Effects of lecithin: cholesterol acyltransferase genotypes, enzyme levels, and activity on high-density lipoprotein levels.

BACKGROUND: Lecithin:cholesterol acyltransferase (LCAT) is one of the key enzymes controlling cholesterol homeostasis and plays a primary role in high-density lipoprotein cholesterol (HDL-C) maturation. OBJECTIVE: The aim of our study was to evaluate the effects of LCAT gene polymorphisms 511C/T (exon4), 4886C/T (rs5923), and 608C/T (rs5922) on LCAT enzyme level, activity, and HDL-C levels. METHODS: The study population was selected from consecutive subjects with low (<35 mg/dL) and high HDL-C levels (>65 mg/dL) seen in our lipid clinic. LCAT polymorphisms were analyzed with a restriction fragment length polymorphism assay. LCAT activity and levels were measured by colorimetric enzymatic and enzyme-linked immunoassay methods, respectively. RESULTS: The 4886C/T polymorphism was the most commonly observed variant of LCAT gene. T-allele frequencies in subjects with low (n = 50) and high (n = 50) HDL-C were 0.54 and 0.37, respectively (P = .019). TT genotype was more common among low HDL-C group (30% vs 14%, P = .05). The effects of LCAT enzyme appeared to depend on the HDL-C level. In subjects with low HDL-C, LCAT enzyme levels correlated positively with body mass index (P < .001, r = 0.544), HDL-C (P = .006, r = 0.404), triglycerides (P = .001, r = 0.487), total cholesterol (P < .001, r = 0.541), and low-density lipoprotein-cholesterol (P = .001, r = 0.477) levels. LCAT activity correlated positively with fasting glucose levels (P = .008, r = 0.390). CONCLUSION: LCAT genotype, enzyme level, and activity modulate HDL-C metabolism, particularly among subjects with low HDL-C levels.

Angiotensin converting enzyme gene polymorphism in Turkish asthmatic patients.

Asthma is a chronic inflammatory disease of the airways. Several candidate genes have been identified with a potential role in the pathogenesis of asthma, including the angiotensin converting enzyme (ACE) gene. We aimed to investigate the frequency of an ACE gene polymorphism in Turkish asthmatic patients and to determine its impact on clinical parameters and disease severity. Ninety-seven asthmatic patients (M/F 25/72, mean age 39 +/- 13 years) and 96 healthy subjects (M/F 26/70, mean age 38 +/- 12 years) were included. At baseline, all participants completed a questionnaire on demographics, symptoms, triggering factors, severity of asthma, and the presence of atopism. Blood samples were obtained from all patients and genomic DNA was isolated. The frequency of the ACE genotypes (I = insertion and D = deletion) among asthmatics and controls were compared: asthmatics showed a 40.2% prevalence of the DD genotype (n = 39), ID was 45.4% (n = 44), and II was 14.4% (n = 14.4). In the control subjects, the frequency of DD was 18.8% (n = 18), ID was 50% (n = 48) and II was 31.3% (n = 30). The DD ACE genotype was significantly more frequent in asthmatics compared with controls (p < 0.001). Asthmatics with the ID ACE genotype showed a higher frequency of drug allergies, although this was not statistically significant (p = 0.08). Asthmatics with the DD genotype appeared to have a higher incidence of asthmatic episode exacerbations due to viral infections, but again this was not statistically significant (p = 0.08). Patients with mild or moderate-severe asthma had similar frequencies of these mutations. We found a higher frequency of the ACE DD gene mutation in Turkish asthmatic patients compared with non-asthmatics, suggesting that this ACE gene polymorphism may be a risk factor for asthma but does not increase the severity of the disease.

Therapeutic efficacy of SJA6017, a calpain inhibitor, in rat spinal cord injury.

