Cloning and characterization of cDNAs encoding steroidogenic acute regulatory protein from freshwater stingrays (Potamotrygon spp.).

The steroidogenic acute regulatory protein (StAR) is critical to the regulated synthesis of steroids in vertebrates. We have isolated cDNA sequences encoding StAR in the freshwater stingrays Potamotrygon hystrix and P. motoro. A single P. hystrix StAR transcript (3376 bp) and two overlapping P. motoro StAR transcripts (1272 and 3365 bp) were isolated. The P. hystrix and P. motoro StAR transcripts contain open reading frames encoding proteins of 284 amino acids that are 99% identical to each other and 56-64% identical to other StAR proteins. Pregnenolone synthesis by green monkey kidney (COS-1) cells transfected with an expression construct encoding a human cholesterol side chain cleavage/adrenodoxin reductase/adrenodoxin fusion protein was increased 16-fold by coexpression with a pCMV5/P. motoro StAR expression construct. Northern blot analysis revealed a single 4000 bp StAR transcript in the P. motoro interrenal gland, but RT-PCR indicates StAR mRNA is also expressed in the brain, gonads, atria, ventricle, gill (female only) and muscle (female only). Expression in extragonadal and extraadrenocortical tissues is an indication that StAR may be critical to processes other than steroidogenesis. The longest P. motoro StAR transcript contains a sequence with great similarity to short interspersed repetitive elements found in other elasmobranchs. This study is the first to isolate and characterize elasmobranch StAR cDNA sequences and to demonstrate the activity of a nonmammalian StAR protein in a heterologous expression system.

Depletion study of trimethoprim and sulphadiazine in milk and its relationship with mastitis pathogenic bacteria strains minimum inhibitory concentrations (MICs) in dairy cows.

Time-related concentrations in milk of a combination of trimethoprim-sulphadiazine (TMP-SDZ) intramammary formulated infusion and its relationship with pathogenic bacteria strains minimum inhibitory concentrations (MICs) isolated from clinical mastitis cows were analysed. The MICs study was performed for Escherichia coli, Staphylococcus aureus and Streptococcus sp. strains. The SDZ concentrations in milk were analysed using high-performance liquid chromatography (HPLC) and TMP using a microbiological assay. Ten lactating cows milked three times daily were used in the time-concentration studies of TMP-SDZ. Milk samples (approximately 20 mL) from the treated mammary quarters were taken at 6, 12, 24, 30 and 36 h after first administration. In order to define the withdrawal time, milk samples from the treated mammary quarters were taken at 24, 36, 48, 72, 84 and 96 h, after finishing the therapy. The MICs fluctuated between 1 and 8 microg/mL. Effective therapeutic concentrations lasted for 36 h when intramammary infusion was repeated three times every 12 h. No TMP was detected in milk for 24 h after finishing therapy. Milk SDZ concentrations were below 0.1 microg/mL in all treated cows after 84 h finishing therapy. At 96 h after finishing therapy, no SDZ milk concentrations were found in six animals, although four animals of the experimental group still had concentrations of 0.07 microg/mL.

Sequence similarities between a novel putative G protein-coupled receptor and Na+/Ca2+ exchangers define a cation binding domain.

cDNA clones encoding a novel putative G protein-coupled receptor have been characterized. The receptor is widely expressed in normal solid tissues. Consisting of 1967 amino acid residues, this receptor is one of the largest known and is therefore referred to as a very large G protein-coupled receptor, or VLGR1. It is most closely related to the secretin family of G protein-coupled receptors based on similarity of the sequences of its transmembrane segments. As demonstrated by cell surface labeling with a biotin derivative, the recombinant protein is expressed on the surface of transfected mammalian cells. Whereas several other recently described receptors in this family also have large extracellular domains, the large extracellular domain of VLGR1 has a unique structure. It has nine imperfectly repeated units that are rich in acidic residues and are spaced at intervals of approximately 120 amino acid residues. These repeats resemble the regulatory domains of Na+/Ca2+ exchangers as well as a component of an extracellular aggregation factor of marine sponges. Bacterial fusion proteins containing two or four repeats specifically bind 45Ca in overlay experiments; binding is competed poorly by Mg2+ but competed well by neomycin, Al3+, and Gd3+. These results define a consensus cation binding motif employed in several widely divergent types of proteins. The ligand for VLGR1, its function, and the signaling pathway(s) it employs remain to be defined.

Genotype-phenotype correlations of mutations and polymorphisms in HSD11B2, the gene encoding the kidney isozyme of 11beta-hydroxysteroid dehydrogenase.

Mutations in the HSD11B2 gene encoding the kidney (11-HSD2) isozyme of 11beta-hydroxysteroid dehydrogenase cause the syndrome of apparent mineralocorticoid excess, a form of salt-sensitive hypertension. Enzymatic activities of mutant enzymes measured in cultured cells are correlated with several parameters of clinical severity including urinary steroid product:precursor ratios, age at diagnosis, birth weight and potassium levels, but not with blood pressure. In normals or in subjects with essential hypertension, sensitivity of blood pressure to salt loading is correlated with activity of renal 11-HSD2, as measured by an increase in the ratio of urinary free cortisol/urinary free cortisone (UFF/UFE), and also correlated with length of a CA repeat polymorphism in the first intron of HSD11B2. A functional explanation for these associations remains to be elucidated.

