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Biomedical subjects

A Kumar

Publications and source records attributed to A Kumar.

At least 253 records · Page 14Linked to original sources
PubMed2001

Cross-resistance to the synthetic retinoid CD437 in a paclitaxel-resistant human ovarian carcinoma cell line is independent of the overexpression of retinoic acid receptor-gamma.

Treatment of ovarian carcinomas with the antimitotic antitumor drug paclitaxel is highly efficacious. However, development of drug resistance presents a major obstacle. The common cellular phenotypes associated with paclitaxel resistance are an increased expression of the drug transport protein P-glycoprotein (P-gp), an alteration in the levels of beta-tubulin isotypes, and/or changes in the drug binding affinity of the microtubules. We established two paclitaxel-resistant human ovarian carcinoma cell lines. The 2008/17/4 cells exhibited a "classic" multidrug-resistant phenotype (overexpression of P-gp associated with cross-resistance to natural product drugs), whereas the 2008/13/4 cells were an atypical multidrug-resistant subline (no overexpression of P-gp). In addition to being paclitaxel resistant (250-fold), the 2008/13/4 cells were also cross-resistant to etoposide (39-fold) and vincristine (460-fold). To identify the alterations in the gene expression profile associated with the development of atypical paclitaxel resistance, we used the Clontech Atlas Human Cancer cDNA Microarray (spotted with 588 genes). The expression of retinoic acid receptor (RAR)-gamma was significantly higher in the paclitaxel-resistant (2008/13/4 and 2008/17/4) cells than in the parental (2008) cells. Northern blotting analysis demonstrated that the expression of RAR-gamma was 7-fold higher in the 2008/13/4 and 2008/17/4 cells than in the 2008 cells, whereas the expression of RAR-alpha and RAR-beta was not observed in any cell line. Whereas the 2008, 2008/13/4, and 2008/17/4 cells were found to resist the antiproliferative effects of all-trans-retinoic acid, the paclitaxel-resistant cells were 6- to 7-fold cross-resistant to the antiproliferative effects of CD437 (a synthetic RAR-gamma-selective agonist; 6-[-(1-admantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid) compared with the sensitivity of the parental cells. To further understand the association of paclitaxel and CD437 resistance with the observed RAR-gamma overexpression, we transfected the 2008 cells with a full-length RAR-gamma cDNA construct. Two transfectants with increased expression of the RAR-gamma mRNA and protein were isolated and subjected to growth inhibition assays in the presence of various concentrations of paclitaxel, etoposide, vincristine, and CD437. The sensitivity of the 2008 transfected clones (displaying increased expression of RAR-gamma) to the cytotoxic effects of paclitaxel, etoposide, vincristine, and CD437 was similar to that observed in the parental 2008 cells. These results suggest that the overexpression of RAR-gamma (observed in the 2008/13/4 and 2008/17/4 cells) by itself is not capable of inducing paclitaxel and CD437 resistance (or resistance to etoposide and vincristine).

Antineoplastic Agents↗
PubMed2001

Supravalvular aortic stenosis: genetic and molecular dissection of a complex mutation in the elastin gene.

We have identified two elastin gene (ELN) mutations located in cis in two related families with supravalvular aortic stenosis (SVAS). These mutations included an in-frame duplication in exon 18 (1034-1057dup) and a single base substitution in exon 26 (1829G-->A) predicted to result in the amino acid substitution R610Q. Haplotype analysis in one of the families identified an individual with a recombination between exon 18 and 26 of the elastin gene. This individual was unaffected and carried the exon 18 insertion mutation but not 1829G-->A. Skin fibroblasts were established from this recombinant normal individual and from an affected individual carrying both of the mutations. Reverse transcription/polymerase chain reaction (RT-PCR) analysis indicated that the expression of the mutant allele was reduced to 12%-27% of the normal allele in the affected but not in the unaffected individual. RNA-blot hybridization and immunoprecipitation experiments revealed reduced steady-state elastin mRNA levels and tropoelastin synthesis in the affected individual. RT-PCR analysis of the mRNA rescued by cycloheximide treatment indicated that mutation 1829G-->A created a cryptic donor splice site within exon 26, resulting in the deletion of four nucleotides at the 3'-end of exon 26 and a frameshift in the mRNA. This frameshift mutation generated a premature termination codon in the domain encoded by exon 28, clearly resulting in nonsense-mediated decay (NMD) of this frameshift RNA product. Despite considerable variability in the molecular nature of mutations responsible for SVAS, the unifying mechanism appears to be the generation of null alleles by NMD leading to elastin haploinsufficiency.