Apoptosis is an important element of the secondary processes that occur after spinal cord injury. Calpain and caspases are key proteases in apoptotic cell death. We evaluated the neuroprotective effects of SJA6017 (a calpain inhibitor) and measured functional recovery in a rat spinal cord injury model. Thirty Wistar albino rats were divided into three groups of 10 animals each: sham-operated (group 1), trauma control (group 2) and trauma-plus-SJA6017 treatment (group 3). Spinal cord trauma was produced in the thoracic region of the animals. Rats in group 3 received SJA6017 1 min after trauma. Treatment efficacy was evaluated after injury using light microscopy and TUNEL staining. Neurological performance was assessed using an inclined plane and a modified version of the Tarlov's grading scale. Group 2 rats showed moderate trauma with widespread edema, hemorrhage, vascular thrombi and necrosis 24 h after injury. Group 3 rats had significantly reduced tissue injury and apoptosis. Tarlov scores revealed that group 3 rats also had ameliorated recovery of limb function. Our results demonstrate that treatment with SJA6017 reduces apoptotic cell death, preserves spinal cord tissue and improves functional outcome. Treating calpain-induced apoptosis with this agent may be a feasible therapeutic strategy for patients with spinal cord injury.

A new silent germline mutation of the TSH receptor: coexpression in a hyperthyroid family member with a second activating somatic mutation.

BACKGROUND: Up to date, three thyroid-stimulating hormone receptor (TSHR) germline variants have been reported for which no functional consequences have been detected by in vitro characterizations. However, familial nonautoimmune hyperthyroidism and hot nodules are clearly associated with constitutively activating TSHR germline mutations. We describe a family with a new TSHR germline mutation that is associated with euthyroidism in 13 family members and hyperthyroidism in 1 family member. METHODS: Mutation analysis of the TSHR gene was performed by denaturing gradient gel electrophoresis. TSHR constructs were characterized by determination of cell surface expression, 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation, and constitutive cAMP activity. RESULTS: A novel TSHR germline mutation (N372T) was found in a man who presented with thyrotoxicosis. The mutation was also detected in 13 family members, all of whom were euthyroid. Interestingly, an additional constitutively active somatic mutation (S281N) was identified on the second parental TSHR allele of the hyperthyroid index patient. Linear regression analysis showed a lack of constitutive activity for N372T. Moreover, coexpression studies of N372T with S281N did not reveal any evidence for a functional influence of N372T on the constitutively active mutation (CAM). CONCLUSIONS: N372T is unlikely to cause altered thyroid function. This is consistent with the finding that only the index patient with the additional somatic mutation S281N was hyperthyroid.

Therapeutic efficacy of Ac-DMQD-CHO, a caspase 3 inhibitor, for rat spinal cord injury.

We investigated the therapeutic efficacy of Ac-DMQD-CHO, a caspase-3 inhibitor, and functional recovery in spinal cord injury in a rat model. Thirty rats were randomized into three groups of 10 each. In groups 2 and 3, spinal cord trauma was produced in the thoracic region. Group 3 rats were treated with Ac-DMQD-CHO. Treatment responses were evaluated based on histopathological and TUNEL staining findings at 24 h and 5 days post-injury. Neurologic performance was assessed during and following treatment. Twenty-four hours after injury, light microscopy examination revealed diffuse hemorrhagic necrosis, edema, vascular thrombi, and polymorphonuclear leukocyte infiltration in group 2 and 3 rats, but cavitation and demyelinization were less prominent in group 3. At this time point, treatment of the rats with Ac-DMQD-CHO significantly reduced the number of apoptotic cells. Traumatic injury to the spinal cord causes apoptosis and administration of Ac-DMQD-CHO decreases apoptosis and improves functional outcome.

Similar prevalence of somatic TSH receptor and Gsalpha mutations in toxic thyroid nodules in geographical regions with different iodine supply in Turkey.

