Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane.

Porcine thyrotropin (TSH) receptors have been purified by Sepharose-TSH affinity chromatography and crosslinked to a 125I-labelled photoactive derivative (N-hydroxysuccinimidyl 4-azidobenzoate; HSAB) of TSH (125I-HSAB-TSH). Purification of the crosslinked complexes on Sephacryl S-300 followed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate showed that the receptor contained two subunits. One subunit (A) with Mr 45 000 was crosslinked to TSH and the other (B) subunit, Mr 25 000, was linked to the A subunit by a disulphide bridge(s). Other, as yet unidentified, subunits may have been non-covalently associated with the A and B subunits. Analysis of reduced and non-reduced crosslinked TSH receptor-125I-HSAB-TSH on Sephacryl S-300 in the presence and absence of detergent indicated that the A subunit was a hydrophilic peptide. This was confirmed in studies of the release into aqueous solution by reducing agent treatment of 125I-HSAB-TSH crosslinked to the TSH receptor A subunit in thyroid membranes. Similar results were obtained with TSH receptors in human thyroid and guinea pig fat cell membranes. These studies suggest that the hydrophilic A subunit of the receptor forms a binding site for TSH on the outside surface of the cell membrane and that the A subunit is linked to the cell membrane by way of a disulphide bridge to the receptor B subunit.     

Detection of 1-phenyl-N-methyl-1,2,3,4-tetrahydroisoquinoline and 1-phenyl-1,2,3,4-tetrahydroisoquinoline in human brain by gas chromatography—tandem mass spectrometry

1-Phenyl-N-methyl-1,2,3,4-tetrahydroisoquinoline and 1-phenyl-1,2,3,4-tetrahydroisoquinoline were detected for the first time in parkinsonian human brain using gas chromatography-tandem mass spectrometry (GC-MS-MS). Since these compounds are structural analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that produces parkinsonism in humans, they might be candidates for endogenous MPTP-like neurotoxins.     

A phylogeny of the chloroplast gene rbcL in the Leguminosae: taxonomic correlations and insights into the evolution of nodulation

Phylogenetic analysis of the chloroplast-encoded rbcL gene in Leguminosae are consistent with previous hypotheses in suggesting that the family as a whole is monophyletic, but that only two of its three subfamilies are natural. The earliest dichotomies in the family appear to have involved tribes Cercideae or Cassieae (subtribe Dialiinae), followed by Detarieae/ Macrolobieae, all of which are members of subfamily Caesalpinioideae. The remainder of the family is divided into two clades: (1) Mimosoideae and the caesalpinioid tribes Caeasalpinieae and Cassieae (subtribes Ceratoniinae and Cassiinae); (2) Papilionoideae. Basal groups within Papilionoideae are, as expected, elements of the grade tribes Sophoreae and Swartzieae. Major clades within Papilionoideae include: (1) a Genistoid Alliance comprising Genisteae, Crotalarieae, Podalyrieae, Thermopsideae, Euchresteae, and also some Sophoreae; (2) a clade marked by the absence of one copy of the chloroplast inverted repeat, with which are associated Robinieae. Loteae, and some Sophoreae; (3) Phaseoleae, Desmodieae. Psoraleeae, and most Millettieae, a group also marked by presence of pseudoracemose inflorescences; and (4) a well-supported clade comprising Aeschynomeneae, Adesmieae, and some Dalbergieae. Nodulation is most parsimoniously optimized on the rbcL strict consensus tree as three parallel gains, occurring in Papilionoideae, the caesalpioioid ancestors of Mimosoideae, and in the genus Chamaecrista (Caesalpinieae: Cassieae).     

Phospholipid/Ca2+-dependent protein kinase activity in human parathyroid adenoma

We have examined the activities of phospholipid/Ca2+-dependent and cyclic AMP-dependent protein kinases of the parathyroid adenomas and the atrophic glands which were resected from three patients with primary hyperparathyroidism. Phospholipid/Ca2+-dependent protein kinase activity of atrophic parathyroid gland was exclusively present in cytosol fraction (90.7 +/- 12.3%). On the other hand, phospholipid/Ca2+-dependent protein kinase activity of parathyroid adenomas was 66.9 +/- 6.4% in cytosol and 33.1 +/- 6.4% in membrane fraction, suggesting a translocation of the enzyme from the cytosol to the membranes. Cyclic AMP-dependent protein kinase activity appeared to be higher in parathyroid adenoma than in atrophic parathyroid gland in both cytosol and membrane fractions.     

Inhibition of TSH binding to chicken thyroid by some cases of LATS (long acting thyroid stimulator)

TSH receptor antibody (TRAb) activity using chicken thyroid receptor (c-TRAb) and porcine thyroid receptor (p-TRAb) was determined by the incubation of 125I-bovine TSH with each receptor. Both c-TRAb and p-TRAb activity in LATS positive and negative Graves' sera were compared. 15 out of 39 LATS positive sera and 4 out of 46 LATS negative sera had positive c-TRAb activity. On the other hand, all LATS positive sera and 33 out of 46 LATS negative sera had positive p-TRAb activity. No relationship between c-TRAb and p-TRAb activity was observed, and there was also no correlation between c-TRAb and LATS activity. Changes in c-TRAb, p-TRAb and LATS activity in the clinical course of patients with Graves' disease were examined. These activities were parallel in some cases, but in others they were not. A weak c-TRAb activity was observed in 4 out of 29 Hashimoto's disease, but all cases with thyroid cancer and subacute thyroiditis showed no activity. Sera with positive c-TRAb activity did not stimulate chicken thyroid in chick bioassay. These results suggest that some cases of TRAb in Graves' disease (mainly LATS) inhibit TSH binding to chicken thyroid receptor (non-mammalian species) in the same way as mammalian thyroid, but may not have any stimulatory action on thyroid hormone synthesis. It is interesting to note that TRAb including LATS have the similar effect on TSH receptor even in nonmammalian species.     

Analysis of oxygen equilibria of the giant hemoglobin from the earthworm Eisenia foetida using the Adair model

Oxygen equilibrium curves of the giant hemoglobin from the earthworm Eisenia foetida were determined at various concentrations of cations. Using the Adair model of 12 oxygenation steps, we succeeded in fitting the data better than the simple concerted model (MWC model). Analysis of the Adair constants (K1 to K12) indicated that the increase in oxygen affinity occurs in the last six steps (K7 to K12) of the oxygen binding and that it is enhanced by increase in Ca2+ concentration. The Hill coefficient (nmax) at pH 7.5 attained a maximum value of 9.76 at 20 mM CaCl2. In the presence of physiological levels of Ca2+ (5 mM), the Bohr effect was similar to that seen in vertebrates. The data were consistent with the release of two Bohr protons being accompanied by the oxygen-linked binding of one Ca2+. Mg2+ and Na+ exerted a similar effect on the hemoglobin, though to a lesser extent. The stoichiometry of Ca2+ binding of the hemoglobin revealed the presence of two classes of binding sites, of which the affinities are high (Ka = 8.8 x 10(3) +/- 103 M-1) and low. The number of high affinity sites per heme was found to be 0.3, comparable to the number of oxygen-linked Ca2+ binding sites.