Analysis of trends in water quality: constructed wetlands in metropolitan Taipei.

Meandering through the most densely populated metropolitan areas of Taipei, Taiwan, the Danshui River and its tributaries have undergone the construction of 14 wetlands since 2004, as a means to improve water quality. This study was conducted to examine the functional capabilities associated with treating non-point source pollution through these riparian wetlands. Trend analysis was used to differentiate dissolved oxygen, biochemical oxygen demand, suspended solids, ammonia, and Escherichia coli, among 13 sampling sites using both functions of a Mann-Kendall test and a seasonal Mann-Kendall test. The results show that water quality in Taipei metropolitan rivers has been improving since increasing the number of constructed wetlands and connecting households to the public sewage system. The concentration of pollutants such as those influencing biochemical oxygen demand have gradually declined in drought seasons because riparian wetlands contribute a base flow to dilute riverine pollutants. This paper indicates that the creation of treatment systems influences dissolved oxygen conditions at the municipal scale, suggesting that constructed wetlands could stabilize water quality during extreme hydrological events and improve water quality particularly in times of drought.

CYP1B1 expression in rat testis and Leydig cells is not inducible by aryl hydrocarbon receptor agonists.

1. Cytochrome P450 1B1 (CYP1B1) is highly expressed in testis, but there is conflicting information regarding the inducibility of testicular CYP1B1 by aryl hydrocarbon receptor (AhR) agonists. 2. To assess AhR-mediated regulation, testicular CYP1B1 expression was measured following treatment of adult rats with 3-methylcholanthrene and various dosages of benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The effect of TCDD on CYP1B1 expression in R2C rat Leydig and MA-10 mouse Leydig cells in culture was also determined. 3. Immunoblot analysis showed that treatment with benzo[a]pyrene at dosages up to 200 mg/kg/day and 3-methylcholanthrene at 25 mg/kg/day did not induce testicular CYP1B1 expression. Treatment with TCDD at dosages of 1, 5 or 100 microg/kg had no effect, but testicular CYP1B1 protein levels were increased by approximately 50% at dosages of 10 and 50 microg/kg. 4. CYP1B1 mRNA levels in MA-10 and CYP1B1 protein levels in R2C cells were not induced by exposure to TCDD (10-1000 nM). 5. Overall, the results indicate that rodent testicular CYP1B1 is not inducible by AhR agonists.

Improved treatment results for childhood acute myeloid leukemia in Taiwan.

To improve treatment results for children with de novo acute myeloid leukemia (AML), we introduced a novel protocol, Taiwan Pediatric Oncology Group-AML-97A, for AML other than acute promyelocytic leukemia (APL), for which modified conventional protocols were used. From January 1, 1997, to December 31, 2002, 141 children younger than 17 years old with de novo AML were enrolled. In total, 117 patients with non-APL AML were treated with induction therapy of idarubicin and cytarabine (Ara-C), postremission therapy with high-dose Ara-C - containing regimens for four monthly courses, and moderate-dose therapy with idarubicin and Ara-C for four monthly courses. The first 19 patients with APL were treated with all-trans retinoic acid, idarubicin and Ara-C, with the remaining five patients receiving all-trans retinoic acid and idarubicin, followed by maintenance therapy for 2 years. Stem cell transplantation was performed in 29 patients in first remission with a similar outcome as chemotherapy alone. The remission rate in the AML-97A study was 90%, the 5-year survival 51 +/- 5.3% (s.e.) and the 5-year event-free survival 50 +/- 4.8%; for APL, these were 100%, 86 +/- 7.0, and 75 +/- 9.8%. For the whole group, the 5-year survival was 57 +/- 4.7% and the 5-year event-free survival 54 +/- 4.4%. The AML-97A regimen was well tolerated.

Cyclophosphamide modulates rat hepatic cytochrome P450 2C11 and steroid 5 alpha-reductase activity and messenger RNA levels through the combined action of acrolein and phosphoramide mustard.