Alleles↗
PubMed2001

Magnetic resonance imaging permits in vivo monitoring of catheter-based vascular gene delivery.

BACKGROUND: Gene therapy is an exciting frontier in modern medicine. To date, most investigations about the imaging of gene therapy have primarily focused on noncardiovascular systems, and no in vivo imaging modalities are currently available for monitoring vascular gene therapy. The purpose of this study was to develop an in vivo imaging tool to monitor a catheter-based vascular gene delivery procedure. METHODS AND RESULTS: We produced gadolinium/blue dye and gadolinium/gene-vector media by mixing Magnevist with a trypan-blue or a lentiviral vector carrying a green fluorescent protein (GFP) gene. The gadolinium was used as an imaging marker for magnetic resonance (MR) imaging to visualize vessel wall enhancement, and the blue dye/GFP was used as a tissue stain marker for histology/immunohistochemistry to confirm the success of the transfer. Using Remedy gene delivery catheters, we transferred the gadolinium/blue dye (n=8) or gadolinium/GFP lentivirus (n=4) into the arteries of 12 pigs, that were monitored under high-resolution MR imaging. The results showed, in all 12 pigs, the gadolinium enhancement of the target vessel walls on MR imaging and the blue/GFP staining of the target vessel tissues with histology/immunohistochemistry. This study shows the potential of using MR imaging to dynamically visualize (1) where the gadolinium/genes are delivered; (2) how the target portion is marked; and (3) whether the gene transfer procedure causes complications. CONCLUSIONS: We present a technical development that uses high-resolution MR imaging as an in vivo imaging tool to monitor catheter-based vascular gene delivery.

Animals↗
PubMed2001

TFIIH inhibits CDK9 phosphorylation during human immunodeficiency virus type 1 transcription.

Tat stimulates human immunodeficiency virus, type 1 (HIV-1), transcription elongation by recruitment of the human transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to the TAR RNA structure. It has been demonstrated further that CDK9 phosphorylation is required for high affinity binding of Tat/P-TEFb to the TAR RNA structure and that the state of P-TEFb phosphorylation may regulate Tat transactivation. We now demonstrate that CDK9 phosphorylation is uniquely regulated in the HIV-1 preinitiation and elongation complexes. The presence of TFIIH in the HIV-1 preinitiation complex inhibits CDK9 phosphorylation. As TFIIH is released from the elongation complex between +14 and +36, CDK9 phosphorylation is observed. In contrast to the activity in the "soluble" complex, phosphorylation of CDK9 is increased by the presence of Tat in the transcription complexes. Consistent with these observations, we have demonstrated that purified TFIIH directly inhibits CDK9 autophosphorylation. By using recombinant TFIIH subcomplexes, our results suggest that the XPB subunit of TFIIH is responsible for this inhibition of CDK9 phosphorylation. Interestingly, our results further suggest that the phosphorylated form of CDK9 is the active kinase for RNA polymerase II carboxyl-terminal domain phosphorylation.

Blotting, Western↗
PubMed2001

A truncated precursor form of prostate-specific antigen is a more specific serum marker of prostate cancer.

