A prospective study of radiation therapy-associated thrombocytopenia.
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Humans↗
Biomedical subjects
K Lee
Publications and source records attributed to K Lee.
Selection for G156A O6-methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and protection from lethality in mice treated with O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea.
A retroviral gene therapy approach was developed to protect early hematopoietic progenitors from 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a stem cell toxin, and O6-benzylguanine (BG), an inhibitor of a key BCNU resistance protein, O6-alkylguanine DNA alkyltransferase (AGT). The retroviral vector MFG was used to transfer the G156A MGMT (deltaMGMT) cDNA, encoding a mutant AGT that is resistant to inhibition by BG, into murine bone marrow-derived hematopoietic progenitors. Following transplantation into lethally irradiated mice, the transduced cells were subjected to in vivo BG and BCNU treatment to examine the ability to enrich for transduced cells expressing deltaAGT. Transplantation of deltaMGMT-transduced cells resulted in deltaAGT expression in 30% of bone marrow nucleated cells 13 weeks after transplantation. After one cycle of BG and BCNU, deltaAGT expression was observed in 60% of bone marrow cells, and the percentage of colony-forming units (culture; CFU-C) containing proviral sequence increased from 67 to 100%. CFU-C obtained from BG and BCNU-treated deltaMGMT animals up to 23 weeks after transplantation were more resistant to combination BG and BCNU than CFU-C from mice transplanted with lacZ-transduced cells and treated with BG and BCNU or from mice transplanted with deltaMGMT-transduced cells and left untreated. The degree of drug resistance in deltaMGMT-transduced hematopoietic progenitors to BG and BCNU was much greater than we observed previously with wild-type MGMT gene transfer and treatment with BCNU alone. Furthermore, whereas 21 of 22 mice transplanted with deltaMGMT-transduced cells survived in vivo BG and BCNU administration, only 3 of 13 mice transplanted with lacZ-transduced progenitors survived similar drug treatment. Thus, deltaMGMT-transduced murine bone marrow cells selectively survive in vivo BG and BCNU exposure, resulting in prolonged enrichment for the transduced cells and protection from mortality induced by this drug combination.
Animals↗
Transcriptional regulation of the human alpha1a-adrenergic receptor gene. Characterization Of the 5'-regulatory and promoter region.
We recently cloned cDNAs encoding three subtypes of human alpha1-adrenergic receptors (alpha1ARs), alpha1a, alpha1b, and alpha1d (Schwinn, D. A., Johnston, G. L., Page, S. O., Mosley, M. J., Wilson, K. H., Worman, N. P., Campbell, S., Fidock, M. D., Furness, L. M., Parry-Smith, D. J., Peter, B., and Bailey, D. S. (1995) J. Pharmacol. Exp. Ther. 272, 134-142) and demonstrated predominance of alpha1aARs in many human tissues (Price, D. T., Lefkowitz, R. J., Caron, M. G., Berkowitz, D., and Schwinn, D. A. (1994) Mol. Pharmacol. 45, 171-175). Several lines of evidence indicate that alpha1aARs are important in clinical diseases such as myocardial hypertrophy and benign prostatic hyperplasia. Therefore, we initiated studies to understand mechanisms underlying regulation of alpha1aAR gene transcription. A genomic clone containing 6.2 kb of 5'-untranslated region of the human alpha1aAR gene was recently isolated. Ribonuclease protection and primer extension assays indicate that alpha1aAR gene transcription occurs at multiple initiation sites with the major site located 696 base pairs upstream of the ATG, where a classic initiator sequence is located. Transfection of luciferase reporter constructs containing varying amounts of 5'-untranslated region into human SK-N-MC neuroblastoma cells indicate that a region extending 125 base pairs upstream from the main transcription initiation site contains full alpha1aAR promoter activity. Furthermore, distinct activator and suppressor elements lie 2-3 and 3-5 kilobase pairs upstream, respectively. Although the alpha1aAR promoter contains neither TATA or CAAT elements, gel shift mobility assays targeting three GC boxes immediately upstream of the main transcription initiation site confirm binding of Sp1. Activity of the alpha1aAR promoter is cell-specific, demonstrating highest activity in cells endogenously expressing alpha1aARs. The human alpha1aAR gene also contains several cis regulatory elements, including several insulin and cAMP response elements. Consistent with these observations, we provide the first evidence that treatment of SK-N-MC cells with insulin and cAMP elevating agents leads to an increase in alpha1aAR expression. In conclusion, these data represent the first characterization of the alpha1aAR gene; our findings should facilitate further studies designed to understand mechanisms regulating alpha1AR subtype-specific expression in healthy and diseased human tissue.
