Isolation and characterisation of a novel rabbit sulfotransferase isoform belonging to the SULT1A subfamily.

Sulfotransferases (SULTs) catalyse the sulfonation of both endogenous and exogenous compounds including hormones, catecholamines, drugs and xenobiotics. While in most occasions, sulfonation is a detoxication pathway, in the case of certain drugs and carcinogens, it leads to metabolic activation. Since, the rabbit has been extensively used for both pharmacological and toxicological studies, the purpose of this study was to further characterise the sulfotransferase system of this animal. In the present study, a novel sulfotransferase isoform (GenBank Accession no. AF360872) was isolated from a rabbit liver cDNA lambdaZAP II library. The full-length sequence of the clone was 1138 bp long and contained a coding region of 888 bp encoding a cytosolic protein of 295 amino acids (deduced molecular weight 34,193 Da). The amino acid sequence of this novel SULT isoform showed >70% identity with members of the SULT1A subfamily of sulfotransferases from other species. Upon expression of the encoded rabbit sulfotransferase in Escherchia coli (E. coli), it was shown that the enzyme was capable of sulfonating both p-nitrophenol (K(m) and Vmax values of 0.15 microM and 897.5 nmol/min/mg protein, respectively) and dopamine (K(m) and V(max) values of 175.3 microM and 151.1 nmol/min/mg protein, respectively). Based on the sequence data obtained and substrate specificity, this new rabbit sulfotransferase was named rabSULT1A1. Immunoblotting was used to demonstrate that rabSULT1A1 protein is expressed in liver, duodenum, jejunum, ileum, colon and rectum.

Stability of sirolimus (rapamycin) in whole blood.

The effects of storage time (0-8 days), temperature (4 degrees C and 30 degrees C in dark and light), and freeze-thaw cycles on the stability of sirolimus in blood were examined. Sirolimus quantification was undertaken using HPLC-electrospray-tandem mass spectrometry. Whole blood samples supplemented with sirolimus (5.0, 15.0, and 30.0 microg/L) and pooled renal and heart transplant samples were found to be stable during the 8 days under all conditions (<10% decrease in concentration). No significant difference was observed in sirolimus concentration between freshly collected patient samples and sirolimus-supplemented samples (5.0, 15.0, and 30.0 microg/L) after three freeze-thaw cycles (p > 0.198). In conclusion, blood samples can be transported with or without cooling for up to 8 days without sirolimus results being compromised. The reanalysis of sirolimus samples, which may entail freeze-thaw cycles, can be undertaken if the number of cycles is three or less.

The 460Trp polymorphism of the human alpha-adducin gene is not associated with isolated systolic hypertension in elderly Australian Caucasians.

The study was undertaken to determine whether polymorphic variants of the alpha-adducin gene are associated with isolated systolic hypertension (ISH) in elderly Australian Caucasians. Participants were classified with ISH (n = 87, systolic blood pressure (SBP) > or =160 mm Hg and diastolic blood pressure (DBP) < or =90 mm Hg) or normotension (n = 124, SBP <140 mm Hg and DBP <90 mm Hg with no family history of hypertension). To collect demographic data, a structured questionnaire was used. DNA was extracted using standard techniques from 211 subjects (age range 61-89, mean age 73 +/- 6.6 years, male: female ratio 1.1:1). Genotypes (gly/gly, trp/gly and trp/trp) were assigned in triplicate by polymerase chain reaction (PCR) followed by electrophoresis, using a laser scanning electrophoresis unit. The validity of the method was confirmed by sequencing. Frequencies of allele distribution in ISH or control groups were determined by Chi-square tests and a stepwise logistic regression model, which controlled for potential confounders, was used to examine any independent association between alpha-adducin genotypes or alleles with ISH and normotensive controls. Mean BP (+/- s.d.) was: 170/79.8 +/- 14.9/8.3 mm Hg and 122.1/ 73.4 +/- 8. 8/7.6 mm Hg in the ISH and normotension groups respectively. The unadjusted allele and genotypes frequencies were not significantly different in the ISH patients groups compared with normotensive controls (chi2 = 1.59, P = 0.45 and chi2 = 1.23, P = 0.28 respectively). In this elderly cohort, after adjustment for potential confounders, no statistically significant association was found between alpha-adducin genotype and SBP (P = 0.65 for homozygotes, P = 0.59, for heterozygotes), DBP (P = 0.49 homozygotes, for heterozygotes P = 0.45) pulse pressure (P = 0.87 homozygotes, for heterozygotes P = 0.95) diagnosis of ISH (P = 0.72 for homozygotes, P = 0.68 for heterozygotes). However age and renal disease predicted the diagnosis of ISH (P = 0.001, P = 0.459, respectively), a large pulse pressure (P < 0.0001, P = 0.033, respectively) and a higher SBP (P < 0.0001, P = 0.025, respectively) in this large cohort of elderly Australian Caucasian volunteers. Journal of Human Hypertension (2000) 14, 199-203.