Suppressive effect of elcatonin, an eel calcitonin analogue, on excessive urinary hydroxyproline excretion in polyostotic fibrous dysplasia (McCune-Albright's syndrome)

A 12-year-old Japanese girl with polyostotic fibrous dysplasia and endocrine concomitants, was treated with elcatonin, a synthetic eel calcitonin analogue, 10 MRC unit/twice a week given by intramuscular injection. Significant decreases in 24 hr urinary content of hydroxyproline and other amino acids from bone collagen were observed during the course of treatment over 5 months. This biochemical result suggests that the synthetic eel calcitonin analogue exhibits the therapeutic effect in patients with polyostotic fibrous dysplasia by inhibiting bone resorption.     

H4-isozyme of lactate dehydrogenase in the solution of sodium chloride--3. The enzymatic activity and the pyruvate inhibition

1. Low enzymatic activities in low pyruvate concentrations and high Km were observed in sodium chloride solutions. 2. The pyruvate inhibition shown by the % activity at 1 mM pyruvate was lower sodium chloride than in 0.1 M sodium phosphate. 3. At 40 degrees C, as compared with results at 20 degrees C, less pyruvate inhibition was observed in phosphate buffer and in sodium chloride solutions. 4. By using the equilibrium constants between dimer and tetramer, a theoretical explanation is proposed for the pyruvate inhibition. In this explanation, it is suggested that the quaternary complex which is composed of tetrameric enzyme, coenzyme and two kinds of pyruvates was the main cause of the pyruvate inhibition.     

Effect of high osmotic media on blood viscosity and red blood cell deformability

Effects of high osmotic media on the shape and deformability of RBC were examined for determining increasing factors of blood viscosity. Dog blood and Urographin (a hypertonic contrast medium) were used; the plasma osmolality was changed by Urografin suspended in blood. The viscosity was measured for normal RBC and glutaraldehyde-treated RBC suspensions with a cell volume concentration. The RBC deformability was evaluated from the difference in viscosity between the two suspensions. It was shown that normal RBC suspension increased the viscosity with increase in osmolality at high shear rate; hardened RBC suspension decreased the viscosity with increase in osmolality. It was concluded that the RBC deformability decreased with increasing osmolality.     

Effects of hemolysis, hematocrit, RBC swelling, and flow rate on light scattering by blood in a 0.26 cm ID transparent tube

The intensity of light scattering by blood in a tube of diameter 0.26 cm was measured with an apparatus devised by us at different angles on an incident cross-sectional plane. Changes in angular distribution of light intensity associated with hemolysis, and changes in hematocrit (Ht), red blood cell (RBC) swelling, and flow rate were plotted on polar coordinates. The dyssymmetry index, defined as the ratio of the intensity of light at 45 degrees to that at 135 degrees, was used to characterize the shape of the diagrams of light scattering. The index decreased with Ht, flow rate, but increased with RBC swelling. It is concluded that light scattering by blood requires intactness of the RBC membrane. Even when the cell membrane is intact, light scattering is subject to changes with the flow rate of blood, presumably due to RBC aggregation.     

Mechanisms for the spontaneous formation of covalently linked polymers of the terminal membranolytic complement protein (C9)

Purified human C9 spontaneously polymerizes upon prolonged incubation at 37 degrees C, and a fraction of these C9 polymers becomes resistant to dissociation by sodium dodecyl sulfate (SDS) and reducing agents. We examined possible mechanisms for this spontaneous covalent linking of C9. The following results are consistent with the conclusion that the formation of the covalently linked C9 polymer involves disulfide linking. 1) In addition to the SDS/dithiothreitol (DTT)-resistant C9 polymer (Mr = 950,000), disulfide-linked C9 dimers and trimers were formed upon incubation of C9 at 37 degrees C for 64 h. 2) The C9 polymer formed upon incubation at 37 degrees C for 64 h was resistant to dissociation by 6 M guanidine hydrochloride, 20 mM DTT but was dissociated by 6 M guanidine thiocyanate alone, yielding disulfide-linked C9 oligomers. 3) The formation of the SDS/DTT-resistant C9 polymer was completely inhibited by 1 mM iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), while DTNB enhanced the formation of disulfide-linked C9 oligomers. 4) A significant amount of free sulfhydryl group was detected in the polymerized C9 samples with various SH-specific reagents, though native C9 reacted with none of these reagents. In addition, inhibition by 1 mM iodoacetamide of C9 disulfide linking inhibited the self-association of C9 as analyzed by gel filtration on TSK-G4000 SW, whereas enhancement by 1mM DTNB of C9 disulfide linking enhanced C9 self-association. Thus, these results indicate that C9 disulfide linking that occurs upon C9 polymerization is an intrinsic property of C9 which is of importance in the formation of the stable C9 polymer structure.     

