Recombinant growth hormone therapy for prepubertal children with idiopathic short stature in Korea: a phase III randomized trial.

PURPOSE: Several studies have evaluated the effects of growth hormone (GH) on auxological and biochemical parameters in children with non-GH-deficient, idiopathic short stature (ISS). This study evaluated the efficacy and safety of Growtropin®-II (recombinant human GH) in Korean patients with ISS. METHODS: This was a 1-year, open-label, multicenter, phase III randomized trial of Growtropin®-II in Korean patients with ISS. In total, 70 prepubertal subjects (39 males, 31 females) between 4 and 12 years of age were included in the study. All patients were naive to GH treatment. RESULTS: Annual height velocity was significantly higher in the treatment group (10.68 ± 1.95 cm/year) than the control group (5.72 ± 1.72, p < 0.001). Increases in height and weight standard deviation scores (SDSs) at 26 weeks were 0.63 ± 0.16 and 0.64 ± 0.46, respectively, for the treatment group, and 0.06 ± 0.15 and 0.06 ± 0.28, respectively, for the control group (p < 0.001). Serum insulin-like growth factor (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) increased significantly in the treatment group at week 26 compared to baseline. However, the SDS for body mass index (BMI) at 26 weeks did not change significantly in either group. Growtropin®-II was well tolerated and safe over 1 year of treatment. CONCLUSIONS: One-year GH treatment for prepubertal children with ISS demonstrated increased annualized velocity, height and weight SDSs, and IGF-1 and IGFBP-3 levels, with a favorable safety profile. Further evaluations are needed to determine the optimal dose, final adult height, and long-term effects of ISS treatment.

Pharmacogenomics of insulin-like growth factor-I generation during GH treatment in children with GH deficiency or Turner syndrome.

Individual responses to growth hormone (GH) treatment are variable. Short-term generation of insulin-like growth factor-I (IGF-I) is recognized as a potential marker of sensitivity to GH treatment. This prospective, phase IV study used an integrated genomic analysis to identify markers associated with 1-month change in IGF-I (ΔIGF-I) following initiation of recombinant human (r-h)GH therapy in treatment-naïve children with GH deficiency (GHD) (n=166) or Turner syndrome (TS) (n=147). In both GHD and TS, polymorphisms in the cell-cycle regulator CDK4 were associated with 1-month ΔIGF-I (P<0.05). Baseline gene expression was also correlated with 1-month ΔIGF-I in both GHD and TS (r=0.3; P<0.01). In patients with low IGF-I responses, carriage of specific CDK4 alleles was associated with MAPK and glucocorticoid receptor signaling in GHD, and with p53 and Wnt signaling pathways in TS. Understanding the relationship between genomic markers and early changes in IGF-I may allow development of strategies to rapidly individualize r-hGH dose.

Congenital erythropoietic porphyria: identification and expression of exonic mutations in the uroporphyrinogen III synthase gene.

Congenital erythropoietic porphyria (CEP), an inborn error of heme biosynthesis, results from the deficient activity of uroporphyrinogen III synthase (URO-synthase). This autosomal recessive disorder is heterogeneous; patients with severe disease are often transfusion dependent, while milder patients primarily have cutaneous involvement. To investigate this phenotypic heterogeneity, exonic point mutations in the URO-synthase gene were identified in unrelated CEP patients. Four missense mutations were identified: (a) an A to G transition of nucleotide (nt) 184 that predicted a Thr to Ala substitution at residue 62 (designated T62A); (b) a C to T transition of nt 197 that encoded an Ala to Val replacement at residue 66 (A66V); (c) a T to C transition of nt 217 that predicted a Cys to Arg substitution at residue 73 (C73R); and (d) a C to T transition of nt 683 that resulted in a Thr to Met replacement at residue 228 (T228M). In addition, a G to A transition of nt 27 that did not change the encoded amino acid (A9A) was detected in an African patient. The T62A, C73R, and T228M alleles did not express detectable enzymatic activity, while the A66V allele expressed residual, but unstable activity. The C73R allele was present in eight of 21 unrelated CEP patients (21% of CEP alleles). In three patients, identification of both alleles permitted genotype-phenotype correlations; the A66V/C73R, T228M/C73R, and C73R/C73R genotypes had mild, moderately severe, and severe disease, respectively. These findings provide the first genotype-phenotype correlations and permit molecular heterozygote detection in this inherited porphyria.

