.

We have reported earlier that cytosolic expression of the full-length human apoptosis inducer Bax-alpha (Bax) in the yeast Saccharomyces cerevisiae suppresses growth and induces mortality in cells containing functional mitochondria. Human Bcl-x(L) overcomes this toxicity. Here we describe that a mutant Bax protein, with a missing membrane anchor region (Bax delta), also inhibits growth and causes cell death in yeast. However, the death inhibitory proteins Bcl-x(L) and Bcl-2 fail to rescue Bax delta-mediated growth inhibition under conditions promoting respiration, although they bind Bax delta in the cell. Results in Jurkat T-cells corroborate that Bcl-x(L) is much less efficient at rescuing mammalian cells from the effect of Bax delta than from full length Bax. We have also inquired if the respiration-dependent toxicity of Bax and Bax delta in yeast is nullified by Bcl-x(L)delta and Bcl-2delta, molecules which lack membrane anchors but bind Bax in the yeast two-hybrid system. It appears that, under conditions which facilitate respiration in yeast, Bcl-x(L)delta and Bcl-2delta are incapable of rescuing both Bax-containing and Bax delta-containing cells. Our results open up the interesting possibility that there might exist proteins, unrelated to the Bcl-2 family, which could negate death induced by a membrane anchor-free form of Bax.

Amino Acid Sequence↗