Induction of hormone-sensitive lipase/cholesteryl esterase gene expression by C/EBPα independently of the PKA pathway in the adrenocortical Y-1 cells.

The effect of C/EBPα on the expression of LIPE gene encoding hormone-sensitive lipase/cholesteryl esterase (HSL) was investigated in Y-1 CCL79 cells. It was found that transfection of these cells with the vector overexpressing C/EBPα increased both the level of LIPE transcript, measured by RT-qPCR, and the luminesce emitted by luciferase reporter gene fused to the -2150 fragment of LIPE promoter. Activation of adenylyl cyclase by forskolin resulted in 2.5-fold increase in the intensity of luminescence and over 3-fold increase in luminescence was observed when the cells were cotransfected with the vector overexpressing C/EBP. The incubation of C/EBP-cotransfected cells with forskolin caused over 6-fold increase in the intensity of luminescence, suggesting that the effects of C/EBPα and forskolin are additive. The analysis of sequence of the proximal LIPE promoter showed multiple binding sites for various transcription factors including C/EBPα site, which is located between nucleotides -46 bp and -59 bp. When the Y-1 cells were transfected with the recombinant vector containing -60 bp fragment of LIPE promoter fused to the luciferase reporter gene and were cotransfected with the vector overexpressing C/EBPα, the luminescence increases about 9-fold indicating that C/EBPα stimulates the expression of LIPE by reacting with its response element. The results indicate that C/EBPα stimulates the expression of LIPE independently of the PKA pathway by binding to a response element situated within the -60 bp fragment of LIPE promoter. This suggests that C/EBPα might be involved in the regulation of LIPE expression and thus cholesterol supply for steroid hormone synthesis.

SF-1 (NR5A1) expression is stimulated by the PKA pathway and is essential for the PKA-induced activation of LIPE expression in Y-1 cells.

In the adrenal cortex, corticotropin induces the expression of several genes encoding proteins involved in the synthesis and intracellular transport of steroid hormones via the protein kinase A (PKA) signalling pathway, and this process is mediated by steroidogenic factor-1 (SF-1). This study was designed to elucidate the influence of the PKA and PKC pathways on the expression of the SF-1 gene in mouse adrenocortical cells, line Y-1. It has also been attempted to answer the question whether or not SF-1 plays a role in the PKA-induced expression of LIPE gene encoding hormone-sensitive lipase/cholesteryl esterase, which supplies cholesterol for steroid hormone synthesis. In this study, we found that stimulation of the PKA pathway caused a significant increase in SF-1 expression, and that this effect was abolished by the PKA inhibitor, H89. Decreased SF-1 gene transcript levels were seen with the simultaneous activation of PKA and PKC, suggesting a possible interaction between the PKA and PKC pathways. It was also observed that SF-1 increased the transcriptional activity of the LIPE gene by interacting with the SF-1 response element located in promoter A. Moreover, transient silencing of SF-1 expression with specific siRNAs abolished PKA-stimulated transcription of the LIPE gene, indicating that SF-1 is an important regulator of LIPE expression in Y-1 cells and thus could play a role in the regulation of the cholesterol supply for adrenal steroidogenesis.

A novel insA2933 causes premature termination of translation and is accompanied by overexpression of truncated androgen receptor gene in a patient with complete androgen insensitivity syndrome.

A patient with a female phenotype, 46,XY karyotype, and a diagnosis of complete androgen insensitivity syndrome (CAIS) was examined. Her mother and three 46,XX sisters were also included in the study. Sequence analysis of the androgen receptor gene (AR) revealed a novel A2933 insertion that alters the Tyr codon to a termination codon (Y857X), resulting in a truncated form of the receptor. Computer simulation revealed major conformational changes in the hydrophobic pocket that accommodates the hormone. An insA2933 results in a truncated receptor incapable of binding the ligand and is responsible for the clinical symptoms of CAIS in the patient. The levels of the AR transcript in peripheral blood leukocytes were higher in the patient than in her heterozygous mother and her heterozygous sister, as well as in the two healthy sisters. It is hypothesized that elevated levels of the AR transcript in the patient might be caused by the inability of the truncated receptor to react with IFI-16, which functions in complex with AR to inhibit the expression of the AR gene.