Oral V2 receptor antagonist (RWJ-351647) in patients with cirrhosis and ascites: a randomized, double-blind, placebo-controlled, single ascending dose study.

BACKGROUND: RWJ-351647 is a selective V2 receptor antagonist that inhibits vasopressin-induced water reabsorption in the kidney. AIM: To investigate the safety and tolerability of RWJ-351647 compared with placebo after single oral dose administration to patients with cirrhosis and ascites, on a stable treatment with furosemide and spironolactone. METHODS: Single oral doses of 1, 2 and 5 mg of RWJ-351647 were administered to 24 patients with ascites on stable concomitant diuretic treatment. RESULTS: RWJ-351647 had a tmax of 1 to 1.1 h and mean half-life of 10.4-17.4 h. There was no affect on the pharmacokinetics of concomitant diuretics. Increases in cumulative urine volume and free water excretion, and a decrease in urine osmolality were noted in a dose-dependent manner reaching the statistical significance at the 5-mg dose. Four patients exhibited a decrease of > 2 kg in weight in the 24 h after dosing. RWJ-351647 was well tolerated, with no evidence of a dose-related increase in adverse events when compared with placebo. No changes in either serum chemistry or plasma AVP (arginine vasopressin) and renin levels were observed despite the observed aquaresis. CONCLUSION: RWJ-351647 is an effective aquaretic causing dose-dependent increases in urine output and free water clearance, when co-administered with conventional diuretics in patients with cirrhosis and ascites.

Plasma and cerebrospinal fluid pharmacokinetics of O6-benzylguanine and analogues in nonhuman primates.

O6-Benzylguanine (BG) is a potent, specific inactivator of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, that enhances the sensitivity of tumor cell lines and tumor xenografts to chloroethylnitrosoureas. To search for BG analogues with greater penetration into the cerebrospinal fluid (CSF), we evaluated plasma and CSF pharmacokinetics of BG, 8-aza-O6-benzylguanine (8-azaBG), O6-benzyl-8-bromoguanine (8-BrBG), O6-benzyl-8-oxoguanine (8-oxoBG), O6-benzyl-8-trifluoromethylguanine (8-tfmBG), and O6-benzyl-2'-deoxyguanosine (B2dG) after i.v. administration of 200 mg/m2 of drug through an indwelling Ommaya reservoir in a nonhuman primate model. BG and its analogues were quantified in plasma and CSF using reverse-phase high-performance liquid chromatography assays. The plasma clearances of the four 8-substituted BG analogues were similar (0.04-0.06 l/h/kg), but half-lives ranged from <2 to >24 h. BG was converted to 8-oxoBG, an equally potent O6-alkylguanine-DNA alkyltransferase inactivator, and the elimination of 8-oxoBG was much slower than that of BG. As a result, the plasma area under the curve of 8-oxoBG was 3.5-fold greater than that of BG. B2dG was metabolized to BG and 8-oxoBG, but this pathway accounted for only 20% of B2dG elimination. The CSF penetration percentages (based on the ratio of AUC(CSF): AUCplasma) for BG, 8-azaBG, 8-oxoBG, 8-tfmBG, 8-BrBG, and B2dG were 3.2, 0.18, 4.1, 1.4, <0.3, and 2.0%, respectively. The CSF penetration of BG and its active metabolite 8-oxoBG is greater than the penetration of 8-azaBG, 8-BrBG, 8-tfmBG, and B2dG.

