ABSTRACT
RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human meti tRNA-derived pol III promoter > 100-fold by modifying the 3' terminus of the transcripts to hybridize to the 5' terminus. This terminal duplex includes the 8 nt leader sequence present in the primary wild-type meti tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed 'TRZ', was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.
Subject(s)
Promoter Regions, Genetic , RNA Polymerase III/genetics , RNA, Catalytic/metabolism , RNA, Messenger/metabolism , RNA, Transfer, Met/genetics , Base Sequence , Blotting, Northern , Cell Line , Gene Expression , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Plasmids , RNA Probes , RNA, Catalytic/genetics , RNA, Messenger/chemistry , Retroviridae/geneticsABSTRACT
Two of the major cell types in bone marrow stroma, macrophages and fibroblasts, have been shown to be important regulators of both myelopoiesis and lymphopoiesis. The enzymology relating to cell-specific metabolism of phenolic metabolites of benzene in isolated mouse bone marrow stromal cells was examined. Fibroblastoid stromal cells had elevated glutathione-S-transferase (4.5-fold) and DT-diaphorase (4-fold) activity relative to macrophages, whereas macrophages demonstrated increased UDP-glucuronosyltransferase (UDP-GT, 7.5-fold) and peroxidase activity relative to stromal fibroblasts. UDP-GT and glutathione-S-transferase activities in macrophages and fibroblasts, respectively, were significantly greater than those in unpurified white marrow. Aryl sulfotransferase activity could not be detected in either bone marrow-derived macrophages or fibroblasts, and there were no significant differences in GSH content between the two cell types. Because UDP-GT activity is high in macrophages, these data suggest that DT-diaphorase levels would be rate limiting in the detoxification of benzene-derived quinones in bone marrow macrophages. The peroxidase responsible for bioactivation of benzene-derived phenolic metabolites in bone marrow macrophages is unknown but has been suggested to be prostaglandin H synthase (PGS). Hydrogen peroxide, but not arachidonic acid, supported metabolism of hydroquinone to reactive species in bone marrow-derived macrophage lysates. These data do not support a major role for PGS in peroxidase-mediated bioactivation of hydroquinone in bone marrow-derived macrophages, although PGS mRNA could be detected in these cells. Similarly, hydrogen peroxide, but not arachidonic acid, supported metabolism of hydroquinone in a human bone marrow homogenate. Peroxidase-mediated interactions between phenolic metabolites of benzene occurred in bone marrow-derived macrophages. Bioactivation of hydroquinone to species that would bind to acid-insoluble cellular macromolecules was increased by phenol and was markedly stimulated by catechol. Bioactivation of catechol was also stimulated by phenol but was inhibited by hydroquinone. These data define the enzymology and the cell-specific metabolism of benzene metabolites in bone marrow stroma and demonstrate that interactions between phenolic metabolites may contribute to the toxicity of benzene in this critical bone marrow compartment.
Subject(s)
Benzene/pharmacokinetics , Bone Marrow/metabolism , Aged , Animals , Arylsulfatases/metabolism , Benzene/toxicity , Biotransformation , Bone Marrow/drug effects , Bone Marrow/enzymology , Bone Marrow Cells , Fibroblasts/enzymology , Fibroblasts/metabolism , Glucuronosyltransferase/metabolism , Glutathione/metabolism , Humans , Hydroquinones/pharmacokinetics , Inactivation, Metabolic , Macrophages/enzymology , Macrophages/metabolism , Male , Mice , NAD(P)H Dehydrogenase (Quinone)/metabolism , Polymerase Chain Reaction , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandin-Endoperoxide Synthases/metabolism , RNA, Messenger/metabolism , Stromal Cells/metabolismABSTRACT
The hemodynamic and metabolic effects of dietary induced hypomagnesemia were studied in two groups of 2-month-old male spontaneously hypertensive rats (SHR). All rats were given distilled water to drink ad libitum and were followed for 2 months. However, control rats (n = 12) were given a regular rat diet to eat, whereas the experimental (hypomagnesemic; HM) rats (n = 12) were given a magnesium-free diet. Metabolic and hemodynamic studies were done at the end of the 2-month observation period in the awake state. HM rats had higher mean arterial pressure, total peripheral resistance, renal vascular resistance, heart rate, UNaV, UKV and serum Na, and lower hematocrit, renal blood flow, serum K and serum Mg than the controls. No differences were observed among the two groups of rats with respect to weight, fluid intake, urine volume, serum calcium, blood urea nitrogen, cardiac index and glomerular filtration rate. In addition, HM rats had widespread myocardial and renal tissue calcification in contrast to controls, which did not show any tissue calcification. We conclude: (1) dietary-induced hypomagnesemia aggravated the hypertension of SHR; (2) it caused widespread tissue calcification; (3) the adverse effects of hypomagnesemia on arterial pressure were possibly produced through calcium-mediated systemic vasoconstriction and increase in peripheral vascular resistance.
