The association between UGT1A1 polymorphisms and treatment toxicities of liposomal irinotecan.

BACKGROUND: Initial dose adjustment is recommended for patients with known UGT1A1∗28 homozygosity for both conventional irinotecan and liposomal irinotecan (nal-IRI). A recent population pharmacokinetic (PK) study showed that Asian patients had a lower prevalence of UGT1A1∗28 homozygosity but a significantly higher maximum blood concentration of SN-38 (SN-38 Cmax) and a higher incidence of grade ≥3 neutropenia after nal-IRI administration than Caucasian patients. The current study investigated the association of UGT1A1 polymorphisms, including the Asian prevalent UGT1A1∗6, PK and toxicities of nal-IRI-based therapy in the Asian population. PATIENTS AND METHODS: A total of 162 patients with nal-IRI-based therapy and available UGT1A1∗6 and UGT1A1∗28 genotyping were included, with 82 Asian patients from six previous phase I or II studies of nal-IRI (cohort 1) and another 80 patients with nal-IRI + 5-fluorouracil/leucovorin every 2 weeks as real-world practice in a single institute in Taiwan (cohort 2). RESULTS: The frequency of UGT1A1∗6 or UGT1A1∗28 homozygosity/compound heterozygosity was 9.3%, with UGT1A1∗6/∗6 in 2.5%, UGT1A1∗28/∗28 in 1.9% and UGT1A1∗6/∗28 in 4.9%. Among the 53 patients in cohort 1 with available PK data, all 7 patients with homozygosity/compound heterozygosity harbored UGT1A1∗6 and had a significantly higher level of median dose-normalized area under the concentration-time curve (AUC) and Cmax of SN-38 than those with single heterozygosity/wild type. Of the entire study population, the incidence of grade ≥3 neutropenia and diarrhea was significantly higher in patients with homozygosity/compound heterozygosity than in those with single heterozygosity/wild type, 73.3% versus 38.1% (P = 0.012, Fisher's exact test) and 33.3% versus 9.5% (P = 0.018, Fisher's exact test), respectively. CONCLUSION: The results suggest that the recommendation of a lower starting dose of nal-IRI for patients with UGT1A1∗28 homozygosity should be extended to include patients with UGT1A1∗6 homozygosity/compound heterozygosity.

FeSe quantum dots for in vivo multiphoton biomedical imaging.

An immense demand in biomedical imaging is to develop efficient photoluminescent probes with high biocompatibility and quantum yield, as well as multiphoton absorption performance to improve penetration depth and spatial resolution. Here, iron selenide (FeSe) quantum dots (QDs) are reported to meet these criteria. The synthesized QDs exhibit two- and three-photon excitation property at 800- and 1080-nm wavelengths and high quantum yield (ca. 40%), which are suitable for second-window imaging. To verify their biosuitability, poly(ethylene glycol)-conjugated QDs were linked with human epidermal growth factor receptor 2 (HER2) antibodies for in vitro/in vivo two-photon imaging in HER2-overexpressed MCF7 cells and a xenograft breast tumor model in mice. Imaging was successfully carried out at a depth of up to 500 µm from the skin using a nonlinear femtosecond laser at an excitation wavelength of 800 nm. These findings may open up a way to apply biocompatible FeSe QDs to multiphoton cancer imaging.

Investigation of multifilament MgB2 superconducting joint technique for development of MRI magnets.

This study presents the investigation of superconducting joints fabricated using multifilament magnesium diboride (MgB2) wires for the development of persistent-current mode magnetic resonance imaging (MRI) magnets. The critical current of the jointed samples decreased with increasing cutting angle because the smaller cutting angle allowed greater exposure of the MgB2 filament, thereby increasing the contact area for the wire-bulk-wire connection. In addition, an appropriate pressing pressure (300 MPa) was necessary to establish the multifilament MgB2 joint without significant degradation of superconducting properties. The resistance of the optimal MgB2 joint, measured using the field-decay technique, was <1.5 × 10-14 Ω. Therefore, the proposed joint technique can be employed for developing multifilament MgB2 MRI magnets operating in the persistent-current mode.

Note: Progress on the use of MgB2 superconducting joint technique for the development of MgB2 magnets for magnetic resonance imaging (MRI).

This note presents a superconducting joint technique for the development of MgB2 magnetic resonance imaging (MRI) magnets. The MgB2 superconducting joint was fabricated by a powder processing method using Mg and B powders to establish a wire-bulk-wire connection. The joint resistance measured using a field-decay method was <10-14 Ω, demonstrating that the proposed joint technique could be employed for developing "next-generation" MgB2 MRI magnets operating in the persistent current mode.