Prostate-specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa) but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia, because both PCa and benign prostatic hyperplasia release PSA into the serum. We have identified previously a truncated form of precursor PSA (pPSA) in prostate tumor extracts consisting of PSA with a serine-arginine pro leader peptide ([-2]pPSA) instead of the normally expressed 7 amino acid pro leader peptide. In the current study we developed monoclonal antibodies to detect [-2]pPSA and other isoforms of pPSA for Western blot analysis. PSA was immunoaffinity purified from 100 to 200 ml of serum from each of five men with biopsy-proven cancer and three biopsy-negative men, all with total PSA levels in the diagnostically relevant range near 10 ng/ml. The truncated [-2]pPSA was estimated to range from 25 to 95% of the free PSA in the five PCa samples but only 6-19% of the free PSA in the biopsy-negative men. Immunohistochemical studies showed positive staining for [-2]pPSA in PCa epithelium and that [-2]pPSA was enriched in cancer cell secretions. In vitro activation studies showed that human kallikrein 2 and trypsin readily activated full-length pPSA but were unable to activate [-2]pPSA to mature PSA. Thus, [-2]pPSA, once formed, is a stable but inactive isoform of PSA. Truncated [-2]pPSA may represent an important new diagnostic marker for the early detection of PCa.

Amino Acid Sequence↗
PubMed2001

Antibodies that neutralize SIV(mac)251 in T lymphocytes cause interruption of the viral life cycle in macrophages by preventing nuclear import of viral DNA.

Previous reports from our lab had shown that sera obtained from SIV(mac)-infected animals neutralized SIV(mac) infectivity in CD4(+) T cells but failed to protect monkey primary macrophages from infection with the virus. However, the antibodies could inhibit completion of the viral life cycle in the macrophages at the postentry stage(s). In this report we examined the mechanisms of the late effect of the antibodies. Using monoclonal antibodies (MAbs), we demonstrated that only antibodies to the SIV envelope protein (KK17 and KK42) but not antibody to the viral core protein (FA2) had the same inhibitory effect as that of the anti-SIV sera. To identify the stage of the viral replication cycle that was inhibited by anti-SIV antibodies in macrophages, we used various PCR techniques to study viral entry/reverse transcription (by amplifying the viral gag gene), viral genome nuclear transport (by amplifying 2-LTR circular forms), viral integration (by Alu-PCR assay), and viral protein expression (by RIPA). We found that in macrophage cultures inoculated with SIV(mac)251 that were preincubated with antienvelope MAbs, viral DNA was detected at 8 h postinoculation but the 2-LTR circular forms and integrated viral DNAs were undetectable, and viral proteins were not expressed in these infected macrophages. These results strongly suggested that anti-SIV antibodies inhibited SIV(mac) replication in macrophages by blocking nuclear transport of viral genomes since viral DNA could not be detected in the nuclei of treated cultures. Furthermore, we showed that although viral replication in macrophages was interrupted by the antibodies, when cocultured with permissive T cells, the viral genomes presented in the cytoplasm of the macrophages could readily transfer to T cells during cell-cell contact. Importantly, this transfer could not be prevented by the antibodies. These results might explain the failure of passive antibody immunization against SIV(mac)251--a critical obstacle in AIDS vaccine development.

Active Transport, Cell Nucleus↗
PubMed2001

Examination of estradiol effects on the rapid estradiol mediated increase in hippocampal synaptic transmission in estrogen receptor alpha knockout mice.

Hippocampal slices from rats exhibit a rapid increase in basal synaptic transmission following 17 beta-estradiol (E(2)) application. In the current study we examined the role of the classic genomic receptor, estrogen receptor alpha (ER alpha), in mediating E(2) effects on synaptic transmission. E(2) (100 pM) increased the extracellular synaptic response in hippocampal slices from gonadectomized male and female mice lacking a functional ER alpha knockout (ER alpha KO) and wild-type (WT) littermates. No sexually dimorphic differences were observed, however, the increase in the field potential was more pronounced in WT mice. ER antagonists did not block E(2) mediated growth of the synaptic response in ER alpha KO mice. The results suggest that the rapid effect of E(2) on synaptic transmission is not mediated by ER alpha, however, ER alpha appears to modulate non-genomic influences on synaptic transmission.

Animals↗
PubMed2001

Recognition of ATGA sequences by the unfused aromatic dication DB293 forming stacked dimers in the DNA minor groove.

