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In JoVE (1)
Other Publications (9)
- AIDS Research and Human Retroviruses
- Biochemical and Biophysical Research Communications
- Journal of Neuroimmune Pharmacology : the Official Journal of the Society on NeuroImmune Pharmacology
- Alcoholism, Clinical and Experimental Research
- Alcoholism, Clinical and Experimental Research
- Journal of Neurovirology
- FEMS Microbiology Letters
- Breast Cancer Research : BCR
- Journal of Hazardous Materials
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Articles by Santosh Kumar in JoVE
JoVE Clinical and Translational Medicine
O α-teste: Rapid livre de células CD4 enumeração Utilizando saliva total
1Department of Medicine, Weill Cornell Medical College, 2Department of Oral Biology, University of Missouri-Kansas City-School of Dentistry, 3Department of Pharmacology and Toxicology, University of Missouri Kansas City- School of Pharmacy, 4Regional Hospital, Bamenda, NWP, Cameroon, 5Mezam Polyclinic HIV/AIDS Treatment Center, Cameroon, 6Institute for Human Genetics and Biochemistry
Um método de enumeração de células CD4, o α-teste, é descrito que utiliza saliva total para fornecer contagens de CD4 rápidos e precisos. As α-teste tostões custos e elimina a necessidade de formação técnica, reagentes caros, tais como anticorpos monoclonais, instrumentação, refrigeração, transporte de amostras, bem como coleta e manuseio de sangue.
Other articles by Santosh Kumar on PubMed
Short Communication: Lack of Immune Response in Rapid Progressor Morphine-dependent and SIV/SHIV-infected Rhesus Macaques is Correlated with Downregulation of TH1 Cytokines
Our previous studies have shown two distinct disease patterns (rapid and normal onset of clinical symptoms) in morphine-dependent SHIV/SIV-inoculated rhesus macaques. We have also shown that control as well as 50% of morphine-dependent macaques (normal progressor) developed humoral and cellular immune responses whereas the other half of the morphine-dependent macaques (rapid progressor) did not develop antiviral immune responses after infection with SIV/SHIV. In the present study, we analyzed the association between cytokine production, immune response, and disease progression. To study the immunological effects of morphine at cytokine levels in the context of a lentiviral infection, we inoculated rhesus macaques with a mixture of SHIV(KU-18), SHIV(89.6)P, and SIV/17E-Fr. These animals were followed for a period of 56 weeks for cytokine level production in plasma. Drug-dependent rapid disease progressors exhibited an increase in IL-18 and IL-1Ra and a decrease in IL-12 levels in the plasma. Morphine-dependent normal progressors and control macaques exhibited an increase in both IL-18 and IL-12, whereas IL-Ra levels remained constant throughout the observation period. These results suggest that rapid disease progression in relation to morphine dependency may be the result of an altered cytokine profile.
Effect of Ethanol on Spectral Binding, Inhibition, and Activity of CYP3A4 with an Antiretroviral Drug Nelfinavir
Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest K(D) (5.9±0.34mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20mM) decreased the K(D) of nelfinavir by >5-fold (0.041±0.007 vs. 0.227±0.038μM). Similarly, 20mM ethanol decreased the IC(50) of nelfinavir by >3-fold (2.6±0.5 vs. 8.3±3.1μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k(cat), it decreased the K(m) of nelfinavir, suggesting a decrease in catalytic efficiency (k(cat)/K(m)). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy.