Organ donation protocols.

Organ transplantation improves survival and quality of life in patients with end-organ failure. Waiting lists continue to grow across the world despite remarkable advances in the transplantation process, from the creation of public engagement campaigns to the development of critical pathways for the timely identification, referral, approach, and treatment of the potential organ donor. The pathophysiology of dying triggers systemic changes that are intimately related to organ viability. The intensive care management of the potential organ donor optimizes organ function and improves the donation yield, representing a significant step in reducing the mismatch between organ supply and demand. Different beliefs and cultures reflect diverse legislations and donation practices amongst different countries, creating a challenge to standardized practices. Maintaining public trust is necessary for continued progress in organ donation and transplantation, hence the urge for a joint effort in creating uniform protocols that ensure transparent practices within the medical community.

DEPDC5 mutations in familial and sporadic focal epilepsy.

BACKGROUND AND AIMS: Mutations in the disheveled, Egl-10 and pleckstrin domain-containing protein 5 (DEPDC5) gene have emerged as an important cause of various familial focal epilepsy syndromes. However, the significance of DEPDC5 mutations in patients with sporadic focal epilepsy has yet to be characterized. MATERIALS AND METHODS: We studied a kindred of familial focal epilepsy with variable foci using whole-exome sequencing. We subsequently studied a cohort of 293 patients with focal epilepsy and sequenced all exons of DEPDC5 using targeted resequencing. RESULTS: We reported a Taiwanese family with a novel splice site mutation which affected mRNA splicing and activated the downstream mammalian target of rapamycin (mTOR) pathway. Among patients with focal epilepsies, the majority (220/293) of these patients had sporadic focal epilepsy without malformation of cortical development. Two (0.9%) of these patients had probably pathogenic mutations in the DEPDC5 gene. DISCUSSION AND CONCLUSIONS: Our finding suggests that DEPDC5 is not only the most common gene for familial focal epilepsy but also could be a significant gene for sporadic focal epilepsy. Since focal epilepsies account for more than 60% of all epilepsies, the effect of mTORC1 inhibitor on patients with focal epilepsy due to DEPDC5 mutations will be an important future direction of research.

Loss of presenilin 2 is associated with increased iPLA2 activity and lung tumor development.

Presenilins are the enzymatic components of γ-secretase complex that cleaves amyloid precursor protein, Notch and ß-catenin, which has critical roles in the development of Alzheimer's disease and cancer cell growth. Therefore, in the present study, we studied the effects and mechanisms of PS2 knockout on lung cancer development and possible mechanisms as a key regulator of lung tumor development. We compared carcinogen-induced tumor growth between PS2 knockout mice and wild-type mice. PS2 knockout mice showed increased urethane (1 mg/g)-induced lung tumor incidence when compared with that of wild-type mice with decreased activity of γ-secretase in the lung tumor tissues. Consequently, iPLA2 activities in lung tumor tissues of PS2 knockout mice were much higher than in tumor tissues of wild-type mice. Furthermore, knockdown of PS2 using PS2 siRNA decreased γ-secretase activity with increased iPLA2 activity in the lung cancer cells (A549 and NCI-H460), leading to increased lung cancer cell growth. PS2 knockout mice and PS2 knockdown lung cancer cells showed increased DNA-binding activities of nuclear factor kappa-beta, signal transducer and activator of transcription 3 (STAT3) and AP-1 which are critical transcriptional factors of iPLA2 than those of PS2 wild-type mice and control lung cancer cells. Taken together, these results suggest that the loss of PS2 could have a critical role in lung tumor development through the upregulation of iPLA2 activity by reducing γ-secretase.

Effects of stromal cell-derived factor 1α delivered at different phases of transient focal ischemia in rats.

