A novel bioassay for anti-thyrotrophin receptor autoantibodies detects both thyroid-blocking and stimulating activity.

Autoantibodies to the thyrotrophin (TSH) receptor (anti-TSHR) are unique, in that they are involved directly in the pathophysiology of certain autoimmune thyroid diseases (AITD). Thyroid-stimulating antibodies (TSAb) act as agonists that activate the thyroid gland and cause Graves' disease. Other anti-TSHR antibodies block TSH and can cause hypothyroidism. Thyroid-blocking antibodies (TBAb) have not been studied as extensively as TSAb. We developed a TBAb bioassay based on a cell line that expresses a chimeric TSHR. The 50% inhibitory concentration of the chimeric Chinese hamster ovary (CHO)-Luc cells was more than five-fold lower compared with the wild-type CHO-Luc cells. We tested the performance of this bioassay using a thyroid-blocking monoclonal antibody K1-70, established an assay cut-off and detected TBAb in 15 of 50 (30%) patients with AITD. Interestingly, the assay detects both TSAb and TBAb and measures the net activity of a mixture of both types of antibodies. There was a high correlation (R(2) 0·9, P < 0·0001) between the results of the TSAb assay and the negative percentage inhibition of the TBAb assay. The TBAb bioassay was approximately 20-fold more sensitive than a commercially available TSHR binding assay (TRAb). In contrast to TRAb, sera with high levels of TBAb activity were able to be diluted several hundred-fold and still exhibit blocking activity above the cut-off level. Thus, this TBAb bioassay provides a useful tool for measuring the activity of anti-TSHR antibodies and may help clinicians to characterize the diverse clinical presentations of patients with AITD.

Biochemical characterization and sequence analysis of the gluconate:NADP 5-oxidoreductase gene from Gluconobacter oxydans.

Gluconate:NADP 5-oxidoreductase (GNO) from the acetic acid bacterium Gluconobacter oxydans subsp. oxydans DSM3503 was purified to homogeneity. This enzyme is involved in the nonphosphorylative, ketogenic oxidation of glucose and oxidizes gluconate to 5-ketogluconate. GNO was localized in the cytoplasm, had an isoelectric point of 4.3, and showed an apparent molecular weight of 75,000. In sodium dodecyl sulfate gel electrophoresis, a single band appeared corresponding to a molecular weight of 33,000, which indicated that the enzyme was composed of two identical subunits. The pH optimum of gluconate oxidation was pH 10, and apparent Km values were 20.6 mM for the substrate gluconate and 73 microM for the cosubstrate NADP. The enzyme was almost inactive with NAD as a cofactor and was very specific for the substrates gluconate and 5-ketogluconate. D-Glucose, D-sorbitol, and D-mannitol were not oxidized, and 2-ketogluconate and L-sorbose were not reduced. Only D-fructose was accepted, with a rate that was 10% of the rate of 5-ketogluconate reduction. The gno gene encoding GNO was identified by hybridization with a gene probe complementary to the DNA sequence encoding the first 20 N-terminal amino acids of the enzyme. The gno gene was cloned on a 3.4-kb DNA fragment and expressed in Escherichia coli. Sequencing of the gene revealed an open reading frame of 771 bp, encoding a protein of 257 amino acids with a predicted relative molecular mass of 27.3 kDa. Plasmid-encoded gno was functionally expressed, with 6.04 U/mg of cell-free protein in E. coli and with 6.80 U/mg of cell-free protein in G. oxydans, which corresponded to 85-fold overexpression of the G. oxydans wild-type GNO activity. Multiple sequence alignments showed that GNO was affiliated with the group II alcohol dehydrogenases, or short-chain dehydrogenases, which display a typical pattern of six strictly conserved amino acid residues.

Incapability of Gluconobacter oxydans to produce tartaric acid.

