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R J Boado

Publications and source records attributed to R J Boado.

At least 37 records · Page 2Linked to original sources
PubMed1998

Ten nucleotide cis element in the 3'-untranslated region of the GLUT1 glucose transporter mRNA increases gene expression via mRNA stabilization.

The GLUT1 glucose transporter gene is regulated at the post-transcriptional level, and a 10 nucleotide (nt) cis-acting element located at nt 2181-2190 of the GLUT1 3'-untranslated region (3'-UTR) increases the transient expression of a luciferase reporter gene. To investigate the role of this mRNA cis-element, stable transfectants expressing luciferase reporter genes were established in rat C6 glioma cells. Insertion of nt 2100-2300 of GLUT1 3'-UTR resulted in a marked increase in the abundance of both reporter gene mRNA and protein compared to the control, in parallel with a 228% increase in the mRNA t1/2 determined with actinomycin D. Deletion of the 10 nt cis-acting element in the GLUT1 3'-UTR reduced the abundance of reporter gene products and the mRNA t1/2 to levels similar to the control clone. Data suggest that the cis-acting element located at nt 2181-2190 of bovine GLUT1 mRNA 3'-UTR is responsible for increased GLUT1 gene expression via enhanced GLUT1 mRNA stabilization.

Animals↗
PubMed1998

GLUT1 glucose transporter: differential gene transcription and mRNA binding to cytosolic and polysome proteins in brain and peripheral tissues.

The ratio of GLUT1 mRNA to actin mRNA in brain was 6- to 13- fold greater than the corresponding ratio in spleen, lung, testis, heart, and skeletal muscle in the rat. However, the ratio of GLUT1 transcription rate to actin transcription rate is comparable in brain and the five other organs. Organ extracts were fractionated into cytosol and polysomes, and ultraviolet light cross-linking studies were performed with these proteins and 32P-labeled GLUT1 mRNA containing the 3'-untranslated region (UTR) generated from transcription plasmids. The cytosol of brain, lung, spleen, C6 glioma cells in tissue culture, and freshly isolated bovine brain capillaries express a pair of 95 kDa and 74 kDa proteins, designated collectively as p88, and the polysome fraction of brain, testis, or C6 glioma cells express a 44-kDa protein, designated p44. In a middle cerebral artery occlusion model, brain cytosol p88 was up-regulated and p44 was down-regulated. These findings are consistent with the hypothesis that GLUT1 gene expression is subject to regulation at the post-transcriptional level.

Animals↗
PubMed1998

Molecular regulation of the blood-brain barrier GLUT1 glucose transporter by brain-derived factors.

Glucose is the crucial metabolic fluid for the brain, and the transport of this nutrient from blood to brain is limited by the blood-brain barrier (BBB) GLUT1 glucose transporter. The activity of this transporter is altered in different pathophysiological conditions including Alzheimer's disease. The expression of the BBB-GLUT1 gene is directed by brain trophic factors, and the brain-derived peptide preparation Cerebrolysin (Cl, EBEWE, Austria), used in the treatment of Alzheimer's disease, increases the BBB-GLUT1 mRNA stability and the expression of the BBB-GLUT1 gene. In the present investigation, Cl markedly increased (p < 0.001) the expression of a BBB-GLUT1 reporter gene, named clone 753, that contains an important regulatory cis-acting element involved in the stabilization of this transcript in brain endothelial cultured cells (ECL). In experiments with a reporter gene lacking this regulatory element, Cl produced only a minimal fraction of the effect observed with clone 753. UV-cross linking/PAGE experiments showed that the GLUT1 transcript reacts with ECL cytosolic proteins to form a RNA/protein complex of approximately 80 kDa. The abundance of this cis/trans acting complex was found to be increased in Cl-treated cells. Overall, data presented here demonstrate that i) Cl increases the expression of a BBB-GLUT1-luciferase reporter gene containing a region of the 3'-untranslated region of BBB-GLUT1 mRNA with important regulatory cis-acting elements involved in the stabilization of this transcript, and ii) the increased expression of this BBB-GLUT1 reporter gene was associated with augmented abundance of a transacting factor that binds to the cis-acting element described in (i), suggesting that this association may be involved in the stabilization of GLUT1 mRNA induced by Cl.