Endogenous stromal cell-derived factor 1α (SDF1α) has been implicated in postischemic tissue repair, suggesting SDF1α as a potential therapeutic molecule to treat stroke patients. In spite of its potential, no data are available regarding the short- and long-term effects of SDF1α when it is delivered at different phases of stroke. In our study, adenovirus expressing SDF1α gene (AV-SDF1α) was introduced into the boundary of the infarcted area either 3 days before or 1 week after ischemia, and behavioral performance was measured over 5 weeks. Immediate behavioral and structural amelioration was evident when AV-SDF1α was injected 3 days before ischemia, which might be the result of SDF1α-mediated neuroprotection as supported by the TUNEL staining and Western blot analysis of active caspase-3. In addition, increase in neurogenesis, neuroblast migration, and neural differentiation was also apparent in the AV-SDF1α-injected brain, which contributed to further amelioration at later time points ("delayed response"). On the contrary, when AV-SDF1α was introduced 1 week post-ischemia (in the subacute phase), significant behavioral recovery became apparent beginning 5 weeks after viral delivery. Taken together, the therapeutic efficacy of SDF1α varied considerably depending on when SDF1α overexpression was initiated; initiating SDF1α overexpression before ischemia exerted both immediate and delayed beneficial effects, whereas initiating overexpression in the subacute phase exerted only a delayed response.

Pitx3-deficient aphakia mice display unique behavioral responses to psychostimulant and antipsychotic drugs.

The dorsal (A9) and ventral striatum (A10) of the midbrain mediate many of the effects of psychoactive drugs that alter emotion, cognition, and motor activity within the contexts of therapy or abuse. Although transgenic and knockout technologies have enabled development of genetic models to dissect contributions of specific dopamine (DA) receptor subtypes to psychoactive drug effects, few models exist that can distinguish contributions of A9 versus A10 circuits. Pitx3 is a transcription factor enriched in DA neurons. Aphakia (ak) mice deficient in Pitx3 show selective loss of nigrostriatal DA, while other DA pathways are relatively spared, and therefore could be a useful tool for investigating the role of this subclass of DA projections. We investigated the effects of stimulants amphetamine, apomorphine, and MK-801 and the antipsychotic drug haloperidol on behavior in ak mice. Whereas wild-type mice showed the characteristic locomotor hyperactivity in response to amphetamine (5 mg/kg) and apomorphine (4 mg/kg), these drugs caused a paradoxical suppression of locomotor hyperactivity in ak mice. MK-801 (0.2 mg/kg) induced hyperactivity was maintained in both wt and ak mice. Additionally, mutant but not wild-type mice were insensitive to the cataleptic effects of haloperidol (1 mg/kg). These studies indicate that the nigrostriatal DA circuit plays a critical role in maintaining normal responsiveness to psychotropic drugs that either stimulate or block DA neurotransmission. We propose that ak mice may represent a valuable genetic model not only to study Parkinson's disease, but also to dissect the pathophysiologic and pharmacotherapuetic mechanisms of other DA-mediated disorders such as attention-deficit hyperactivity disorder, drug abuse and schizophrenia.

Glucocorticoid receptor represses the Dex-mediated induction of human androgen response element-linked Luc activity.

A human androgen response element (hARE), identified within intron 8 of the human sterol regulatory element-binding protein cleavage-activating protein, interacts with both glucocorticoid receptor (GR) and androgen receptors (AR). The aim of this study was to test the hypothesis that human GR (hGR) might modulate the expression of a hARE-linked reporter gene by dexamethasone (Dex). The hypothesis was tested by: a) co-transfecting HepG2 cells with a hGR and a luciferase (Luc)-reporter gene for performing in vitro investigations and b) by their co-injection into the tail vein of mice for in vivo investigation. In vitro co-transfected cells and the in vivo co-injected mice were then treated with Dex. Our results have led us to concluded that both transfection and injection of the hGR leads to a repression in the Dex-mediated induction of hARE-linked Luc activity both in vitro and in vivo settings. These findings suggest that this assay system allows screening of drug candidates affecting to a signal transduction pathway of the GR and AR and may help in the future discovery and analysis of novel and selection of GR and AR agonists.

Tau overexpression in transgenic mice induces glycogen synthase kinase 3beta and beta-catenin phosphorylation.

The abnormal phosphorylations of tau, GSK3beta, and beta-catenin have been shown to perform a crucial function in the neuropathology of Alzheimer's disease (AD). The primary objective of the current study was to determine the manner in which overexpressed htau23 interacts and regulates the behavior and phosphorylation characteristics of tau, GSK3beta, and beta-catenin. In order to accomplish this, transgenic mice expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE/htau23) were created. Transgenic mice evidenced the following: (i) tendency toward memory impairments at later stages, (ii) dramatic overexpression of the tau transgene, coupled with increased tau phosphorylation and paired helical filaments (PHFs), (iii) high levels of GSK3beta phosphorylation with advanced age, resulting in increases in the phosphorylations of tau and beta-catenin, (iv) an inhibitory effect of lithium on the phosphorylations of tau, GSK3beta, and beta-catenin, but not in the non-transgenic littermate group. Therefore, the overexpression of NSE/htau23 in the brains of transgenic mice induces abnormal phosphorylations of tau, GSK3beta, and beta-catenin, which are ultimately linked to neuronal degeneration in cases of AD. These transgenic mice are expected to prove useful for the development of new drugs for the treatment of AD.