Cyclophosphamide treatment of adult male rats leads to sustained decreases in several liver microsomal cytochrome P450 (CYP) activities, including CYP 2C11-catalyzed cyclophosphamide activation, via a process that is associated with a feminization of the overall pattern of liver enzyme expression (G. A. LeBlanc and D. J. Waxman, Cancer Res., 50:5720-5726, 1990). The present study compares the effects of cyclophosphamide and its isomeric analogue ifosphamide on the gender-dependent expression of hepatic CYP 2C11 and steroid 5 alpha-reductase in adult male rats and also examines the role of the cyclophosphamide metabolites acrolein and phosphoramide mustard in feminizing the expression of these liver enzymes. Ifosphamide (a) suppressed the male-specific CYP 2C11 mRNA and CYP 2C11-catalyzed liver microsomal testosterone 2 alpha-hydroxylation and cyclophosphamide and ifosphamide 4-hydroxylation and (b) elevated the female-dominant liver enzyme steroid 5 alpha-reductase and its mRNA 7-9 days after drug treatment, both occurring in a manner similar to that of cyclophosphamide, but requiring a 50% higher dose (180 mg/kg, single i.p. injection) to achieve these effects. This pattern of response could not be achieved by treatment of rats with acrolein or with cyclophosphamide analogues that decompose to acrolein without formation of phosphoramide mustard. In contrast, phosphoramide mustard treatment (100 mg/kg) did modulate microsomal CYP 2C11 and steroid 5 alpha-reductase activities. Treatment with a lower dose (50 mg/kg) of phosphoramide mustard or with the acrolein precursor 4-hydroperoxydechlorocyclophosphamide (200 mg/kg) alone did not affect liver enzyme expression, whereas the combination of these agents produced an overall pattern of response that was similar to that conferred by cyclophosphamide. These studies establish that ifosphamide is less potent than cyclophosphamide in modulating the pattern of cytochrome P450 and steroid 5 alpha-reductase expression and that phosphoramide mustard is responsible for the modulation of liver enzyme expression by cyclophosphamide, with acrolein potentiating the modulating activity of the mustard.

Enhanced cyclophosphamide and ifosfamide activation in primary human hepatocyte cultures: response to cytochrome P-450 inducers and autoinduction by oxazaphosphorines.

The anticancer oxazaphosphorine prodrugs cyclophosphamide and ifosfamide are activated in human liver by a 4-hydroxylation reaction catalyzed by multiple cytochrome P450 (CYP) enzymes. In the present study, we used a cultured human hepatocyte model to identify possible inducers of the CYP-catalyzed activation of these two anticancer prodrugs. Treatment of primary cultures of human hepatocytes with phenobarbital, dexamethasone, or rifampin elevated hepatocyte microsomal oxazaphosphorine 4-hydroxylation by up to 200-400% of control for both drug substrates. These inductions were associated with corresponding increases in immunoreactive CYP2B6, CYP2C8, CYP2C9, and CYP3A4, all previously shown to catalyze oxazaphosphorine activation. Rifampin (1 microM, 96-h exposure) was a particularly potent inducer of ifosfamide and cyclophosphamide 4-hydroxylation, as well as of CYP3A protein levels and CYP3A-dependent testosterone 6beta-hydroxylation. CYP3A4, CYP2C8, and CYP2C9 protein levels were also increased by exposure of the hepatocytes to cyclophosphamide or ifosfamide (50 microM), which thereby enhanced their own rates of 4-hydroxylation in the cultured hepatocytes. In one human hepatocyte culture that contained the polymorphically expressed CYP3A5 in addition to the more widely expressed CYP3A4, only CYP3A4 was induced by cyclophosphamide, ifosfamide, and rifampin. These studies: (a) demonstrate an underlying metabolic basis for the clinically important oxazaphosphorine autoinduction pharmacokinetics seen with these drugs in cancer patients; and (b) identify rifampin and other CYP inducers as potentially useful for increasing the rates of cyclophosphamide 4-hydroxylation and ifosfamide 4-hydroxylation in human liver in a manner that could favorably impact the clinical pharmacokinetics of these anticancer prodrugs.

Differential activation of cyclophosphamide and ifosphamide by cytochromes P-450 2B and 3A in human liver microsomes.

