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Biomedical subjects

Ajit Kumar

Publications and source records attributed to Ajit Kumar.

At least 19 recordsLinked to original sources
PubMed2007

Ellagic acid peracetate is superior to ellagic acid in the prevention of genotoxicity due to aflatoxin B1 in bone marrow and lung cells.

Earlier observations carried out in our laboratory highlighted the mode of action of acetoxy 4-methylcoumarins and quercetin pentaacetate in preventing the genotoxicity of aflatoxin B1 (AFB1). We have extended the observation to an acetoxy biscoumarin i.e. ellagic acid peracetate (EAPA), which unlike ellagic acid (EA) has demonstrated time-dependent inhibition of liver microsomes catalysed AFB1-epoxidation as measured by AFB1 binding to DNA. EAPA was more potent than EA in preventing bone marrow and lung cells from AFB1-induced genotoxicity. EAPA was acted upon by microsomal acetoxy drug:protein transacetylase (TAase) leading to modulation of the catalytic activity of certain functional proteins (cytochrome P450, NADPH cytochrome c reductase and glutathione S-transferase), possibly by way of protein acetylation.

Acetyltransferases↗
PubMed2006

Specificities of acetoxy derivatives of coumarins, biscoumarins, chromones, flavones, isoflavones and xanthones for acetoxy drug: protein transacetylase.

The earlier work carried out in our laboratory led to the identification of a novel rat liver microsomal enzyme termed as acetoxy drug: protein transacetylase (TAase), catalyzing the transfer of acetyl group from polyphenolic acetates (PA) to functional proteins. In this paper, we have reported the comparison of the specificities of acetoxy derivatives of coumarins, biscoumarins, chromones, flavones, isoflavones and xanthones with special reference to the phenyl moiety/bulky group on the pyran ring of PA. The results clearly indicated that compounds having phenyl moieties, when used as the substrates, resulted in a significant reduction of TAase catalyzed activity. The alteration in TAase catalyzed activation of NADPH cytochrome c reductase and inhibition of benzene-induced micronuclei in bone marrow cells by PA were in tune with their specificities to TAase.

Acetylation↗
PubMed2006

Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function.

BACKGROUND: The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv) has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. RESULTS: Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. CONCLUSION: The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

Active Transport, Cell Nucleus↗
PubMed2006

Biochemical characterization of a low molecular weight aspartic protease inhibitor from thermo-tolerant Bacillus licheniformis: kinetic interactions with Pepsin.

The present article reports a low molecular weight aspartic protease inhibitor, API, from a newly isolated thermo-tolerant Bacillus licheniformis. The inhibitor was purified to homogeneity as shown by rp-HPLC and SDS-PAGE. API is found to be stable over a broad pH range of 2-11 and at temperature 90 degrees C for 2 1/2h. It has a Mr (relative molecular mass) of 1363 Da as shown by MALDI-TOF spectra and 1358 Da as analyzed by SDS-PAGE . The amino acid analysis of the peptide shows the presence of 12 amino acid residues having Mr of 1425 Da. The secondary structure of API as analyzed by the CD spectra showed 7% alpha-helix, 49% beta-sheet and 44% aperiodic structure. The Kinetic studies of Pepsin-API interactions reveal that API is a slow-tight binding competitive inhibitor with the IC(50) and Ki values 4.0 nM and (3.83 nM-5.31 nM) respectively. The overall inhibition constant Ki* value is 0.107+/-0.015 nM. The progress curves are time-dependent and consistent with slow-tight binding inhibition: E+I -->/<-- (k(4), k(5)) EI -->/<-- (k(6), k(7)) EI*. Rate constant k(6)=2.73+/-0.32 s(-1) reveals a fast isomerization of enzyme-inhibitor complex and very slow dissociation as proved by k(7)=0.068+/-0.009 s(-1). The Rate constants from the intrinsic tryptophanyl fluorescence data is in agreement with those obtained from the kinetic analysis; therefore, the induced conformational changes were correlated to the isomerization of EI to EI*.

Adaptation, Physiological↗
PubMed2006

Expectation and the placebo effect in Parkinson's disease patients with subthalamic nucleus deep brain stimulation.