Cloning, Molecular↗
Characterization of the inward current induced by metabotropic glutamate receptor stimulation in rat ventromedial hypothalamic neurones.
1. Whole-cell patch clamp recordings were made from rat ventromedial hypothalamic neurones in slices of brain tissue in vitro. Bath application of 50 microM (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) depolarized all neurones tested by activation of an inward current of approximately 55 pA at -60 mV. 2. The inward current elicited by 1S,3R-ACPD was unaffected by K+ channel blockade with external Cs+, Ba2+ or TEA. However, the current was significantly reduced by replacement of the external NaCl with either Tris-HCl or LiCl. 3. The 1S,3R-ACPD-induced current was reduced by the heavy metal ions Ni2+ or La3+ and also by the Na(+)-Ca2+ exchange current inhibitor 3',4'-dichlorobenzamil. 4. The effects of 1S,3R-ACPD were mimicked by the group I metabotropic agonist 3,5-dihydroxyphenylglycine (DHPG) but not by the group III selective agonist, L-2-amino-4-phosphonobutanoate (L-AP4). Furthermore, the effects of 1S,3R-ACPD were inhibited by the metabotropic antagonists alpha-methyl-4-carboxyphenylglycine (MCPG) and 1-aminoindan-1,5-dicarboxylic acid (AIDA) but not by the presynaptic metabotropic receptor antagonists alpha-methyl-4-phosphonophenylglycine (MPPG) or alpha-methyl-4-tetrazolylphenylglycine (MTPG). 5. Photorelease of caged GDP beta S inside neurones irreversibly blocked the 1S,3R-ACPD-induced current whilst photolysis of caged GTP gamma S inside neurones irreversibly potentiated this current. 6. The PLC inhibitor U-73,122 significantly reduced the size of the inward current induced by 1S,3R-ACPD. This effect was not mimicked by the inactive analogue U-73,343. 7. Flash photolysis of the caged calcium chelator diazo-2 inside neurones diminished the response to 1S,3R-ACPD. 8. It is concluded that group I metabotropic glutamate receptors depolarize neurones in the VMH by activation of a Na(+)-Ca2+ exchange current through a G-protein coupled increase in intracellular Ca2+.
Animals↗
Discrimination of single-nucleotide polymorphisms in human DNA using peptide nucleic acid probes detected by MALDI-TOF mass spectrometry.
Human genomic and mitochondrial DNA contain large numbers of single-nucleotide polymorphisms (SNPs), many of which are linked to known diseases. Rapid and accurate genetic screening for important SNPs requires a general methodology which is easily implemented. We present here an approach to SNP discrimination based on high-specificity hybridization of peptide nucleic acid (PNA) probes to PCR-amplified DNA. The assay is directly applied to polymorphisms located within hypervariable region 1 of the human mitochondrial genome and type 1 suballeles of the human leukocyte antigen DQ alpha gene. Captured, single-stranded DNA molecules prepared by PCR amplification are hybridized with PNA probes in an allele-specific fashion. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is then used for rapid, precise, and unambiguous detection and identification of the hybridized PNA probes. Since PNA oligomers bind strongly to complementary DNA under minimal salt conditions, the use of PNA probes is compatible with MALDI-TOFMS. The unparalleled ability of MALDI-TOFMS analysis in terms of molecular weight resolution and accuracy, in conjunction with the highly specific PNA hybridization afforded by this method, offers promise for development into a multiplexed, high-throughput screening technique.