A LC-MS/MS Method for Concurrent Determination of Nicotine Metabolites and Role of CYP2A6 in Nicotine Metabolism in U937 Macrophages: Implications in Oxidative Stress in HIV + Smokers
Nicotine, the major constituent of tobacco, is predominantly metabolized by liver CYP2A6 into cotinine and many other compounds, including nicotine-derived nitrosamine ketone (NNK), which is known to cause oxidative stress. We have recently shown that CYP2A6 is highly expressed in U937 monocyte-derived macrophages. In this study we investigated the role of CYP2A6 in nicotine metabolism and oxidative stress in U937 macrophages. To study nicotine metabolism, we developed a highly sensitive LC-MS/MS method for simultaneous quantitative determination of nicotine, cotinine, and NNK. The LC-MS/MS analysis was carried out by multiple reaction monitoring mass transitions with m/z of 163.2/130.1, 177.4/98.3, and 208.4/122.1 for nicotine, cotinine, and NNK, respectively. The calibration curves were linear within 3.3-1028.1 ng/ml for nicotine and 0.3-652.6 ng/ml for cotinine and NNK. This novel method was then applied to quantify nicotine metabolites, cotinine and NNK, in nicotine-treated U937 macrophages. Cotinine and NNK initially formed at 30 min, followed by a peak at 2-3 h. The role of CYP2A6 in nicotine metabolism in U937 macrophages was further confirmed by using CYP2A6-selective inhibitor, tryptamine, which significantly decreased cotinine (70%) and completely inhibited NNK formations. Finally, we showed that nicotine-treated macrophages increase the formation of oxidant at 30-60 min, which is consistent with the initial formation of cotinine and NNK. In conclusion, we have developed a new LCMS/MS method for concurrent determination of nicotine metabolites and analyzed the role of CYP2A6 in nicotine metabolism and oxidative stress in U937 macrophages, which may have implications in viral replication among HIV + smokers.
Effect of Alcohol on Drug Efflux Protein and Drug Metabolic Enzymes in U937 Macrophages
ATP-binding cassette (ABC) proteins and cytochrome P450 (CYP) enzymes regulate the bioavailability of HIV-1 antiretroviral therapeutic drugs, non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs). They are also involved in regulating, and responding to, oxidative stress in various tissues and organs including liver. This study is designed to assess the effect of alcohol on the ABCC1 and CYP enzymes involved in the metabolism of NNRTIs and PIs (CYP2B6, CYP2D6, and CYP3A4) and oxidative stress (CYP1A1, CYP2A6, and CYP2E1) in U937 macrophages. The U937 cell line has been utilized as an in vitro model of human macrophages.
Differential Effects of Ethanol on Spectral Binding and Inhibition of Cytochrome P450 3A4 with Eight Protease Inhibitors Antiretroviral Drugs
Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver, which metabolizes approximately 50% of the marketed drugs including antiretroviral agents. CYP3A4 induction by ethanol and its impact on drug metabolism and toxicity is known. However, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known, except that we have recently shown that ethanol facilitates the binding of a protease inhibitor (PI), nelfinavir, with CYP3A4. The current study was designed to examine the effect of ethanol on spectral binding and inhibition of CYP3A4 with all currently used PIs that differ in physicochemical properties.
Methamphetamine Toxicity and Its Implications During HIV-1 Infection
Over the past two decades methamphetamine (MA) abuse has seen a dramatic increase. The abuse of MA is particularly high in groups that are at higher risk for HIV-1 infection, especially men who have sex with men (MSM). This review is focused on MA toxicity in the CNS as well as in the periphery. In the CNS, MA toxicity is comprised of numerous effects, including, but not limited to, oxidative stress produced by dysregulation of the dopaminergic system, hyperthermia, apoptosis, and neuroinflammation. Multiple lines of evidence demonstrate that these effects exacerbate the neurodegenerative damage caused by CNS infection of HIV perhaps because both MA and HIV target the frontostriatal regions of the brain. MA has also been demonstrated to increase viral load in the CNS of SIV-infected macaques. Using transgenic animal models, as well as cultured cells, the HIV proteins Tat and gp120 have been demonstrated to have neurotoxic properties that are aggravated by MA. In addition, MA has been shown to exhibit detrimental effects on the blood-brain barrier (BBB) that have the potential to increase the probability of CNS infection by HIV. Although the effects of MA in the periphery have not been as extensively studied as have the effects on the CNS, recent reports demonstrate the potential effects of MA on HIV infection in the periphery including increased expression of HIV co-receptors and increased expression of inflammatory cytokines.
