Risk and cause in medical science.
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Animals↗
Biomedical subjects
R Kumar
Publications and source records attributed to R Kumar.
Acute myeloblastic leukemia in adults.
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Adolescent↗
Epidermal growth factor receptor ectodomain in the urine of patients with squamous cell carcinoma.
In this study we determined the levels of the epidermal growth factor receptor (EGFR) in the urine of patients with squamous cell carcinoma compared to levels in the urine of normal volunteers and patients with nonsquamous cell carcinoma. A 24-h urine specimen was collected from 50 normal volunteers, 50 patients with nonsquamous cell carcinoma, and 42 patients with squamous cell carcinoma. An ELISA using mAbs to the external domain of the EGFR was used to measure levels of the receptor in the urine samples. Measurement of the EGFR ectodomain in the 24-h urine specimens showed detectable levels in 15 (36%) of 42 squamous cell carcinoma patients compared to 3 (6%) of 50 controls and 8 (16%) of 50 nonsquamous patients. It was also observed that 10 (53%) of 19 patients with metastatic squamous cell carcinoma had detectable EGFR ectodomain levels compared to 5 (22%) of 23 squamous cell patients with localized disease. Thus, we concluded that the EGFR ectodomain was detectable in the urine in a significantly higher number of patients with squamous cell carcinoma than normal volunteers or patients with nonsquamous cell carcinoma.
3T3 Cells↗
Inhibitory effect of curcumin on epidermal growth factor receptor kinase activity in A431 cells.
We explored the mechanism of antigrowth action of Curcumin by investigating its effect on epidermal growth factor (EGF) receptor intrinsic kinase activity in the human epidermoid carcinoma A431 cells. The short-term treatment of cells with Curcumin inhibited EGF receptor intrinsic kinase activity up to 90% in a dose- and time-dependent manner, and also inhibited EGF-induced tyrosine phosphorylation of EGF receptors. The observed early effects of Curcumin were mediated via a cellular mechanism(s), and preceded the period when inhibition of cell growth occurred.
Carcinoma, Squamous Cell↗
Structure determination of aquomet porcine hemoglobin at 2.8 A resolution.
Considerable attention is currently focused on hemoglobins from lower mammals, such as the pig, for potential use in cell-free blood substitute preparations safe for use in humans. As the first step in the elucidation of structure-function relationships in porcine hemoglobin, we have determined the three-dimensional structure of aquomet porcine hemoglobin at 2.8 A resolution. Overall, the porcine hemoglobin tetramer is structurally similar to that of human oxyhemoglobin, and the r.m.s. deviation of all backbone atoms (minus five residues at the amino and carboxyl termini of each subunit) is 0.8 A. This similarity is not unexpected given that human and porcine hemoglobins exhibit 85% sequence identity. However, regions of subtle structural differences are implicated in subtle functional differences between the two proteins, such as the 20 to 25% inhibition of the alkaline Bohr effect and the accompanying reduction in oxygen-linked chloride binding observed for porcine hemoglobin. The structural similarity of these two mammalian hemoglobins also rationalizes the novel hybridization behavior of pig and human subunits in transgenic pigs expressing both porcine and human hemoglobins in porcine erythrocytes.
Amino Acid Sequence↗
Synthesis, in vitro biological stability, and anti-HIV activity of 5-halo-6-alkoxy(or azido)-5,6-dihydro-3'-azido-3'-deoxythymidine diastereomers as potential prodrugs to 3'-azido-3'-deoxythymidine (AZT).