DNA↗
Assessing Extreme Models of the Stober Synthesis Using Transients under a Range of Initial Composition
29Si-NMR, conductimetry, and photon correlation spectroscopy are used to monitor the temporal profile of intermediate concentrations in Stober synthesis (i.e., ammonia-catalyzed hydrolysis of tetraethoxysilane in a batch reactor). Extreme models of the process are assessed by examining the effect of initial composition on these transients (over a wider range of composition than attempted previously). The trends with initial composition suggest that the nucleation is rate-limited by the hydrolysis of the singly hydrolyzed monomer, the product of which probably phase separates. Moreover, the trends are consistent with the aggregation model discussed by G. H. Bogush and C. F. Zukoski (J. Colloid Interface Sci. 142, 1, 19 (1991) and by M. T. Harris (Ph.D. dissertation, Univ. of Tennessee, 1992). The trends are not consistent with a growth model without aggregation. Copyright 1997 Academic Press. Copyright 1997Academic Press
Journal Article↗
Activation of c-Jun N-terminal kinase antagonizes an anti-apoptotic action of Bcl-2.
Bcl-2 is an intracellular membrane-associated protein that prevents cell death induced by a variety of apoptotic stimuli. A mechanism by which Bcl-2 exerts an anti-cell death effect is, however, not fully understood. In the present study, Bcl-2 suppressed cell death of N18TG neuroglioma cells caused by various apoptotic stresses, including etoposide, staurosporine, anisomycin, and ultraviolet irradiation. Concomitantly, Bcl-2 disrupted a signaling cascade to the c-Jun N-terminal kinase activation induced by the apoptotic stresses. Bcl-2 also prevented the etoposide-induced stimulation of MEKK1. Furthermore, overexpression of c-Jun N-terminal kinase antagonized the death-protective function of Bcl-2. These data suggest that suppression of the c-Jun N-terminal kinase signaling pathway may be critical for Bcl-2 action.
Animals↗
In vivo determination of RNA structure-function relationships: analysis of the 790 loop in ribosomal RNA.
The 790 loop is a conserved hairpin located between positions 786 and 796 of Escherichia coli 16 S rRNA that is required for ribosome function. Using a novel genetic approach, all positions in the loop were simultaneously mutated and functional mutant sequences were selected in vivo. This "instant evolution" experiment revealed that approximately 190 of the 262,144 possible mutant sequences were functional. Analysis of functional mutant sequences allowed discrimination between nucleotides directly involved in protein synthesis and those involved primarily in loop structure. Among the functional mutant sequences, positions 789 and 791 were invariant and extensive covariation was observed among the nucleotides at the base of the loop at positions 787, 788, 794 and 795. NMR and thermodynamic analyses of model 790 hairpins in vitro revealed weak pairing interactions between positions 787 and 795 and between positions 788 and 794 consistent with the in vivo mutational analysis. Functional analysis of site-directed mutants containing all possible nucleotide combinations at positions 787 and 795 in vivo showed that stable base-pairs at these positions prevent subunit association.
Chloramphenicol↗
Troglitazone inhibits type 2KATP channel activity and depolarises tolbutamide-sensitive neurones in the rat ventromedial hypothalamus.
Patch-clamp recording techniques were used to examine the effect of troglitazone upon visually identified neurones contained in slices from the rat ventromedial hypothalamus. Bath application of 1-50 microM troglitazone depolarised tolbutamide-sensitive neurones in a poorly reversible manner. In outside-out patches, troglitazone inhibited KATP channel activity when added to the bath solution. It is concluded that troglitazone is a novel modulator of the type 2 KATP channel.