The dependence of tartaric acid production by Gluconobacter oxydans ssp. oxydans ATCC 19357 and G. oxydans ssp. suboxydans ATCC 621 on vanadate was investigated. It was found with both organisms that tartaric acid could only be produced in a medium containing vanadate (NH(4)VO(3)). A proposed intermediate of the tartaric acid metabolism in G. oxydans, 5-ketogluconic acid, was tested on its reactivity in the presence of the oxidizing catalyst vanadate. It could be shown that 5-ketogluconic acid and the catalyst vanadate, but not the activity of G. oxydans, were responsible for the formation of tartaric acid. G. oxydans was not able to produce tartaric acid by itself. The stereochemical identity of the formed tartaric acid could be identified as the L-(+)-type. Oxalic acid was formed from 5-ketogluconic acid with vanadate in the absence and in the presence of G. oxydans. The ratio of oxalic acid to tartaric acid was 1:1.

Purification of the pyruvate dehydrogenase multienzyme complex of Zymomonas mobilis and identification and sequence analysis of the corresponding genes.

The pyruvate dehydrogenase (PDH) complex of the gram-negative bacterium Zymomonas mobilis was purified to homogeneity. From 250 g of cells, we isolated 1 mg of PDH complex with a specific activity of 12.6 U/mg of protein. Analysis of subunit composition revealed a PDH (E1) consisting of the two subunits E1alpha (38 kDa) and E1beta (56 kDa), a dihydrolipoamide acetyltransferase (E2) of 48 kDa, and a lipoamide dehydrogenase (E3) of 50 kDa. The E2 core of the complex is arranged to form a pentagonal dodecahedron, as shown by electron microscopic images, resembling the quaternary structures of PDH complexes from gram-positive bacteria and eukaryotes. The PDH complex-encoding genes were identified by hybridization experiments and sequence analysis in two separate gene regions in the genome of Z. mobilis. The genes pdhAalpha (1,065 bp) and pdhAbeta (1,389 bp), encoding the E1alpha and E1beta subunits of the E1 component, were located downstream of the gene encoding enolase. The pdhB (1,323 bp) and lpd (1,401 bp) genes, encoding the E2 and E3 components, were identified in an unrelated gene region together with a 450-bp open reading frame (ORF) of unknown function in the order pdhB-ORF2-lpd. Highest similarities of the gene products of the pdhAalpha, pdhAbeta, and pdhB genes were found with the corresponding enzymes of Saccharomyces cerevisiae and other eukaryotes. Like the dihydrolipoamide acetyltransferases of S. cerevisiae and numerous other organisms, the product of the pdhB gene contains a single lipoyl domain. The E1beta subunit PDH was found to contain an amino-terminal lipoyl domain, a property which is unique among PDHs.

Ovarian hyperstimulation for in-vitro fertilization controlled by GnRH agonist administered in combination with human menopausal gonadotrophins.

Surges of luteinizing hormone (LH) in serum that result in luteinization, but occur prematurely with respect to the diameter of the leading follicle, frustrate attempts to induce multiple follicular maturation for in-vitro fertilization (IVF) in a number of women. We examined the possibility of blocking premature LH surges by the administration of D-TRP6-LH-RH, a potent agonistic analogue of gonadotrophin-releasing hormone (GnRH). Six patients who had repeatedly shown premature LH surges were treated for 10 days, beginning between days 1 and 3 of the cycle with daily s.c. injections of 500 micrograms D-Trp6-LH-RH followed by a daily injection of 100 micrograms of the analogue until the day of administration of human chorionic gonadotrophin (HCG). When pituitary and ovarian suppression had occurred, ovarian stimulation with human menopausal gonadotrophin was started and adjusted in dose according to the ovarian response. HCG was injected when the dominant follicle had reached a diameter of at least 18 mm and oestradiol levels were above 300 pg for each follicle greater than 15 mm. Oocyte collection was performed 36 h later via laparoscopy, followed by IVF and embryo transfer. The six patients studied to date responded to therapy and treatment could be completed up to embryo transfer. Two patients became pregnant; one of the pregnancies, however, resulted in abortion. Combined treatment with GnRH analogue for suppression of pituitary gonadotrophin secretion followed by the administration of gonadotrophins thus seems to be a promising method for ovarian stimulation in patients who frequently exhibit premature LH discharges and therefore fail to complete treatment.