Sonia Q. Doi

Differential effects of kindled and electrically induced seizures on a glutamate receptor (GluR1) gene expression.

To address the question of whether the mode of seizure induction contributes to the effects of seizures on glutamate receptor gene expression, we examined rat dorsal hippocampal slides by in situ hybridization after kindling by electrical stimulation of the amygdala, or after electrically induced tonic-clonic seizures. Levels of a glutamate receptor subtype (GluR1) mRNA were analyzed at three periods post kindled seizures and found to be decreased only in brains that were obtained 24 h after the last kindled seizure. This downregulation of GluR1 mRNA was transient and was observed only in animals that had behavioral manifestations after being electrically stimulated. It is probable that maintenance of the kindled state cannot be explained by a long-lasting change in GluR1 gene expression. Repeated electroshock-induced seizures increased GluR1 mRNA levels in the hippocampus. Our results show that mode of induction is an important determinant of the effects of seizures on the levels of expression of a glutamate receptor gene.

Induction of constitutive heat shock protein 73 mRNA in the dentate gyrus by seizures

We examined the effects of generalized seizures on heat shock protein (hsp) mRNA induction in the rat brain using in situ hybridization. Seizures induced by electroconvulsive shock, electrical or cocaine kindling caused a selective induction of the constitutive hsp 73 gene in the dentate gyrus. In these seizure paradigms, not thought to induce widespread tissue damage, neither the heat-inducible hsp 72 gene nor a member of the hsp 90 family (hsp 84) were induced. Hsp 73 may play a role in the adaptation and/or in the maintenance of dentate granule cell integrity following seizures.

Influence of the CYP3A4/5 genetic score and ABCB1 polymorphisms on tacrolimus exposure and renal function in Brazilian kidney transplant patients.

Background Polymorphisms in genes encoding transport proteins and metabolizing enzymes involved in tacrolimus (TAC) disposition may be important sources of individual variability during treatment. Objective The aim of this study was to investigate the effect of combined CYP3A4 and CYP3A5 variants, using a CYP3A4/5 genetic score, and ABCB1 polymorphisms on therapeutic TAC monitoring and their relationship with clinical outcomes. Material and methods Brazilian kidney transplant recipients (n=151), who received TAC over 3 months after transplantation, were genotyped for CYP3A4 rs2242480 (g.20230G>A), CYP3A5 rs15524 (g.31611C>T) and rs776746 (g.6986A>G), ABCB1 rs1128503 (c.1236C>T), rs1045642 (c.3435C>T), and rs2032582 (c.2677G>T/A) polymorphisms. Results Frequencies of CYP3A4 g.20230A, CYP3A5 g.31611C, and g.6986A were 0.37, 0.26, and 0.28, respectively. These alleles were associated with TAC rapid metabolization and were used for CYP3A4/5 genetic score construction. A higher CYP3A4/5 genetic score was associated with higher TAC dose and lower concentrations for dose administered (Co/D, P<0.05). Ninety days after transplantation, the presence of two or more rapid metabolization alleles contributed toward 27.7% of Co/D variability and was associated with a lower estimated glomerular filtration rate values (P<0.05). For ABCB1, the frequencies of c.1236T, c.3435T, and c.2677T/A alleles were 0.42, 0.42, and 0.33/0.04. At 30 days after transplantation, patients carrying ABCB1 c.1236TT+c.3435TT+(c.2677TT+TA) genotypes had higher TAC Co/D than those with common or heterozygous genotypes (P<0.05). Conclusion The results show the impact of the CYP3A4/5 genetic score on TAC exposure and renal function in Brazilian patients. Furthermore, ABCB1 polymorphisms, in a combined analysis, influenced TAC Co/D at 30 days after transplantation.

Transforming growth factor-β1 regulation of C-type natriuretic peptide expression in human vascular smooth muscle cells: dependence on TSC22D1