The homeodomain transcription factor Pitx3 facilitates differentiation of mouse embryonic stem cells into AHD2-expressing dopaminergic neurons.

The A9 dopaminergic (DA) neuronal group projecting to the dorsal striatum is the most vulnerable in Parkinson's disease (PD). We genetically engineered mouse embryonic stem (ES) cells to express the transcription factors Nurr1 or Pitx3. After in vitro differentiation of Pitx3-expressing ES cells, the proportion of DA neurons expressing aldehyde dehydrogenase 2 (AHD2) increased, while the total number of DA neurons remained the same. The highest levels of AHD2 expression were observed in mouse A9 DA neurons projecting to the dorsal striatum. Furthermore, real-time PCR analyses of in vitro differentiated Pitx3-expressing ES cells revealed that genes highly expressed in A9 DA neurons were up-regulated. When transplanted into the mouse striatum, Pitx3-expressing cells generated an increased proportion of AHD2-expressing DA neurons. Contrastingly, in Nurr1-expressing ES cells, increases of all midbrain DA markers were observed, resulting in a higher total number of DA neurons in vitro and in vivo, whereas the proportion of AHD2-expressing DA neurons was not changed. Our data, using gain-of-function analysis of ES cells, suggest that Pitx3 may be important for specification and/or maintenance of A9-like neuronal properties, while Nurr1 influences overall midbrain DA specification. These findings may be important for modifying ES cells to generate an optimal cell source for transplantation therapy of PD.

Targeting brain stem centers of cardiovascular control using adenoviral vectors: impact of promoters on transgene expression.

Adenoviral vectors (AVV) are widely used as tools for exploring gene function in studies of the central autonomic control, but the cellular specificity of the promoters commonly used in these vectors has not been studied. We evaluated AVV with four "wide-spectrum" promoters, human cytomegalovirus promoter (HCMV), synapsin-1 promoter (Syn1), tubulin-alpha1 promoter (Talpha1), and neuron-specific enolase (NSE) for their ability to express enhanced green fluorescent protein (EGFP) within the dorsal vagal complex and the adjacent brain stem. They were compared with the PRSx8 promoter, specifically designed to target catecholaminergic neurons. AdHCMVEGFP, AdSyn1EGFP-WHE (woodchuck hepatitis enhancer element), AdTalpha1EGFP, and AdNSEEGFP were unable to drive expression of EGFP in dopamine beta-hydroxylase-immunoreactive neurons of the A2 cell group, although the adjacent dorsal vagal motonucleus and especially hypoglossal motoneurons did express high levels of EGFP. AdPRSx8EGFP efficiently drove EGFP expression in the A2 cell group but also in choline acetyltransferase-positive vagal motoneurons. However, catecholaminergic neurons could be selectively and efficiently transduced via a retrograde route by injecting the vector into their target areas. Thus AVV with "wide-spectrum" promoters have strikingly different activity in the diverse cellular populations within brain stem cardiovascular control centers. The PRSx8 promoter is a valuable tool for the study of the role of catecholaminergic neurons.

Viral vectors as tools for studies of central cardiovascular control.

During the last few years physiological genomics has been the most rapidly developing area of physiology. Given the current ease of obtaining information about nucleotide sequences found in genomes and the vast amount of readily available clones, one of the most pertinent tasks is to find out about the roles of the individual genes and their families under normal and pathological conditions. Viral gene delivery into the brain is a powerful tool, which can be used to address a wide range of questions posed by physiological genomics including central nervous mechanisms regulating the cardio-vascular system. In this paper, we will give a short overview of current data obtained in this field using viral vectors and then look critically at the technology of viral gene transfer.

No association of the TGF-beta1 gene polymorphisms with the renal progression in autosomal dominant polycystic kidney disease (ADPKD) patients.

