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The influence of adenosine 3':5'-monophosphate (cyclic AMP) and ATP on the activity of cholesteryl esterase has been studied in bovine adrenal cortex. It has been shown that cyclic AMP and ATP stimulated the activity of cholesteryl esterase in vitro. The increase in cholesteryl esterase activity was dependent upon the presence of cyclic-AMPdependent protein kinase. After removal of protein kinase from cholesteryl esterase preparation no stimulation upon the addition of cyclic AMP and ATP was observed. The addition of purified protein kinase, which was virtually free from cholesteryl esterase activity resulted in approx. 3-fold stimulation of cholesteryl esterase activity in the presence of cyclic AMP and ATP. Without cyclic AMP and ATP no stimulation of partially purified cholesterol esterase by protein kinase was observed. Preincubation of crude chotesteryl esterase in the presence of 5 mM MgCl2. caused a marked decrease in cholesteryl esterase activity. Under these conditions cyclic AMP and ATP protected the enzyme against inactivation Preincubation of partially purified cholesteryl esterase with [γ-32P]ATP, cyclic AMP and cyclic AMP-dependent protein kinase resulted in an increase in cholesteryl esterase activity With a concomitant transfer of 32P-labelled phosphate into the protein fraction. Incubation of the phosphorylated protein fraction in the presence of 5 mM MgCl2 revealed a decrease in cholesteryl esterase activity with a concomitant release of 32P-labelled phosphate from phosphorylated protein. The decrease in cholesteryl esterase activity and the release of 32P-labelled phosphate from phosphorylated protein exhibited similar kinetics. It is postulated that the bovine adrenal cortex cholesteryl esterase is activated by cyclic AMPdependent protein kinase and that the activation involves a transfer of a terminal phosphate group from ATP onto the cholesteryl esterase molecule. [ABSTRACT FROM AUTHOR]

Abstract: 11Beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) deficiency causes sodium retention and severe hypertension by allowing glucocorticoids access to the non-selective mineralocorticosteroid receptor. Understanding regulation of the HSD11B2 gene is thus of fundamental importance in hypertension research. A number of studies have suggested that second messenger pathways may be important in this regard. In the present study we show that HSD11B2 expression in human renal epithelial P58 cells is regulated at the mRNA and protein level, and that protein kinases A (PKA) and C (PKC) are involved in this process. PKA stimulation resulted in almost two-fold increase while PKC activation in almost two-fold decrease in the HSD11B2 mRNA and protein level. Western blot analysis revealed a dimeric form of 11beta-HSD2 of about 80kDa. Arginine vasopressin (AVP), acting through the AVP2 receptor, as well as 11beta-HSD2 substrates, corticosterone and dexamethasone, up-regulate HSD11B2 expression, suggesting their role as possible factors affecting blood pressure. We show that the activators of the PKA pathway induce, while activators of PKC pathway repress the expression of HSD11B2 in human renal epithelial cells. AVP, acting via the PKA pathway, might be a physiological stimulator of the HSD11B2 expression. The 11beta-HSD2 substrates, both natural (corticosterone) and synthetic (dexamethasone), might protect the mineralocorticosteroid-target cells against cortisol excess. [Copyright &y& Elsevier]

PAX9 is a paired domain transcription factor that plays a critical role in odontogenesis. All mutations of PAX9 identified to date have been associated with nonsyndromic form of tooth agenesis. The present report describes an unusual novel mutation in PAX9 identified in a family with severe molar oligodontia. This heterozygous deletion combined with 24 bp insertion (including a 5′ splice site) is localized in the second exon beyond the highly conserved paired box sequence, and might result either in a premature termination of translation at aa 210 or in an aberrant splicing, leading to a frameshift and premature termination of translation at aa 314. Real-time PCR analysis revealed no mutated transcript in cultured lymphocytes of one of the affected individuals indicating that the novel mutation might result in rapid degradation of the mutated transcript leading to haploinsufficiency of PAX9. Our results support the view that mutations in PAX9 constitute a causative factor in nonsyndromic oligodontia.European Journal of Human Genetics (2006) 14, 173–179. doi:10.1038/sj.ejhg.5201536; published online 7 December 2005 [ABSTRACT FROM AUTHOR]

