Guest-induced conformational change of β-cyclodextrin capped with an environmentally sensitive chromophore

A capped cyclodextrin, 6-deoxy-6-(p-hydroxy-m-nitrophenacylthio)-β-cyclodextrin, was prepared in order to detect any conformational change of the host upon the guest binding. The association constant between the cyclodextrin and 1-adamantanecarboxylate, cyclohexanecarboxylate, p-methylbenzoate, 3,3-dimethylbutyrate, or 2,2-dimethylpropionate was enhanced 20, 5.3, 3.7, 2.3, or 2.0 times, respectively, by chromophore capping. The changes in the electronic, NMR, and circular dichroism spectra as well as pK_a of this cyclodextrin upon binding of the guest strongly indicate a conformational change around the chromophoric moiety of the cyclodextrin.     

The role of cyclic AMP in aldosterone production by isolated zona glomerulosa cells.

The role of cyclic AMP in the regulation of aldosterone production by adrenocorticotropic hormone (ACTH), angiotensin II (A II), potassium, and serotonin was examined in collagenase-dispersed adrenal glomerulosa cells. The ability of 8-bromo cyclic AMP and choleragen to stimulate maximum aldosterone production indicated that cyclic AMP could act as second messenger for certain of the aldosterone-stimulating factors. The actions of ACTH and choleragen on aldosterone and cyclic AMP production were correlated in dog and rat cells, and a similar relation was seen during stimulation of rat cells by serotonin. In contrast, A II and potassium did not cause changes in cyclic AMP formation while stimulating aldosterone production. Intracellular and receptor-bound cyclic AMP were increased 3-fold by 10(-7) M ACTH but not by A II. Addition of a phosphodiesterase inhibitor increased the magnitude of the cyclic AMP response to ACTH but did not change the lack of stimulation by A II or potassium. In dog cells, the effects of A II and potassium on aldosterone production were partially additive to those of ACTH, choleragen, and 8-bromo cyclic AMP. In contrast, no additivity was observed between A II and potassium, or between combinations of the cyclic AMP-dependent stimuli. These results indicate that the actions of ACTH on aldosterone secretion are mediated by cyclic AMP formation, whereas A II and potassium stimulate aldosterone production through an independent mechanism. The lack of additivity between steroid responses to A II and potassium suggests that these factors could share a common mode of action on steroidogenesis in zona glomerulosa cells.     

Freeze-Fracture images on filipin-sterol complexes in the thyroid follicle epithelial cell of mice with special regard to absence of cholesterol at the site of micropinocytosis

Summary In order to clarify the distribution of cholesterol in the plasma-and cyto-membranes of the thyroid follicle cell, freeze-fracture images of the filipin-treated tissues of normal and TSH-treated mice were observed. The filipin-sterol complexes, 25 to 30 nm protuberances or pits are distributed densely and almost homogeneously on the fractured plasma membrane, though the small depressions showing aggregates of intramembrane particles lack the complexes. Each depression corresponds to the coated pit, which might be an initial site for micropinocytosis of the luminal colloid. The limiting membranes of all the large colloid droplets reabsorbed are generally very rich in the complexes, but some small regions on the limiting membrane of the droplet are less in their density. The membranes of the rough endoplasmic reticulum, of the nucleus and of the Golgi apparatus are almost free from the complexes, though small clusters consisting of 2–5 complexes are rarely scattered. In thin sections, the membranes which are rich in the filipinsterol complexes become obscure in their fine structure after treatment with filipin for 12–14 h.This study was supperted by grants from the Japan Ministry of Education     

A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.     

Comparison of osteoporosis and calcium intake between Japan and the United States.

The number of osteoporotic females in Japan with vertebral bone mineral density measured by dual energy x-ray absorptiometry, defined as less than -3 SD of the peak bone mass, is approximately 10,000,000, corresponding to 8% of the whole population of Japan. While this value approximately corresponds to the prevalence of low bone mineral density in the United States, the incidence of hip fracture appears to be much less in Japan than in the United States, 50,000 per 125,000,000 per year compared with 250,000 for a population twice as large. This seems to be paradoxical because of the lower bone mineral density and lower calcium intake in Japan, with 400-500 mg/day mainly as soybean products, small fish with bones, and vegetables. The difference in hip fracture incidence, however, may not actually be as wide as it seems when the larger number of bedridden elderly subjects in Japan is taken into consideration. In these bedridden subjects with severe immobilization osteoporosis, hip fracture is only prevented by the fact that they are not ambulatory. Life-style difference may also offer an explanation. Sitting on a tatami mattress on completely flexed knees with frequent standing up, along with other household work, in a narrow home space may ensure a marked development of hip musculature and also provide skill in balancing oneself to prevent fails. The difference in fracture incidence should be analyzed from various angles to obtain a firm ground for the future prevention of hip fracture due to osteoporosis. Although osteoporosis universally affects all races and nationalities, conspicuous differences may be encountered in the severity of its manifestations and complications.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible role of sodium-hydrogen antiport in acetylcholine-induced relaxation of rat aorta.