Furamidine and related diamidines represent a promising series of drugs active against widespread parasites, in particular the Pneumocystic carinii pathogen. In this series, the phenylfuranbenzimidazole diamidine derivative DB293 was recently identified as the first unfused aromatic dication capable of forming stacked dimers in the DNA minor groove of GC-containing sequences. Here we present a detailed biochemical and biophysical characterization of the DNA sequence recognition properties of DB293. Three complementary footprinting techniques using DNase I, Fe(II)-EDTA, and an anthraquinone photonuclease were employed to locate binding sites for DB293 in different DNA restriction fragments. Two categories of sites were identified by DNase I footprinting: (i) 4/5 bp sequences containing contiguous A.T pairs, such as 5'-AAAA and 5'-ATTA; and (ii) sequences including the motif 5'-ATGA.5'-TCAT. In particular, a 13-bp sequence including two contiguous ATGA motifs provided a highly preferential recognition site for DB293. Quantitative footprinting analysis revealed better occupancy of the 5'-ATGA site compared to the AT-rich sites. Preferential binding of DB293 to ATGA sites was also observed with other DNA fragments and was confirmed independently by means of hydroxyl radical footprinting generated by the Fe(II)-EDTA system, as well as by a photofootprinting approach using the probe anthraquinone-2-sulfonate (AQS). In addition, this photosensitive reagent revealed the presence of sites of enhanced cutting specific to DB293. This molecule, but not other minor groove binders such as netropsin, induces specific local structural changes in DNA near certain binding sites, as independently shown by DNase I and the AQS probe. Recognition of the ATGA sequence by DB293 was investigated further using melting temperature experiments and surface plasmon resonance (SPR). The use of different hairpin oligonucleotides showed that DB293 can interact with AT sites via the formation of 1:1 drug-DNA complexes but binds much more strongly, and cooperatively, to ATGA-containing sequences to form 2:1 drug-DNA complexes. DB293 binds strongly to ATGA sequences with no significant context dependence but is highly sensitive to the orientation of the target sequence. The formation of 2:1 DB293/DNA complexes is abolished by reversing the sequence 5'-ATGA-->3'-ATGA, indicating that directionality plays an important role in the drug-DNA recognition process. Similarly, a single mutation in the A[T-->G]GA sequence is very detrimental to the dimer interactions of DB293. From the complementary footprinting and SPR data, the 5'-ATGA sequence is identified as being a highly favored dimer binding site for DB293. The data provide clues for delineating a recognition code for diamidine-type minor groove binding agents, and ultimately to guide the rational design of gene regulatory molecules targeted to specific sites of the genetic material.

Antifungal Agents↗
PubMed2001

Electronic and magnetic structures of Sr(2)FeMoO(6).

We have investigated the electronic and magnetic structures of Sr(2)FeMoO(6) employing site-specific direct probes, namely x-ray absorption spectroscopy with linearly and circularly polarized photons. In contrast to some previous suggestions, the results clearly establish that Fe is in the formal trivalent state in this compound. With the help of circularly polarized light, it is unambiguously shown that the moment at the Mo sites is below the limit of detection (<0.25 mu(B)), resolving a previous controversy. We also show that the decrease of the observed moment in magnetization measurements from the theoretically expected value is driven by the presence of mis-site disorder between Fe and Mo sites.

Journal Article↗
PubMed2001

Novel dications with unfused aromatic systems: trithiophene and trifuran derivatives of furimidazoline.

We report the synthesis, interaction with DNA, topoisomerase II inhibition, and cytotoxicity of two novel unfused aromatic dications derived from the antimicrobial agent furimidazoline. The central diphenylfuran core of furimidazoline has been replaced with a trithiophene (DB358) or a trifuran (DB669) unit and the terminal imidazoline groups were preserved. The strength and mode of binding of the drugs to nucleic acids were investigated by complementary spectroscopic techniques including spectrophotometric, surface plasmon resonance, circular and linear dichroism measurements. The trifuran derivative forms intercalation complexes with double-stranded DNA, whereas the mode of binding of the trithiophene derivative varies depending on the drug/DNA ratio, as independently confirmed by NMR spectroscopic studies performed with (A-T)7 and (G-C)7 oligomers. Two-dimensional NMR data provided a molecular model for the binding of DB358 within the minor groove of the AATT sequence of the decanucleotide d(GCGAATTCGC)(2). DNase I footprinting experiments confirmed the sequence-dependent binding of DB358 to DNA. The trithiophene derivative interacts preferentially with AT-rich sequences at low concentrations, but can accomodate GC sites at higher concentrations. DNA relaxation assays revealed that DB358 stimulated DNA cleavage by topoisomerase II, in contrast to DB669. The substitution of N-alkylamidines for the imidazoline terminal groups abolished the capacity of the drug to poison topoisomerase II. At the cellular level, flow cytometry analysis indicated that DB358, which is about six times more cytotoxic than the trifuran analogue, induced a significant accumulation of HL-60 human leukemia cells in the G2/M phase. The incorporation of thiophene heterocycles appears as a convenient procedure to limit the strict AT selectivity of dications containing an extended unfused aromatic system and to design cytotoxic DNA intercalating agents acting as poisons for human topoisomerase II.

