Browsing by Subject "Chromatography"
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Item Analysis of sodium pump gene expression in microdissected nephrons using competitive RT-PCR and a novel HPLC technique(Texas Tech University, 1996-12) Hayward-Lester, Amanda LHypertension is determined by the interaction of multiple genetic and environmental factors, making it difficult to elucidate any single genetic determinant. Biochemical markers such as intraerythrocytic sodium concentration, erythrocytic ouabain binding-site density and passive sodium leak indicate that abnormal membrane cation flux segregates with some hypertension subtypes. The sodium pump, Na+, K+ ATP-ase (NKA) may therefore participate in the development of hypertension. It is a multi-subunit cell membrane protein which translocates sodium and potassium ions with hydrolysis of ATP. It is inhibited by ouabain and regulated by phosphorylation. The alpha subunit (of which there are four isoforms encoded by different genes) is currently ascribed all catalytic function, while the beta and gamma subunits may have regulatory roles. We examined NBCA alpha and gamma subunit gene expression in spontaneously hypertensive rats (SHR) and Wistar Kyoto controls (WKY). Both prehypertensive and adult SHR exhibit abnormal renal sodium retention. Solution hybridization studies in adult SHR revealed a decrease in alpha 1 expression in kidney. Kidney is a heterogeneous tissue whose functional unit, the nephron, may be divided into 12 distinct segments. To examine NKA expression in individual segments requires an assay allowing quantitation of NKA alpha and gamma isoform RNA in microdissected tissue samples. We combined competitive RT-PCR with a novel ion-paired reversed phase HPLC to produce rapid, accurate and precise measurement of gene expression in a single-tube assay. The ability of HPLC to resolve heteroduplex molecules formed between native and competitor products proved essential. Assay validation confirmed absolute quantification is possible if competitors have identical reverse-transcription efficiency to the native RNA. We used the assay to examine qualitative and quantitative expression of NKA subunits in normotensive Sprague-Dawley, prehypertensive and adult SHR and WKY. Qualitative analysis revealed alpha 1 and gamma expression in all segments examined. Expression of the other alpha isoforms was not detected. Quantitative analysis in the prehypertensive SHR revealed that a selective alteration in alpha 1 expression in proximal convoluted tubule may explain the results obtained in whole kidney and suggest an attempted feedback response by SHR to reduce sodium reabsorption.Item Isolation and effects of citrus limonoids on cytochrome p450 inhibition, apoptotic induction and cytotoxicity on human cancer cells.(Texas A&M University, 2007-04-25) Poulose, Shibu M.This dissertation illustrates an efficient purification method for citrus limonoids and flavonoids, while examining their effects on cytochrome P450 inhibition and apoptotic induction on human neuroblastoma (SH-SY5Y) and colonic adenocarcinoma (Caco-2) cells. The first study developed a bulk purification method for limonoids, from seeds and molasses of citrus fruits, using a combination of chromatographic techniques. This also resulted in an efficient purification method for naringin and hesperidin from citrus byproducts. The second study investigated the inhibitory effects of purified limonoids and flavonoids on the enzymatic activities of different isoforms of human cytochrome P450. O-Dealkylase and hydroxylase activities of CYP1A2, CYP1B1, CYP3A4 and CYP19, using specific substrates such as ethoxyresorufin (ethoxyresorufin O-dealkylase, EROD), methoxyresorufin (methoxyresorufin O-dealkylase, MROD), and dibenzylfluorescein (DBF), were found to be significantly (P < 0.001) reduced at micromolar levels. A kinetic analysis showed competitive and non-competitive modes of inhibition by limonoids, on CYP19 hydroxylase activity. The results corroborate the active role of limonoids in the redox cycling mechanisms. The third study examined the antioxidant and apoptotic inducing ability of limonoid glucosides on human neuroblastoma cells. Four limonoid glucosides, LG (17beta-D glucopyranoside limonin), OG (obacunone 17beta-D glucopyranoside), NAG (nomilinic acid 17beta-D glucopyranoside), and DNAG (deacetylnomilinic acid 17beta-D glucopyranoside), have shown superoxide scavenging at millimolar levels. Micromolar amounts of LG and OG induced rapid necrosis of SH-SY5Y cells. Cytotoxicity was correlated (P = 0.046) to a concentration and timedependent increase in caspase 3/7 activity. Analyses of DNA content during the S phase of the cell cycle indicated reductions of 86.6% for LG and 82.3% for OG as compared to untreated. The results validate the antineoplastic distinctiveness of limonoid glucosides. In the fourth study, cytotoxic effects of limonoid aglycones and glucosides were assessed on human SH-SY5Y neuroblastoma and colon carcinoma (CaCo-2) cell lines and compared with the non-cancerous Chinese hamster ovary (CHO) cells. Significant (P < 0.001) cytotoxic effects were observed only on cancerous cells, over 24 to 36 h. The study revealed a marked increase in the DNA content of aneuploidic cells, which results in cell cycle arrest. The results confirm that glycosides are the most active apoptotic inducing form.Item Profiling the natural products from the cancer-plant Sutherlandia frutescens reveal disrupted sterol homeostasis(2013-08) Thomas, Crista D.; Nes, William David; Shaw, Robert W.From the non-saponifiable fraction of the “cancer bush”- Sutherlandia frutescens was isolated 12 sterols and a pentacyclice triterpenoid mixture of a- and b-amyrin. These natural products derived from the isoprenoid biosynthesis pathway were characterized by a combination of chromatographic and spectral methods. The major sterols of physiological significance to accumulate in stems and leaves at 1% dry weight of original sample were cycloartenol and sitosterol in a ratio of approximately 1 to 1. The notably high accumulation of cycloartenol in this plant is very uncommon but nonetheless is at a level which coincides with the high level of the cancer preventing cycloartane glycosides known as sutherlandiosides. We surmise that developmental of regulation of phytosterol biosynthesis has occurred to block cycloartenol conversion to sitosterol affording redirection of cycloartenol into the glycoside derivatives.