C-type natriuretic peptide (CNP) possesses nitric oxide-like signaling mechanisms and actions in the vasculature, including the inhibition of fibrosis and vascular remodeling through counterregulation of transforming growth factor-β (TGF-β) signaling. The leucine zipper protein transforming growth factor stimulated clone 22 domain 1 (TSC22D1), cloned via its presumed binding to a GC-rich element in the CNP promoter, was the first protein to be described as a CNP transcription factor, but the lack of supporting evidence since its discovery and its lack of a classical DNA-binding site have left in question its role in the regulation of CNP by TGF-β and other factors. To define a specific role for TSC22D1 in CNP transcription, we have examined the effects of the profibrotic growth factors TGF-β1 and PDGF-BB on CNP mRNA expression in cultured human vascular smooth muscle cells (SMC) in which TSC22D1 expression was suppressed with small interfering RNA. Results showed that TGF-β and PDGF-BB significantly increased CNP expression in all three SMC types. Twenty-four-hour TGF-β-induced elevations in CNP were strongly correlated with changes in TSC22D1 mRNA levels, and both genes exhibited their greatest response to TGF-β1 in coronary artery SMC. Furthermore, siRNA suppression of TSC22D1 expression in coronary artery and aortic SMC by ∼90% resulted in 45-65% reductions of both PDGF- and TGF-β-stimulated CNP expression, respectively. These results support a postulated role of TSC22D1 as an enhancer of CNP transcription and suggest that TGF-β-induced upregulation of CNP expression in SMC may be mediated in part by increased transcription of TSC22D1.

A method to detect the G894T polymorphism of the NOS3 gene. Clinical validation in familial hypercholesterolemia

Abstract An endothelial nitric oxide synthase gene (NOS3) polymorphism in exon 7 (G894T), resulting in Glu298Asp substitution at protein level, has been associated with myocardial infarction, hypertension and coronary atherosclerosis in some populations. This polymorphism is usually identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, the procedures described to date do not eliminate the possibility of misclassification and either require confirmation by DNA sequencing or are timeconsuming. In this study, a PCR-RFLP procedure to detect the G894T polymorphism at the NOS3 was optimized by the introduction of a constitutive cleavage site in the amplification product. This cleavage site provides an internal control for enzymatic activity to avoid mistyping. The method was validated by the study of 35 white unrelated individuals with familial hypercholesterolemia and 70 controls. The frequency of the variant allele (T) was similar between both groups (27% vs. 22%, NS), and comparable to the frequency found in other white populations. However, future studies are necessary to confirm these data. In summary, the optimized procedure for detection of the G894T NOS3 polymorphism is rapid, simple, and does not require confirmatory tests. Using this method, we found no association between this polymorphism and familial hypercholesterolemia.

Clinical and morphologic findings of familial goiter in bongo antelope (Tragelaphus eurycerus).

Inherited defects of thyroglobulin synthesis resulting in congenital goiter are well described in certain breeds of domestic ungulates and in human beings. Goiter associated with synthesis of an abnormal thyroglobulin and the presence of thyroidal albumin was identified in five closely related bongo antelopes (Tragelaphus eurycerus). The goiter had an adult onset, and the affected bongos appeared to remain euthyroid with normal serum T3 and T4 values, normal serum cholesterol concentrations, and nonelevated concentrations of circulating thyroid stimulating hormone (TSH). Goitrous bongos had significant reproductive difficulties, including reduced cyclic activity and prolonged gestations, but were otherwise normal. Over the course of the disease, the thyroid glands greatly enlarged (up to 10 × 20 cm) and became polycystic. Microscopically, there was an admixture of giant colloid-filled follicles and follicles of normal size lined with variable follicular epithelium ranging from squamoid to mildly to moderately hyperplastic. The pathogenesis of goiter in the bongo may reflect a mixture of genetic predisposition coupled with environmental factors, including a period of exposure to a goitrogen.

Detection of the TLR4 1196C>T Polymorphism by Mismatched-Polymerase Chain Reaction Using Plasmid DNA as Internal Control in Restriction Fragment Length Polymorphism Assays

BACKGROUND Restriction fragment length polymorphism (RFLP) is a common molecular assay used for genotyping, and it requires validated quality control procedures to prevent mistyping caused by impaired endonuclease activity. We have evaluated the usefulness of a plasmid-based internal control in RFLP assays. RESULTS Blood samples were collected from 102 individuals with acute myocardial infarction (AMI) and 108 non-AMI individuals (controls) for DNA extraction and laboratory analyses. The 1196C > T polymorphism in the toll-like receptor 4 (TLR4) gene was amplified by mismatched-polymerase chain reaction (PCR). Amplicons and pBluescript II SK- plasmid were simultaneously digested with endonuclease HincII. Fragments were separated on 2% agarose gels. Plasmid was completely digested using up to 55.2 nmL/L DNA solutions and 1 microL PCR product. Nevertheless, plasmid DNA with 41.4 nM or higher concentrations was incompletely digested in the presence of 7 microL PCR product. In standardized conditions, TLR4 1196C>T variant was accurately genotyped. TLR4 1196T allele frequency was similar between AMI (3.1%) and controls (2.0%, p = 0.948). TLR4 SNP was not associated with AMI in this sample population. In conclusion, the plasmid-based control is a useful approach to prevent mistyping in RFLP assays, and it is validate for genetic association studies such as TLR4 1196C>T.