Synthesis and cytotoxicity of 2-methyl-4, 9-dihydro-1-substituted-1H-imidazo[4,5-g]quinoxaline-4,9-diones and 2,3-disubstituted-5,10-pyrazino[2,3-g]quinoxalinediones.

Series of 2-methyl-4,9-dihydro-1H-imidazo[4,5-g]quinoxaline-4, 9-diones and 5,10-pyrazino[2,3-g]quinoxalinediones have been synthesized from 6,7-dichloro-5,8-quinoxalinedione for developing new anticancer drugs. Intercalation complex of 2-methyl-4, 9-dihydro-1-methyl-1H-imidazo[4,5-g]quinoxaline-4,9-dione with GC/GC base pairs by a computer graphics-aided method was parallel to the axis of the helix. The interaction energies of synthetic compounds were low. 2-Methyl-4,9-dihydro-1-(4-bromophenyl)-1H-imidazo[4, 5-g]quinoxaline-4,9-dione, which has the lowest interaction energy of the test compounds, was identified as being more cytotoxic against human gastric adenocarcinoma cells (MKN 45) than adriamycin and cis-platin in vitro using the MTT assay. The IC50 value of this compound was 1.30 microM, whereas those of adriamycin and cis-platin were 3.13 and 86.5 microM, respectively. The cytotoxicity of 2, 3-diethyl-5,10-pyrazino[2,3-g]quinoxalinedione was comparable to that of adriamycin in the in vitro assay. However these compounds showed loss of activity on human lung adenocarcinoma cells (PC 14) and human colon adenocarcinoma cells (colon 205). These results suggest that 2-methyl-4,9-dihydro-1-(4-bromophenyl)-1H-imidazo[4, 5-g]quinoxaline-4,9-dione, with the lowest interaction energy, might be an excellent and selective antitumor agent against MKN 45.

Localized proton MR spectroscopy in infants with urea cycle defect.

SUMMARY: Urea cycle defect is an inborn error of ammonium metabolism caused by a deficient activity of the enzymes involved in urea synthesis. Localized short-TE proton MR spectroscopy, performed in two infants who had citrullinemia and ornithine transcarbamylase deficiency, respectively, showed a prominent increase of glutamine/glutamate and lipid/lactate complex in both cases. N-acetylaspartate, total creatine, and myo-inositol were decreased in the infant with citrullinemia. Proton MR spectroscopy provided useful information for the diagnosis and understanding of the pathophysiology of urea cycle enzyme defect.

Molecular and clinical characterization of Korean patients with congenital lipoid adrenal hyperplasia.

Congenital lipoid adrenal hyperplasia (CLAH) is a rare autosomal recessive disorder of steroid biosynthesis, caused by a molecular defect in the steroidogenic acute regulatory protein (StAR). Patients with CLAH usually manifest severe salt wasting, hypovolemia, enlargement of the adrenal glands and complete female external genitalia irrespective of the gonadal sex. CLAH seems to be more common in Koreans and Japanese than in other ethnic populations, with a preponderance of the mutation of a glutamine to a stop codon at the 258th amino acid residue (Q258X) in the StAR gene. Clinical findings of five unrelated Korean patients with CLAH and their molecular defects in the StAR gene are described, and the gene frequency of the Q258X mutation in the Korean population is investigated. All patients developed hypovolemic shock due to severe salt wasting in the first three months after birth. All were hyperpigmented, and three of five phenotypic females were genetic males. The basal ACTH level was extremely high in all patients with the minimal concentrations of all adrenal and gonadal steroids. The Q258X mutation was identified in 9 out of 10 alleles, indicating that the genetic defect in the StAR gene of Korean patients with CLAH is highly homogeneous probably due to a founder effect. The carrier frequency of the Q258X mutation in the normal Korean population has been estimated as approximately 1 in 250 with the allele frequency of 1 in 500. However, the confidence limits of the gene frequency for the mutant allele are wide (0.5 to 8.0 among 1,000 alleles). This implies that the carrier frequency could be lower, down to 1/1,000, or higher, up to 16/1,000.