Identification of mutations in the HVR1 and PKR-BD regions in HCV-infected patients resistant to PEG-IFNα/RBV therapy.

The identification of mutations in the HVR1 region of hepatitis type C virus (HCV) is time-consuming and expensive, and there is a need for a rapid, inexpensive method of screening for these mutations to predict the ineffectiveness of pegylated interferon alpha combined with ribavirin (PEG-IFNα/RBV) therapy. The project was designed to evaluate the usefulness of the high resolution melting (HRM) technique to screen for mutation in the cDNAs encoding the HVR1 and protein kinase R-binding domain (PKR-BD) regions in a group of 36 patients infected with HCV and resistant to 12 months of combined therapy with PEG-IFNα/RBV. Viral RNA was isolated, reverse transcribed, and the fragments encoding the HVR1 and PKR-BD regions were polymerase chain reaction (PCR)-amplified, cloned, sequenced, and the melting profiles and the melting temperature (Tm) were determined by the HRM technique. After the treatment, the melting profiles of HVR1 cDNAs revealed a dominant peak corresponding to the Tm of about 85 °C (HCVs85) in almost all patients. One or more minor peaks were also observed, indicating the existence of cDNA(s) of different Tm. The HMR analysis suggested four typical forms of response to treatment. These suppositions were supported by sequencing. The HRM analysis revealed no changes in the melting profiles of PKR-BD cDNAs in the same patient before and after the therapy, suggesting that, within 12 months of treatment, new mutations were not introduced in PKR-BD. These findings were substantiated by sequencing. The HRM technique can be applied for the rapid screening for mutations in the cDNAs encoding the HVR and PKR-BD regions of HCV. We suggest that the detection of HCVs85 peak before the IFNα/RBV therapy might predict the ineffectiveness of treatment.

Application of a high-resolution melting technique for the rapid detection of partial replacement of HCV-1b by HCV-1a after PEG-IFNα/RBV therapy.

A modified method which can be used for the rapid screening of mutations in the protein kinase R-binding domain (PKR-BD) region and the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) is described. This method is based on a high-resolution melting (HRM) technique used for genotyping single nucleotide polymorphisms and allows the detection of single nucleotide substitutions in the DNA sequence by measuring its Tm. The modified method, in addition to precisely measuring the Tm, allows the recording of the melting curve of the investigated cDNA fragment, which can provide provisional information about the number of different quasi-species present in the sample. The HRM analysis of the amplified cDNAs encoding the PKR-BD and HVR1 allowed the detection of partial replacement of HCV-1b by HCV-1a subspecies in one of our patients, as well as evaluation of the effectiveness of pegylated interferon α/ribavirin (PEG-IFNα/RBV) therapy. The HRM technique has never been used for the rapid screening of sequence variations in these regions and may be used for a similar purpose in any viral genome.

A rare heterozygous TRAF6 variant is associated with hypohidrotic ectodermal dysplasia.

BACKGROUND: Mutations in the genes encoding components of the tumour necrosis factor (TNF)-α-like pathway cause hypohidrotic ectodermal dysplasia (HED). It has been postulated that the TNF receptor-associated factor 6 (TRAF6) is also involved in this pathway. OBJECTIVES: To investigate mutations in the TRAF6 gene in an individual with HED. METHODS: Genetic analysis was performed on TRAF6 in a patient with HED, her parents, her sister and 150 ethnically matched, healthy individuals. RESULTS: In the patient, sequencing analysis of one DNA strand revealed a deletion of eight nucleotides (c.1074-1081delCAATTTG) in the 5' fragment of the last exon of TRAF6, while no deletion was detected in the other DNA strand indicating a heterozygous mutation. No such sequence abnormality was detected in the patient's parents and her sister. CONCLUSION: This is the first report of a heterozygous TRAF6 sequence variant associated with symptoms typical of HED.