To determine whether the degree to which a patient with Parkinson's disease expects therapeutic benefit from subthalamic nucleus-deep brain stimulation (STN-DBS) influences the magnitude of his or her improved motor response, 10 patients with idiopathic Parkinson's and bilateral STN-DBS were tested after a 12-hour period off medication and stimulation. Four consecutive UPDRS III scores were performed in the following conditions: (a) stimulation OFF, patient aware; (b) stimulation OFF, patient blind; (c) stimulation ON, patient aware; and (d) stimulation ON, patient blind. Statistical significance (P = 0.0001) was observed when comparing main effect ON versus OFF (mean ON: 32.55; mean OFF: 49.15). When the stimulation was OFF, patients aware of this condition had higher UPDRS motor scores than when they were blinded (mean: 50.7 vs. 47.6). With the stimulation ON, UPDRS motor scores were lower when the patients were aware of the stimulation compared with when they were blinded (mean: 30.6 vs. 34.5). The interaction between these levels was significant (P = 0.049). This variation was important for bradykinesia and was not significant for tremor and rigidity. The authors conclude that the information about the condition of the stimulation enhanced the final clinical effect in opposite directions. The results presented support the role of expectation and placebo effects in STN-DBS in Parkinson's disease patients.

Adult↗
PubMed2005

Acetoxy drug: protein transacetylase catalyzed activation of human platelet nitric oxide synthase by polyphenolic peracetates.

An enhanced intracellular level of Nitric oxide (NO) is essential to ameliorate several pathological conditions of heart and vasculature necessitating the activation of NOS. We have projected in this report the acetylation of eNOS by polyphenolic peracetates (PA) catalyzed by the novel enzyme acetoxy drug: protein transacetylase (TAase) discovered in our laboratory as an unambiguous way of activating NOS which results in the manifestation of physiological action. The human platelet was chosen as the experimental system in order to validate the aforementioned proposition. PA caused profound irreversible activation of platelet NADPH cytochrome c reductase mediated by TAase. The convincing biochemical evidences are presented to show that PA could cause acetylation of the reductase domain of NOS leading to the activation of eNOS in tune with their specificities to platelet TAase. As a result, the enhanced level of NO due to activation of platelet eNOS by PA was found to inhibit the ADP-induced platelet aggregation. The present studies highlight for the first time the role of PA as the novel potent agent for enhancing the intracellular NO levels.

Acetates↗
PubMed2005

Mechanism of biochemical action of substituted 4-methylcoumarins. Part 11: Comparison of the specificities of acetoxy derivatives of 4-methylcoumarin and 4-phenylcoumarin to acetoxycoumarins: protein transacetylase.

Our earlier observations led to the identification of a microsomal enzyme termed as acetoxy drug: protein transacetylase (TAase) catalyzing the transfer of acetyl groups from acetylated polyphenols to the receptor proteins. TAase was conveniently assayed by the irreversible inhibition of cytosolic glutathione S-transferase (GST) by the model acetoxycoumarin, 7,8-diacetoxy-4-methylcoumarin (1). The specificities of the acetoxy group on the benzenoid ring and position of the pyran carbonyl group of the coumarin with respect to oxygen heteroatom for the catalytic activity of TAase were also reported earlier. In this communication, we have demonstrated that the acetoxy coumarins and acetoxy dihydrocoumarins having a methyl group instead of a phenyl ring at the C-4, when used as the substrates, resulted in enhancement of TAase activity, while the saturation of double bond at C-3 and C-4 position had no effect on TAase activity. A comparison of the optimized structures of 1 and 7,8-diacetoxy-4-phenylcoumarin (2) suggested that the observed influence may be due to out of plane configuration of the phenyl ring at C-4. Further, the TAase-catalyzed activation of NADPH cytochrome c reductase and inhibition of aflatoxin B1 (AFB1)-DNA binding by acetoxy 4-phenylcoumarins and dihydrocoumarins were significantly lower as compared to those caused by acetoxy 4-methylcoumarins.

Acetylation↗
PubMed2005

Novel thiocoumarins as inhibitors of TNF-alpha induced ICAM-1 expression on human umbilical vein endothelial cells (HUVECs) and microsomal lipid peroxidation.