A new class of 5-halo-6-alkoxy(or azido)-5,6-dihydro-3'-azido-3'-deoxythymidines was investigated as potential anti-AIDS drugs. These 5,6-dihydro derivatives, which are also potential prodrugs to 3'-azido-3'-deoxythymidine (AZT), were designed in an effort to enhance the duration of action, lipophilicity, and cephalic delivery to the central nervous system. The 5-halo-6-alkoxy(or azido)-5,6-dihydro-3'-azido-3'-deoxythymidines, which differ in configuration at the C-5 and C-6 positions, were synthesized by the regiospecific addition of XR (X = Br, Cl, I; R = alkoxy, azido) to the 5,6-olefinic bond of AZT. The 5-halo-6-methoxy-5,6-dihydro derivatives of AZT are more lipophilic (P = 3.3-18.8 range) than the parent compound AZT (P = 1.29). These 5-halo-6-methoxy-5,6-dihydro compounds, like AZT, did not undergo glycosidic bond cleavage upon incubation with Escherichia coli thymidine phosphorylase. Regeneration of the 5,6-olefinic bond to give AZT, upon incubation of the 5-halo-6-methoxy-5,6-dihydro compounds with glutathione, mouse blood, or mouse liver homogenate, was dependent upon the nature of the 5-halo substituent (I > Br). No 5,6-olefinic bond regeneration was observed for the 5-chloro analogs. The ability of these 5-halo-6-alkoxy (or azido)-5,6-dihydro-3'-azido-3'-deoxythymidines to protect CEM cells against HIV-induced cytopathogenicity was evaluated. Structure-activity studies showed that the C-5 substituent (I, Br, Cl) was a determinant of anti-HIV-1 activity where the potency order was I > or = Br > Cl. In the 5-bromo series of compounds, the C-6 substituent was also a determinant of activity where 6-OMe and 6-OEt substituents exhibited a greater potency than the corresponding 6-i-PrO, 6-(1-octyloxy), 6-(1-hexadecyloxy), and 6-azido analogs. All of the 5-chloro-6-substituted-5,6-dihydro compounds were inactive, except for the approximately equipotent 6-OMe and 6-azido diastereomeric mixtures which were 2-3 log units less active than the reference drug AZT. The configuration at the C-5 and C-6 positions also influenced potency where the activity of the 5R,6R-diastereomer was generally greater than that of the corresponding 5S,6S-diastereomer.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs.
The prokaryotic mRNA ribosome binding site (RBS) usually contains part or all of a polypurine domain UAAGGAGGU known as the Shine-Dalgarno (SD) sequence found just 5' to the translation initiation codon. It is now clear that the SD sequence is important for identification of the translation initiation site on the mRNA by the ribosome, and that as a result, the spacing between the SD and the initiation codon strongly affects translational efficiency (1). It is not as clear, however, whether there is a unique optimal spacing. Complications involving the definition of the spacing as well as secondary structures have obscured matters. We thus undertook a systematic study by inserting two series of synthetic RBSs of varying spacing and SD sequence into a plasmid vector containing the chloramphenicol acetyltransferase gene. Care was taken not to introduce any secondary structure. Measurements of protein expression demonstrated an optimal aligned spacing of 5 nt for both series. Since aligned spacing corresponds naturally to the spacing between the 3'-end of the 16S rRNA and the P-site, we conclude that there is a unique optimal aligned SD-AUG spacing in the absence of other complicating issues.
Base Sequence↗
Synthesis and antiviral (HIV-1, HBV) activities of 5-halo-6-methoxy(or azido)-5,6-dihydro-3'-fluoro-3'-deoxythymidine diastereomers. Potential prodrugs to 3'-fluoro-3'-deoxythymidine.