OBJECTIVE: Differences in iodine intake could account for the variable prevalences reported for somatic TSH receptor (TSHR) mutations in toxic thyroid nodules (TTNs). However, this question has not been settled, since no study has yet determined the TSHR mutation prevalence in regions with different iodine supplies in the same population using the same methodology. Therefore, we studied the prevalence of somatic TSHR mutations in TTNs from patients living in iodine-deficient or -sufficient regions in Turkey. DESIGN AND METHODS: We screened 74 TTNs for somatic TSHR mutations. Exons 9 and 10 of the TSHR and 7 and 8 of the Gsalpha were screened by denaturing gradient gel electrophoresis. Determination of X-chromosome inactivation was used for clonality analysis. RESULTS: TSHR mutations were identified in 52 (70.2%) of 74 TTNs. A Gsalpha mutation was identified in one TTN. Three new TSHR mutations were detected (A627V, I640K, I486N). No significant difference between frequencies of TSHR mutations in iodine deficient/sufficient regions was found. The frequency of non-random X-chromosome inactivation was similar in iodine-sufficient or -deficient regions and in TSHR mutation positive or negative hot nodules. CONCLUSIONS: These findings suggest that TTNs in iodine deficient/sufficient areas predominantly arise from aberrant growth of a single cell. Our results suggest that neither the prevalence of TSHR mutations nor that of monoclonal TTNs is related to iodine supply.

Effect of hormone replacement therapy on plasma lipoproteins and apolipoproteins, endothelial function and myocardial perfusion in postmenopausal women with estrogen receptor-alpha IVS1-397 C/C genotype and established coronary artery disease.

UNLABELLED: Effect of hormone replacement (HRT) therapy on plasma lipoproteins and apolipoproteins, endothelial function and myocardial perfusion in postmenopausal women with estrogen receptor-alpha (ER-alpha) IVS1-397 C/C genotype and established coronary artery disease. BACKGROUND/AIMS: Associations between various ER-alpha polymorphisms and clinical phenotypes have been studied, including lipid levels and coronary atherosclerosis. We studied 48 postmenopausal women to determine the effect of ER-alpha IVS1-397 polymorphism on the response to treatment with HRT. METHODS: The study had a randomized, double-blind, placebo-controlled and crossover design. Patients were divided into two groups according to ER-alpha IVS1-397 polymorphism: CC genotype (n = 9); CT or TT genotype (n = 39). HRT was given continuously for 4 weeks, with 4-week washout periods between the treatment periods. Brachial artery Doppler and Tl-201 scintigraphy were performed at the end of each treatment period. RESULTS: HRT lowered total cholesterol, LDL-c and Apo-B levels from baseline values (all p < 0.05) and to a similar degree in CC and CT/TT genotype patients. HRT increased estradiol, HDL-c and Apo A-1 levels relative to baseline values, but to a greater degree in CC patients (p = 0.04, 0.05 and 0.04 by ANOVA, respectively). HRT increased peak forearm blood flow, brachial artery diameter during reactive hyperemia and endothelium-dependent dilation in both groups, but to a greater degree in CC patients (p = 0.03, 0.03 and 0.04 by ANOVA, respectively). Summed stress and rest scores were also more markedly reduced in CC patients (p = 0.04 and 0.05, respectively). The increase in estradiol levels was strongly correlated with the improvement in endothelium-dependent dilation (r = 0.66, p < 0.01), which in turn showed negative correlation with summed stress (r = -0.62, p < 0.01) and rest scores (r = -0.52, p < 0.05) in the CC genotype group. CONCLUSION: These data suggest that the improvement in endothelium-dependent dilation and the reduction in perfusion abnormalities by increasing estradiol levels with HRT in postmenopausal women with coronary artery disease may differ with respect to different genotypes, the effect being more prominent in those patients with ER-alpha IVS1-397 CC genotype.

A case of McCune-Albright syndrome associated with Gs alpha mutation in the bone tissue.