Different coumarin/thiocoumarin derivatives, that is, 7-hydroxy-4-methylcoumarin, 7,8-dihydroxy-4-methylcoumarin, 7-acetoxy-4-methylcoumarin, 7,8-diacetoxy-4-methylcoumarin, 7-hydroxy-4-methylthiocoumarin, 7,8-dihydroxy-4-methylthiocoumarin, 7-acetoxy-4-methylthiocoumarin and 7,8-diacetoxy-4-methylthiocoumarin were synthesized and evaluated for their effects on TNF-alpha induced expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and on NADPH-catalyzed rat liver microsomal lipid peroxidation with a view to identify modulators for expression of cell adhesion molecules and to establish structure-activity relationship. We found that dihydroxy and diacetoxy derivatives of thiocoumarin were more potent in comparison to the corresponding coumarin derivatives in inhibiting TNF-alpha-induced expression of ICAM-1. However, coumarin derivatives were found to be more potent in comparison to the corresponding thiocoumarins in inhibiting microsomal lipid peroxidation. We have also tested the intermediate compounds 7,8-dibenzyloxy-4-methylcoumarin and 7,8-dibenzyloxy-4-methylthiocoumarin for their inhibitory activity on TNF-alpha-induced ICMA-1 expression. We found that dibenzyloxy-4-methylthiocoumarin is better than dibenzyloxy-4-methylcoumarin. The mechanisms underlying the observed activities of coumarins and thiocoumarins have been discussed with reference to their structures. Such structure-function relationship studies may help in developing molecules with better anti-inflammatory and anti-oxidant activities.

Animals↗
PubMed2005

Synthesis of novel amino and acetyl amino-4-methylcoumarins and evaluation of their antioxidant activity.

The six novel 4-methylcoumarins bearing different functionalities such as amino, hydroxy, N-acetyl, acetoxy and nitro have been synthesized and confirmed on the basis of their spectral data (1H-, 13C-NMR, UV, IR and EI mass). They were examined for the first time for their effect on NADPH dependent liver microsomal lipid peroxidation in vitro, and the results were compared with other model 4-methylcoumarin derivatives to establish the structure-activity relationship. Our studies demonstrated that amino group is an effective substitute for the hydroxyl group for antioxidant property and produced a dramatic inhibition of lipid peroxidation. Ortho dihydroxy and ortho hydroxy-amino coumarins were found to possess highest antioxidant and radical scavenging activities.

Acetylation↗
PubMed2004

Clustering of Parkinson disease: shared cause or coincidence?

BACKGROUND: The spatial and temporal pattern of excessive disease occurrence, termed clustering, may provide clues about the underlying etiology. OBJECTIVE: To report the occurrence of 3 clusters of Parkinson disease (PD) in Canada. DESIGN AND PATIENTS: We determined the population groups containing the clusters, geographical limits, and duration of exposure to the specific environments. We tested whether there was an excessive presence of Parkinson disease by calculating the probability of the observed cases occurring under the null hypothesis that the disease developed independently and at random in cluster subjects. Results of genetic testing for mutations in the alpha-synuclein, parkin, tau genes, and spinocerebellar ataxia genes (SCA2 and SCA3) were negative. RESULTS: The probabilities of random occurrence (P values) in the 3 clusters were P = 7.9 x 10 (-7)for cluster 1, P = 2.6 x 10 (-7)for cluster 2, and P = 1.5 x 10 (-7)for cluster 3. CONCLUSIONS: Our findings indicate an important role for environmental causation in Parkinson disease. A possible role exists for environmental factors such as viral infection and toxins in the light of current evidence.

Adult↗
PubMed2004

Tremor induced by thalamic deep brain stimulation in patients with complex regional facial pain.

We report on two patients who developed a new postural and action tremor after chronic stimulation of the contralateral thalamus (VPM nucleus) during treatment of a complex regional facial pain syndrome. The tremor was only present during deep brain stimulation (DBS) and was suppressed with adjustment of the stimulation parameters. Tremor was seen only with low frequency stimulation (50 Hz or lower) and disappeared with higher stimulation frequencies. In addition to being an unusual side effect of thalamic DBS, we believe that this phenomenon affords insight into one possible mechanism underlying essential tremor (ET). A central oscillatory mechanism involving the olivocerebellar complex and the thalamus, which is a part of the cerebro-cerebello-cerebral circuit, is thought to play an important role in the genesis of ET. Induction of a tremor resembling ET in our patients indicates an active role for low frequency stimulation. A plausible explanation for this is that low frequency stimulation in the thalamic area enhances the output of the tremor-producing network. This leads credence to the concept of central oscillations in a "tremor circuit," of which the thalamus is a part, as being important in ET.