BACKGROUND: Two genetic loci, PKD I and PKD2, have been identified as being responsible for ADPKD, and PKD1 is known to be associated with a poor prognosis. However, the presence of an intrafamilial study clinical diversity suggests that there are disease-modifying loci. Because the mechanism ofthe renal failure in ADPKD includes a cystic growth and tubulointerstitial atrophy and fibrosis, we studied the associations between 2 polymorphisms in the TGF-beta1 gene, which are known to be associated with chronic tubulointerstitial inflammation, and ADPKD progression in Korean patients. PATIENTS AND METHODS: One hundred and twenty-five individuals who had ADPKD and 47 normal control subjects were genotyped by PCR-RFLP, the T869C (Leu10Pro) variant of TGF-beta gene leader sequence was discriminated with MspA1I and the G915C (Arg25Pro) variants with Bg1I. Statistical significances were determined using the Chi-square test. RESULTS: The distribution of the alleles for the TGF beta1 Leu10Pro polymorphism in ADPKD was: T 54%, C 46%, which was similar to the Korean (56: 44, p = 0.887) and Western controls (65: 35). In addition, no differences were found between the ESRD and the non-ESRD groups (p = 0.888) or the early hypertension and the normotension groups (p = 0.249). The distribution of alleles for the TGF beta1 Arg25Pro polymorphism showed only the GG type which was different from the Western population controls (G:C = 90:10, p = 0.000). CONCLUSIONS: Our results suggest that the polymorphism at Arg25Pro of TGF-beta1 in the Korean population has an allele distribution different from that ofthe Western population and that the polymorphism at Leu10Pro of TGF-beta1 has no association with the renal progression in Korean ADPKD patients.

Three novel mutations of the PKD1 gene in Korean patients with autosomal dominant polycystic kidney disease.

Mutations at the PKD1 locus account for 85% of cases of the common genetic disorder called autosomal dominant polycystic kidney disease (ADPKD). Screening for mutations of the PKD1 gene is complicated by the genomic structure of the 5'-duplicated region encoding 75% of the gene. To date, more than 90 mutations of the PKD1 gene have been reported in the European and American populations, and relatively little information is available concerning the pattern of mutations present in the Asian populations. We looked for mutations of the PKD1 gene in 51 unrelated Korean ADPKD patients, using polymerase chain reaction (PCR) with primer pairs located in the 3' single-copy region of the PKD1 gene and by single-strand conformation polymorphism (SSCP) analysis. We found three novel mutations, a G to A substitution at nucleotide 11012 (G3601S), a C to A substitution at nucleotide 11312 (Q3701X), and a C to T substitution at nucleotide 12971 (P4254S), and a single polymorphism involving a G to C substitution at nucleotide 11470 (L3753L). These mutations were not found in control individuals, and no other mutations in the 3' single-copy region of the PKD1 gene of patients with these mutations were observed. In particular, P4254S segregated with the disease phenotype. The clinical data of affected individuals from this study, and of previously reported Korean PKD1 mutations, showed that patients with frameshift or nonsense mutations were more prone to develop end-stage renal failure than those with missense mutations. Our findings indicate that many different PKD1 mutations are likely to be responsible for ADPKD in the Korean population, as in the Western population.

Xenobiotic response in humanized double transgenic mice expressing tetracycline-controlled transactivator and human CYP1B1.

The cytochrome P450 enzymes (P450s or CYPs) are a superfamily of hemeproteins that catalyze the monooxygenation of a wide range of endobiotic and xenobiotic substrates. A typical strategy in toxicological research and testing involves applying a toxicant at high doses for a short period to homogeneous animals under controlled conditions. However, the conditions of this approach have very little in common with actual human exposure. Transgenic (Tg) mice carrying human genes encoding a drug-metabolizing enzyme (CYP) offer a solution to many of the difficulties in the evaluation of chemical toxicity. It has been demonstrated that the expression of human CYP transgenes under the control of mammalian-inducible promoters exhibits relatively poor fold increases after induction. In this study, we used the tetracycline-regulated (tet) promoter system to increase the expression of the human CYP1B1 (hCYP1B1) gene in the tissues of transgenic mice. By mating two lineages of transgenic mice, double transgenic (dTg) mice expressing both tTA and hCYP1B1 genes under the control of the tet promoter were successfully produced, into which the two transgenes were introduced in an embryo. The expression pattern of tTA-driven hCYP1B1 transgene featured a fold induction of more than 3 to 12 in the brain, heart, and lung and 2- to 4-fold induction in the liver, kidney, and intestine upon doxycycline removal. Immunohistochemical staining with hCYP1B1 antibody was also increased by the removal of doxycycline. In addition, the activities of CYP liver microsomes in the dTg mice without doxycycline showed an increase compared to that in the dTg mice treated with doxycycline. The level of activities correspond to the levels of human CYP1B1 protein expression in the Tg mice (-dox) that was increased by 2-fold induction as compared to that of the dTg mice with doxycycline. Thus, overproduction in Tg can be purified and the activity of purified human CYP1B1 can be characterized by alterations to the coding sequence in order to solve the physiological function of this enzyme in a humanized in vivo system. It is also possible to examine the activity of purified human CYP1B1 using several environmental toxicants such as procarcinogens.