The present study identifies the specific human cytochrome P-450 (CYP) enzymes involved in hydroxylation leading to activation of the anticancer drug cyclophosphamide and its isomeric analogue, ifosphamide. Substantial interindividual variation (4-9-fold) was observed in the hydroxylation of these oxazaphosphorines by a panel of 12 human liver microsomes, and a significant correlation was obtained between these 2 activities (r = 0.85, P < 0.001). Enzyme kinetic analyses revealed that human liver microsomal cyclophosphamide 4-hydroxylation and ifosphamide 4-hydroxylation are best described by a 2-component Michaelis-Menten model composed of both low Km and high Km P-450 4-hydroxylases. To ascertain whether one or more human P-450 enzymes are catalytically competent in activating these oxazaphosphorines, microsomal fractions prepared from a panel of human B-lymphoblastoid cell lines stably transformed with individual P-450 complementary DNAs were assayed in vitro for oxazaphosphorine activation. Expressed CYP2A6, -2B6, -2C8, -2C9, and -3A4 were catalytically competent in hydroxylating cyclophosphamide and ifosphamide. Whereas CYP2C8 and CYP2C9 have the characteristics of low Km oxazaphosphorine 4-hydroxylases, CYP2A6, -2B6, and -3A4 are high Km forms. In contrast, CYP1A1, -1A2, -2D6, and -2E1 did not produce detectable activities. Furthermore, growth of cultured CYP2A6- and CYP2B6-expressing B-lymphoblastoid cells, but not of CYP-negative control cells, was inhibited by cyclophosphamide and ifosphamide as a consequence of prodrug activation to cytotoxic metabolites. Experiments with P-450 form-selective chemical inhibitors and inhibitory anti-P-450 antibodies were then performed to determine the contributions of individual P-450s to the activation of these drugs in human liver microsomes. Orphenadrine (a CYP2B6 inhibitor) and anti-CYP2B IgG inhibited microsomal cyclophosphamide hydroxylation to a greater extent than ifosphamide hydroxylation, consistent with the 8-fold higher activity of complementary DNA-expressed CYP2B6 with cyclophosphamide. In contrast, troleandomycin, a selective inhibitor of CYP3A3 and -3A4, and anti-CYP3A IgG substantially inhibited microsomal ifosphamide hydroxylation but had little or no effect on microsomal cyclophosphamide hydroxylation. By contrast, the CYP2D6-selective inhibitor quinidine did not affect either microsomal activity, while anti-CYP2A antibodies had only a modest inhibitory effect. Overall, the present study establishes that liver microsomal CYP2B and CYP3A preferentially catalyze cyclophosphamide and ifosphamide 4-hydroxylation, respectively, suggesting that liver P-450-inducing agents targeted at these enzymes might be used in cancer patients to enhance drug activation and therapeutic efficacy.

Fault tolerant control of multivariable processes using auto-tuning PID controller.

Fault tolerant control of dynamic processes is investigated in this paper using an auto-tuning PID controller. A fault tolerant control scheme is proposed composing an auto-tuning PID controller based on an adaptive neural network model. The model is trained online using the extended Kalman filter (EKF) algorithm to learn system post-fault dynamics. Based on this model, the PID controller adjusts its parameters to compensate the effects of the faults, so that the control performance is recovered from degradation. The auto-tuning algorithm for the PID controller is derived with the Lyapunov method and therefore, the model predicted tracking error is guaranteed to converge asymptotically. The method is applied to a simulated two-input two-output continuous stirred tank reactor (CSTR) with various faults, which demonstrate the applicability of the developed scheme to industrial processes.

Experimental tumor therapy in mice using the cyclophosphamide-activating cytochrome P450 2B1 gene.