The orphan nuclear receptor, steroidogenic factor 1 (SF-1), plays a major role in adrenal and gonadal development, as well as in sexual differentiation. It has been demonstrated that the expression of a number of genes regulated by SF-1 is inhibited by the transforming growth factor β (TGF-β). To date, however, the influence of TGF-β on the expression of SF-1 gene has not been reported. A Northern blot analysis with the use of a radiolabeled cDNA probe, and immunodetection with antibodies directed against SF-1, demonstrated that the Sf-1 transcript and the SF-1 protein levels were lowered by TGF-β in Y-1 adrenocortical cells, both in untreated and adenylyl cyclase activator, forskolin-treated cells. An examination of the Sf-1 transcript stability in the presence of actinomycin D revealed no influence of TGF-β on the rate of Sf-1 mRNA decay. Inhibition of Sf-1 expression by TGF-β was abolished by cycloheximide, suggesting that the growth factor inhibitory effect requires ongoing protein synthesis. We conclude that in Y-1 cells TGF-β inhibits the expression of SF-1 gene at a transcriptional level, and we postulate that the inhibitory effect of TGF-β on steroid hormone synthesis in the adrenal cortex could be due to an attenuated transcription of Sf-1 . [ABSTRACT FROM AUTHOR]

Tooth agenesis constitutes the most common anomalies of dental development in man. Despite this, little is known about the genetic defects responsible for this complex condition. To date, the only genes associated with the non-syndromic form of tooth agenesis are MSX1 and PAX9 , which encode transcription factors that play a critical role during tooth development. This paper aims to review current literature about the molecular mechanisms responsible for selective tooth agenesis in humans. [ABSTRACT FROM AUTHOR]

Although the estrogenic properties of numerous chloroorganic pesticides have been widely recognized, population studies do not give clear results indicating the link between the exposure to these compounds and breast cancer development. Because of the weak affinity of these pesticides to estrogen receptors, they probably act by affecting the expression of CYP genes encoding cytochromes P450 engaged in the metabolism of environmental as well as natural estrogens. To examine the possible correlation between environmental estrogen levels in adipose tissue and breast cancer stage, grade, receptor status and onset of the disease, adipose tissue was isolated from 54 breast cancer patients and 23 healthy individuals. Clinical characteristics were obtained from the medical records, while the information concerning exposure to environmental estrogens where obtained from questionnaires. The environmental estrogens were identified and quantified by GC-chromatography. The data was analyzed with the use of Student t-test and Spearman correlation. The levels of most environmental estrogens did not differ between the patients and the controls, except the β -HCH (β -hexachlorocyclohexane) level, which was higher in the patients than in the healthy individuals. Significantly higher levels of DDE (1,1-bis(4-chlorophenyl)-2,2-dichloroethene) and DDT (1,1,1-trichloro-2,2-bis(4-chlorophenol)ethane) (P < 0.05) were observed in the patients with late onset of the disease which was probably due to the time of exposure. Moreover, in the patients exposed to environmental estrogens, significantly higher concentrations of DDD (1,1-bis(4-chlorophenyl)-2,2-dichloroethane) were found (P < 0.05). We also evidenced that estrogen-independent cancer was more frequent in the patients exposed to numerous risk factors in which higher levels of HCB (hexachlorobenzene), γ -HCH (γ -hexachlorocyclohexane), DDD and DDT in adipose tissue were detected. Breast cancer development is probably related to the accumulation of DDT and its derivatives, but the effect appears only in older patients. We postulate that environmental estrogens acting together with other risk factors might influence the progress and exacerbate the prognosis of breast cancer. [ABSTRACT FROM AUTHOR]