Automated spherical aberration correction in scanning confocal microscopy.

Mismatch between the refractive indexes of immersion media and glass coverslips introduces spherical aberrations in microscopes especially for high numerical aperture objectives. This contribution demonstrates an automated adjustment of the coverslip correction collar in scanning confocal microscopy to compensate for spherical aberrations due to coverslip thickness mismatch. With a motorized coverslip correction collar, the adjustment procedure consists of xz image scans, image processing, correction quality evaluation, the mismatch estimation, and eventually the optimal adjustment of the correction collar. For fast correction with less photodamage, coarse-fine Gaussian fitting algorithms are proposed and evaluated with various specimen for their estimation accuracy. The benefits of the proposed automated correction are demonstrated for various coverslips with biological specimens, showing the optimized resolution of the confocal microscope.

A pharmacogenomic approach to the treatment of children with GH deficiency or Turner syndrome.

OBJECTIVE: Individual sensitivity to recombinant human GH (r-hGH) is variable. Identification of genetic factors contributing to this variability has potential use for individualization of treatment. The objective of this study was to identify genetic markers and gene expression profiles associated with growth response on r-hGH therapy in treatment-naïve, prepubertal children with GH deficiency (GHD) or Turner syndrome (TS). DESIGN: A prospective, multicenter, international, open-label pharmacogenomic study. METHODS: The associations of genotypes in 103 growth- and metabolism-related genes and baseline gene expression profiles with growth response to r-hGH (cm/year) over the first year were evaluated. Genotype associations were assessed with growth response as a continuous variable and as a categorical variable divided into quartiles. RESULTS: Eleven genes in GHD and ten in TS, with two overlapping between conditions, were significantly associated with growth response either as a continuous variable (seven in GHD, two in TS) or as a categorical variable (four more in GHD, eight more in TS). For example, in GHD, GRB10 was associated with high response (≥ Q3; P=0.0012), while SOS2 was associated with low response (≤ Q1; P=0.006), while in TS, LHX4 was associated with high response (P=0.0003) and PTPN1 with low response (P=0.0037). Differences in expression were identified for one of the growth response-associated genes in GHD (AKT1) and for two in TS (KRAS and MYOD1). CONCLUSIONS: Carriage of specific growth-related genetic markers is associated with growth response in GHD and TS. These findings indicate that pharmacogenomics could have a role in individualized management of childhood growth disorders.

Clinical characterization and molecular classification of 12 Korean patients with pseudohypoparathyroidism and pseudopseudohypoparathyroidism.

CONTEXT: Pseudohypoparathyroidism (PHP) is defined as resistance toward parathyroid hormones. PHP and pseudopseudohypoparathyroidism (PPHP) are rare disorders resulting from genetic and epigenetic aberrations within or upstream of the GNAS locus. This study investigated the clinical characteristics and performed a molecular analysis of PHP and PPHP. METHODS: A total of 12 patients with (P)PHP from 11 unrelated families (4 with PHP-Ia, 6 with PHP-Ib, and 2 with PPHP) were characterized using both clinical and molecular methods. Clinical features included the presenting symptoms, Albright hereditary osteodystrophy features, and resistance to hormones. Comprehensive analysis of the GNAS and STX16 loci was undertaken to investigate the molecular defects underlying (P)PHP. RESULTS: All PHP-Ib patients displayed hypocalcemic symptoms. All PHP-Ia patients showed resistance toward TSH, in addition to PTH. In most patients with PHP, when the diagnosis of PHP was first established, hypocalcemia and hyperphosphatemia were associated with a significant increase in serum PTH levels. One patient with PHP-Ia was diagnosed with growth hormone deficiency and showed a good response to human recombinant growth hormone therapy. 6 patients with PHP-Ia and PPHP showed 5 different mutations in the GNAS gene. 5 patients with PHP-Ib displayed a loss of differentially methylated region (DMR) imprints of the maternal GNAS. One PHP-Ib patient showed a de novo microdeletion in STX16 and a loss of methylation of exon A/B on the maternal allele. No patients revealed paternal disomy among 4 patients with PHP-Ib. CONCLUSIONS: Identification of the molecular causes of PHP and PPHP explains their distinctive clinical features and enables confirmation of the diagnosis and exact genetic counseling.

