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E Santha

Publications and source records attributed to E Santha.

10 recordsLinked to original sources
PubMed2002

Alternative non-coding exons support serotonin transporter mRNA expression in the brain and gut.

A number of studies in recent years have linked polymorphisms within the serotonin transporter (5HTT) gene to affective disorders and anxiety traits. The human 5HTT mRNA is alternatively spliced, and the splice variants are equally expressed in the human placental cell line and dorsal raphe. In this study, using 5' rapid amplification of cDNA ends, we show that the rat 5HTT mRNA is alternatively spliced, leading to three distinct mRNAs differing in the 5' untranslated region. To determine whether the three alternatively spliced mRNA species that contain one of the following untranslated regions (i) exon 1A, 63 bp (ii) exon 1A + 1B, 125 bp or (iii) exon 1C, 101 bp, were expressed in a tissue-specific manner, we used RT-PCR and exon-specific oligonucleotide hybridization. Our results suggest two of the variants (1A + 1B and 1A) may utilize the same promoter; however, they are not equally expressed. While in the adult CNS and adrenal medulla, the shorter mRNA consisting of exon 1A was considerably more abundant, in the stomach and heart, the two variants were equally expressed. The third splice variant exon 1C is only expressed in the gut and to a lesser extent in the heart. The data from this study suggest the splice variant consisting of exon 1C may utilize a distinct promoter compared to the other two.

5' Untranslated Regions↗
PubMed2001

Hydrophilic, pro-drug analogues of T138067 are efficacious in controlling tumor growth in vivo and show a decreased ability to cross the blood brain barrier.

The novel anticancer compound T138067 is an irreversible inhibitor of tubulin polymerization. Amides 3-6 were synthesized using standard methodologies and determined to be significantly less lipophilic than T138067 based on logP calculations. Tubulin polymerization and [(3)H]-T138067 competition assays revealed that these amides are pro-drugs for parent aniline 2. Amides 3-5 showed no detectable signs of crossing the blood brain barrier, while amide 6 was found in extremely small amounts (12 ng/g of brain tissue). Aniline 2, which was formed in vivo from these amides, was found in significantly smaller amounts (approximately 20 to >5000 times) in the brain than when 2 was administered directly. The in vivo efficacy of amide 6 approached that of T138067 and was better tolerated when administered to athymic nude mice bearing MX-1 human mammary tumor xenografts.

Aniline Compounds↗
PubMed2001

Low temperature prevents potentiation of norepinephrine release by phenylephrine.

The effect of 1-phenylephrine (1-PE), an alpha(1)-receptor agonist, was investigated on the release of tritiated norepinephrine ([3H]NE). Pairs of guinea pig vasa deferentia were loaded with [3H]NE, superfused continuously, and stimulated electrically. 1-PE (10, 100 microM) enhanced the basal release of tritium in concentration-dependent manner. The stimulation-evoked release of radioactivity was significantly increased by 100 microM 1-PE. Both basal and stimulation-evoked release by 1-PE were reduced by desipramine (10 microM), a monoamine uptake inhibitor. The effect of 1-PE on basal release was independent on extracellular Ca(2+) concentration ([Ca(2+)](o)) and alpha(1)-adrenoceptor blockade. However, the 1-PE-induced release was temperature dependent: at low temperature 1-PE failed to increase either basal or stimulation-evoked release of NE. Using three different temperatures (7, 12, 17 degrees C, respectively), it was found that basal release was blocked at all three temperature values but the stimulation-evoked release was inhibited only at the lower values. The effect of 1-PE on the NE release appears to involve a desipramine-, and temperature-sensitive process. These results suggest that a non-receptorial and direct carrier-mediated mechanism is involved in NE releasing effect of 1-PE.

Adrenergic Uptake Inhibitors↗
PubMed1999

Selective, covalent modification of beta-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumors.

Microtubules are linear polymers of alpha- and beta-tubulin heterodimers and are the major constituents of mitotic spindles, which are essential for the separation of chromosomes during mitosis. Here we describe a synthetic compound, 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067), which covalently and selectively modifies the beta1, beta2, and beta4 isotypes of beta-tubulin at a conserved cysteine residue, thereby disrupting microtubule polymerization. Cells exposed to T138067 become altered in shape, indicating a collapse of the cytoskeleton, and show an increase in chromosomal ploidy. Subsequently, these cells undergo apoptosis. Furthermore, T138067 exhibits cytotoxicity against tumor cell lines that exhibit substantial resistance to vinblastine, paclitaxel, doxorubicin, and actinomycin D. T138067 is also equally efficacious in inhibiting the growth of sensitive and multidrug-resistant human tumor xenografts in athymic nude mice. These observations suggest that T138067 may be clinically useful for the treatment of multidrug-resistant tumors.

Animals↗
PubMed1998

Isolation and structural and genetic analysis of the mouse enkephalin gene and its d(AC/TG)n repeats.