A Catabolic Pathway for the Degradation of Chrysene by Pseudoxanthomonas Sp. PNK-04
The chrysene-degrading bacterium Pseudoxanthomonas sp. PNK-04 was isolated from a coal sample. Three novel metabolites, hydroxyphenanthroic acid, 1-hydroxy-2-naphthoic acid and salicylic acid, were identified by TLC, HPLC and MS. Key enzyme activities, namely 1-hydroxy-2-naphthoate hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-1,2-dioxygenase, were noted in the cell-free extract. These results suggest that chrysene is catabolized via hydroxyphenanthroic acid, 1-hydroxy-2-naphthoic acid, salicylic acid and catechol. The terminal aromatic metabolite, catechol, is then catabolized by catechol-1,2-dioxygenase to cis,cis-muconic acid, ultimately forming TCA cycle intermediates. Based on these studies, the proposed catabolic pathway for chrysene degradation by strain PNK-04 is chrysene → hydroxyphenanthroic acid → 1-hydroxy-2-naphthoic acid → 1,2-dihydroxynaphthalene → salicylic acid → catechol →cis,cis-muconic acid.
Evidence That GTP-binding Domain but Not Catalytic Domain of Transglutaminase 2 is Essential for Epithelial-to-mesenchymal Transition in Mammary Epithelial Cells
ABSTRACT: INTRODUCTION: The expression of proinflammatory protein tissue transglutaminase (TG2) is frequently upregulated in multiple cancer cell types. However, the exact role of TG2 in cancer cells is not well understood. We recently initiated studies to determine the significance of TG2 in cancer cells and observed that sustained expression of TG2 resulted in epithelial-to-mesenchymal transition (EMT) and promoted stem cell (CSC) traits in mammary epithelial cells. These results suggested that TG2 could serve as a promising therapeutic target for overcoming chemoresistance and inhibiting metastatic spread of cancer cells. METHODS: Using various mutant constructs, we analyzed the activity of TG2 that is essential for promoting the EMT/CSC phenotype. RESULTS: Our results suggest that catalytically inactive TG2 (TG2-C277S) is as effective as wild-type TG2 (TG2-WT) in inducing the EMT/CSC in mammary epithelial cells. In contrast, overexpression of a GTP-binding-deficient mutant (TG2-R580A) was completely incompetent in this regard. Moreover, TG2-dependent activation of the proinflammatory transcription factor, NF-kappaB, is deemed essential for promoting the EMT/CSC phenotype in mammary epithelial cells. CONCLUSIONS: Our results suggest that transamidation activity of TG2 is not essential for promoting its oncogenic functions and provide strong rationale for developing small molecule inhibitors to block GTP-binding pockets of TG2. Such inhibitors may have great potential for inhibiting the TG2-regulated pathways and for reversing drug resistance and inhibiting metastasis of cancer cells.
Generation of Continuous Packed Bed Reactor with PVA-alginate Blend Immobilized Ochrobactrum Sp. DGVK1 Cells for Effective Removal of N,N-dimethylformamide from Industrial Effluents
Effective removal of dimethylformamide (DMF), the organic solvent found in industrial effluents of textile and pharma industries, was demonstrated by using free and immobilized cells of Ochrobactrum sp. DGVK1, a soil isolate capable of utilizing DMF as a sole source of carbon, nitrogen. The free cells have efficiently removed DMF from culture media and effluents, only when DMF concentration was less than 1% (v/v). Entrapment of cells either in alginate or in polyvinyl alcohol (PVA) failed to increase tolerance limits. However, the cells of Ochrobactrum sp. DGVK1 entrapped in PVA-alginate mixed matrix tolerated higher concentration of DMF (2.5%, v/v) and effectively removed DMF from industrial effluents. As determined through batch fermentation, these immobilized cells have retained viability and degradability for more than 20 cycles. A continuous packed bed reactor, generated by using PVA-alginate beads, efficiently removed DMF from industrial effluents, even in the presence of certain organic solvents frequently found in effluents along with DMF.