Anti-HIV Agents↗
PubMed2001

Differential effect of IL-4 and IL-13 on CD44 expression in the Burkitt's lymphoma B cell line BL30/B95-8 and in Epstein-Barr virus (EBV) transformed human B cells: loss of IL-13 receptors on Burkitt's lymphoma B cells.

IL-4 and IL-13, cytokines with similar biological effects may influence growth and progression of B-cell tumors through regulation of key cell surface molecules important in intercellular communications. In this study, we demonstrate that IL-4 and IL-13 exhibited differential effects on CD23 and CD44 expression and binding to hyaluronan in BL30/B95-8, a Burkitt's lymphoma (BL), and MK3.31, an Epstein-Barr virus transformed normal human B cell line (B-LCL). Studies conducted to understand the molecular mechanisms underlying this differential effect show that IL-4 induced phosphorylation of JAK1, JAK3, and STAT6 in BL30/B95-8 cells and of JAK3 and STAT6 in MK 3.31 cells. In contrast, IL-13 failed to induce the phosphorylation of JAK kinases or STAT6 proteins in these cell lines. The inability of BL30/B95-8 cells to respond to IL-13 was attributed to the loss of expression of IL-13R subunits alpha1 and alpha2, a finding confirmed for a number of other BL cell lines examined.

B-Lymphocytes↗
PubMed2001

Occurrence of Campylobacter jejuni in vegetables.

In order to understand the importance of vegetables in the transmission of thermophilic Campylobacter, 56 samples of different vegetables were screened. Out of these, 2 samples (1 spinach and 1 fenugreek) revealed the presence of Campylobacter jejuni biotype I. Both the isolates were enteropathogenic in rat ileal loop test.

Animals↗
PubMed2001

Stimulation of myelin gene expression in vitro and of sciatic nerve remyelination by interleukin-6 receptor-interleukin-6 chimera.

Induction of myelin gene expression denotes the last stage of differentiation of myelinating glial cells. Following peripheral nerve transection, Schwann cells (SC) lose myelin gene expression and proliferate, resembling premyelinating embryonic SC (eSC). We show that a fusion protein of the soluble interleukin-6 receptor to interleukin-6 (IL6RIL6), a potent activator of the gp130 signaling receptor, is an inducer of MBP and Po gene products in rat E18 embryonic dorsal root ganglia (DRG) 3 day cultures. Cells whose growth is dependent on the IL6RIL6 chimera were isolated from DRG. These cells (designated CH cells) express Krox-20, as do promyelinating and myelinating SC (mSC). IL6RIL6 induces Po and MBP in CH cells and their cocultures with neurons. In addition, IL6RIL6 leads to a disappearance of Pax-3, a marker of eSC and nonmyelinating Schwann cells (nmSC). Glial fibrillary acidic protein, present in nmSC, is not significantly induced by IL6RIL6. The CH cells acquire glial morphology when exposed to IL6RIL6 and cover axons in cocultures. In a sciatic nerve-derived SC line, IL6RIL6 also induces Po and triggers a rapid attachment along axons. In vivo administration of IL6RIL6 intraperitoneally to rats after sciatic nerve transection and resuture increases 4-fold the number of myelinated nerve fibers (MF) measured on day 12, 2.5-5 mm distal to the suture. The stimulation by IL6RIL6 treatment is highest (7.1-fold) at the more distant 5 mm site, and the thickness of myelin sheaths is increased. Compared to known SC growth factors, the gp130 activator IL6RIL6 appears to combine both in vitro mitogenic effects and promotion of myelin gene expression.

