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1.  Characterization of rat serum amyloid A4 (SAA4): A novel member of the SAA superfamily 
The serum amyloid A (SAA) family of proteins is encoded by multiple genes, which display allelic variation and a high degree of homology in mammals. The SAA1/2 genes code for non-glycosylated acute-phase SAA1/2 proteins, that may increase up to 1000-fold during inflammation. The SAA4 gene, well characterized in humans (hSAA4) and mice (mSaa4) codes for a SAA4 protein that is glycosylated only in humans. We here report on a previously uncharacterized SAA4 gene (rSAA4) and its product in Rattus norvegicus, the only mammalian species known not to express acute-phase SAA. The exon/intron organization of rSAA4 is similar to that reported for hSAA4 and mSaa4. By performing 5′- and 3′RACE, we identified a 1830-bases containing rSAA4 mRNA (including a GA-dinucleotide tandem repeat). Highest rSAA4 mRNA expression was detected in rat liver. In McA-RH7777 rat hepatoma cells, rSAA4 transcription was significantly upregulated in response to LPS and IL-6 while IL-1α/β and TNFα were without effect. Luciferase assays with promoter-truncation constructs identified three proximal C/EBP-elements that mediate expression of rSAA4 in McA-RH7777 cells. In line with sequence prediction a 14-kDa non-glycosylated SAA4 protein is abundantly expressed in rat liver. Fluorescence microscopy revealed predominant localization of rSAA4-GFP-tagged fusion protein in the ER.
doi:10.1016/j.bbrc.2014.07.054
PMCID: PMC4145149  PMID: 25044109
Rat SAA; Luciferase assay; Promoter activity; Inflammation
2.  Characterization of rat serum amyloid A4 (SAA4): A novel member of the SAA superfamily 
Highlights
•The full length rat SAA4 (rSAA4) mRNA was characterized by rapid amplification of cDNA ends.•rSAA4 mRNA has 1830 bases including a GA dinucleotide tandem repeat in the 5′UTR.•Three consecutive C/EBP promoter elements are crucial for transcription of rSAA4.•rSAA4 is abundantly expressed in the liver on mRNA and protein level.
The serum amyloid A (SAA) family of proteins is encoded by multiple genes, which display allelic variation and a high degree of homology in mammals. The SAA1/2 genes code for non-glycosylated acute-phase SAA1/2 proteins, that may increase up to 1000-fold during inflammation. The SAA4 gene, well characterized in humans (hSAA4) and mice (mSaa4) codes for a SAA4 protein that is glycosylated only in humans. We here report on a previously uncharacterized SAA4 gene (rSAA4) and its product in Rattus norvegicus, the only mammalian species known not to express acute-phase SAA. The exon/intron organization of rSAA4 is similar to that reported for hSAA4 and mSaa4. By performing 5′- and 3′RACE, we identified a 1830-bases containing rSAA4 mRNA (including a GA-dinucleotide tandem repeat). Highest rSAA4 mRNA expression was detected in rat liver. In McA-RH7777 rat hepatoma cells, rSAA4 transcription was significantly upregulated in response to LPS and IL-6 while IL-1α/β and TNFα were without effect. Luciferase assays with promoter-truncation constructs identified three proximal C/EBP-elements that mediate expression of rSAA4 in McA-RH7777 cells. In line with sequence prediction a 14-kDa non-glycosylated SAA4 protein is abundantly expressed in rat liver. Fluorescence microscopy revealed predominant localization of rSAA4-GFP-tagged fusion protein in the ER.
doi:10.1016/j.bbrc.2014.07.054
PMCID: PMC4145149  PMID: 25044109
Rat SAA; Luciferase assay; Promoter activity; Inflammation
3.  Graz Endocrine Causes of Hypertension (GECOH) study: a diagnostic accuracy study of aldosterone to active renin ratio in screening for primary aldosteronism 
Background
Primary aldosteronism (PA) affects approximately 5 to 10% of all patients with arterial hypertension and is associated with an excess rate of cardiovascular complications that can be significantly reduced by a targeted treatment. There exists a general consensus that the aldosterone to renin ratio should be used as a screening tool but valid data about the accuracy of the aldosterone to renin ratio in screening for PA are sparse. In the Graz endocrine causes of hypertension (GECOH) study we aim to prospectively evaluate diagnostic procedures for PA.
Methods and design
In this single center, diagnostic accuracy study we will enrol 400 patients that are routinely referred to our tertiary care center for screening for endocrine hypertension. We will determine the aldosterone to active renin ratio (AARR) as a screening test. In addition, all study participants will have a second determination of the AARR and will undergo a saline infusion test (SIT) as a confirmatory test. PA will be diagnosed in patients with at least one AARR of ≥ 5.7 ng/dL/ng/L (including an aldosterone concentration of ≥ 9 ng/dL) who have an aldosterone level of ≥ 10 ng/dL after the saline infusion test. As a primary outcome we will calculate the receiver operating characteristic curve of the AARR in diagnosing PA. Secondary outcomes include the test characteristics of the saline infusion test involving a comparison with 24 hours urine aldosterone levels and the accuracy of the aldosterone to renin activity ratio in diagnosing PA. In addition we will evaluate whether the use of beta-blockers significantly alters the accuracy of the AARR and we will validate our laboratory methods for aldosterone and renin.
Conclusion
Screening for PA with subsequent targeted treatment is of great potential benefit for hypertensive patients. In the GECOH study we will evaluate a standardised procedure for screening and diagnosing of this disease.
doi:10.1186/1472-6823-9-11
PMCID: PMC2671510  PMID: 19351411
4.  Molecular Mechanism of Terbinafine Resistance in Saccharomyces cerevisiae 
Antimicrobial Agents and Chemotherapy  2003;47(12):3890-3900.
Ten mutants of the yeast Saccharomyces cerevisiae resistant to the antimycotic terbinafine were isolated after chemical or UV mutagenesis. Molecular analysis of these mutants revealed single base pair exchanges in the ERG1 gene coding for squalene epoxidase, the target of terbinafine. The mutants did not show cross-resistance to any of the substrates of various pleiotropic drug resistance efflux pumps tested. The ERG1 mRNA levels in the mutants did not differ from those in the wild-type parent strains. Terbinafine resistance was transmitted with the mutated alleles in gene replacement experiments, proving that single amino acid substitutions in the Erg1 protein were sufficient to confer the resistance phenotype. The amino acid changes caused by the point mutations were clustered in two regions of the Erg1 protein. Seven mutants carried the amino acid substitutions F402L (one mutant), F420L (one mutant), and P430S (five mutants) in the C-terminal part of the protein; and three mutants carried an L251F exchange in the central part of the protein. Interestingly, all exchanges identified involved amino acids which are conserved in the squalene epoxidases of yeasts and mammals. Two mutations that were generated by PCR mutagenesis of the ERG1 gene and that conferred terbinafine resistance mapped in the same regions of the Erg1 protein, with one resulting in an L251F exchange and the other resulting in an F433S exchange. The results strongly indicate that these regions are responsible for the interaction of yeast squalene epoxidase with terbinafine.
doi:10.1128/AAC.47.12.3890-3900.2003
PMCID: PMC296195  PMID: 14638499

Results 1-4 (4)