3' Untranslated Regions↗
PubMed1998

Drug delivery of antisense molecules to the brain for treatment of Alzheimer's disease and cerebral AIDS.

Antisense oligonucleotides (ODNs) and peptide nucleic acids (PNAs) are potential therapeutics for eradication of malignancies, viral infections, and other pathologies. However, ODNs and PNAs in general are unable to cross cellular membranes and blood-tissue barriers, such as the blood-brain barrier (BBB), which is only permeable to lipophilic molecules of molecular weight <600 Da. Cellular delivery systems based on conjugates of streptavidin (SA) and the OX26 monoclonal antibody directed to the transferrin receptor may be employed as a universal carrier for the transport of mono-biotinylated peptides, ODNs, or PNAs. 3'-Biotinylation of phosphodiester (PO)-ODN produces complete protection of ODN against serum and cellular 3'-exonucleases, facilitating the conjugation to avidin-based delivery systems and maintaining the activation of RNase H. These delivery systems markedly increased the cellular uptake and antisense efficacy of 3'-biotinylated ODNs in models of Alzheimer's disease and HIV-AIDS. In vivo brain delivery studies demonstrated that 3'-protected PO-ODNs and PO-phosphorothioate(PS)-ODN hybrids containing a single PO linkage are subjected to endonuclease degradation in vivo. On the contrary PS-ODNs, which were also protected at 3'-terminus by biotinylation, are metabolically stable in vivo and resistant to exo/endonuclease degradation. However, because of the strong binding of these oligomers to plasma protein, PS-ODNs are poorly transported into the brain through the BBB by the OX26-SA delivery vector following intravenous administration. PNAs are also resistant to exo/endonuclease and protease degradation, and these molecules biotinylated at the amino terminal group were transported into the brain by the OX26-SA delivery system with brain uptake levels comparable to that of morphine. Using the rev gene of HIV as a model target, RNase protection assays and cell-free translation arrest showed that the PNA-OX26-SA conjugate maintained active recognition and inactivation of target mRNA, respectively. The overall experimental evidence suggests that PNA-OX26-SA conjugates represent optimal antisense molecules for drug delivery to the brain.

AIDS Dementia Complex↗
PubMed1998

Up-regulation of blood-brain barrier short-form leptin receptor gene products in rats fed a high fat diet.

Leptin is a 16-kDa protein synthesized in adipose tissue that produces a satiety effect in the CNS. Leptin may gain access to the brain via receptor-mediated transport through the blood-brain barrier (BBB), and the BBB leptin receptor (OBR) may regulate the availability of circulating leptin to brain cells. The aim of the present study was twofold: first, to identify the OBR isoform expressed at the BBB, i.e., short, or "a," and long, or "b," form; and second, to compare the abundance of the BBB OBR mRNA and protein between control and high fat-fed rats. RT-PCR with isoform-specific primers showed that OBRa is the most abundant isoform at the BBB. BBB OBRa transcript content was markedly increased in high fat-fed rats compared with controls (11-fold), and no changes were observed in the expression of the internal standard control actin. The high fat feeding induction of OBR mRNA was correlated with an increase in the immunoreactive BBB OBR determined by immunocytochemistry using an all-isoform reactive antibody in high fat-fed obese rats. This investigation demonstrates (a) the OBRa is the principal leptin receptor expressed at the BBB and (b) this BBB OBR isoform is up-regulated by a high fat diet.

Animals↗
PubMed1997

The 5'-untranslated region of GLUT1 glucose transporter mRNA causes differential regulation of the translational rate in plant and animal systems.

The blood-brain barrier GLUT1 glucose transporter is under post-transcriptional regulation, and the 5'-untranslated region (5'-UTR) of the GLUT1 mRNA increases its translational rate in mammalian cells. To obtain more insight into the mechanism of translational control of GLUT1, the present investigation studied the translational efficiency of capped full-length synthetic human (h) and rabbit (rab) GLUT1 mRNA and both 5'- and 3'-UTR deleted hGLUT1 mRNAs in both mammalian and plant cell free translation systems. Translation efficiency of both h- and rabGLUT1 mRNA was increased 3- to 6-fold in rabbit retyculocyte lysate (RRL) compared with wheat germ extract (WGE). Confirming previous observations, deletion of 5'- and 5'/-3'-UTR markedly reduced the translation efficiency of the h-GLUT1 transcript in RRL. On the contrary, these deletions markedly increased the translation of GLUT1 in WGE. The present data provide additional evidence suggesting that the 5'-UTR of the GLUT1 mRNA contains cis-acting elements involved in the translational activation of the GLUT1 gene in mammalian cells and that factors involved in this cis/trans-acting interaction are either absent or down-regulated in plant systems.