Most malignant tumors of the central nervous system do not respond well to chemotherapy. The anticancer drug cyclophosphamide (CPA) is largely ineffective against these neoplasms as its conversion to DNA-alkylating, cytotoxic metabolites is restricted primarily to the liver and these metabolites do not readily cross the blood-brain barrier. Here, we show that brain tumor cells can be sensitized to the cytotoxic effects of CPA, both in culture and in vivo, by introduction of the hepatic enzyme responsible for the activation of CPA, cytochrome P450 2B1. Stable transfection of rat C6 glioma cells with the P450 2B1 gene rendered the cultured tumor cells sensitive to CPA. Further, C6 cells bearing this gene were more sensitive than parental cells to the cytotoxic action of CPA when grown subcutaneously in the flanks of athymic mice. Murine fibroblasts producing a retrovirus vector encoding P450 2B1 and expressing this enzyme were then prepared and grafted into the brains of athymic mice seeded with rat C6 gliomas. Intrathecal administration of CPA prevented the development of meningeal neoplasia and led to partial regression of the parenchymal tumor mass. By contrast, C6 glioma-bearing mice receiving fibroblasts expressing the Escherichia coli lacZ gene and CPA exhibited extensive meningeal tumors and parenchymal solid brain tumors. The in situ activation of CPA by cytochrome P450 2B1 provides a novel approach not only for brain tumor gene therapy, but also for negative, drug-conditional selection of other defined cell populations.

Identification of the polymorphically expressed CYP2C19 and the wild-type CYP2C9-ILE359 allele as low-Km catalysts of cyclophosphamide and ifosfamide activation.

Cyclophosphamide and ifosfamide are alkylating agent prodrugs that require activation by cytochrome P450 (CYP) to manifest their cancer chemotherapeutic activity. The present study investigates the activity of four individual human CYP2C enzymes and their allelic variants in cyclophosphamide and ifosfamide activation as an initial attempt to gain insight into the underlying basis for the large interpatient differences in the clinical pharmacokinetics and metabolism of these anticancer drugs. Recombinant CYP2C8, CYP2C19, two allelic variants of CYP2C18, and six variants of CYP2C9 expressed in a yeast cDNA expression system were each enzymatically active, as judged by the ability of the isolated microsomes to catalyse 7-ethoxycoumarin O-deethylation after reconstitution with purified NADPH-cytochrome P450 reductase and cytochrome b5. With cyclophosphamide as substrate, CYP2C19 had the lowest apparent Km, followed by CYP2C9, CYP2C18 and CYP2C8, whereas in the case of ifosfamide, the rank order was: Km CYP2C19 < CYP2C18 < CYP2C9 < CYP2C8. CYP2C18 had the highest in vitro intrinsic clearance/catalytic efficiency (apparent Vmax/Km) in cyclophosphamide and ifosfamide activation, followed by 2C19 > 2C9 approximately 2C8. Examination of a panel of CYP2C allelic variants revealed that CYP2C18-Thr385 had both a higher Vmax and a higher apparent Km toward cyclophosphamide than CYP2C18-Met385 with no difference in catalytic efficiency, whereas with ifosfamide the Thr385 allele exhibited a strikingly lower apparent Km resulting in a six-fold higher catalytic efficiency. In the case of CYP2C9, a Ile359 to Leu mutation associated with poor metabolism of the hypoglycemic drug tolbutamide decreased catalytic efficiency toward cyclophosphamide by increasing the apparent Km, whereas the same mutation reduced the efficiency of this P450 toward ifosfamide by decreasing the Vmax. Substitution of CYP2C9-Gly417 by Asp resulted in a two-fold lower catalytic efficiency for cyclophosphamide metabolism but a three-fold higher efficiency for ifosfamide metabolism. A His276 to Gly substitution resulted in an increase in both Vmax and apparent Km with no net change in catalytic efficiency for either oxazaphosphorine. Mutations at CYP2C9 residues 144 and 358 had little or no effect. Thus (a) wild type CYP2C19 and CYP2C9 are relatively low Km catalysts of cyclophosphamide and ifosfamide activation, and (b) all four human CYP2C enzymes activate these two anticancer prodrugs with varying efficiencies and with striking differences among naturally occurring allelic variants in the case of CYP2C9 and CYP2C18.

Molecular cloning, genomic characterization and expression of novel human alpha1A-adrenoceptor isoforms.

We have isolated and characterized from human prostate novel splice variants of the human alpha1A-adrenoceptor, several of which generate truncated products and one isoform, alpha(1A-4), which has the identical splice site as the three previously described isoforms. Long-PCR on human genomic DNA showed that the alpha(1A-4) exon is located between those encoding the alpha(1A-1) and alpha(1A-3) variants. CHO-K1 cells stably expressing alpha(1A-4) showed ligand binding properties similar to those of the other functional isoforms as well as agonist-stimulated inositol phosphate accumulation. Quantitative PCR analyses revealed that alpha(1A-4) is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.