A new class of 5-halo-6-methoxy(or azido)-5,6-dihydro-3'-fluoro-3'- deoxythymidines (4-13) were investigated as potential anti-AIDS drugs. These 5,6-dihydro derivatives, which are also potential prodrugs to 3'-fluoro-3'-deoxythymidine (FLT), were designed to have properties which would enhance their duration of action, lipophilicity, and cephalic delivery to the central nervous system. The 5-halo-6-methoxy(or azido)-5,6-dihydro-3'-fluoro-3'-deoxythymidines, which differ in configuration at the C-5 and C-6 positions, were synthesized by the regiospecific addition of XR (X = Br, Cl, I; R = OMe, N3) to the 5,6-olefinic bond of FLT. These 5-halo-6-methoxy-5,6-dihydro derivatives are more lipophilic (P = 1.5-5.15 range) than the parent compound FLT (P = 0.5). Regeneration of the 5,6-olefinic bond to give FLT, upon incubation of the 5-halo-6-methoxy-5,6-dihydro compounds with glutathione, was dependent on the nature of the 5-halo substituent (I > Br > Cl). The ability of these 5-halo-6-methoxy(or azido)-5,6- dihydro compounds (4-13) to protect CEM cells against HIV-induced cytopathogenicity was evaluated. The C-5 halo substituent was a determinant of anti-HIV-1 activity where the approximately equipotent 5-iodo and 5-bromo were generally more potent than the 5-chloro derivatives of FLT. Compounds having the (5S,6S)-configuration were more potent than the corresponding (5R,6R)-diastereomer. The most potent anti-HIV-1 agents, which included the (5R,6R)-5-Br,6-OMe (4), (5S,6S)-5-Br,6-OMe (5), and (5S,6S)-5-I,6-OMe (10) derivatives of FLT, exhibited comparable activities to the reference drugs AZT and FLT. Although (5R,6R)-5-bromo-6-methoxy-5,6-dihydro-3'-fluoro-3'-deoxythymidine (4) inhibited hepatitis B virus replication at a 5-6-fold higher concentration (EC50) than the reference drug 2',3'-dideoxycytidine (DDC), it was 3-5-fold less cytotoxic (CC50) than DDC.
Antiviral Agents↗
Cell cycle-dependent modulation of alpha-interferon-inducible gene expression and activation of signaling components in Daudi cells.
To investigate cell cycle-dependent expression of interferon (IFN)-induced genes and signaling pathways, we examined the modulation of IFN-inducible gene expression and p91 activation, and JAK-1 and Tyk-2 kinases' activation in human Burkitt's lymphoma Daudi cells which were synchronized at different points in the cell cycle progression. We observed that each one of 100-, 67-, and 46-kDa 2'-5'-oligoadenylate synthetase (2-5(A)-synthetase) proteins had a unique expression pattern in different phases of the cell cycle which was different from the expression of 2-5(A)-synthetase proteins in exponentially growing Daudi cells. The cell cycle-dependent changes in the overall levels of expression of 2-5(A)-synthetase proteins correlated well with the induction of expression of 2-5(A)-synthetase mRNA as well as the appearance of IFN stimulatory gene factor-3 and expression and activation of p91 in cells released from the G1/S block in the absence of exogenous IFN-alpha. In addition, cells from the G1/S phase exhibited increased tyrosine phosphorylation of JAK-1 and Tyk-2 kinases and p91 by IFN-alpha as compared with asynchronized cells. Enhanced stimulation of activation of JAK-1, Tyk-2, and p91 by IFN-alpha was progressively reduced from G1/S to G2/M to pass G2/M. These findings demonstrate that the levels of constitutive and inducible expression of IFN-alpha-inducible genes, activation of p91, and tyrosine phosphorylation of JAK-1 and Tyk-2 kinases are differentially influenced by the state of the cell cycle.
2',5'-Oligoadenylate Synthetase↗
Electron-correlation effects on the properties of LaxSr1-xTiO3.
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Journal Article↗
High-efficiency synthesis of human alpha-endorphin and magainin in the erythrocytes of transgenic mice: a production system for therapeutic peptides.