Adenosine Triphosphate↗
Release of lactate by the lung in acute lung injury.
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Biological Transport, Active↗
Implications of early systemic therapy on the incidence of endogenous fungal endophthalmitis.
OBJECTIVE: In the past, evidence of endogenous fungal endophthalmitis has been used as a guide to initiating potentially toxic antifungal therapy in patients with systemic fungal infections. Recently, however, a trend has developed to provide patients with antifungal therapy at the first evidence of fungal infection. The authors' study evaluates the incidence of endogenous fungal endophthalmitis in this setting. DESIGN: The design is a retrospective review of the medical records of patients examined by the inpatient ophthalmology consultation service to rule out endogenous fungal endophthalmitis between January 1994 and April 1996 at the University of Michigan Hospitals, Ann Arbor, Michigan. PARTICIPANTS: Two hundred fourteen eyes of 107 patients with a diagnosis of systemic fungal infection were studied. INTERVENTION: A review of medical records was performed. MAIN OUTCOME MEASURES: The findings of the ocular examination, the presence of risk factors for disseminated fungal infection, the type of antifungal therapy, and the source and identity of the isolated fungus were recorded. RESULTS: The majority of patients examined had either fungal growth from blood cultures or evidence of deep tissue fungal infection. All patients in the study were at risk for fungal disease with each having at least one risk factor for disseminated fungal infection. Of the patients examined, 93.4% already were receiving systemic antifungal therapy at the time of ophthalmologic consultation. Only 3 (2.8%) of the 107 patients examined had chorioretinal findings consistent with early endogenous fungal endophthalmitis. None had intravitreous involvement, and the ocular findings did not change the course of therapy. CONCLUSIONS: Early systemic treatment of deep tissue fungal infection appears to dramatically decrease the incidence of endogenous fungal endophthalmitis.
Administration, Oral↗
Regulation of cAMP response element binding protein (CREB) binding in the mammalian clock pacemaker by light but not a circadian clock.
Mammalian circadian rhythms are considered to be regulated by a clock pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. The molecular mechanism of entrainment and oscillation of circadian rhythm are not well understood but photic induction of immediate-early gene (IEG) expression in the SCN is thought to play a role. Here we show that under 12 h light:12 h dark (LD) condition, the cAMP response element binding protein (CREB) binding to cAMP responsive promoter element (CRE) of NMDAR1/zeta1 promoter region in the SCN is higher during the light than the dark by electro-mobility shift assay (EMSA). When animals are placed in constant dark, CREB DNA binding activity in the SCN is low and does not vary with circadian time when compared with cortex nuclear extract as a control. Most significantly, photic induction of CREB binding activity in the SCN occurs at all circadian times tested, indicating that CREB DNA binding in the SCN is not gated by the endogenous clock. These results implicate the role of CREB in photic neuronal signaling in the SCN and suggest that CREB DNA binding activities may not be regulated by a circadian clock.
Animals↗
[Prevention and treatment of erectile disorders in sickle cell disease].
Priapism is a frequent and serious cause of morbidity in males with sickle cell disease. Acute priapism (AP) is preceeded in two-thirds of the cases by repeated minor events called stuttering priapism (SP). Since 1994, we have used a specific approach to prevent the commonly devastating effects of AP, using the alpha adrenergic agent etilefrine. Treatment of AP has been simplified (drainage without aspiration followed by one or two intracavernous injections (ICI) of 10 mg of etilefrine, until detumescence). For SP lasting more than one hour or causing pain, we use oral etilefrine and/or self ICI. This strategy was effective in five patients seen having AP, 21 patients with SP; it is simple, cheap, and avoids surgical procedure and transfusion. Moreover, erectile dysfunction, present in three patients, has been treated safely by ICI of protaglandins.
Adolescent↗
Epidemiological evidence for the disruption of ionized calcium homeostasis in the elderly.