Pharmacogenetics: the therapeutic drug monitoring of the future?

Genetic variability in drug response occurs as a result of molecular alterations at the level of drug-metabolising enzymes, drug targets/receptors, and drug transport proteins. In this paper, we discuss the possibility that therapeutic drug monitoring (TDM) in the future will involve not the mere measurement and interpretation of drug concentrations but will include both traditional TDM and pharmacogenetics-oriented TDM. In contrast to traditional TDM, which cannot be performed until after a drug is administered to the patient. pharmacogenetics-oriented TDM can be conducted even before treatment begins. Other advantages of genotyping over traditional TDM include, but are not limited to, the following: (i) it does not require the assumption of steady-state conditions (or patient compliance) for the interpretation of results; (ii) it can often be performed less invasively (with saliva, hair root or buccal swab samples); (iii) it can provide predictive value for multiple drugs [e.g. a number of cytochrome P450 (CYP) 2D6, CYP2C 19 or CYP2C9 substrates] rather than a single drug; (iv) it provides mechanistic, instead of merely descriptive, information; and (v) it is constant over an individual's lifetime (and not influenced by concurrent drug administration, alteration in hormonal levels or disease states). Pharmacogenetic information can be applied a priori for initial dose stratification and identification of cases where certain drugs are simply not effective. However, traditional TDM will still be required for all of the reasons that we use it now. In current clinical practice, pharmacogenetic testing is performed for only a few drugs (e.g. mercaptopurine, thioguanine, azathioprine, trastuzumab and tacrine) and in a limited number of teaching hospitals and specialist academic centres. We propose that other drugs (e.g. warfarin, phenytoin, codeine, oral hypoglycaemics, tricyclic antidepressants, aminoglycosides, digoxin, cyclosporin, cyclophosphamide, ifosfamide, theophylline and clozapine) are potential candidates for pharmacogenetics-oriented TDM. However, prospective studies of phaymacogenetics-oriented TDM must be performed to determine its efficacy and cost effectiveness in optimising therapeutic effects while minimising toxicity. In the future, in addition to targeting a patient's drug concentrations within a therapeutic range, pharmacists are likely to be making dosage recommendations for individual drugs on the basis of the individual patient's genotype. As we enter the era of personalised drug therapy, we will be able to identify not only the best drug to be administered to a particular patient, but also the most effective and safest dosage from the outset of therapy.

Growth hormone regulation and developmental expression of rat hepatic CYP3A18, CYP3A9, and CYP3A2.

The present study investigated the role of growth hormone (GH) in hepatic CYP3A18 and CYP3A9 expression in prepubertal and adult male rats. For comparison, the effects of GH on CYP3A2 expression were also measured. Initial experiments demonstrated that CYP3A18 mRNA levels were greater during puberty and adulthood than during the prepubertal period, CYP3A9 mRNA was not expressed until puberty and its expression increased in adulthood, and CYP3A2 mRNA levels were relatively constant from prepuberty to adult life. Hypophysectomy, which results in the loss of multiple pituitary factors including GH, increased CYP3A2 and CYP3A18 mRNA expression 3- to 4-fold, but it did not affect CYP3A9 mRNA levels or CYP3A-mediated testosterone 2beta- or 6beta-hydroxylase activity in adult rats. GH administered as twice daily s.c. injections (0.12 microg/g body weight) to hypophysectomized or intact adult rats did not affect CYP3A18 or CYP3A9 mRNA expression. The same treatment decreased CYP3A2 mRNA and protein and testosterone 2beta- and 6beta-hydroxylase activity levels in intact but not hypophysectomized rats. However, in intact prepubertal rats, intermittent GH administration decreased CYP3A18 and CYP3A2 mRNA levels, but a higher dosage (3.6 microg/g) was required to suppress CYP3A2. Overall, the present study demonstrated that: (a) the constitutive expression of CYP3A18, CYP3A9, and CYP3A2 does not require the presence of GH, (b) CYP3A18 is more sensitive than CYP3A9 to GH modulation in adult rats; and (c) CYP3A2 is less sensitive to the suppressive influence of GH during the prepubertal period than during adult life.