Atrial natriuretic factor (ANF) binds to thyrotropin-regulated receptors in FRTL-5 cells and increases thyroglobulin mRNA

Thyrotropin (TSH) regulation of atrial natriuretic factor (ANF) receptors was studied in the rat thyroid follicular cell line, FRTL-5. Exposure of FRTL-5 cells to 1 mU/ml TSH for 7 days resulted in a tenfold increase in ANF receptors (Bmax = 188 fmol/mg protein) compared with control (Bmax = 18 fmol/mg protein), without affecting binding affinity. An identical treatment of porcine thyrocytes with TSH resulted in a 50% decrease in ANF binding sites. Displacement binding studies indicated that > 80% of the ANF receptors in FRTL-5 cells belong to the ANF-R1 (guanylate cyclase-coupled) receptor subtype. By contrast, > 98% of the ANF receptors in porcine thyrocytes were of the ANF-R2, or clearance, receptor subtype. Intracellular cGMP content was increased thirty-sixfold in FRTL-5 cells by 1 microM ANF, but only 2.5-fold in porcine thyrocytes. cAMP levels were unaffected by ANF in either cell type. Northern blot analysis of poly A mRNA extracted from FRTL-5 cells incubated 2 days in the presence of 100 nM ANF indicated a twofold increase in thyroglobulin mRNA content compared with control. These findings suggest that the ANF-R1 receptor, preferentially expressed in FRTL-5 cells and regulated by TSH, might play a role in regulating thyroid hormone production.

FRTL-5 Rat Thyroid Cells Release Thyroglobulin Sequestered in Exosomes: A Possible Novel Mechanism for Thyroglobulin Processing in the Thyroid

Exosomes are 30–100 nm, membrane-bound vesicles containing specific cellular proteins, mRNAs, and microRNAs that take part in intercellular communication between cells. A possible role for exosomes in thyroid function has not been fully explored. In the present study, FRTL-5 rat thyroid cells were grown to confluence and received medium containing either thyroid stimulating hormone (TSH), exogenous bovine thyroglobulin (bTg), or neither additive for 24 or 48 hours followed by collection of spent medium and ultracentrifugation to isolate small vesicles. Transmission electron microscopy and Western blotting for CD9 indicated the presence of exosomes. Western blotting of exosome extract using a monoclonal anti-Tg antibody revealed a Tg-positive band at ~330 kDa (the expected size of monomeric Tg) with a higher density in TSH-treated cells compared to that in untreated cells. These results are the first to show that normal thyroid cells in culture produce exosomes containing undegraded Tg.

Association of the PPP3CA c.249G>A variant with clinical outcomes of tacrolimus-based therapy in kidney transplant recipients

Background The effects of genetic variants related to the pharmacodynamic mechanisms of immunosuppressive drugs on their therapeutic efficacy and safety have been poorly explored. This study was performed to investigate the influence of the PPP3CA c.249G>A variant on the clinical outcomes of kidney transplant recipients. Patients and methods A total of 148 Brazilian patients received tacrolimus (TAC)-based immunosuppressive therapy for 90 days post-kidney transplantation. The PPP3CA rs3730251 (c.249G>A) polymorphism was determined by real-time polymerase chain reaction. Single-nucleotide polymorphism (SNP) data for CYP3A5 rs776746 (CYP3A5*3C; g.6986A>G) were used to eliminate the confounding effects of this variant. Results The PPP3CA c.249G>A SNP did not influence early TAC exposure, renal function, or other laboratory parameters, including levels of urea, creatinine, glucose, and lipids, and blood counts. This variant also did not account for the cumulative incidence of biopsy-confirmed acute rejection or delayed graft function. Regarding adverse events, PPP3CA c.249A allele carriers initially had a 3.05-fold increased probability of treatment-induced blood and lymphatic system disorders compared with c.249GG genotype individuals (95% confidence interval: 1.10–8.48, p=0.032). However, this result was not maintained after adjusting for body weight and CYP3A5*3C SNP status (p=0.086). Conclusion The PPP3CA c.249G>A variant does not influence the clinical outcomes of Brazilian patients in the early phase of TAC-based immunosuppressive regimen.