Transcription of LIPE gene encoding hormone-sensitive lipase/cholesteryl esterase is regulated by SF-1 in human adrenocortical cells: involvement of protein kinase A signal transduction pathway.

The study was designed to elucidate the influence of the protein kinase A (PKA) signal transduction pathway on transcription of the LIPE gene encoding hormone-sensitive lipase/cholesteryl esterase (HSL) in H295R cells. HSL is one of the key enzymes involved in steroid hormone synthesis, and ACTH, with mediation of the PKA pathway, increases its activity. However, the mode of regulation of LIPE gene expression by ACTH remains unknown. It was found that stimulation of the PKA pathway by the adenylyl cyclase activator, forskolin, caused a twofold increase in LIPE transcript accompanied by appreciable rise in the protein product of the gene and cortisol output. RNA polymerase II inhibitor abolished, and protein synthesis inhibitor attenuated this effect. Forskolin and PKA catalytic subunit increased transcriptional activity of LIPE promoter A in cells transfected with the luciferase reporter vector. Overexpression of steroidogenic factor-1 (SF-1) increased LIPE promoter activity, while transient silencing of SF-1 expression with specific siRNAs abolished forskolin-stimulated LIPE transcription. It is concluded that ACTH via the PKA pathway stimulates expression of SF-1, which activates transcription of LIPE presumably by interaction with putative binding sequences within promoter A. A novel mechanism contributing to the long-term effect of ACTH on adrenal steroidogenesis is proposed: ACTH stimulates transcription of SF-1, which interacts with the putative SF-1-binding sequences within the promoter and activates LIPE transcription. An increased level of HSL results in an enhanced supply of cholesterol required for steroid hormone synthesis.

The correlation of homocysteine-thiolactonase activity of the paraoxonase (PON1) protein with coronary heart disease status.

Homocysteine (Hcy)-thiolactonase (HTase) activity of the paraoxonase-1 (PON1) protein detoxifies Hcythiolactone in human blood and could thus delay the development of atherosclerosis. We investigated a hypothesis that HTase activity is associated with coronary heart disease. We studied HTase activities and PON1 genotypes in a group of 475 subjects, 42.5% of whom were healthy and 57.5% had coronary heart disease (CHD). We found that HTase activity was positively correlated with total cholesterol (r=0.254, P<0.0001), LDL cholesterol (0.149, P=0.016), ApoB (r=0.167, P=0.006), ApoA1 (0.140, P=0.023), and HDL cholesterol (0.184, P=0.002) in a group of CHD cases (n=270) but not in controls (n=202). Mean HTase activity was significantly higher in CHD cases than in controls (4.57 units vs. 3.30 units, P <10(-5)). The frequencies of the PON1-192 genotypes in CHD cases were similar to those in controls. HTase activity was not different between patients receiving statins and those not treated with statins. Multiple regression analysis shows that CHD status, PON1 genotype, and total cholesterol are determinants of HTase activity in humans. Our results suggest that HTase activity of the PON 1 protein is a predictor of CHD.

A correlation between the heterogeneity of hypervariable region 1 of E2 glycoprotein of Hepatitis C virus (HCV) and HCV antibody profile: a case study.

A correlation between the heterogeneity of hypervariable region 1 (HVR1) of E2 glycoprotein (gp) and Hepatitis C virus (HCV) antibody profile was investigated. Of 6 patients studied two were in acute phase, two in chronic phase and two showed signs of long-time HCV infection, i.e. liver cirrhosis. All the patients exhibited a vigorous antibody response to viral proteins C, NS3, NS4 and NS5. An antibody response to HVR1 of E2 was found in one patient in acute phase and in one or two patients in chronic phase. Such a response was not found in the two patients with liver cirrhosis. Single-stranded conformation polymorphism (SSCP) and sequence analyses of HVR1 of E2 showed the lowest HVR1 heterogeneity in patients in acute phase and the highest one in those in chronic phase, while the long-time carriers of the virus showed an intermediate heterogeneity. This may reflect a specific interplay between the virus and immune system. The HVR1 heterogeneity may rise in the course of infection as a means of evading the immune pressure. Then, when an organism is unable to clear the virus, because the responses to HVR1 epitopes are weakened or exhausted, a population of less heterogeneous HVR1 variants may be established.