Animals↗
PubMed1997

Site-directed deletion of a 10-nucleotide domain of the 3'-untranslated region of the GLUT1 glucose transporter mRNA eliminates cytosolic protein binding in human brain tumors and induction of reporter gene expression.

The posttranscriptional regulation of GLUT1 glucose transporter gene expression may be mediated by specific interactions between cytosolic trans-acting factors and regulatory cis-elements within the 3'-untranslated regions (UTRs) of the GLUT1 mRNA. Recent studies demonstrate that experimental and human brain tumors express an 80-kDa protein that reacts with a specific sequence around nucleotide 2,200 within the GLUT1 mRNA 3'-UTR. The 80-kDa protein is selectively expressed in hemangioblastoma, a tumor characterized by overexpression of GLUT1. The enhancer role of this GLUT1 3'-UTR cis-element was confirmed in the present studies using the luciferase expression vector pGL2 and site-directed deletion. Transfection of C6 glioma cells with pGL2 (containing nucleotides 2,100-2,300 of the bovine GLUT1 3'-UTR inserted at the Pfl MI site within the luciferase 3'-UTR) results in a fivefold increase in luciferase gene expression. Deletion of nucleotides 2,181-2,190 of the bovine GLUT1 3'-UTR, i.e., the putative binding site of the 80-kDa protein, completely eliminated the enhancement of luciferase activity in the transfected cells. Luciferase mRNA containing the putative cis-element inserted in the 3'-UTR was transcribed, and after UV crosslinking, this mRNA complexed with the 80-kDa protein in cytosol of either C6 cells or hemangioblastoma. In contrast, this complex was undetected with either luciferase control mRNA or 10 nucleotide-deleted RNA. The present study provides evidence that nucleotides 2,181-2,190 of the bovine GLUT1 mRNA 3'-UTR forms a complex with brain tumor cytosolic proteins that serves to increase GLUT1 gene expression at the posttranscriptional level.

Animals↗
PubMed1996

Brain-derived peptides increase the expression of a blood-brain barrier GLUT1 glucose transporter reporter gene.

The brain-derived peptide preparation Cerebrolysin (C1; EBEWE, Austria) increases the stability of blood-brain barrier (BBB)-GLUT1 transcript. To determine if the increase in BBB-GLUT1 mRNA stability is associated with an augmentation of gene expression, the present investigation studied the effect of C1 on the expression of a BBB-GLUT1-luciferase reporter gene in brain endothelial cultured (ECL) cells. Dose response studies showed that C1 markedly increased the expression of luciferase when the BBB-GLUT1-reporter gene was used. On the contrary, C1 produced no changes in the expression pattern of the control reporter gene, which lacks the GLUT1 regulatory sequence. Desensitization of the protein kinase C (PKC) receptor with the phorbol ester TPA, or inhibition with either 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) or staurosporine, had no effect on the increased levels of luciferase induced by C1. Transfection efficiency was determined by measuring intracellular levels of the expression vector using a quantitative polymerase chain reaction (PCR) assay. The data presented here demonstrate that C1 increases BBB-GLUT1 gene expression in ECL cells through a mechanism that appears to be independent of activation of PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Differential expression in glioblastoma multiforme and cerebral hemangioblastoma of cytoplasmic proteins that bind two different domains within the 3'-untranslated region of the human glucose transporter 1 (GLUT1) messenger RNA.