Complex effects of inhibitors on cyclic GMP-stimulated cyclic nucleotide phosphodiesterase

We have investigated the effects of several phosphodiesterase inhibitors on the activity of a cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver supernatant. Theophylline, RO 20-1724, and MY 5445 were not effective inhibitors. With 0.5 microM [3H]cGMP as substrate or with 0.5 microM [3H]cAMP in the presence of 1 microM cGMP, activity was inhibited by papaverine, dipyridamole, isobutylmethylxanthine (IBMX), and cilostamide. With 0.5 microM [3H]cAMP as substrate, however, only cilostamide was inhibitory; papaverine, dipyridamole, and IBMX increased activity. The increase was dependent on both drug and substrate concentration with maximal stimulation (150-180%) at concentrations of cAMP between 0.5 and 2.5 microM. At higher cAMP concentrations, the three drugs were inhibitory; inhibition was maximal at approximately 40 microM and decreased at higher cAMP concentrations. Inhibition of cGMP hydrolysis was maximal at approximately 3 microM and decreased at higher concentrations. Papaverine, IBMX, dipyridamole, and cilostamide inhibited [3H] cGMP hydrolysis competitively with Ki values of 3, 6.5, 7, and 11.5 microM, respectively. Papaverine, IBMX, or dipyridamole reduced the Hill coefficient for cAMP hydrolysis from 1.8 to 1.1-1.2, and Lineweaver-Burk plots were linear or nearly linear. With cilostamide, however, Lineweaver-Burk plots remained curvilinear. Thus, three competitive inhibitors, papaverine, dipyridamole, and IBMX, can mimic substrate and effect allosteric transitions that increase catalytic activity, whereas another, cilostamide, apparently cannot. Differences in the actions of these inhibitors presumably reflect differences in the molecular requirements for effective interaction at catalytic and allosteric sites on phosphodiesterase, i.e. differences in the structure of these sites.     

Effects of W-7 on catecholamine release and 45Ca2+ uptake in cultured adrenal chromaffin cells.

Effects of N-(6-aminohexyl)-5-chloro-1-naph-thalenesulfonamide (W-7), a calmodulin antagonist, on catecholamine (CA) release and ⁴⁵Ca²⁺ uptake were studied using cultured bovine adrenal chromaffin cells. W-7 inhibited the carbamylcholine (CCh)-evoked CA release and ⁴⁵Ca²⁺ uptake in a concentration-dependent manner. The inhibitory effect of W-7 on CCh-evoked CA release was not overcome either by an increase in extracellular calcium or CCh concentration. Although W-7 inhibited the high K⁺-evoked CA release and ⁴⁵Ca²⁺ uptake, potency of the drug was approximately 50–100 fold less than when inhibiting the CCh-evoked CA release and ⁴⁵Ca²⁺ uptake. The inhibitory effects of W-7 were observed both in norepinephrine release and epinephrine release. Moreover, W-7 inhibited the CCh-evoked ⁴⁵Ca²⁺ efflux. These results suggest that the inhibition of CA release by W-7 in adrenal chromaffin cells is mainly due to its inhibition of calcium uptake. W-7 may influence the linkage between acetylcholine-receptor and calcium uptake with higher potency than depolarization-dependent calcium entry.     

Cadaverine Involved in Urate Degrading Activity (Uricase Activity) in Soybean Radicles

Cadaverine, a 5-carbon diamine, was identified as the cofactorof uricase activity previously found in soybean seedlings. Thesubstance purified from freeze dried hypocotyls was subjectedto liquid chromatography, mass spectrometry, ¹H- and ¹³C-nuclearmagnetic resonance spectrometry for identification. The concentrationsof cadaverine in 3-day-old radicles and hypocotyls were 2.37mM and 5.09 mM, respectively. Other polyamine concentrationswere low. Biogenic polyamines (cadaverine, putrescine, spermidineand spermine) functioned as cofactors, whereas conjugated polyamines(tyramine and histamine) and amino acids had no effect. Theaddition of catalase to the assay system counteracted the effectof cadaverine. Peroxide at appropriate concentrations actedlike cadaverine with an identical K_m value, suggesting thaturate degrading activity can be ascribed to the diamine oxidase-peroxidasesystem. (Received October 19, 1982; Accepted December 23, 1982)     

An inducible lactone hydrolase yielding 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid from pulvinic acid

The metabolism of vulpinic acid by an unclassified soil micro-organism was studied. A new compound, 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid (DHOHA) was isolated from the reaction mixture of a cell-free preparation and pulvinic acid. The existence of a hydrolase which catalyses the conversion of vulpinic acid to pulvinic acid was detected in cell-free preparation, and an inducible lactone hydrolase capable of converting pulvinic acid to DHOHA was purified 130-fold and characterized. This enzyme had a MW of ca 34 000, a K_m for pulvinic acid at pH optimum (pH 7.0) less than 10 ^{? 6} M, pI = 5.0, and was inhibited by p-chloromercuriphenylsulfonate and diethylpyrocarbonate. The enzyme was highly specific for pulvinic acid. The initial degradative steps proposed for this organism are vulpinic acid → pulvinic acid → DHOHA.     

Whole body autoradiographic distribution of exogenously administered renin in mice

The distribution of exogenously administered renin was investigated using whole body autoradiography. Purified renin from mouse submaxillary gland (SR) was labeled with radioactive iodine (125I). This labeled renin (125I-SR) and Na125I were administered into the tail vein of male ddY mice, in doses of 10.2 and 16.4 mu Ci/30 g body weight, respectively. Mice were killed by an overdose of ether, and autoradiography was performed on whole body sections. To separate free 125I liberated from 125I-SR, sections were treated with perchloric acid. A major accumulation of 125I-SR, acid-insoluble, was evident in the renal cortex, whereas the hepatic accumulation of 125I-SR was minor. Radioactivity in the thyroid and submaxillary glands, in the stomach, and in urine was also apparent, but disappeared after acid treatment, except in the thyroid glands. Radioactivity in the brain, intestinal content, spleen, and adrenal glands was nil. These autoradiograms provide the first evidence that exogenously administered renin is mainly distributed in the renal cortex.