The syndrome of McCune-Albright syndrome (MAS) is clasically defined as a triad presentation with the findings of polyostotic fibrous dysplasia, café-au-lait spots, and sexual precocity. However, not all patients present with complete symptoms. A 52-year-old man was diagnosed as having a variant of McCune-Albright syndrome with the following findings: polyostotic fibrous dysplasia, acromegaly due to pituitary tumor and subclinical hyperthyroidism due to toxic multinodular goiter. Sexual precocity and café-au-lait spots were not noted. Acromegaly was confirmed by laboratory examination (IGF-1, glucose suppression test and TRH stimulation test). Long acting somatostatin analogue was used as treatment. Although the pituitary tumor could not be removed due to technical problems, mass lesions on the cranium were removed subtotally. Histopathological evaluation demonstrated that the lesion complied with fibrous dysplasia. Genomic DNAs were isolated from the craniofacial bones and peripheral leucocytes of the patient. After amplifying the related regions, Gs alpha (Gs alpha) gene was analysed by automatic DNA sequence analysis. An activating mutation of the Gs alpha gene (Arg 201 Cys) was found in the genomic DNA isolated from the bone tissue of the patient, but not in the genomic DNA isolated from the blood. We described a case of MAS associated with Gs alpha mutation in the bone tissue, presenting with polyostotic fibrous dysplasia, subclinical hyperthyroidism and acromegaly.

Stable transmission and expression of the hepatitis B virus total genome in hybrid transgenic mice until F10 generation.

The aim of the present study was the generation of transgenic mice carrying the complete Hepatitis B Virus (HBV) genome and investigation of the presence of Hepatitis B surface antigen (HBsAg) expression through successive generations. Transgenic mice were generated by microinjecting HBV genome into fertilized eggs. Integration and expression of HBsAg in transgenic mice were analyzed by genomic DNA PCR, Southern and slot blots and enzyme-linked immunosorbent assay (ELISA). Expression was also confirmed by Western blotting and RT-PCR. Histological changes in liver tissue of transgenic mice were examined by HE staining. The HBV genome was transmitted to the F10 generation and the presence of HBV X gene transcripts was confirmed by RT-PCR analysis using liver cDNAs from the F10 generation mice. During an observation period of 2.5 years, mice were sacrificed and their organs subjected to histopathological examination. In the liver, slight histopathologic alterations were observed but none of these lineages had any hepatocellular carcinoma (HCC). HBV DNA can be stably transmitted and expressed in the transgenic mice until F10 generation. However, although we showed the presence of X gene transcripts in liver tissues of F10 generation mice by RT-PCR in these animals, long-term expression of the HBV complete genome and expression of X protein in hepatocytes did not cause neoplasia during the life span and HCC. These transgenic mice should be useful for detailed studies of the replication and expression of HBV and for physiological studies of HBV genome.

Mutations in the thyrotropin receptor signal transduction pathway in the hyperfunctioning thyroid nodules from multinodular goiters: a study in the Turkish population.

Many studies have been carried out to determine G(s) alpha and TSHR mutations in autonomously functioning thyroid nodules. Variable prevalences for somatic constitutively activating TSHR mutations in hot nodules have been reported. Moreover, the increased prevalence of toxic multinodular goiters in iodine-deficient regions is well known. In Turkey, a country with high incidence rates of goiter due to iodine deficiency, the frequency of mutations in the thyrotropin receptor signal transduction pathway has not been evaluated up to now. In the present study, a part of the genes of the TSHR, G(s)alpha and the catalytic subunit of the PKA were checked for activating mutations. Thirty-five patients who underwent thyroidectomy for multinodular goiters were examined. Genomic DNAs were extracted from 58 hyperactive nodular specimens and surrounding normal thyroid tissues. Mutation screening was done by single-strand conformational polymorphism (SSCP) analysis. In those cases where a mutation was detected, the localization of the mutation was determined by automatic DNA sequencing. No G(s)alpha or PKA mutations were detected, whereas ten mutations (17%) were identified in the TSHR gene. All mutations were somatic and heterozygotic. In conclusion, the frequency of mutations in the cAMP signal transduction pathway was found to be lower than expected in the Turkish population most likely because of the use of SSCP as a screening method and sequencing only a part of TSHR exon 10.