Influence of dietary zinc deficiency during development on hepatic CYP2C11, CYP2C12, CYP3A2, CYP3A9, and CYP3A18 expression in postpubertal male rats.

The present study investigated the effect of dietary zinc deficiency during the developmental period on hepatic cytochrome P450 (CYP) expression in postpubertal male rats. Twenty-one-day-old weanling male Wistar rats were randomly assigned to one of the following dietary groups: zinc-adequate (31 mg zinc/kg diet); marginal zinc-deficient (3 mg zinc/kg diet); severe zinc-deficient (1 mg zinc/kg diet); or pair-fed control for either the marginal or severe zinc-deficient group. All rats were killed at 63 days of age. Compared with the corresponding pair-fed controls, marginal zinc deficiency decreased CYP2C11-mediated testosterone 2alpha- and 16alpha-hydroxylase activities by 43 and 42%, respectively, whereas severe zinc deficiency reduced each of these activities by approximately 60%. The decrease in CYP2C11 activity was accompanied by a reduction in CYP2C11 protein and mRNA levels, as assessed by immunoblot and reverse transcription-polymerase chain reaction (RT-PCR) assays, respectively. Additional RT-PCR analysis indicated that severe zinc deficiency decreased CYP3A2 and CYP3A18 mRNA levels by 49 and 43%, respectively, whereas it increased CYP2C12 (253%) and CYP3A9 (238%) mRNA expression. Plasma testosterone concentration was decreased by 67% in the marginal zinc-deficient group when compared with the corresponding pair-fed control group. By comparison, it was below the limit of quantification (0.2 ng/mL) in the severe zinc-deficient rats. Overall, these results indicate that dietary zinc deficiency during the developmental period feminized the hepatic gene expression of the sexually dimorphic CYP2C11, CYP3A2, CYP3A18, CYP2C12, and CYP3A9 in postpubertal male rats.

Impact of tamoxifen on peripubertal androgen imprinting of rat hepatic cytochrome P450 2C11, cytochrome P450 3A2, and steroid 5 alpha-reductase.

Expression of sex-dependent rat hepatic cytochromes P450 and steroid 5 alpha-reductase is regulated mainly by the sex-specific pattern of growth hormone (GH) secretion and is subject to androgen imprinting. Since tamoxifen suppresses GH pulse amplitude and nadir levels, we investigated the effect of tamoxifen on peripubertal testosterone imprinting of hepatic CYP2C11, CYP3A2, CYP2A1, and steroid 5 alpha-reductase. Prepubertal tamoxifen administration (5 mg once daily s.c. on days 28 and 29 of age) to non-ovariectomized female Sprague-Dawley rats did not affect hepatic microsomal CYP2C11-dependent testosterone 2 alpha-hydroxylase, CYP3A-mediated testosterone 6 beta-hydroxylase, CYP2A1-dependent testosterone 7 alpha-hydroxylase, or steroid 5 alpha-reductase activity in adult rats. Testosterone treatment (5 mumol/kg, s.c., once daily) of intact female rats during either puberty (days 35-49 of age) or adult life (days 69-77 of age) had no effect on these enzyme activities in adult (78-day-old) female rats, but the same treatment given during both of these periods induced the male-specific testosterone 2 alpha- and 6 beta-hydroxylase activities and suppressed the female-predominant testosterone 7 alpha-hydroxylase and steroid 5 alpha-reductase activities, indicating that peripubertal testosterone administration imprints the adult androgen responsiveness but not the basal levels of these enzyme activities in non-ovariectomized female rats. However, peripubertal androgen imprinting of the basal levels of testosterone 2 alpha-hydroxylase and steroid 5 alpha-reductase activities was observed in female rats administered tamoxifen prepubertally. Tamoxifen pretreatment also enhanced testosterone imprinting of the adult androgen responsiveness of testosterone 2 alpha- and 6 beta-hydroxylase and steroid 5 alpha-reductase activities. The enhanced testosterone hydroxylase activities were, however, not associated with an increase in microsomal NADPH-cytochrome P450 reductase activity, but were accompanied by elevated hepatic CYP2C11 and CYP3A2 protein levels. Overall, the present study indicates that prepubertal tamoxifen administration does not interfere with the normal sex differentiation of the gender-dependent hepatic cytochromes P450 and steroid 5 alpha-reductase, but this drug modulates peripubertal androgen imprinting of CYP2C11, CYP3A2, and steroid 5 alpha-reductase in adult female rats.