The decreased extracellular Na concentration (25mM) attenuated vasodilator effect of acetylcholine (ACh) in norepinephrine-treated aortic ring. This attenuation was greater in the low Na medium substituted by Li, which can exchange intracellular H through Na-H antiport, as compared with that substituted by choline, which cannot. 10 microM amiloride canceled the difference between the two low Na mediums. Thus the inhibition of Na-H antiport may counteract the suppressive effect of decreased intracellular Na on ACh vasodilation, suggesting a possible role of Na-H antiport in a release of vasoactive substance(s) from endothelial cells.     

Direct inhibitory effect of amino-terminal parathyroid hormone fragment [PTH(1-34)] on PTH secretion from bovine parathyroid primary cultured cells in vitro

We have examined the possibility of direct inhibitory effect of PTH(1-34) on PTH secretion in bovine parathyroid cells. As low as 10(-12) M PTH(1-34) completely inhibited low calcium (0.5 mM Ca2+)-stimulated PTH secretion by these cells. In the presence of 1.25 mM Ca2+, 10(-12) M PTH(1-34) inhibited PTH secretion by about 14.3% of the basal value, while 10(-11) M or higher concentration of PTH(1-34) showed potent inhibitory effects equivalent to the inhibitory action of high calcium concentration (2.5 mM Ca2+) on PTH secretion. At 2.5 mM Ca2+, as much as 10(-9) M PTH(1-34) failed to inhibit PTH secretion further. These results suggest that PTH(1-34) might directly, not via calcium concentration, inhibit PTH secretion by parathyroid cells and that a cooperative mechanism could exist between calcium and PTH(1-34) to inhibit PTH secretion.     

Message amplification phenotyping of an inherited delta-aminolevulinate dehydratase deficiency in a family with acute hepatic porphyria

The molecular basis of the enzymatic defect responsible for acute hepatic porphyria due to delta-aminolevulinate dehydratase (ALAD) deficiency was investigated in a family including a proband with the acute disease. In order to delineate the mutation in the proband, cDNA for deficient ALAD was synthesized from the proband's cells. The ALAD phenotype was studied by message amplification phenotyping with total RNA extracted from lymphoblastoid cells of the proband and his family members. Two independent mutant alleles of ALAD were identified in the proband's cells. One mutant allele was shown to result in an amino acid substitution at residue 274 (Ala274----Thr). Message amplification phenotyping studies have also permitted us to define the ALAD phenotype of each subject in the family. This is the first mutation to be recognized in the human ALAD gene.     

Fine structural studies on the reabsorption of colloid and fusion of colloid droplets in thyroid glands of TSH-treated mice

Summary The mechanism of the luminal colloid reabsorption and the fate of reabsorbed colloid droplets were studied ultracytochemically in epithelial cells of thyroid cells of TSH-treated mice. The luminal colloid is reabsorbed by micropinocytosis as well as phagocytosis into the follicle epithelial cell. Almost all the pinocytotic pits and vesicles are coated and often closely associated with actin filaments demonstrated by use of heavy meromyosin (HMM). This suggests the involvement of the actin filament system in making and transporting coated vesicles for micropinocytosis of the luminal colloid. Freeze-fracture images show aggregates of intramembrane particles on the P-face of the small depressions corresponding to the initial site for coated pits.The reabsorbed colloid droplets fuse with one another and with lysosomes. At the initial stage of this fusion, the limiting membranes of adjoining droplets fuse in a limited area to become pentalaminar, and then become trilaminar. Eventually, the membranes at the fusion point disappear, and the contents of both droplets become continuous. Freeze-fracture images reveal the disappearance of the intramembrane particles at the initial site where the fusion occurs.Examination of thin-sectioned tissue treated by rapid-freeze substitution fixation, shows clearly delineated cell organelles, and the rounded mitochondria have a characteristically high electron-dense matrix. Just beneath the limiting membrane of each colloid droplet, there always exists a low electron-dense layer about 10 nm thickness. The lysosomes are sometimes seen wrapped around the colloid droplet.This study was supported by grants (No. 56370002, No. 00544016) from the Japan Ministry of Education