Two novel mutations in the sixth transmembrane segment of the thyrotropin receptor gene causing hyperfunctioning thyroid nodules.

Autonomously functioning thyroid nodules (AFTNs) can present as hyperfunctioning adenomas or toxic multinodular goiters. In the last decade, a large number of activating mutations have been identified in the thyrotropin receptor (TSHR) gene in autonomously functioning thyroid nodules. Most have been situated close to, or within the sixth transmembrane segment and third intracellular loop of the TSHR where the receptor interacts with the Gs protein. In this study we describe two novel mutations in the sixth transmembrane segment of the TSHR causing hyperfunctioning thyroid nodules. Genomic DNAs were isolated from four hyperfunctioning thyroid nodules, normal tissues and peripheral leukocytes of two patients with toxic multinodular goiter. After amplifying the related regions, TSHR and G(s)alpha genes were analyzed by single-strand conformation polymorphism (SSCP) analysis. The precise localization of the mutations was identified by automatic DNA sequence analysis. Functional studies were done by site-directed mutagenesis and transfection of a mutant construct into COS-7 cells. We identified two novel TSHR mutations in two hyperfunctioning thyroid nodules: Phe631Val in the first patient and Iso630Met in the second patient. Both mutant receptors display an increase in constitutive stimulation of basal cyclic adenosine monophosphate (cAMP) levels compared to the wild-type receptor. This confirms that these mutant receptors cause hyperfunctioning thyroid nodules.

Does a Leu 512 Arg thyrotropin receptor mutation cause an autonomously functioning papillary carcinoma?

In the last decade, studies were first done to determine the frequency of Gsalpha and later thyrotropin receptor (TSHR) mutations in benign autonomously functioning thyroid nodules (AFTN). Different frequencies ranging from 0% to 38% for GSp mutations and from 20% to 86% for TSHR mutations were found. There were only some limited case reports related to TSHR genetic alterations in malignant AFTN. Their role in autonomously functioning thyroid carcinomas is not well established. We present a patient who had thyroidectomy for toxic multinodular goiter and a papillary carcinoma was demonstrated histopathologically. Genomic DNA was isolated from two solid areas in the hot nodule and peripheral leukocytes of the patient. After amplifying the related regions, TSHR and GSalpha genes were analyzed by single-strand conformation polymorphism (SSCP) analysis. The precise localization of the mutations was identified by automatic DNA sequence analysis. An activating mutation of the TSHR gene (Leu 512 Arg) was found in the autonomously functioning papillary carcinoma. It is believed that this mutation causes constitutive activation of the cyclic adenosine monophosphate (cAMP) signal transduction pathway and thereby causes thyrotoxicosis and a hot thyroid nodule in an autonomously functioning papillary carcinoma.

Effect of BCG vaccination on cytokine mRNA expression in atopic children with asthma.

BACKGROUND: To investigate whether a preexisting T(H2)-type immune response could be suppressed by BCG immunization in atopic children with asthma. METHODS AND RESULTS: We have used PCR to amplify reverse transcribed (RT) IFN-gamma and IL-5 mRNA expressed by peripheral blood mononuclear cells (PBMCs) in response to in vitro phytohemagglutinin A, purified protein derivative and Dermatophagoides pteronyssinus II stimulation from nine atopic children, both before and 8 weeks after BCG vaccination. We have demonstrated that IFN-gamma expression was induced in response to all stimulants (IFN-gamma/beta-actin) after the vaccination, whereas there was no expression before (P<0.001). Although there was a tendency to diminish in the expression of IL-5 mRNA in response to the stimulants, only PHA rendered a statistically significant decrease after the vaccination. CONCLUSIONS: These results provide some evidence of TH1 dominance after BCG administration in atopic children.