Chemical synthesis of peptides, though feasible, is hindered by considerations of cost, purity, and efficiency of synthesizing longer chains. Here we describe a transgenic system for producing peptides of therapeutic interest as fusion proteins at low cost and high purity. Transgenic hemoglobin expression technology using the locus control region was employed to produce fusion hemoglobins in the erythrocytes of mice. The fusion hemoglobin contains the desired peptides as an extension at the C end of human alpha-globin. A protein cleavage site is inserted between the C end of the alpha-globin chain and the N-terminal residue of the desired peptide. The peptide is recovered after cleavage of the fusion protein with enzymes that recognize this cleavage signal as their substrate. Due to the selective compartmentalization of hemoglobin in the erythrocytes, purification of the fusion hemoglobin is easy and efficient. Because of its compact and highly ordered structure, the internal sites of hemoglobin are resistant to protease digestion and the desired peptide is efficiently released and recovered. The applicability of this approach was established by producing a 16-mer alpha-endorphin peptide and a 26-mer magainin peptide in transgenic mice. Transgenic animals and their progeny expressing these fusion proteins remain health, even when the fusion protein is expressed at > 25% of the total hemoglobin in the erythrocytes. Additional applications and potential improvements of this methodology are discussed.
Amino Acid Sequence↗
Human and rat osteoblast-like cells express stathmin, a growth-regulatory protein.
We used the differential display polymerase chain reaction method of Liang and Pardee (Science (1992) 257:967-971) to identify a growth-regulatory protein, stathmin or oncoprotein 18, in human and rat osteoblast-like cells. When messenger ribonuleic acid from human osteosarcoma (Saos-2) cells treated with 10(-8)M 1,25-dihydroxyvitamin D3 was compared with that of cells treated with vehicle alone by this method, we observed an increase in the intensity of a band that on subsequent DNA sequence analysis was found to encode stathmin. We examined normal human osteoblast-like cells, transformed human osteoblast-like cells (HOBIT), human osteosarcoma cells (Saos-2), and rat osteoblast cell (ROS 17/2.8, ROS 25/1) lines for the presence of the messenger RNA for stathmin. All osteoblast-like cells examined expressed the RNA for this protein. Western analysis of the protein of these cells with a polyclonal antibody directed against stathmin showed the presence of a M(r) 19,000 band. We conclude that human and rat osteoblast-like cells express stathmin and that this protein could play a role in regulation of the growth of these cells in response to various hormonal stimuli.
Animals↗
Brief report: inhibition of renal phosphate transport by a tumor product in a patient with oncogenic osteomalacia.
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Animals↗
Synthesis, biotransformation, pharmacokinetics, and antiviral properties of 5-ethyl-5-halo-6-methoxy-5,6-dihydro-2'-deoxyuridine diastereomers.
Diastereomers of 5-ethyl-5-halo-6-methoxy-5,6-dihydro-2'-deoxyuridine were synthesized by the regiospecific addition of XOMe (X = Br, Cl) to the 5,6-olefinic bond of 5-ethyl-2'-deoxyuridine (EDU). 5-(1-Hydroxyethyl)-2'-deoxyuridine (HEDU) was identified as a metabolite of the 5-bromo-5-ethyl-6-methoxy-5,6-dihydro-2'-deoxyuridine diastereomers (BMEDU). The concentration of EDU and 5-ethyluracil (EU) in blood was higher after i.v. administration of the bromo diastereomers (BMEDU) to rats, relative to the concentration of EDU and EU after injection of the chloro (CMEDU) diastereomers. The CMEDU diastereomers were found to be oxidized less extensively to HEDU, were more stable to glycosidic bond cleavage, and were converted more slowly to EDU, than the BMEDU compounds. These BMEDU and CMEDU diastereomers exhibited pharmacokinetics characterized by a biphasic decline in plasma concentration. All diastereomers exhibited a characteristic second maximum blood concentration (Cmax), which was attributed to reabsorption after biliary excretion. All of these 5-ethyl-5-halo-6-methoxy-5,6-dihydro compounds, with the exception of (5S,6S)-BMEDU, had higher AUC values (ranging from 0.32 to 1.20 microM.hr.mL-1) and lower plasma clearances (10-36 mL.min-1) relative to the AUC values (0.14 microM.hr.mL-1) and plasma clearance (85 mL.min-1) of EDU. These BMEDU and CMEDU diastereomers are more lipophilic (log P = -0.42 to 0.40 range) than EDU (log P = -1.09), which should enhance their ability to cross the blood-brain barrier. These 5,6-dihydro compounds showed higher levels (11-22%) of binding to bovine serum albumin than EDU (7%). The BMEDU compounds exhibited equipotent in vitro antiviral activity to EDU against HSV-1 and HSV-2, whereas the CMEDU analogs were inactive. The (5S,6R)-CMEDU diastereomer was equipotent to ganciclovir in the human cytomegalovirus assay.
