Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1

SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 by to +78 by is required for the core promoter activity. No consensus TATA or CHAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis. (C) 2008 Elsevier B.V. All rights reserved.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

Purpose: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease.

Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1

SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 by to +78 by is required for the core promoter activity. No consensus TATA or CHAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis. (C) 2008 Elsevier B.V. All rights reserved.

Array-based gene expression, CGH and tissue data defines a 12q24 gain in neuroblastic tumors with prognostic implication

Neuroblastoma has successfully served as a model system for the identification of neuroectoderm-derived oncogenes. However, in spite of various efforts, only a few clinically useful prognostic markers have been found. Here, we present a framework, which integrates DNA, RNA and tissue data to identify and prioritize genetic events that represent clinically relevant new therapeutic targets and prognostic biomarkers for neuroblastoma.

Isolation and properties of catechol-cleaving yeasts from coir rets

A suitable method for the selective isolation of catechol-cleaving yeasts from coir rets has been worked out. The yeast strains, all belonging toDebaryomyces hansenii, were found to demand biotin as an essential vitamin. The organism has the ability to grow on catechol, phenol and some related compounds as sole source of carbon. It tolerates 0.4% catechol and 0.26% phenol. Evidence was obtained that the catechol-cleaving enzyme of the isolates is a pyrocatechase. Some properties of the cell-free catechol oxygenase are described.

The Isolation and Characterization of β-N-Oxalyl-L-α,β-Diaminopropionic Acid: A Neurotoxin from the Seeds of Lathyrus sativus

A neurotoxic compound has been isolated from the seeds of Lathyrus sativus in 0.5% yield and characterized as β-N-oxalyl-L-α,β-diaminopropionic acid. The compound is highly acidic in character and forms oxalic acid and diaminopropionic acid on acid hydrolysis. The compound has a specific rotation of -36.9° and has apparent pK values in the order of 1.95, 2.95, and 9.25, corresponding to the two carboxyl and one amino functions, respectively. The compound has been synthesized by reacting an aqueous methanolic solution of the copper complex of L-α,β-diaminopropionic acid prepared at pH 4.5-5.0 with dimethyl oxalate under controlled pH conditions and isolating the compound by chromatography on a Dowex 50-H+ column after precipitating the copper. The compound induced severe neurological symptoms in day-old chicks at the level of 20 mg/chick, but not in rats or mice. It also inhibited the growth of several microorganisms and of the insect larva Corcyra cephalonica Staint. L-Homoarginine had no neural action in chicks. It is suggested that the neurotoxic compound is species specific in its action and may be related to "neurolathyrism" associated with the human consumption of L. sativus seeds.

Part I: Enzyme replacement versus neurotrophic factor viral vector-mediated gene therapy of Parkinson’s disease, Part II: The effects of orally dosed tolcapone and entacapone on dopamine metabolism in the dorsal striatum and nucleus accumbens in rats

