Relative potency in acute and chronic suppressive effects of prednisolone and betamethasone on the hypothalamic-pituitary-adrenal axis in man

The relative potency in the hypothalamic-pituitary-adrenal (HPA) suppression of both prednisolone and betamethasone was examined in an acute study with normal volunteers and in a chronic study with glucocorticoid-treated patients. Circadian rhythm of plasma cortisol was studied after a single dose administration of 5 to 30 mg prednisolone or 0.5 to 3.0 mg betamethasone at 8:00 hr. Morning-rise of plasma cortisol occurred on the morning after the administration of 30 mg or less prednisolone but no morning rise was noted after the administration of 1.0 mg or more betamethasone. Plasma ACTH was slightly elevated on the morning after 30 mg prednisolone administration but showed low levels throughout the night after 3.0 mg betamethasone administration. Plasma cortisol responsiveness to ACTH was examined in patients before and during therapy with either prednisolone or betamethasone. The basal cortisol level was not suppressed and the responsiveness to ACTH remained nearly normal during long-term 5 mg prednisolone therapy, but these were completely suppressed during long-term 5 mg betamethasone therapy. The responsiveness to ACTH was nearly normal in patients receiving alternate-day therapy with prednisolone in such large doses as 50 or 60 mg every other day, but was completely suppressed in patients receiving 1.0 mg betamethasone every other day. The relative potency of betamethasone in acute and chronic suppressive effects on the HPA system seems to be much stronger than that of prednisolone in equivalent doses with comparable anti-inflammatory effects. It is also suggested that the alternate-day therapy with such long-acting steroids as betamethasone are useless in preventing HPA suppression.     

Structure and intramolecular dynamics of a photoactive pigment-protein eosin-casein complex

The structure of eosin--casein complex was studied by triplet label method. Quantitative data on the quantum--mechanic exchange interaction between eosin centres and external quenchers were obtained. The dynamic state of water--protein matrix at -20 degrees to -180 degrees C with eosin as fluorescence and phosphorescence labels and natural chromophores of protein--tryptophane was studied.     

Nucleotide specificity of cardiac sarcoplasmic reticulum. Inhibition of GTPase activity by ATP analogue in fluorescein isothiocyanate-modified calcium ATPase.

Unlike skeletal muscle sarcoplasmic reticulum, canine cardiac sarcoplasmic reticulum hydrolyzes GTP in ways that are similar and different from ATP hydrolysis. Also, ATP and ATP analogues inhibit GTPase activity noncompetitively with a Ki compatible with the high affinity ATP-binding site (c.f. Tate, C.A., Bick, R.J., Blaylock, S., Youker, K., Scherer, N.M., and Entman, M.L. (1989) J. Biol. Chem. 264, 7809-7813). This suggested that ATP and GTP may enter the reaction pathway at separate nucleotide-binding sites on the CaATPase. To test this hypothesis, cardiac sarcoplasmic reticulum was incorporated with fluorescein isothiocyanate (FITC), which apparently binds at or near the ATP-binding site of the enzyme, preventing ATP binding. After FITC incorporation, calcium-dependent ATPase activity, but not GTPase activity, was completely inhibited. Adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), but not guanyl-5'-yl imidodiphosphate, protected against FITC incorporation and the inhibition of calcium-dependent ATPase activity; at least 100 microM AMP-P(NH)P was required for some protection. Despite FITC incorporation, AMP-P(NH)P still inhibited the GTPase activity with a Ki of 3-7 microM. Direct photo-affinity labeling with either 0.2 microM [alpha-32P]ATP or 0.2 microM [alpha-32P]GTP demonstrated that FITC incorporation did not prevent ATP or GTP binding. The mechanism of FITC inhibition of calcium-dependent ATPase activity was related to the prevention of all calcium-dependent, but not calcium-independent, reactions with both nucleotides.     

Notes on the morphology,ecology and life cycles of Fukaurahydra anthoformis and Hataia parva (Hydrozoa,Athecata)

Fukaurahydra anthoformis and Hataia parva are solitary athecate hydroids occurring in northern Japan. New information on the external morphology, nematocysts, ecology, and life cycles of these species is presented. It is noteworthy that H. parva bears stenoteles, which are generally not found among the families of Filifera. Neither species produces free medusae. The eggs are fertilized in the female gonophores, from which unciliated larvae are released. These larvae do not swim and soon attach to a substrate. After attachment the larvae become covered by a sheath to form cysts. The cysts rest on a substrate without any outer change for several months. As the water temperature drops in autumn to early winter the cysts begin to hatch, forming tiny polyps after the larva creeps out from the chitinous sheath. Cyst formation proves to be common also in other solitary hydroids, most of which are inhabitants of cool or cold waters.     

The extent of N epsilon-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid.