A novel c.C2754 > T transition in the androgen receptor gene introduces the premature termination codon Q798X and results in a truncated form of the receptor.

A genetic analysis of a female with a 46,XY genotype and typical symptoms of the complete androgen insensitivity syndrome (CAIS) was conducted. The patient was diagnosed with an abdominal tumor due to the presence of a Sertoli cell adenoma in both gonads. Multiple temperature single-stranded conformational polymorphism (MSSCP) and sequence analyses of the androgen receptor gene revealed a c.C2754 > T mutation in exon 6. This mutation, which has not been previously reported, alters a Gln codon to a termination codon (Q798X). This results in the interruption of the amino acid sequence of the androgen receptor within the ligand-binding domain between helices VII and VIII. The truncated form of the receptor is devoid of 123 amino acids at the carboxyl end, a major part of the ligand-binding domain, and the AT2 sequence responsible for the activation of the transcription. It was concluded that the novel c.C2754 > T transition rendered the androgen receptor incapable of both ligand binding and activating the transcription, and was the cause of CAIS in the patient.

Determinants of homocysteine-thiolactonase activity of the paraoxonase-1 (PON1) protein in humans.

Homocysteine (Hcy)-thiolactonase (HTase) activity of the paraoxonase-1 (PON1) protein detoxifies Hcy-thiolactone in human blood and could thus delay the development of atherosclerosis. To gain insight into physiological role(s) of the PON1 protein, we studied HTase activities and PON1 genotypes in a group of 184 subjects, 32.6% of whom were healthy, 27.7% had angiographically proven coronary artery disease but did not have myocardial infarction (CAD), and 39.7% had myocardial infarction (MI). We found that the hydrolytic activities of the serum PON1 protein towards Hcy-thiolactone and the organophosphate paraoxon substrates were strongly correlated. PON1-192-RR and PON1-55-LL genotypes were associated with high HTase activity. HTase activity was negatively correlated with age (beta = -0.135, p =0.002), plasma total Hcy (in 192-QR subjects only; r = -0.46, p = 0.001), and positively correlated with total cholesterol (beta = 0.169, p<0.001), but not with HDL cholesterol. Mean HTase activities were similar in CAD subjects, MI subjects, and in healthy controls. However, the frequency of the PON1-192-RR genotype tended to be lower in CAD subjects than in controls (2% vs 10.0%, p = 0.057) and higher in MI subjects that in CAD subjects (10.9% vs 2.0%, p = 0.001). The R-allele was marginally associated with CAD (26.7% in controls vs 17.6% in CAD, p = 0.146) and significantly associated with MI (17.6% in CAD vs 31.5% in MI, p = 0.018). Multiple regression analysis suggests that PON1 genotype, total Hcy, total cholesterol, and age are major determinants of HTase activity in humans.

Determination of the CCR2-64I/CCR5-59653T haplotype linkage disequilibrium in a sample of Polish population.

It has been reported that different polymorphisms in the regulatory regions of CCR5 and in the CCR2 gene of the chemokine receptors are associated with AIDS. We decided to determine the congruence between CCR5-59653T and CCR2-64I alleles in a group of 281 persons. Frequencies of combined CCR5-59653T and CCR2-64I haplotypes were examined in a group of 281 persons. Among 281 individuals 26 (9.3%) and 24 (8.5%) respectively were carriers of the CCR5-59653T and the CCR2-64I alleles. We also found that 24 persons (8.5%) were carriers of combined CCR2-64I/CCR5-59653T allele. The calculation of the congruence revealed that 92.0% of individuals exhibited the same genotype for both CCR2-64I and CCR5-59653T polymorphisms. Our results confirm that linkage between CCR5-59653T and CCR2-64I alleles is not absolute.