Animals↗
Synthesis and antiviral activity of 3'-azido-3'-deoxythymidine triphosphate distearoylglycerol: a novel phospholipid conjugate of the anti-HIV agent AZT.
Phospholipid conjugates of 3'-azido-3'-deoxythymidine (AZT) show activity against the human immunodeficiency virus (HIV) in vitro. In a previous report (K.Y. Hostetler, L.M. Stuhmiller, B.H.M. Lenting, H. van den Bosch and D.D. Richman (1991), J. Biol. Chem. 265, 6112-6117) the syntheses and anti-HIV activities of AZT mono- and diphosphate diglyceride have been described. We now report on the synthesis, characterization and biological activity of 3'-azido-3'-deoxythymidine triphosphate distearoylglycerol (AZTTP-DSG). The compound was prepared by the condensation of AZT diphosphate with distearoylphosphatidic acid morpholidate in anhydrous pyridine at room temperature and purified by means of high-performance liquid chromatography using a silica column. Characterization was performed with 31P-NMR and IR analyses and determination of the fatty acid, phosphorus and nucleoside content of the product. AZTTP-DSG inhibited HIV-1 replication in both CEM and HT4-6C cells at a level intermediate in potency between its mono- and diphosphate analogs. The IC50 values of AZTTP-DSG were 0.33 and 0.79 microM in these two cell lines, respectively. In addition, AZTTP-DSG was less toxic to CEM cells in vitro than the other AZT liponucleotides and reduced viable cell numbers in this cell type by 50% at 1000 microM. Initial studies on the metabolism of AZTTP-DSG revealed that both AZT and AZT monophosphate were liberated from the lipid pro-drug by a rat liver mitochondrial enzyme preparation. These phospholipid derivatives of AZT nucleotides represent pro-drugs for the intracellular delivery of phosphorylated antiviral nucleoside analogs.
Antiviral Agents↗
Interferon-alpha inhibits cyclin E- and cyclin D1-dependent CDK-2 kinase activity associated with RB protein and E2F in Daudi cells.
The state of phosphorylation of retinoblastoma (RB) protein is regulated by CDK2 and CDC2 kinases. In the studies presented here, we have investigated the effect of interferon-alpha (IFN-alpha) on cyclin-dependent kinases in Daudi cells which were synchronized at different points of the cell cycle progression. We observed that the IFN-alpha enhances the expression of underphosphorylated RB protein in Daudi cells released from the G1/S, and this was closely associated with the inhibition of CDK2 kinase and not CDC2 kinase activity. The observed IFN-alpha-sensitive CDK2 kinase activity was dependent on Cyclin E and cyclin D1 but not on cyclin A and was physically associated with transcriptional factors: RB and E2F. In addition, treatment of G1/S Daudi cells with IFN-alpha also inhibited the ability of CDK2 enzyme to phosphorylate the RB protein in vitro. These results suggest possible involvement of cell cycle kinases in IFN-alpha action.
Burkitt Lymphoma↗
Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations.