Adult↗
PubMed2004

Coordination of transcription factor phosphorylation and histone methylation by the P-TEFb kinase during human immunodeficiency virus type 1 transcription.

The human immunodeficiency virus type 1 (HIV-1) Tat protein recruits positive transcription elongation factor b (P-TEFb) to the transactivation response (TAR) RNA structure to facilitate formation of processive transcription elongation complexes (TECs). Here we examine the role of the Tat/TAR-specified cyclin-dependent kinase 9 (CDK9) kinase activity in regulation of HIV-1 transcription elongation and histone methylation. In HIV-1 TECs, P-TEFb phosphorylates the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) and the transcription elongation factors SPT5 and Tat-SF1 in a Tat/TAR-dependent manner. Using in vivo chromatin immunoprecipitation analysis, we demonstrate the following distinct properties of the HIV-1 transcription complexes. First, the RNAP II CTD is phosphorylated at Ser 2 and Ser 5 near the promoter and at downstream coding regions. Second, the stable association of SPT5 with the TECs is dependent upon P-TEFb kinase activity. Third, P-TEFb kinase activity is critical for the induction of methylation of histone H3 at lysine 4 and lysine 36 on HIV-1 genes. Flavopiridol, a potent P-TEFb kinase inhibitor, inhibits CTD phosphorylation, stable SPT5 binding, and histone methylation, suggesting that its potent antiviral activity is due to its ability to inhibit several critical and unique steps in HIV-1 transcription elongation.

Animals↗
PubMed2003

The Tat/TAR-dependent phosphorylation of RNA polymerase II C-terminal domain stimulates cotranscriptional capping of HIV-1 mRNA.

The HIV type 1 (HIV-1) Tat protein stimulates transcription elongation by recruiting P-TEFb (CDK9/cyclin T1) to the transactivation response (TAR) RNA structure. Tat-induced CDK9 kinase has been shown to phosphorylate Ser-5 of RNA polymerase II (RNAP II) C-terminal domain (CTD). Results presented here demonstrate that Tat-induced Ser-5 phosphorylation of CTD by P-TEFb stimulates the guanylyltransferase activity of human capping enzyme and RNA cap formation. Sequential phosphorylation of CTD by Tat-induced P-TEFb enhances the stimulation of human capping enzyme guanylyltransferase activity and RNA cap formation by transcription factor IIH-mediated CTD phosphorylation. Using an immobilized template assay that permits isolation of transcription complexes, we show that Tat/TAR-dependent phosphorylation of RNAP II CTD stimulates cotranscriptional capping of HIV-1 mRNA. Upon transcriptional induction of latently infected cells, accumulation of capped transcripts occurs along with Ser-5-phosphorylated RNAP II in the promoter proximal region of the HIV-1 genome. Therefore, these observations suggest that Tat/TAR-dependent phosphorylation of RNAP II CTD is crucial not only in promoting transcription elongation but also in stimulating nascent viral RNA capping.

Binding Sites↗
PubMed2003

[11C]DTBZ-PET correlates of levodopa responses in asymmetric Parkinson's disease.