Animals↗
PubMed2001

Calcineurin links Ca2+ dysregulation with brain aging.

Brain aging is associated with altered Ca(2+) regulation. However, many Ca(2+) signal transduction mechanisms have not been explored in the aged brain. Here, we report that cytosolic expression and activity of the Ca(2+)-dependent protein phosphatase calcineurin (CaN) increases in the hippocampus during aging. CaN changes were paralleled by increased activation, but not expression, of CaN-regulated protein phosphatase 1 and a reduction in the phosphorylation state of CaN substrates involved in cell survival (i.e., Bcl-2-associated death protein and cAMP response element-binding protein). The age-related increase in CaN activity was not attributable to the inability of CaN to translocate to the membrane and was reduced by blocking L-type Ca(2+) channels. Finally, increased CaN activity correlated with memory function as measured with the Morris water escape task. The results suggest that altered regulation of CaN is one of the processes that could link Ca(2+) dyshomeostasis to age-related changes in neural function and cognition.

Aging↗
PubMed2001

Genomic organization and characterization of the promoter region of murine GSTM2 gene.

In this study, we have isolated the genomic clone of the murine GSTM2 gene and determined its sequence. Consistent with the class mu genefamily, the mGSTM2 gene consists of eight exons. The exon-intron boundaries and the distribution of coding sequences within the exons of the known GST class mu family members were found to follow a similar pattern suggesting that various members of this family have originated from a single primordial gene by duplication and the structure has been closely maintained through evolution. By primer extension, the start of transcription was determined to be 40 bp upstream of the initial AUG codon. To gain an understanding of the mGSTM2 regulation, we have also cloned and analyzed its promoter region. Maximal activity was observed in a 170 bp 5'-flanking region. The activity was decreased by 3-fold in a 402 bp 5'-flanking region suggesting the presence of repressor elements. While no TATA box was identified, the presence of an SP1 site at position -38 was noted. Deletion of this SP1 site completely abrogated promoter activity. The promoter contained eight putative Myb responsive elements and its transcriptional activity was upregulated by t-Myb but not by c-Myb.

Animals↗
PubMed2001

Extranasopharyngeal angiofibroma arising from the nasal septum.

Extra nasopharyngeal origin of angiofibroma is very rare. The nasal septum is a very rare site of extra nasopharyngeal angiofibroma with only two cases reported in the medical literature. We report here a case of a vascular mass arising from the nasal septum of an 8 year old boy. Histopathology confirmed it to be a case of angiofibroma. A review is also made of the other reported cases of angiofibroma arising from the nasal cavity. The likely theory of origin of the tumor and the management is also discussed.

Angiofibroma↗
PubMed2001

An unexpected extended conformation for the third TPR motif of the peroxin PEX5 from Trypanosoma brucei.

A number of helix-rich protein motifs are involved in a variety of critical protein-protein interactions in living cells. One of these is the tetratrico peptide repeat (TPR) motif that is involved, amongst others, in cell cycle regulation, chaperone function and post-translation modifications. So far, these helix-rich TPR motifs have always been observed to be a compact unit of two helices interacting with each other in antiparallel fashion. Here, we describe the structure of the first three TPR-motifs of the peroxin PEX5 from Trypanosoma brucei, the causative agent of sleeping sickness. Peroxins are proteins involved in peroxisome, glycosome and glyoxysome biogenesis. PEX5 is the receptor of the proteins targeted to these organelles by the "peroxisomal targeting signal-1", a C-terminal tripeptide called PTS-1. The first two of the three TPR-motifs of T. brucei PEX5 appear to adopt the canonical antiparallel helix hairpin structure. In contrast, the third TPR motif of PEX5 has a dramatically different conformation in our crystals: the two helices that were supposed to form a hairpin are folded into one single 44 A long continuous helix. Such a conformation has never been observed before for a TPR motif. This raises interesting questions including the potential functional importance of a "jack-knife" conformational change in TPR motifs.

Amino Acid Motifs↗