Part I: Parkinson’s disease is a slowly progressive neurodegenerative disorder in which particularly the dopaminergic neurons of the substantia nigra pars compacta degenerate and die. Current conventional treatment is based on restraining symptoms but it has no effect on the progression of the disease. Gene therapy research has focused on the possibility of restoring the lost brain function by at least two means: substitution of critical enzymes needed for the synthesis of dopamine and slowing down the progression of the disease by supporting the functions of the remaining nigral dopaminergic neurons by neurotrophic factors. The striatal levels of enzymes such as tyrosine hydroxylase, dopadecarboxylase and GTP-CH1 are decreased as the disease progresses. By replacing one or all of the enzymes, dopamine levels in the striatum may be restored to normal and behavioral impairments caused by the disease may be ameliorated especially in the later stages of the disease. The neurotrophic factors glial cell derived neurotrophic factor (GDNF) and neurturin have shown to protect and restore functions of dopaminergic cell somas and terminals as well as improve behavior in animal lesion models. This therapy may be best suited at the early stages of the disease when there are more dopaminergic neurons for neurotrophic factors to reach. Viral vector-mediated gene transfer provides a tool to deliver proteins with complex structures into specific brain locations and provides long-term protein over-expression. Part II: The aim of our study was to investigate the effects of two orally dosed COMT inhibitors entacapone (10 and 30 mg/kg) and tolcapone (10 and 30 mg/kg) with a subsequent administration of a peripheral dopadecarboxylase inhibitor carbidopa (30 mg/kg) and L- dopa (30 mg/kg) on dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens of freely moving rats using dual-probe in vivo microdialysis. Earlier similarly designed studies have only been conducted in the dorsal striatum. We also confirmed the result of earlier ex vivo studies regarding the effects of intraperitoneally dosed tolcapone (30 mg/kg) and entacapone (30 mg/kg) on striatal and hepatic COMT activity. The results obtained from the dorsal striatum were generally in line with earlier studies, where tolcapone tended to increase dopamine and DOPAC levels and decrease HVA levels. Entacapone tended to keep striatal dopamine and HVA levels elevated longer than in controls and also tended to elevate the levels of DOPAC. Surprisingly in the nucleus accumbens, dopamine levels after either dose of entacapone or tolcapone were not elevated. Accumbal DOPAC levels, especially in the tolcapone 30 mg/kg group, were elevated nearly to the same extent as measured in the dorsal striatum. Entacapone 10 mg/kg elevated accumbal HVA levels more than the dose of 30 mg/kg and the effect was more pronounced in the nucleus accumbens than in the dorsal striatum. This suggests that entacapone 30 mg/kg has minor central effects. Also our ex vivo study results obtained from the dorsal striatum suggest that entacapone 30 mg/kg has minor and transient central effects, even though central HVA levels were not suppressed below those of the control group in either brain area in the microdialysis study. Both entacapone and tolcapone suppressed hepatic COMT activity more than striatal COMT activity. Tolcapone was more effective than entacapone in the dorsal striatum. The differences between dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens may be due to different properties of the two brain areas.

A method for the preferential isolation of actinomycetes from soils

A successful plate-method for the preferential isolation of actinomycetes from soils is described. The principles underlying it are: (1) the inhibition of growth of non-sporulating bacteria by pre-incubation at a high temperature (110 C) for 10 min, and (2) limiting the spreading growth of sporeforming bacteria and fungi by the use of dried plates. The majority of the 191 species isolated by this method from 82 soil samples were shown to be pectinolytic.

Isolation and partial characterization of sheep plasma α1-mucoprotein

1. 1. Sheep plasma α1-mucoprotein was isolated in an electrophoretically homogeneous state by a combination of ammonium sulphate saturation, isoelectric precipitation and preparative agar electrophoresis in a yield of approx. 150 mg/l of plasma. 2. 2. The mucoprotein was water-soluble, non-coagulable on heating at 100°, not precipitable by 1.8 M perchloric acid, 10% trichloroacetic acid but precipitable by saturated ammonium sulphate solution, 0.6 M sulfosalicylic acid and 5% phosphotungstic acid in 2 N HCl. It had E1 cm1 % value of 9.57 at 278 mμ in water, refractive-index index increment 1.9·10-4 (g/l) in water, isoelectric point at pH 4.45 (sodium acetate-acetic acid buffer) and was homogeneous in pH range 4.0-11.5 but at pH values 2.6 and 3.5 showed some dissociation. 3. 3. The mucoprotein had the following chemical composition: Nitrogen, 12.4%; polypeptide, 77.4%; total hexose (only mannose and galactose), 7.1%; fucose, 1.0%; glucosamine, 4.9% and sialic acid, 4.8%. It had no N-terminal amino acid.

Isolation of Salmonella typhimurium mutants affecting the plaque morphology of phage P22

Salmonella typhimurium mutants affecting the plaque morphology of P22 and other phages have been isolated. Using one such bacterial mutant phage mutants making turbid plaques have been isolated.

Genetic variation in the LDL receptor-related protein 5 (LRP5) gene : Association with bone health and metabolic parameters