Ionized calcium (Ca2+), phosphate, albumin, total calcium, and pH measurements taken from participants in a large population-based epidemiological study were examined to determine the change in physiological variation with age for persons over 43 years old. Only Ca2+ showed a statistically significant increase in SD with age (p < 0.0001). The Ca2+ coefficients of variation (CV) increased from 2.92% in the youngest age group (43-54 years) to 3.69% in the oldest age group (75-86 years of age). In females, the increase in Ca2+ variability was nearly complete by age 55. Males also showed a significant (p = 0.006) increase in SD between the 43-54 age group and the 55-64 age group, however, Ca2+ variability did not plateau after age 55 in men as it did in women. In the 43-54 (p = 0.04) and 55-64 (p = 0.03) age group men showed significantly better physiological control of Ca2+ than women. Phosphate showed a slight decrease in CV with age. These data suggest that Ca2+ homeostasis is disrupted in the same age groups that are most vulnerable to osteoporosis.
Adult↗
Effect of englitazone on KATP and calcium-activated non-selective cation channels in CRI-G1 insulin-secreting cells.
1. The effects of englitazone sodium, an antidiabetic agent, on ion channel activity in the CRI-G1 insulin secreting cell line was examined by use of the patch clamp technique. 2. Application of englitazone to the outside of CRI-G1 cells in the whole-cell recording configuration produced concentration-dependent inhibition of KATP currents with an IC50 value of 8 microM. The inhibition of the K+ current was not affected by the removal of Mg2+ ions from or the addition of trypsin to the solution bathing the intracellular surface of the cell membrane. 3. Englitazone also inhibited KATP channel activity in recordings from inside out excise membrane patches. The concentration-dependence of inhibition was identical to that observed in whole-cell recordings and was voltage-independent. Single channel recordings confirmed that neither the absence or presence of Mg2+ ions nor the addition of trypsin at the intracellular surface of the membrane influenced the inhibition of KATP channels by englitazone. 4. Englitazone also inhibited Ca(2+)-activated non-selective cation (NSCa) channels in inside-out patches in a concentration-dependent and voltage-independent manner with an IC50 value of 10 microM. In comparison, the non-sulphonylurea KATP channel blocker ciclazindol produced a slight voltage-dependent inhibition of the NSCa channel at a concentration of 20 microM. 5. In whole-cell recordings englitazone, at a relatively high concentration (50 microM) in comparison with that required to block KATP and NSCa channels, inhibited voltage-activated Ca2+ currents by 33% but did not inhibit voltage-activated K+ and Na+ currents. 6. It is concluded that englitazone is a novel blocker of NSCa and KATP channels. The inhibition of KATP channels occurs following procedures that dissociate sulphonylurea receptor coupling to the channel. The equipotent and voltage-independent inhibition of NSCa and KATP channels by englitazone may indicate a common mechanism of block.
Adenosine Triphosphate↗
Catecholamine modulatory effects of nepicastat (RS-25560-197), a novel, potent and selective inhibitor of dopamine-beta-hydroxylase.