Objective. Transforming growth factor β (TGF-β) is a potent inhibitor of 17α-hydroxylase/17,20 lyase activity and CYP17 gene expression. We investigated the mechanism how CYP17 is inhibited by TGF-β in adrenocortical cells. Methods. H295R cells were culture and incubated with TGF-β, transcription inhibitor (DRB), activin receptor-like kinase 5 ALK5 (TβRII) inhibitor (SB431542), mitogen activated kinases inhibitors (PD98059 and SB203580), subsequently using reverse transcription and quantitative PCR (RT-qPCR) we determined CYP17 expression. Results. TGF-β significantly decreased the level of cytochrome P450c17 mRNA and this inhibitory effect of TGF-β on CYP17 expression required activin receptor-like kinase 5 (ALK5) and on-going transcription. Mitogen activated kinases MEK1 and p38 MAPK are not involved it the inhibitory effect of TGF-β on CYP17 expression. Conclusion. We concluded that the TGF-β-dependent decrease of 17α-hydroxylase/17,20 lyase activity in the H295R cells is caused by inhibition of CYP17 transcription and is mediated by the ALK5 receptor. [ABSTRACT FROM AUTHOR]

Large differences in relation to dental size, number, and morphology among and within modern human populations and between modern humans and other primate species have been observed. Molecular studies have demonstrated that tooth development is under strict genetic control, but, the genetic basis of primate tooth variation remains unknown. The PAX9 gene, which codes for a paired domain-containing transcription factor that plays an essential role in the development of mammal dentition, has been associated with selective tooth agenesis in humans and mice, which mainly involves the posterior teeth. To determine whether this gene is polymorphic in humans, we sequenced !2.1 kb of the entire four-exon region (exons 1, 2, 3 and 4; 1,026 bp) and exon-intron (1.1 kb) boundaries of 86 individuals sampled from Asian, European, and Native American populations. We provided evidence that human PAX9 polymorphisms are limited to exon 3 only and furnished details about the distribution of a mutation there in 350 Polish subjects. To investigate the pattern of selective pressure on exon 3, we sequenced ortholog regions of this exon in four species of New World monkeys and one gorilla. In addition, orthologous sequences of PAX9 available in public databases were also analyzed. Although several differences were identified between humans and other species, our findings support the view that strong purifying selection is acting on PAX9. New World and Old World primate lineages may, however, have different degrees of restriction for changes in this DNA region. [ABSTRACT FROM AUTHOR]

The study aimed at the analysis of polymorphisms in the gene coding for the nicotinic acetylcholine receptor α4 subunit ( CHRNA4) and the evaluation of the extent of the oxidative damage to DNA (8-oxo2dG), as well as the level of proteins participating in DNA repair (p53, PARP) and DNA degradation (Bax:Bcl-2, 85-kDa fragment) in the peripheral blood lymphocytes of the patients suffering from Alzheimer's disease (AD) and in the healthy individuals of the control group. In the AD patients the increased levels of oxidized guanine were demonstrated in DNA, accompanied by the elevated expression of p53, Bax, PARP, and of a 85-kDa protein subunit as well as an augmented ratio of Bax:Bcl-2. Also, the level of Bcl-2 protein was decreased. In the AD patients with the CHRNA4 polymorphisms the highest level of 8-oxo2dG and of proteins involved in DNA repair were documented in patients with polymorphisms in exon 5, in contrast to the patients with polymorphisms in intron 5. In the former patients, levels of pro- and antiapoptotic proteins remained at the same level. Both CHRNA4 polymorphisms and the extent of dementia seem to affect the levels of DNA oxidative damage as well as to activate factors that participate in the DNA degradation and its repair. [ABSTRACT FROM AUTHOR]

Summary: Purpose: To identify mutations of the neuronal nicotinic acetylcholine receptor α4 subunit gene (CHRNA4 ) responsible for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) in a group of white patients. Methods: A group of 47 patients from 21 unrelated families with ADNFLE were screened for mutations in CHRNA4 . Clinical features and EEG findings in the patients were consistent with those reported in the literature for other affected families. The entire gene was amplified from genomic DNA by polymerase chain reaction (PCR) followed by multitemperature single-strand conformation polymorphism analysis (MSSCP) and sequencing. Results: A c.851C>T transition in exon 5 of CHRNA4 was identified in three affected individuals from two generations of the same family, but not in the remaining patients or in 100 healthy volunteers. This mutation caused an S284L substitution in the transmembrane domain M2 segment of the α4 subunit of the neuronal nicotinic acetylcholine receptor. The same mutation had previously been detected in a single Japanese family with ADNFLE, and in an Australian woman with a sporadic form of NFLE. Conclusions: This is the first report of an occurrence of c.851C>T transition in a white family with ADNFLE. [ABSTRACT FROM AUTHOR]