Apparent cortisone reductase deficiency: a functional defect in 11beta-hydroxysteroid dehydrogenase type 1.

A 36-yr-old woman was referred to the endocrine clinic for investigation of oligomenorrhea, hirsutism, and acne. She was plethoric and overweight with central fat distribution. Plasma cortisol was normal, but her adrenal glands were enlarged (CT scan). Urinary tetrahydrocortisone excretion rate was consistently high, raising the possibility of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) deficiency. In addition, 5beta- reduction of cortisol and cortisone was markedly enhanced. The levels of all cortisol metabolites were suppressed normally with dexamethasone, but conversion of oral cortisone acetate to plasma cortisol was delayed and subnormal compared with that of healthy volunteers. This was accompanied by a larger than normal increase in plasma cortisone concentration. Thus, the defect appears to be in 11beta-HSD1 activity and not in 5beta-reductase activity. Three close relatives of the subject showed no comparable abnormalities, and analysis of the coding region and exon/intron boundaries of the 11beta-HSD1 gene of the case revealed no differences from the consensus sequence. The defect may lie outside the coding region. Alternatively, some other inherited or acquired defect may lead to inhibition of this enzyme system.

Mutants of 11beta-hydroxysteroid dehydrogenase (11-HSD2) with partial activity: improved correlations between genotype and biochemical phenotype in apparent mineralocorticoid excess.

Mutations in the kidney isozyme of human 11-hydroxysteroid dehydrogenase (11-HSD2) cause apparent mineralocorticoid excess, an autosomal recessive form of familial hypertension. We studied 4 patients with AME, identifying 4 novel and 3 previously reported mutations in the HSD11B2 (HSD11K) gene. Point mutations causing amino acid substitutions were introduced into a pCMV5/11HSD2 expression construct and expressed in mammalian CHOP cells. Mutations L179R and R208H abolished activity in whole cells. Mutants S180F, A237V, and A328V had 19%, 72%, and 25%, respectively, of the activity of the wild-type enzyme in whole cells when cortisol was used as the substrate and 80%, 140%, and 55%, respectively, of wild-type activity when corticosterone was used as the substrate. However, these mutant proteins were only 0.6% to 5.7% as active as the wild-type enzyme in cell lysates, suggesting that these mutations alter stability of the enzyme. In regression analyses of all AME patients with published genotypes, several biochemical and clinical parameters were highly correlated with mutant enzymatic activity, demonstrated in whole cells, when cortisol was used as the substrate. These included the ratio of urinary cortisone to cortisol metabolites (R(2)=0.648, P<0.0001), age at presentation (R(2)=0.614, P<0.0001), and birth weight (R(2)=0.576, P=0.0004). Approximately 5% conversion of cortisol to cortisone is predicted in subjects with mutations that completely inactivate HSD11B2, suggesting that a low level of enzymatic activity is mediated by another enzyme, possibly 11-HSD1.

Functional analysis of four CYP21 mutations from spanish patients with congenital adrenal hyperplasia.

Deleterious mutations in the CYP21 (steroid 21-hydroxylase) gene cause congenital adrenal hyperplasia (CAH). These mutations usually result from recombinations between CYP21 and an adjacent pseudogene, CYP21P, including deletions and transfers of deleterious mutations from CYP21P to CYP21 (gene conversions). Additional rare mutations that are not gene conversions account for 5-10% of 21-hydroxylase deficiency alleles. Recently, four novel CYP21 point mutations leading to amino acid changes were identified in a population of 57 Spanish families with CAH. A nonsense mutation, K74X, was also identified. The enzymatic activities of 21-hydroxylase mutants G90V, G178A, G291C, and R354H were examined in transiently transfected CHOP cells using progesterone and 17alpha-hydroxyprogesterone as substrates. The G90V, G291C, and R354H mutations effectively eliminated 21-hydroxylase activity. However, the G178A mutant retained significant activity when 17alpha-hydroxyprogesterone was the substrate. These results correlate well with the identification of G90V, G291C, and R354H in patients with severe "salt-wasting" disease and G178A in a patient with the milder simple virilizing form.

Expression of human kidney 11beta-hydroxysteroid dehydrogenase (11-HSD2) in bacteria.

The kidney isozyme of 11beta-hydroxysteroid dehydrogenase (11-HSD2) protects the mineralocorticoid receptor from spurious activation by glucocorticoids. To explore structure-function relationships, human 11-HSD2 cDNA was subcloned into the bacterial expression vector, pET25b. E. coli transformed with wild-type cDNA produced active enzyme that retained biochemical characteristics of the native protein. The addition of 6 histidine residues to the C-terminus of the wild-type enzyme (11-HSD2/His) increased activity 2-fold. Whereas wild-type activity was almost completely sedimented following 100,000g centrifugation, 10-30% of total activity of 11-HSD2/His remained in the supernatant. The 11-HSD2 isozyme normally contains three N-terminal hydrophobic domains. Mutant 11-HSD2/His possessing a single hydrophobic domain retained partial activity, but elimination of all domains inactivated the enzyme. Thus, the N-terminal hydrophobic domains are essential for complete activity of 11-HSD2 but association with an intact cell membrane is not.