Levodopa effectively improves motor symptoms of Parkinson's disease. However, the beneficial effects of levodopa often erode over time with the emergence of response fluctuations. Although these response changes have been recognized from the early levodopa era, their mechanisms remain poorly understood. We investigated the role of dopamine (DA) terminal loss in the development of motor fluctuations by employing PET with [11C](+/-)dihydrotetrabenazine ([11C]DTBZ) as an in vivo marker for DA nerve terminals. Levodopa response was characterized by analysing the time-response curve to a single dose of levodopa with a finger-tapping test. PET scans were performed in 11 patients with asymmetric Parkinson's disease (age: 61.12 +/- 7.97 years; duration of Parkinson's disease: 10.55 +/- 4.53 years; mean +/- SD). Each patient performed finger-tapping tests for up to 5 h after taking a therapeutic dose of levodopa. Results showed significantly lower [11C]DTBZ binding potential (BP; Bmax/Kd) and baseline tapping rates on the more affected putamen and corresponding body side, respectively, than on the other (P = 0.003 for the former, P = 0.013 for the latter). Among the variables describing the time-response curve, the duration and early decay time were significantly shorter on the more affected side (P = 0.051 and P = 0.021, respectively). Latency to the onset and latency to 50% Emax (the magnitude of the levodopa response) were significantly longer on the more-affected side (P = 0.013 and P = 0.004, respectively). Emax was not significantly different between the two sides. The asymmetry (difference from the more affected to less affected side) of [11C]DTBZ BP in the putamen showed a highly significant correlation with the corresponding asymmetry of the estimated EC50 (levodopa concentration producing 50% of the maximal response; P = 0.022; r = -0.727), a marginally significant correlation with that of latency to the onset (P = 0.065; r = -0.583) and no significant correlation with that of the magnitude, duration or early decay time. This pattern of changes in levodopa response from the less affected to more affected side was similar to that from stable to fluctuating responders except for the latency to onset. These findings suggest a pathogenetic role for DA terminal loss in the development of motor fluctuations. However, the absence of a significant correlation between the early decay of levodopa response and DA terminal density suggests that DA terminal loss alone cannot account for the development of motor fluctuations. Therefore, our study suggests that both levodopa treatment and DA terminal loss contribute to the pathogenesis of motor fluctuations.

Aged↗
PubMed2003

Nuclear protein phosphatase-1 regulates HIV-1 transcription.

We recently reported that protein phosphatase 1 (PP1) dephosphorylates RNA polymerase II C-terminal repeats and regulates HIV-1 transcription in vitro. Here we provide evidence that PP1 is also required for Tat-induced HIV-1 transcription and for viral replication in cultured cells. Inhibition of PP1 by overexpression of nuclear inhibitor of PP1 (NIPP1) inhibited Tat-induced HIV-1 transcription in transient transfection assays. A mutant of NIPP1 that was defective in binding to PP1 did not have this effect. Also the co-expression of PP1 gamma reversed the inhibitory effect of NIPP1. Adeno-associated virus-mediated delivery of NIPP1 significantly reduced HIV-1 transcription induced by Tat-expressing adenovirus in CD4+ HeLa cells that contained an integrated HIV-1 promoter (HeLa MAGI cells). In addition, infection of HeLa MAGI cells with adeno-associated virus-NIPP1 prior to the infection with HIV-1 significantly reduced the level of HIV-1 replication. Our results indicate that PP1 might be a host cell factor that is required for HIV-1 viral transcription. Therefore, nuclear PP1 may represent a novel target for anti-HIV-1 therapeutics.

Cell Nucleus↗
PubMed2003

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 10: identification of inhibitors for the liver microsomal acetoxycoumarin: protein transacetylase.

The quantitative structure-activity relationship (QSAR) studies conducted by us earlier revealed the cardinal role of the pyran ring carbonyl group in the acetoxy polyphenolic compounds for the acetoxy polyphenol:protein transacetylase (TAase) activity. Hence, an attempt was made to examine whether such substrate analogues of benzopyran acetates which lack in the pyran ring carbonyl group, such as 7-acetoxy-2,3-dihydro-2,2-dimethylbenzopyran (BPA), cetachin pentaacetate (CPA) and hematoxylin pentaacetate (HPA) could inhibit the 7,8-diacetoxy-4-methylcoumarin (DAMC):protein (glutathione-S-transferase) transacetylase activity. These compounds were indeed found to remarkably inhibit the TAase activity in a concentration dependent manner and exerted their inhibitory action very rapidly. Further BPA, CPA and HPA were found to abolish the TAase mediated activation of NADPH cytochrome C reductase as well as the inhibition of liver microsome catalyzed aflatoxin B(1) (AFB(1))-DNA binding by DAMC very effectively. These results strongly suggest that the acetoxybenzopyrans merit as potent inhibitors of TAase.

Aflatoxin B1↗