Clinical phenotype and mutation spectrum of the CYP21A2 gene in patients with steroid 21-hydroxylase deficiency.

Steroid 21-hydroxylase deficiency is caused by inactivating mutations in the CYP21A2 gene. This paper reports on the mutation spectrum and the genotype-phenotype correlation of 21-hydroxylase deficiency. 72 unrelated patients with congenital adrenal hyperplasia (CAH) were included. Molecular analysis of CYP21A2 was performed, via the multiplex ligation-dependent probe amplification (MLPA) analysis and sequence-specific differenzial PCR amplification of the CYP21A2 and CYP21A1P genes, using 4 pair-wise sequence-specific primers, followed by sequencing of the entire CYP21A2 gene. Large gene deletions were identified in 45 (31.3%) of the 144 unrelated CAH alleles, whereas the most frequent point mutations were intron 2 splice mutations (c.293-13A>G) (41/144, 28.5%). The MLPA analysis successfully identified 23 of 72 patients (31.9%) with single copy deletion in CYP21A2. This paper describes a rapid and accurate method for the molecular diagnosis of 21-hydroxylase deficiency, which relies on the identification of point mutations and structural rearrangements within the CYP21A2 gene.

Successful liver transplantation for a child with life-threatening recurrent bleeding episodes due to congenital factor X deficiency: a case report.

Factor X (FX) deficiency is a rare, autosomal-recessive coagulation disorder. Diagnosis can be confirmed by a factor X assay. Although fresh frozen plasma and prothrombin complex concentrates have been used as a temporary treatment of bleeding symptoms and preparation for surgery, frequent transfusion has its risk and prothrombin complex is not available in Korea. We report the first pediatric case of successful liver transplantation for the correction of a severe congenital FX deficiency in a child with recurrent life-threatening hemorrhagic episodes.

Two cases of citrullinaemia presenting with stroke.

Urea cycle disorders are known to be unusual causes of stroke. We report two infant cases of citrullinaemia that both initially presented with a stroke and suggest that urea cycle disorders should be included in the differential diagnosis of unexplained stroke during infancy and childhood.

Characteristics of CADASIL in Korea: a novel cysteine-sparing Notch3 mutation.

OBJECTIVE: To elucidate the phenotype, genotype, and MRI findings of Korean patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and mutation carriers. METHODS: The authors studied 40 members of nine unrelated Korean CADASIL families. After genetic analysis of Notch3, clinical and MRI findings were correlated in 27 mutation carriers. RESULT: Notch3 mutation sites were C174R (one family, n = 3), R133C (one family, n = 3), R587C (one family, n = 1), R544C (two families, n = 5), and R75P (four families, n = 15). The clinical features were typical of CADASIL, but the frequency of migraine in the Korean population appears low. MRI abnormalities were found in 54% of the mutant carriers, the most common being white matter hyperintensities. The prevalence of lacunes and microbleeds increased with patient age. Anterior temporal areas were less often involved in subjects with R75P mutations than in those where mutations occurred in other sites (p = 0.02). Gradient echo imaging identified microbleedings in 33% of mutation carriers (64% of those with abnormal MRI), whereas diffusion-weighted MRI showed abnormal findings in only one patient. Neurologic disability was related to the number of lacunar infarcts and the lesion volume of white matter hyperintensities (p < 0.001) whereas MMSE score was related to the number of lacunar infarcts (p < 0.005). CONCLUSIONS: Although Korean cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) mutation carriers show similar clinical and MRI findings, these abnormalities appear less frequently than in other populations. Relatively frequent microbleedings on gradient echo imaging suggest that treatment should be individualized according to MRI findings. The novel mutation of R75P, not involving a cysteine residue, is related to less frequent involvement of the anterior temporal area, thus broadening the spectrum of CADASIL.