The glucose transporter 1 (GLUT1) protein is underexpressed in human glioblastoma multiforme and is overexpressed in human cerebral hemangioblastoma. To gain in-sight into possible posttranscriptional mechanisms regulating the expression of the GLUT1 protein in human brain tumors, cytosolic proteins were prepared from these two tumors and used in RNase T1 protection assays that employed [32P]human GLUT1 synthetic RNA prepared from transcription plasmids. Gel shift mobility assays and ultra-violet light cross-linking studies demonstrated the formation of specific RNA/protein complexes that migrated with a mol mass of 120, 44, and 41 kD. RNase T1 mapping and oligodeoxynucleotide competition studies showed that the 120 kD complex was comprised of an RNA fragment that localized to nucleotides 2186-2203 of the GLUT1 mRNA. The 44 kD complex contained an adenosine-uridine-rich RNA fragment that localized to nucleotides 1885-1906 of the human GLUT1 mRNA, and the formation of this complex was inhibited by synthetic RNA enriched in adenosine-uridine sequences. The 44 kD complex was selectively downregulated in hemangioblastoma as compared to glioblastoma multiforme. These studies demonstrate that human brain tumors have differential regulation of cytosolic proteins that specifically interact with two different domains in the 3'-untranslated region of the GLUT1 mRNA, which may serve to mediate the posttranscriptional regulation of GLUT1 gene expression in these tumors.

Base Sequence↗
PubMed1996

Cis-element/cytoplasmic protein interaction within the 3'-untranslated region of the GLUT1 glucose transporter mRNA.

The posttranscriptional regulation of glucose transporter GLUT1 gene expression may be mediated by specific interactions of cytosolic proteins and regulatory cis-elements within the untranslated regions (UTRs) of the GLUT1 mRNA. These putative cis/trans interactions were examined in the present studies with RNase T1 protection assays using 32P-labeled GLUT1 3'-UTR prepared from transcription plasmids and cytosolic proteins from C6 rat glioma cells. RNase T1 mapping studies localized a cis-element to nucleotides 2,170-2,207 on the bovine GLUT1 mRNA 3'-UTR. Ultraviolet cross-linking of RNA/protein complexes identified two complexes having molecular masses of 88 and 44 kDa. Competition studies with synthetic RNA and oligodeoxynucleotides showed the 88-kDa complex reacted with nucleotides 2,180-2,197 and that the 44-kDa complex reacted with sequences within nucleotides 1,717-2,132 of the bovine GLUT1 mRNA. The GLUT1 3'-UTR between nucleotides 2,100 and 2,300 was generated by polymerase chain reaction and subcloned at a unique Pfl/MI site within the 3'-UTR of a luciferase gene within the mammalian expression vector pGL2. Transfection of C6 rat glioma cells with the luciferase expression vector containing this portion of the GLUT1 3'-UTR resulted in a sixfold increase in luciferase gene expression in C6 cells. The identification of these cis/trans mechanisms provides support for the hypothesis that the posttranscriptional regulation of GLUT1 gene expression may be mediated by the interaction of specific cytosolic proteins with the GLUT1 mRNA 3'-UTR.

Animals↗
PubMed1996

Evidence for translational control elements within the 5'-untranslated region of GLUT1 glucose transporter mRNA.

Recent studies have indicated that the blood-brain barrier GLUT1 glucose transporter is under post-transcriptional regulation. To begin functional mapping of the GLUT1 transcript, in the present investigation we studied the translational efficiency of capped full-length synthetic GLUT1 mRNA, and both 5'- and 3'-untranslated regions (UTRs) deleted GLUT1 mRNAs. Deletion of 5'- and 5'-/3'-UTRs markedly reduced the translation efficiency of the human (h) GLUT1 transcript in the rabbit reticulocyte lysate (RRL), and this effect was not modified by addition of microsomes to the translation system. The putative role of these hGLUT1 5'-UTR cis-acting elements was studied using the luciferase expression vector pGL2. DNA corresponding to the hGLUT1 5'-UTR generated by PCR was subcloned at the HindIII site of the pGL2 located upstream of the luciferase 5'-UTR. Transfection of brain endothelial cultured cells with pGL2 containing most of the hGLUT1 5'-UTR (nucleotides 1-171) markedly increased the expression of luciferase, and disruption of luciferase-leading sequence with an unrelated 171-nucleotide fragment decreased its expression. Insertion of nucleotides 1-96 of the hGLUT1 5'-UTR retained most of the stimulatory effect, and nucleotides 123-171 produced 64% of maximal induction. On the contrary, clones containing nucleotides 79-171 and 154-171 of bGLUT1 5'-UTR had marginal effects on luciferase expression. The present data provide evidence suggesting that the 5'-UTR of the GLUT1 mRNA contains cis-acting elements involved in the translational control of the GLUT1 gene in mammalian cells.