The application of cationic liposome reagents has advanced DNA and mRNA transfection research in vitro, and data are accumulating which show their utility for in vivo gene transfer. However, chemical structure-activity data leading to a better mechanistic understanding of their biological activity is still limited. Most of the cationic lipid reagents in use today for this application are formulated as liposomes containing two lipid species, a cationic amphiphile and a neutral phospholipid, typically dioleoylphosphatidylethanolamine (DOPE). The studies reported here examine the effects of some systematic chemical structural changes in both of these lipid components. Cationic and neutral phospholipids were formulated together as large multilamellar vesicles (MLV) or small sonicated unilamellar vesicles (SUV) in water, and each formulation was assayed quantitatively in 96-well microtiter plates under 64 different assay conditions using COS.7 cells and an RSV-beta-galactosidase expression plasmid. The cationic lipid molecules used for these studies were derived from a novel series of 2,3-dialkyloxypropyl quaternary ammonium compounds containing a hydroxyalkyl moiety on the quaternary amine. A homologous series of dioleylalkyl (C18:1) compounds containing increasing hydroxyalkyl chain lengths on the quaternary amine were synthesized, formulated with 50 mol % DOPE, and assayed for transfection activity. The order of efficacy was ethyl > propyl > butyl > pentyl > 2,3-dioleyloxypropyl-1-trimethyl ammonium bromide (DOTMA). DOTMA, which is commercially available under the trademark Lipofectin Reagent, lacks a hydroxyalkyl moiety on the quaternary amine. A homologous series of hydroxyethyl quaternary ammonium derivatives with different alkyl chain substitutions were synthesized, formulated with 50 mol % DOPE, and assayed in the transfection assay. The order of transfection efficacy was dimyristyl (di-C14:0) > dioleyl (di-C18:1) > dipalmityl (di-C16:0) > disteryl (di-C18:0). The addition of 100 microM chloroquine in the transfection experiment enhanced the activity of the dioleyl compound by 4-fold and decreased the activity of the dimyristyl compound by 70%. For each of the compounds and formulations examined in this report, large multilamellar vesicles (MLV; diameter 300-700 nm) were more active than small unilamellar vesicles (SUV; diameter 50-100 nm). The neutral phospholipid requirements for transfection activity in COS.7 cells with these cationic lipid molecules were examined.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Differential expression of an E-selectin ligand (SLex) by two Chinese hamster ovary cell lines transfected with the same alpha (1,3)-fucosyltransferase gene (ELFT).
The mammalian cDNA encoding alpha (1,3)-fucosyltransferase (alpha (1,3)Fuc-T) termed ELAM-1 ligand fucosyltransferase (ELFT) or Fuc-TIV was previously cloned by three groups who reported different results from transfection studies Goelz et al. (Goelz, S. E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi-Rosso, G., and Lobb, R. (1990) Cell 63, 1349-1356) found that Chinese hamster ovary (CHO) cells expressing the ELFT cDNA had alpha (1,3)Fuc-T activity and were able to bind to E-selectin. In contrast, Lowe et al. (Lowe, J. B., Kukowska-Latallo, J. F., Nair, R. P., Larsen, R. D., Marks, R. M., Macher, B. A., Kelly, R. J., and Ernst, L. K. (1991) J. Biol. Chem. 266, 17467-17477) and Kumar et al. (Kumar, R., Potvin, B., Muller, W. A., and Stanley, P. (1991) J. Biol. Chem. 266, 21777-21783) found no binding to E-selectin of CHO transfectants expressing the same alpha (1,3)Fuc-T gene; nor did the latter transfectants synthesize a known E-selectin ligand, sialylated Lex (SLex), although they had substantial alpha (1,3)Fuc-T activity. We now show that these discrepant results were due to a difference between the parental CHO cell lines. Following transfection of ELFT cDNA into Pro-5 or dihydrofolate reductase (DHFR)- CHO cells, only the DHFR- transfectants expressed SLex and bound to E-selectin. Indirect evidence from monoclonal antibody and lectin binding studies indicates that the range of carbohydrate structures synthesized by the Pro-5 and DHFR- CHO cell lines differs. Since DHFR-/ELFT transfectants expressed cell surface SLex but transferred fucose poorly to sialylated substrates in vitro, ELFT may be able to fucosylate a complex carbohydrate missing from Pro-5 cells. Alternatively, either CHO line may have an activity (such as an alpha (2,3)-sialyltransferase), that modifies alpha (1,3)-fucosylated lactosamines.
Animals↗