Genetic heterogeneity in Korean families with autosomal-dominant polycystic kidney disease (ADPKD): the first Asian report.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease in adults, and the prevalence of this disease within the chronic haemodialysis patient population is known to be approximately 2% in Korea. So far, three genetic locus have been identified as being responsible for ADPKD, and approximately 85% of the cases in Western countries are related to the PKD1 gene. However, little information is available concerning the pattern of linkage analysis in Asian populations. METHODS: 48 families with hereditary renal cysts were recruited by consent and their molecular genetic characteristics were studied. Linkage analysis was done with microsatellite markers (PKD1: SM7, UT581, AC2.5, KG8, D16S418; PKD2: D4S423, D4S1534, D4S1542, D4S1544, D4S2460). Genomic DNA polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) gel run were performed, and the resultant allele patterns were compared with sonographic findings. RESULTS: The results of this study showed that the ratio PKD1:PKD2 was 31:8, and that the PKD2 families exhibited a tendency toward a milder renal prognosis than the PKD1 families. CONCLUSION: We confirmed the applicability of linkage analysis for ADPKD in the Korean population, and our data confirmed a similar incidence of PKD1 (79%) and PKD2 (21%) in Korean patients as in the Western population.

A high-efficiency synthetic promoter that drives transgene expression selectively in noradrenergic neurons.

Gene promoter systems that drive high-level, long-term, and cell-specific transgene expression are of great interest because of their potential utility for gene therapy. To generate an efficient promoter system specific for noradrenergic (NA) neurons, we multimerized an NA-specific cis-regulatory element (PRS2) identified in the human dopamine beta-hydroxylase (hDBH) promoter, and combined it with a minimal promoter (containing a TATA box and transcription start site). Forms of this synthetic promoter that contain 8 or more copies of PRS2 were >50 times more effective than the 1.15-kb hDBH promoter at driving reporter gene expression in cell lines originated from NA neurons. Neither the synthetic promoter nor the 1.15-kb hDBH promoter drove reporter gene expression in nonneuronal cells. Microinjections of an adenoviral vector containing the synthetic promoter directly into rat brain caused more strict NA-specific reporter gene expression than that caused by a vector containing the 1.15-kb hDBH promoter when the targeted region contained large numbers of NA neurons (locus coeruleus). Furthermore, the vector containing the synthetic promoter caused less nonspecific ("leaky") reporter gene expression than that caused by the vector containing the 1.15-kb hDBH promoter when the targeted region was devoid of NA neurons (cerebellum, dentate gyrus). Together, these studies provide in vitro and in vivo evidence that this novel synthetic promoter can target transgene expression to NA neurons even more efficiently and selectively than the naturally occurring, 1.15-kb hDBH promoter.

Stimulation of GLUT-1 glucose transporter expression in response to hyperosmolarity.

Glucose transporter isoform-1 (GLUT-1) expression is stimulated in response to stressful conditions. Here we examined the mechanisms mediating the enhanced expression of GLUT-1 by hyperosmolarity. GLUT-1 mRNA, GLUT-1 protein, and glucose transport increased after exposure of Clone 9 cells to 600 mosmol/l (produced by addition of mannitol). The stimulation of glucose transport was biphasic: in the early phase (0-6 h) a approximately 2.5-fold stimulation of glucose uptake was associated with no change in the content of GLUT-1 mRNA, GLUT-1 protein, or GLUT-1 in the plasma membrane, whereas the approximately 17-fold stimulation of glucose transport during the late phase (12-24 h) was associated with increases in both GLUT-1 mRNA (approximately 7.5-fold) and GLUT-1 protein content. Cell sorbitol increased after 3 h of exposure to hyperosmolarity. The increase in GLUT-1 mRNA content was associated with an increase in the half-life of the mRNA from 2 to 8 h. A 44-bp region in the proximal GLUT-1 promoter was necessary for basal activity and for the two- to threefold increases in expression by hyperosmolarity. It is concluded that the increase in GLUT-1 mRNA content is mediated by both enhanced transcription and stabilization of GLUT-1 mRNA and is associated with increases in GLUT-1 content and glucose transport activity.