Ligand efficacy and potency at recombinant alpha2 adrenergic receptors: agonist-mediated [35S]GTPgammaS binding.

Alpha-2 adrenergic receptors (alpha2 AR) mediate incorporation of guanosine 5'-O-(gamma-thio)triphosphate ([35S]GTPgammaS) into isolated membranes via receptor-catalyzed exchange of [35S]GTPgammaS for GDP. In the current study, we used [35S]GTPgammaS incorporation to characterize the intrinsic activity and potency of agonists and antagonists at the cloned mouse alpha2a/d and human alpha2a, alpha2b, and alpha2c ARs. Full agonists increased [35S]GTPgammaS binding to membranes by 2- to 3-fold. Antagonists did not increase [35S]GTPgammaS binding but competitively inhibited agonist-stimulated [35S]GTPgammaS binding. Compounds with intrinsic activities less than that of the full agonists norepinephrine (NE) or epinephrine (EPI) were capable of antagonizing agonist-stimulated [35S]GTPgammaS binding. The agonistic properties of a number of alpha2 AR ligands were characterized at each alpha2 AR subtype. The rank order of agonist potency for selected compounds at the human receptors (with intrinsic activity compared with NE, defined as 1.0) was: alpha2a: Dexmedetomidine (0.73) > guanabenz (0.38) > UK-14304 (1.02) > clonidine (0.32) > ST-91 (0.63) > NE (1.00). alpha2b: Dexmedetomidine (1.10) > clonidine (0.18) > guanabenz (0.71) > NE (1.00) > ST-91 (0.44) > UK-14304 (0.59). alpha2c: Dexmedetomidine (1.03) > NE (1.00) > UK-14304 (0.75) > ST-91 (0.32) > or = clonidine (0.23) >> guanabenz (0). This report provides a functional characterization of adrenergic receptor ligands at human and mouse alpha2a/d AR. It also illustrates the utility of [35S]GTPgammaS incorporation as a functional marker of receptor activation.

Inhibitory effect of xylamine on the uptake of [3H]norepinephrine into primary astrocyte cultures.

Primary astrocyte cultures from neonatal rat brains show a Na+-dependent, desipramine-sensitive uptake of [3H]norepinephrine ([3H]NE). Xylamine, a nitrogen mustard, attenuated this uptake of [3H]NE into the astrocytes (IC50 = 0.8 microM). The dose-dependent reduction of [3H]NE uptake by xylamine indicated competitive kinetics. However, xylamine lost the effect if the active transport inhibitor, ouabain or iodoacetate, was also incubated during the pretreatment period of if Na+ was absent. The activity of Na(+)-K(+)-ATPase in astrocytes was not modified by xylamine at the concentrations sufficient to block the uptake of [3H]NE. These findings suggest that xylamine has the ability to compete with the transport of [3H]NE into astrocytes through an effect on the carrier instead of an action on enzymatic activity.

Activation of alpha 1-adrenoceptors to lower cerebrocortical neuropeptide Y (NPY)-like immunoreactivity in rats receiving pargyline treatment.

Role of neuropeptide Y (NPY) in noradrenergic neurotransmission has been mentioned as co-transmitter in both central and peripheral nervous system. Cerebral NPY content was changed by drugs influencing endogenous norepinephrine (NE) in rats. In an attempt to understand this mechanism, the present study was carried out using the radioimmunoassay of NPY. Values of NPY-like immunoreactivity (NPY-ir) were reduced in rats receiving the treatment of pargyline, the inhibitor of monoamine oxidase, with an elevation of catecholamine in parallel. This action was abolished by pretreatment with a mixture of phentolamine, propranolol and haloperidol at concentration sufficient to block the receptors. However, it was not influenced by treatment with haloperidol alone. Cerebrocortical NPY-ir was lowered in rats receiving an intracerebroventricular (i.c.v.) injection of methoxamine, the agonist of alpha 1-adrenoceptors. This action was prevented by prazosin via an i.c.v. injection at the dose sufficient to block alpha 1-adrenoceptors. Moreover, decrease of cerebrocortical NPY-ir by pargyline was also reversed by similar treatment of prazosin. The data obtained suggests that activation of alpha 1-adrenoceptors by endogenous NE which was increased by pargyline may lower the contents of NPY in cerebrocortex of the rat.