Animals↗
PubMed1996

Pharmacokinetics and blood-brain barrier transport of [3H]-biotinylated phosphorothioate oligodeoxynucleotide conjugated to a vector-mediated drug delivery system.

Antisense phosphorothioate oligodeoxynucleotides (PS-ODNs) are potential neuropharmaceuticals should these agents be made transportable through the blood-brain barrier (BBB) in vivo. The present studies report on attempts to enhance brain uptake of systemically administered 3'-biotinylated PS-ODN (bio-PS-ODN) by conjugation to a complex of streptavidin (SA) and the OX26 monoclonal antibody to the rat transferrin receptor. This antibody undergoes receptor-mediated transcytosis through the BBB and the OX26/SA conjugate mediates BBB transport of biotinylated therapeutics. The brain uptake of unconjugated [3H]-bio-PS-ODN approximated that of [14C]sucrose, a plasma volume marker that is not significantly transported through the BBB. Conjugation of [3H]-bio-PS-ODN to the OX26/SA vector resulted in a marked increase in BBB transport and the permeability-surface area (PS) product of the conjugate was 4.0 microliters/min/g. However, when the bio-PS-ODN/OX26-SA conjugate was injected intravenously in anesthetized rats, the BBB PS product of the conjugate was reduced 23-fold to a value of 0.173 +/- 0.006 microliters/min/g. The marked inhibition of vector-mediated transport of the bio-PS-ODN after intravenous injection was due to avid plasma protein binding of PS-ODNs, as has been demonstrated with protein binding assays and internal carotid artery perfusion studies. In conclusion, although PS-ODNs have the advantage of increased metabolic stability and resistance to endonucleases in vivo, the BBB transport of antisense PS-ODN therapeutics conjugated to the brain drug delivery vector OX26/SA is markedly attenuated due to plasma protein-binding effects.

Animals↗
PubMed1995

Brain-derived peptides regulate the steady state levels and increase stability of the blood-brain barrier GLUT1 glucose transporter mRNA.

The blood-brain barrier (BBB) GLUT1 glucose transporter gene expression is known to be regulated by putative brain trophic factors. Therefore, the present study investigated the effect of a brain-derived peptide rich preparation containing a neurotrophic factor-like action [Cerebrolysin (Cl), EBEWE, Austria]. In cultures of brain capillary endothelial cells, Cl induced a transient increase in the abundance of BBB-GLUT1 relative to actin measured by reverse transcription-polymerase chain reaction during the first 2 h of incubation, whereas a significant reduction in the GLUT1 transcript was observed at 20 and 48 h. In addition, Cl abolished the fall in GLUT1 levels induced by actinomycin D. The present data suggest that brain-derived factors in Cl are able to modulate the expression of the BBB-GLUT1 gene increasing the BBB-GLUT1 transcript stability.

Amino Acids↗
PubMed1995

Vector-mediated delivery of a polyamide ("peptide") nucleic acid analogue through the blood-brain barrier in vivo.

Polyamide ("peptide") nucleic acids (PNAs) are molecules with antigene and antisense effects that may prove to be effective neuropharmaceuticals if these molecules are enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier in vivo. The model PNA used in the present studies is an 18-mer that is antisense to the rev gene of human immunodeficiency virus type 1 and is biotinylated at the amino terminus and iodinated at a tyrosine residue near the carboxyl terminus. The biotinylated PNA was linked to a conjugate of streptavidin (SA) and the OX26 murine monoclonal antibody to the rat transferrin receptor. The blood-brain barrier is endowed with high transferrin receptor concentrations, enabling the OX26-SA conjugate to deliver the biotinylated PNA to the brain. Although the brain uptake of the free PNA was negligible following intravenous administration, the brain uptake of the PNA was increased at least 28-fold when the PNA was bound to the OX26-SA vector. The brain uptake of the PNA bound to the OX26-SA vector was 0.1% of the injected dose per gram of brain at 60 min after an intravenous injection, approximating the brain uptake of intravenously injected morphine. The PNA bound to the OX26-SA vector retained the ability to bind to synthetic rev mRNA as shown by RNase protection assays. In summary, the present studies show that while the transport of PNAs across the blood-brain barrier is negligible, delivery of these potential neuropharmaceutical drugs to the brain may be achieved by coupling them to vector-mediated peptide-drug delivery systems.