Enkephalins, the endogenous opioids, mediate a wide variety of intercellular communications through ontogeny and their involvement has been suggested in drug addiction and alcohol abuse as well as in various neuropsychiatric disorders. In order to generate a genetic model, we have isolated the mouse enkephalin (mENK) gene, analyzed its regulatory region and compared its structure to the well characterized rat ENK (rENK) gene. We analyzed 2600 bp and found 3 highly homologous regions: The highest level (98%) of positional and sequence homology between mice and rats was in the TATA/proximal regulatory region. This region contains all the inducible regulatory elements (enkCRE1, NF1, AP-2, NFkappaB, etc.) and also an octamer-like element at -543 bp. This high homology is interrupted in both mice and rats by the typically polymorphic d(AC/TG)n and d(TC/GA)n dinucleotide repeats positioned between nucleotides -670 and -950. The position and orientation of these repetitive elements differ substantially in the two species. Genomic PCR analysis of the d(AC/TG)n repeat in various mouse strains, including aberrant behavioral or neurological phenotypes, showed lack of polymorphism at this repeat. The positional and sequence homologies between the rat and the mouse ENK genes decrease in more upstream regions due to the presence of nonhomologues repetititve DNA sequences.

Animals↗
PubMed1996

Differential mechanisms involved in the effect of nicotinic agonists DMPP and lobeline to release [3H]5-HT from rat hippocampal slices.

In the present study we investigated the effect of different nicotinic agonists (dimethylphenyl-piperazinium-iodide (DMPP), (-)nicotine, cytisine, (-)-lobeline, and (-)epibatidine) and antagonists (mecamylamine and dihydro-beta-erythroidine) on the release of [3H]5-HT from hippocampal slices. The nicotinic agonists DMPP and lobeline and electrical field stimulation, released [3H]5-HT from the hippocampus; other nicotinic agonists, such as (-)-nicotine, cytisine, and (-)-epibatidine had no effect. Unlike lobeline-induced release of [3H]5-HT, the effect of DMPP (10 and 40 microM) was antagonized by mecamylamine (20 and 10 microM). The effect of DMPP was [Ca2+]o-independent. In experiments carried out at 7 degrees C, i.e. the membrane carrier proteins are inhibited and the release by lobeline was abolished while the DMPP-induced release of 5-HT was rather potentiated. It is proposed that the effect of DMPP and lobeline, to enhance the release of [3H]5-HT from the hippocampus, was mediated by two different mechanisms. While DMPP-induced 5-HT release can be linked to a non-classical nAChR activation ([Ca2+]o-independence), the effect of lobeline was likely mediated by uptake carriers.

Alkaloids↗
PubMedDate not supplied

Protein-DNA interactions during phenotypic differentiation.

We have been studying the molecular mechanism of neuronal differentiation through which the multipotent precursor becomes limited to the final transmitter phenotype. Here we focused on the role of the 5' proximal regulatory cassette (-190; +53 bp) of the rat enkephalin (rENK) gene in the developmental regulation of the enkephalin phenotype. Several well characterized cis-elements, including AP2, CREB, NF1, and NFkB, reside on this region of the rENK gene. These motifs were sufficient to confer activity-dependent expression of the gene during neurodifferentiation when it was tested using transient transfection assays of primary developing spinal cord neurons treated with tetrodotoxin (TTX). This region was then used as a DNA probe in mobility shift assays, with nuclear proteins derived from phenotypically and ontogenetically distinct brain regions. Only a few low abundance protein-DNA complexes were detected and only with nuclear proteins derived from developing but not from adult brain. The spatiotemporal pattern of these complexes did not show correlation with enkephalin expression which was assessed by RT-PCR. We employed synthetic probes corresponding to consensus as well as ENK-specific sequences of the individual motifs to identify the nature of the observed bands. Although both consensus NF1 and enkCRE1(NF1) formed complexes with nuclear proteins derived from the striatum and cortex at various ages, the appearance of the bands was not correlated with ENK expression. Surprisingly, no complexes were detected if other ENK-specific motifs were used as probes. We also tested nuclear extracts derived from forskolin-induced and control C6 glioma cells, again using the whole proximal regulatory cassette as well as individual motifs. These experiments showed the formation of elaborate protein-DNA bands. There was no direct correlation between the appearance of bands and forskolin-induced ENK expression. Unexpectedly, all ENK-specific motifs formed specific and highly abundant protein-DNA complexes when nuclear extracts from the human tumor cell line (HeLa), which does not express ENK, were used. Based on these observations, we concluded that: 1. Interactions between the proximal regulatory cassette and additional probably far distant regions of the rENK gene and their binding proteins may be necessary to confer developmentally regulated, cell-specific expression of the ENK gene; and 2. Inducibility of the gene by common cis-elements can be governed by this region; however, the cell-specificity of the induction remains elusive.

Animals↗