Immunocytochemical study of small round cell tumors in routinely processed specimens.

Fourteen commercially available monoclonal antibodies were used to examine, by the indirect immunoperoxidase technique, 20 formaldehyde solution-fixed, routinely processed bone marrow biopsy and surgical biopsy specimens from a group of small round cell tumors with bone marrow involvement, including 6 neuroblastomas, 4 rhabdomyosarcomas, 3 Ewing's sarcomas, 3 Burkitt's lymphomas, and 4 small-cell carcinomas of the lung. Eight of 14 monoclonal antibodies worked well on the routinely processed sections. Antidesmin (D33) (Dako Corp, Santa Barbara, Calif) and anti-muscle actin (HHF35) showed specific immunostaining in all 4 rhabdomyosarcomas included in this study. Anti-epithelial membrane antigen immunoreactivity was positive in all small-cell carcinomas of the lung. Anti-leukocyte common antigen (Dako) showed positive immunostaining in lymphomas and did not immunoreact with nonhematopoietic tumors. However, it was important to be aware that some lymphomas may not have been labeled with anti-leukocyte common antigen. All 6 neuroblastomas showed a positive immunoreactivity with anti-chromogranin A (LK2H10) and a weak reaction with antisynaptophysin (SY38). Panels of commercially available monoclonal antibodies, including anti-leukocyte common antigen, anti-epithelial membrane antigen, antidesmin (Dako), anti-muscle actin, and anti-chromogranin A, appear to be most useful in the differential diagnosis and staging of small round cell tumors on routinely processed biopsy specimens.

Prediction of bone fracture by bone mineral density in Taiwanese.

BACKGROUND AND PURPOSE: This study evaluated whether lumbar spine bone mineral density (BMDL) and the rate of change of BMDL could predict the risk of bone fracture in Taiwanese. METHODS: In 1989, a baseline survey was carried out in Lin-Kou Township. BMDL was measured using dual photon absorptiometry in 404 healthy volunteers. In 1994/95, a second survey was conducted to reexamine the BMDL and fracture history as well as the risk factors associated with fracture in these subjects. Fractures were classified according to the causes of the injury as severe or mild trauma. RESULTS: Of the 404 original examinees, 381 (79%) participated in the second survey. Twenty-six subjects had experienced a total of 32 fracture incidents during their lifetime and 10 subjects had experienced fracture during the observation period. After adjusting for the effect of body mass index, gender, and age, each standard deviation of decrease in BMDL was found to be associated with a significant 2.38-fold increase in the risk of mild trauma fracture, and a BMDL below the fracture threshold was associated with a 3.93-fold increase of fracture risk. Higher BMDL change rates were found in subjects with a history of fracture. CONCLUSION: This study indicates the importance of maintaining the BMDL above the fracture threshold (1 g/cm2). The strategy for fracture prevention should be directed at prevention of age-related bone loss.

Thanatophoric dysplasia type I.

Thanatophoric dysplasia is a sporadic, nearly always lethal congenital skeletal dysplasia. It is characterized by shortening of the limbs, a severely small thorax, macrocephaly, and platyspondyly. There are two major subtypes: a short, curved femur characterizes type I, and a straighter femur with cloverleaf skull characterizes type II. Recently, mutations in the fibroblast growth factor receptor 3 (FGFR-3) gene have been identified in both subtypes, which suggest that thanatophoric dysplasia is a genetically homogenous skeletal disorder. Most affected neonates die of respiratory failure, due to narrow thorax with pulmonary hypoplasia. Antenatal sonographic diagnosis is feasible in the second trimester of pregnancy, but differentiating thanatophoric dysplasia from non-lethal skeletal disorders is very important. At the present time, however, prenatal genetic screening seems unpractical.