Prenatal molecular evaluation of six fetuses in four unrelated Korean families with ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) deficiency, an X-linked inborn error of the urea cycle, leads to the accumulation of ammonia, causing neurologic deficits. Clinical management for the patients with OTC deficiency is frustrating and requires a burdensome medical regimen, since they may have impairment and recurrent episodes of hyperammonemia in spite of intensive care. Therefore, prenatal diagnosis of the affected fetus is important in genetic counselling for the family at high risk. In this study, mutations in the OTC gene of three obligate heterozygotes and a proband have been identified in four unrelated families: R141Q, R320X, H214Y, M205T. Each mutation altered restriction recognition sites; TaqI for R141Q, NlaIII for M205T, RsaI for H214Y, BclI for R320X. Based on their molecular defects, prenatal diagnoses of 6 fetuses including one set of fraternal twins were successfully made at the ninth to eleventh week of gestation by polymerase chain reaction (PCR)-restriction digestion using genomic DNA from chorionic villus sampling (CVS). We predicted the outcome of all fetuses prenatally. Among six, four were females and two were males, which were determined by PCR amplification of the sex determining region of the Y chromosome (SRY) gene. Each carried a wild type allele for the corresponding mutant allele. They were also tested postnatally for the mutations to be unaffected.

Comparison of enzyme and DNA analysis in a Tay-Sachs disease carrier screening program.

Tay-Sachs disease (GM2 gangliosidosis, type 1; TSD) is an autosomal recessive GM2 gangliosidosis resulting from the deficient activity of the lysosomal hydrolase beta-hexosaminidase A (Hex A). With a carrier frequency estimated at 1 in 25, it is a common lysosomal disorder in the Ashkenazi Jewish population. Tay-Sachs disease has provided the prototype for the prevention of severe recessive genetic diseases. Molecular analysis of the Hex A gene (HEXA) of Ashkenazi Jewish individuals affected with Tay-Sachs disease revealed that three common mutations cause the infantile and adult onset forms of the disease; a four base insertion in exon 11, a splice junction mutation in intron 12 and a point mutation in exon 7 (G269S). A study was undertaken to determine whether mutation analysis would be useful in TSD screening programs in identifying carriers and clarifying the status of individuals whose enzyme assays are inconclusive. Ashkenazi Jewish individuals who had been diagnosed as carriers, inconclusives by enzyme assay and non-carriers with low normal enzyme levels in the Mount Sinai Tay-Sachs Disease Prevention Program were examined for the presence of the three mutations using polymerase chain reaction (PCR) and allele specific oligonucleotide (ASO) hybridization. The insertion mutation was present in 29 of 34 carriers and 2 of 36 inconclusive individuals, the splice junction mutation was found in 4 of 34 carriers and the G269S mutation was found in 1 of 34 carriers. Of the 313 non-carrier individuals with normal enzyme activity in the lower normal range, one was positive for the splice junction mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of new mutations in the ornithine transcarbamylase (OTC) gene in Korean families.