Bone mass accrual and maintenance are regulated by a complex interplay between genetic and environmental factors. Recent studies have revealed an important role for the low-density lipoprotein receptor-related protein 5 (LRP5) in this process. The aim of this thesis study was to identify novel variants in the LRP5 gene and to further elucidate the association of LRP5 and its variants with various bone health related clinical characteristics. The results of our studies show that loss-of-function mutations in LRP5 cause severe osteoporosis not only in homozygous subjects but also in the carriers of these mutations, who have significantly reduced bone mineral density (BMD) and increased susceptibility to fractures. In addition, we demonstrated for the first time that a common polymorphic LRP5 variant (p.A1330V) was associated with reduced peak bone mass, an important determinant of BMD and osteoporosis in later life. The results from these two studies are concordant with results seen in other studies on LRP5 mutations and in association studies linking genetic variation in LRP5 with BMD and osteoporosis. Several rare LRP5 variants were identified in children with recurrent fractures. Sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses revealed no disease-causing mutations or whole-exon deletions. Our findings from clinical assessments and family-based genotype-phenotype studies suggested that the rare LRP5 variants identified are not the definite cause of fractures in these children. Clinical assessments of our study subjects with LPR5 mutations revealed an unexpectedly high prevalence of impaired glucose tolerance and dyslipidaemia. Moreover, in subsequent studies we discovered that common polymorphic LRP5 variants are associated with unfavorable metabolic characteristics. Changes in lipid profile were already apparent in pre-pubertal children. These results, together with the findings from other studies, suggest an important role for LRP5 also in glucose and lipid metabolism. Our results underscore the important role of LRP5 not only in bone mass accrual and maintenance of skeletal health but also in glucose and lipid metabolism. The role of LRP5 in bone metabolism has long been studied, but further studies with larger study cohorts are still needed to evaluate the specific role of LRP5 variants as metabolic risk factors.

Hiekkatympösen (Hebeloma cylindrosporum) L-aminohappo-oksidaasi -geenin eristäminen

Pohjoisella havumetsävyöhykkeellä typpi on usein kasvien kasvua rajoittava tekijä. Metsämaan typpivarannot koostuvat pääasiassa orgaaniseen ainekseen sitoutuneista typpiyhdisteistä, erityisesti aminohapoista. Ektomykorritsasienet osallistuvat metsämaassa tapahtuvaan typenkiertoon hajottamalla orgaanisia typpiyhdisteitä ja kuljettamalla niitä kasvien käytettäväksi. Sienisolun sisällä tapahtuvasta aminohappojen mineralisaatiosta tiedetään toistaiseksi melko vähän. Aminohappo-oksidaasit katalysoivat aminohappojen mineralisaatiota. Eräissä ektomykorritsaa muodostavien kantasienten suvuissa on osoitettu L-aminohappo-oksidaaseja (LAO). Toistaiseksi LAO-geeniä ei tunneta kantasienistä. Työssä kuvattiin ensimmäistä kertaa LAO-geeni kantasienistä. Hiekkatympösen LAO1- geenin cDNA:n 5´ ja 3´ päiden emäsjärjestykset määritettiin RACE-PCR -menetelmällä, josta saatujen sekvenssien perusteella suunniteltiin alukkeet koko geenin cDNA:n ja genomisen DNA:n monistamiseksi. Genomisen DNA ja cDNA -sekvenssien perusteella määritettiin hiekkatympösen LAO1-geenin rakenne. Hiekkatympösen LAO1-geeni koostuu viidestä eksonista ja neljästä intronista. Hiekkatympösen LAO1-geenin yläpuoliselta alueelta löydettiin typpimetabolian säätelyyn osallistuvan proteiinin sitoutumiskohta. LAO1-geeniä edeltävä geenin osittainen genominen DNA-sekvenssi määritettiin. Kangaslohisienen genomissa LAO1-geeniä edeltävä geeni oli ennustettu pyruvaattidekarboksylaasiksi. Lisäksi työssä määritettiin hiekkatympösen toisen LAOhomologin cDNA:n osittainen emäsjärjestys. Työssä tunnistettiin myös toisen kantasienen, kangaslohisienen, LAO-geeni. LAO-geeniksi tunnistettu kangaslohisienen geenimalli oli aiemmin ennustettu NCBI:n tietokannassa toiminnaltaan tuntemattomaksi proteiiniksi. Proteiinien sukupuun perusteella hiekkatympösen ja kangaslohisienen LAO:n kantamuoto on kahdentunut. Työstä saatu tutkimustulos tuo täysin uutta tietoa molekyylibiologian tasolla ektomykorritsasienten aminohappojen katabolisista reaktioista. Aminohappojen mineralisaation seurauksen muodostuneet ammoniumionit saattavat olla merkittävä typen lähde myös maan muille mikrobeille ja kasveille. On mahdollista, että ektomykorritsasienten LAO-entsyymi on yksi merkittävä tekijä metsämaan typenkierrossa.