Animals↗
PubMed1995

Upregulation of blood-brain barrier GLUT1 glucose transporter protein and mRNA in experimental chronic hypoglycemia.

An in vivo model of chronic hypoglycemia was used to investigate changes in blood-brain barrier (BBB) glucose transport activity and changes in the expression of GLUT1 mRNA and protein in brain microvasculature occurring as an adaptive response to low circulating glucose levels. Chronic hypoglycemia was induced in rats by constant infusion of insulin via osmotic minipumps; control animals received infusions of saline. The criterion for chronic hypoglycemia was an average blood glucose concentration of < 2.3 mmol/l (42 mg/dl) after 5 days. The average blood glucose concentration at the end of the experimental period in the rats selected for study was 2.0 +/- 0.1 mmol/l (36 +/- 1 mg/dl) vs. 4.9 +/- 0.1 mmol/l (88 +/- 1 mg/dl) in the controls. Internal carotid artery perfusion studies demonstrated an increase in the BBB permeability-surface area (PS) product of 40% (P < 0.0005) in the chronically hypoglycemic animals as compared with controls. Western blotting of solubilized isolated brain capillaries demonstrated a 51% increase (P < 0.05) in immunoreactive BBB GLUT1 in the chronically hypoglycemic rats, and Northern blotting of whole-brain poly(A+) mRNA revealed a 50% increase in the GLUT1-to-actin ratio in the insulin-treated group (P < 0.05). Northern blotting analysis of microvessel-depleted total brain poly(A+) showed that the increase in GLUT1 mRNA in the chronically hypoglycemic rats was restricted to the BBB. The present study demonstrates increased expression of GLUT1 mRNA and protein at the BBB in chronic hypoglycemia and suggests that this increase is responsible for the compensatory increase in BBB glucose transport activity that occurs with chronically low circulating blood glucose levels.

Animals↗
PubMed1995

Pharmacokinetics and organ clearance of a 3'-biotinylated, internally [32P]-labeled phosphodiester oligodeoxynucleotide coupled to a neutral avidin/monoclonal antibody conjugate.

The pharmacokinetics and organ uptake of a 3'-biotinylated, [32P] internally labeled 36-mer phosphodiester oligodeoxynucleotide (PO-ODN) were measured after intravenous injection in the anesthetized adult rat. The PO-ODN was antisense to the tat gene of the human immunodeficiency virus, and was 3'-biotinylated to a) protect against serum and tissue 3'-exonuclease activity, and b) facilitate coupling to a neutral avidin-based transcellular drug delivery vector. The latter was comprised of a covalent conjugate of neutral avidin (NLA) and the OX26 murine monoclonal antibody to the rat transferrin receptor. The PO-ODN was internally labeled at the 21-nucleotide position to prevent rapid hydrolysis [32P] label by serum and tissue 5'-phosphatases. The uptake of the 3'-bio-[32P21]PO-ODN by brain, heart, kidney, lung, and liver was measured. The studies show that the unconjugated 3'-bio-[32P21]PO-ODN was rapidly removed from plasma, with a mean residence time of 22 +/- 1 min and a systemic clearance of 9.2 +/- 0.5 ml/min/kg. Large amounts of [32P] radioactivity were recovered in the urine following the injection of the PO-ODN, and when this fraction was included in the calculation of the renal clearance parameter, the renal clearance was 20-fold higher, indicating the principal site of organ clearance of the unconjugated PO-ODN was the kidney. Conjugation of the 3'-bio-PO-ODN to the NLA-OX26 vector reduced the systemic clearance 50%, owing to a > 10-fold reduction in renal clearance. Following conjugation of the 3'-bio-PO-ODN to the NLA-OX26 vector, the major clearance organ was the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