Ornithine transcarbamylase (OTC) deficiency, an X-linked inborn error of the urea cycle, is known to be heterogeneous genetically as well as phenotypically. Molecular defects of Korean patients with OTC deficiency have not been reported. To investigate molecular lesions resulting in OTC deficiency, the OTC genes of unrelated symptomatic or asymptomatic female heterozygotes were amplified exon by exon and analysed by direct sequencing of double-stranded DNA. Three new mutations, two missense and one nonsense, were detected: (1) a C-to-T transition in codon 44 in exon 2 replacing a threonine by an isoleucine (T44I) was found in a late-onset symptomatic female patient but not in her asymptomatic mother; (2) a C-to-T transition in codon 214 creating a new RsaI recognition site in exon 6 and substituting tyrosine for histidine (H214Y) was identified in an asymptomatic female carrier whose son developed acute neonatal onset of OTC deficiency; (3) a C-to-T transition in codon 320 (arginine) abolishing a TaqI recognition site and creating a new BclI site in exon 9 with generation of a stop codon (R320X) leading to premature termination in the enzyme. This nonsense mutation was found in a symptomatic female patient and her asymptomatic mother whose son died of OTC deficiency during the neonatal period. In addition, we found a G-to-A transition in codon 141 in exon 5 causing substitution of glutamine for arginine (R141Q) in a female obligate heterozygote whose previous three sons succumbed to acute neonatal-onset OTC deficiency. This missense mutation has been described previously and is known to eliminate a TaqI recognition site in exon 5.

Hydroxymethylbilane synthase: complete genomic sequence and amplifiable polymorphisms in the human gene.

Acute intermittent porphyria (AIP), an autosomal dominant inborn error of heme biosynthesis, results from the half-normal activity of the heme biosynthetic enzyme hydroxymethylbilane synthase (HMB-synthase). Heterozygous individuals are prone to life-threatening acute neurologic attacks, which are precipitated by certain drugs and other metabolic, hormonal, and nutritional factors. Since the biochemical diagnosis of heterozygous individuals has been problematic, recent efforts have focused on the identification of mutations and diagnostically useful restriction fragment length polymorphisms (RFLPs) in the HMB-synthase gene. To facilitate these endeavors, the human HMB-synthase gene, including 1.1 kg of the 5' flanking region, was isolated and completely sequenced in both orientations. The 10,024-bp gene contained 15 exons ranging in size from 39 to 438 bp and 14 introns ranging from 87 to 2913 bp. All intron/exon boundaries conformed to the GT/AG consensus rule. There were six Alu repetitive elements, one of the J and five of the Sa subfamilies. Analysis of the 1.1-kb 5' flanking region revealed putative regulatory elements for the housekeeping promoter including AP1, AP4, SP1, TRE, ENH, and CAC. This region contained 10 HpaII sites and had an overall GC content of 54%. Intron 1, which contained the erythroid-specific promoter, had putative regulatory motifs for NF-1, NF-E1, NF-E1(b), NF-E2, AP1, AP4, TOPO, CAAC, CAC, CAAT, and TATA. The locations and variant nucleotides for the known RFLPs in intron 1 were identified [MspI, nucleotide 1345 G/A; PstI, 1500 C/T; ApaLI, 2377 C/A; and BstNI, 2479 G/A] and improved polymerase chain reaction (PCR)-based detection methods for each were established.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxic effects of pyridino[2,3-f]indole-4,9-diones on human tumor cell lines.

The cytotoxicities of pyridino[2,3-f]indole-4,9-dione derivatives were examined against human lung tumor cell lines (A 549), human ovarian tumor cell lines (SK-OV-3), human melanoma tumor cell lines (SK-MEL-2), human CNS tumor cell lines (XF 498) and human colon tumor cell lines (HCT 15) in vitro using a Sulforhodamine B assay. 3-Ethoxycarbonyl-1-(2-methoxyethyl)-2-methyl-1H-pyridino[2,3-f]ind ole-4,9-dione (5) showed excellent cytotoxicity against XF 498 and HCT 15. The ED50 values of 5 were 0.006 microg/ml against XF 498 and 0.073 microg/ml against HCT 15, while those of doxorubicin were 0.012 and 0.264 microg/ml, respectively. 1-Benzyl-3-ethoxycarbonyl-2-methyl-1H-pyridino[2,3-f]indole-4,9-di one (7) (ED50 value 0.065 microg/ml) was also significantly more cytotoxic against HCT 15 compared with doxorubicin.