Tooth development is regulated through a series of reciprocal interactions between the dental epithelium and mesenchyme and requires protein products of a number of genes. It has been reported that selective tooth agenesis is associated with mutations in human MSX and PAX9 genes. Mutational analysis of the two genes was performed in 25 individuals with familial or sporadic form of permanent tooth agenesis. Single-stranded conformational polymorphism analysis revealed no mutations in the entire coding sequence of the MSX1 gene. In PAX9 , a novel, heterozygous G151A transition in the sequence encoding the paired domain of the PAX9 protein was detected in a patient with agenesis of third molars, second premolars and incisors, but not in her parents, the remaining patients or 162 individuals with normal dentition. This is the first de novo mutation described in PAX9 . Our results support the view that mutations in PAX9 could constitute a causative factor of oligodontia. We hypothesize that the G151A transition in PAX9 might be responsible for the sporadic form of tooth agenesis in this patient. [ABSTRACT FROM AUTHOR]

The regulation of 11β-hydroxysteroid dehydrogenase type II (11βHSD2) expression at the level of specific mRNA and 11βHSD2 protein was investigated in primary culture of renal epithelial cells of the rat. It has been shown that treatment of the SE cells with adenylyl cyclase activator, forskolin, known to stimulate the protein kinase A (PKA) pathway, resulted in an increase in 11βHSD2 mRNA content in these cells. Semi-quantitative RT-PCR revealed that the effect of forskolin was attenuated by the addition of phorbol ester, tetradecanoyl phorbol acetate (TPA), an activator of the protein kinase C (PKC) pathway, whereas TPA on its own slightly reduced the basal level of 11βHSD2 expression judging from the content of specific mRNA. Measurements of [[SUP35]S]-methionine incorporation into immunoprecipitable 11βHSD2 revealed an increased synthesis of this protein in renal epithelial cells treated with forskolin. Phorbol ester TPA markedly reduced the effect of forskolin on the synthesis of 11βHSD2 and attenuated the basal level of synthesis of this protein. It is concluded that in renal epithelial cells in primary culture, stimulation of PKA pathway results in the induction of 11βHSD2 both at a specific mRNA and effect is markedly reduced by activation of PKC pathway. [ABSTRACT FROM AUTHOR]

The article describes the case of a boy with X-linked hypohidrotic ectodermal dysplasia (XLHED). The boy was subjected to DNA sampling isolated from peripheral blood leukocytes and an amplified exon 8 of ED1 revealed a hemizygous mutation that resulted from a deletion of tumor necrosis factor (TNF) homology sequence. It is speculated that mRNA decay preceded mRNA mutation.

Objective: To conduct clinical, genetic, and molecular diagnostics of two sisters with typical symptoms of complete androgen insensitivity syndrome. Design: Case report. Setting: Research laboratory at a university of medical science. Patient(s): Two patients with 46,XY karyotype and a female phenotype were diagnosed because of primary amenorrhea. Their sister with 46,XX karyotype, her daughter, and five other family members including their mother also were examined. Intervention(s): Orchiectomy, estrogen substitution therapy. Main Outcome Measure(s): Cancer prophylaxis. Result(s): Multiple-temperature single-stranded conformation polymorphism and sequence analyses of the androgen receptor gene (AR) revealed a c.C2812T transition in exon 7 in the two sisters. Their mother and the third sister (46,XX) were carriers of the same mutation. This mutation, which previously had never been reported, resulted in Pro817Leu substitution in the ligand-binding domain of the androgen. Computer simulation of structural changes generated by Pro817Leu substitution revealed appreciable conformational changes in the region responsible for dimerization of the receptor. Conclusion(s): The novel c.C2812T transition that might impair dimerization of the receptor is responsible for the clinical symptoms of complete androgen insensitivity syndrome in the affected individuals. Molecular analysis of AR proved to be very useful for genetic counseling of the unaffected sister, who was a carrier of the same mutation. [Copyright &y& Elsevier]