The autoxidation of ascorbic acid (ASA) leads to the formation of compounds which are capable of glycating and crosslinking proteins in vitro. When the soluble crystallins from bovine lens were incubated with ASA in the presence of sodium cyanoborohydride, a single major adduct was observed, whose appearance correlated with the loss of lysine. When polylysine was reacted with equivalent amounts of ASA under the same conditions, this product represented half of the total lysine content after four weeks of incubation at 37 degrees C. This adduct was isolated and identified as N epsilon-(carboxymethyl)lysine (CML) by TLC, GC/MS and amino acid analysis. Several oxidation products of ASA were each reacted with polylysine in the presence of sodium cyanoborohydride to identify the reactive species. CML was the major adduct formed with either ASA and dehydroascorbic acid (DHA). Markedly diminished amounts were seen with L-2,3-diketogulonic acid (DKG), and L-threose, while no CML was formed with L-threo-pentos-2-ulose (L-xylosone). In the absence of sodium cyanoborohydride the yield of CML was similar with each of the ASA autoxidation products and required oxygen. Reactions with [1-14C]ASA gave rise to [14C]CML, but only with NaCNBH3 present. At least two routes of CML formation appear to be operating depending upon whether NaCNBH3 is present to reduce the putative Schiff base formed between lysine and DHA.     

Acid secretion and the H,K ATPase of stomach.

The regulation of acid secretion was clarified by the development of H2-receptor antagonists in the 1970s. It appears that gastrin and acetylcholine exert their effects on acid secretion mainly by stimulation of histamine release from the enterochromaffin-like (ECL) cell of the fundic gastric mucosa. The isolated ECL cell of rat gastric mucosa responds to gastrin/cholecystokinin (CCK), acetylcholine, and epinephrine with histamine release and to somatostatin and R-alpha-methyl histamine by inhibition of histamine release. Histamine and acetylcholine stimulate the parietal cell by elevation of cAMP or [Ca]i by activation of H2 or M3 receptors, respectively. These independent pathways converge to activate the gastric acid pump, the H+,K+ ATPase. Activation is a function of the association of the ATPase with a potassium chloride transport pathway that occurs in the membrane of the secretory canaliculus of the parietal cell. Hence the secretory canaliculus is the site of acid secretion, the acid being pumped into the lumen of the canaliculus. The pump is composed of two subunits, a large catalytic and a smaller glycosylated protein. This final step of acid secretion has become the target of drugs also designed to inhibit acid secretion. The target domain of the benzimidazole class of acid pump inhibitors is the extracytoplasmic domain of the pump that is secreting acid, and the target amino acids are the cysteines present in this domain. The secondary structure of the pump can be analyzed by determining trypsin-sensitive bonds in intact, cytoplasmic-side-out vesicles of the ATPase, and it has been shown that the alpha subunit has at least eight membrane-spanning segments. Omeprazole, the first acid pump inhibitor, forms a disulfide bond with cysteines in the extracytoplasmic loop between the fifth and sixth membrane-spanning segment and to a cysteine in the extracytoplasmic loop between the seventh and eight segments, preventing phosphorylation of the pump by ATP. As a result of the effective and long-lasting inhibition of acid secretion by the acid pump inhibitor, superior clinical results have been found in all forms of acid-related disease.     

Kinetic properties of a L-cysteine desulfhydrase-deficient mutant in the enzymatic formation of L-cysteine from D,L-ATC

Summary A mutant strain lacking in activity of L-cysteine desulfhydrase, a L-cysteine-decomposing enzyme, was screened after UV-treatment ofPseudomonas sp. CU6. The properties of the two strains, original and mutant, were compared on the basis of parameter values estimated from kinetic simulations of the enzymatic formation of L-cysteine from D,L-ATC. Both strains suffered from product inhibition, though inhibition was less for the mutant strain.     

Production of recombinant human glucagon in the form of a fusion protein in Escherichia coli; recovery of glucagon by sequence-specific digestion

Summary Recombinant human glucagon was succesfully produced with a high level of expression in Escherichia coli as a fusion protein with human interferon . The synthetic gene was designed to release glucagon, which does not contain glutamic acid residues, from fusion protein with the Staphylococcus aureus strain V8 protease that specifically cleaves the peptide bond on the carboxyl side of the glutamic acid residue. The resulting glucagon was purified to homogeneity by a combination of C₁₈ reverse-phase HPLC and ion-exchange HPLC. The yield of intact glucagon obtained from 11 of culture was approximately 12 mg. The structure of recombinant human glucagon was confirmed by HPLC and amino acid composition/sequence analyses. Offprint requests to: J. Ishizaki     

Plant thioredoxin h: an animal-like thioredoxin occurring in multiple cell compartments

Thioredoxin h has been purified to electrophoretic homogeneity from spinach roots using a procedure devised for leaves. The root thioredoxin (h2 form) differed from chloroplast and animal thioredoxins in showing an atypical active site (Cys-Ala-Pro-Cys) but otherwise resembled animal thioredoxin in structure. Sequence data for a total of 72 residues of spinach root thioredoxin h2 (about 69% of the primary structure) showed 43-44% identity with rabbit and rat thioredoxin. Analysis of cell fractions from the endosperm of germinating castor beans revealed that thioredoxin h occurs in the cytosol, endoplasmic reticulum, and mitochondria. The present findings demonstrate a similarity between plant thioredoxin h and animal thioredoxins in structure and intracellular location and raise the question of whether these proteins have similar functions.