1. Inhibitory modulation of sympathetic nerve function may have a favourable impact on the progression of congestive heart failure. Nepicastat is a novel inhibitor of dopamine-beta-hydroxylase, the enzyme which catalyses the conversion of dopamine to noradrenaline in sympathetic nerves. The in vitro pharmacology and in vivo catecholamine modulatory effects of nepicastat were investigated in the present study. 2. Nepicastat produced concentration-dependent inhibition of bovine (IC50 = 8.5 +/- 0.8 nM) and human (IC50 = 9.0 +/- 0.8 nM) dopamine-beta-hydroxylase. The corresponding R-enantiomer (RS-25560-198) was approximately 2-3 fold less potent than nepicastat. Nepicastat had negligible affinity (> 10 microM) for twelve other enzymes and thirteen neurotransmitter receptors. 3. Administration of nepicastat to spontaneously hypertensive rats (SHRs) (three consecutive doses of either 3, 10, 30 or 100 mg kg-1, p.o.; 12 h apart) or beagle dogs (0.05, 0.5, 1.5 or 5 mg kg-1, p.o.; b.i.d., for 5 days) produced dose-dependent decreases in noradrenaline content, increases in dopamine content and increases in dopamine/noradrenaline ratio in the artery (mesenteric or renal), left ventricle and cerebral cortex. At the highest dose studied, the decreases in tissue noadrenaline were 47%, 35% and 42% (in SHRs) and 88%, 91% and 96% (in dogs) in the artery, left ventricle and cerebral cortex, respectively. When tested at 30 mg kg-1, p.o., in SHRs, nepicastat produced significantly greater changes in noradrenaline and dopamine content, as compared to the R-enantiomer (RS-25560-198), in the mesenteric artery and left ventricle. 4. Administration of nepicastat (2 mg kg-1, b.i.d, p.o.) to beagle dogs for 15 days produced significant decreases in plasma concentrations of noradrenaline and increases in plasma concentrations of dopamine and dopamine/noradrenaline ratio. The peak reduction (52%) in plasma concentration of noradrenaline and the peak increase (646%) in plasma concentration of dopamine were observed on day-6 and day-7 of dosing, respectively. 5. The findings of this study suggest that nepicastat is a potent, selective and orally active inhibitor of dopamine-beta-hydroxylase which produces gradual modulation of the sympathetic nervous system by inhibiting the biosynthesis of noradrenaline. This drug may, therefore, be of value in the treatment of cardiovascular disorders associated with over-activation of the sympathetic nervous system, such as congestive heart failure.
Animals↗
The effects of neuroleptic and tricyclic compounds on BKCa channel activity in rat isolated cortical neurones.
1. The actions of several neuroleptic and tricyclic compounds were examined on the large conductance Ca(2+)-activated K+ (BKCa) channel present in neurones isolated from the rat motor cortex. 2. Classical neuroleptic compounds including chlorpromazine and haloperidol applied to the intracellular surface of inside-out patches produced a concentration-dependent reduction in BKCa channel activity. Similar effects were observed when these compounds were applied to the extracellular surface of outside-out patches. 3. In contrast, the atypical neuroleptic compounds clozapine and sulpiride did not affect BKCa channel activity (100 nM-1 mM) in either inside-out or outside-out patches, while 10 microM pimozide produced 73% of the inhibition produced by 10 microM chlorpromazine. 4. BKCa channel activity was also unaffected by application of structurally related tricyclic compounds including the anti-cholinesterase tacrine and the anti-epileptic carbamazepine. The tricyclic antidepressant drug amitriptyline was found to inhibit BKCa channel activity but was much less effective than the classical neuroleptic compounds. 5. It is concluded that compounds belonging to the classical neuroleptic group of drugs inhibit BKCa channel activity in the rat motor cortex in a structurally-specific manner. This observation may be of clinical significance as it may contribute to some of the side effects associated with classical neuroleptic drug therapy.
Animals↗
Antidiabetic sulphonylureas stimulate acetylcholine release from striatal cholinergic interneurones through inhibition of K(ATP) channel activity.
The sulphonylureas tolbutamide and glibenclamide were shown to stimulate acetylcholine release from rat striatal slices. To determine the mechanism of this effect, whole-cell patch-clamp recordings were made from large neurones within the striatum that displayed morphological, electrophysiological, and pharmacological characteristics typical of cholinergic interneurones. Dialysis of these neurones with a pipette solution containing low concentrations of ATP produced a gradual hyperpolarisation that could be reversed by bath application of the sulphonylureas. In voltage-clamp studies, these compounds were shown to act through the inhibition of a potassium conductance. It is concluded that cholinergic interneurones within the rat striatum express sulphonylurea-sensitive ATP-sensitive potassium channel activity. These channels are probably cytoprotective and may prove to be novel sites of therapeutic modulation.
Acetylcholine↗