Regulation of 11beta-hydroxysteroid dehydrogenase type II expression in the renal epithelial cells.

The regulation of 11beta-hydroxysteroid dehydrogenase type II (11betaHSD2) expression at the level of specific mRNA and 11betaHSD2 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 11betaHSD2 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 11betaHSD2 expression judging from the content of specific mRNA. Measurements of [35S]-methionine incorporation into immunoprecipitable 11betaHSD2 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 11betaHSD2 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 11betaHSD2 both at a specific mRNA and at a protein level and that this effect is markedly reduced by activation of PKC pathway.

The effect of high molecular weight dextran sulfate on the production of interleukin-8 in monocyte cell culture.

It has been demonstrated that high molecular weight dextran sulfate (HMDS) is involved in the activation of immune cells. We have shown that HMDS increases the concentration of interleukin (IL)-8 in the medium of monocyte cell culture, in a dose-dependent fashion, whereas under the same conditions, low molecular weight dextran sulfate (LMDS) does not exhibit any effect on IL-8 biosynthesis. The effect of HMDS on IL-8 production is additive to that of IL-1beta and tumor necrosis factor-a (TNFalpha). Flow cytometric analysis revealed the biosynthesis of IL-8 in monocytes incubated in the presence of the HMDS. We hereby postulate that HMDS induces IL-8 biosynthesis in monocyte cell culture.

Decreased density of the CCR5 receptor on the surface of CD4+ lymphocytes and monocytes/macrophages is associated with the CCR5-59653T transition in the promoter region.

We compared the density of the CCR5 receptor on the surface of CD4+ lymphocytes and monocytes/macrophages of the homozygote (CCR5-59653C) and the heterozygote (CCR5-59653T), bearing CCR2-64V alleles. Flow cytometric analysis revealed lower density of the CCR5 receptor on the surface of CD4+ lymphocytes and monocytes/macrophages of the heterozygote than in the same cells of the homozygote. Our observation might explain slower replication of HIV and the delay in progression to AIDS in the individuals bearing CCR5-59653T transition.

Study of the family of a patient with male-limited precocious puberty (MPP) due to T1193C transition in exon 11 of LH receptor gene.

Molecular diagnostics of the LHR gene was conducted in a 5-year-old boy with clinical symptoms and hormonal profile typical of precocious puberty. His parents and 4 sisters were also diagnosed. Single-strand conformation polymorphism analysis under temperature gradient conditions (Multitemperature SSCP) of 3 overlapping fragments of exon 11 of LHR gene revealed a mutation in the fragment spanning nucleotides 1072 to 1804. This mutation was found in the patient, in his mother and in his 4 sisters, and was confirmed by digestion with the use of restriction enzyme Bbr Cl. Direct sequencing revealed a heterozygous T1193C transition in the DNA fragment of the patient and in one of the alleles of his mother's and sister's DNA. This mutation causes Met398Thr substitution in the second transmembrane helix and results in a constitutive activation of LH receptor. This is the second identical mutation detected in Poland and one of the 7 identified so far in the world population.

Additive effect of tunicamycin and dextran sulfate on the binding of monoclonal antibody to the V2 domain of the envelope glycoprotein 120 of human immunodeficiency virus type 1.

Using flow cytometry or immunoprecipitation analysis in cells chronically infected with HIV-1 IIIB Supt-1, we noticed an additive effect of tunicamycin and low molecular weight dextran (LMDS) on the binding of the G3-4 monoclonal antibody to monomeric and oligomeric forms of glycoprotein 120 (gp120). The inhibition of glycosylation by tunicamycin reduced the number of monomeric and oligomeric forms of gp120. The inhibition of the binding of the G3-4 antibody to monomeric and oligomeric forms of gp120 was more pronounced in the presence of LMDS. We also found that the G3-4 antibody can not recognise the nascent polypeptide chain of the envelope glycoprotein.