Membrane-associated NAD+ glycohydrolase from rabbit erythrocytes is solubilized by phosphatidylinositol-specific phospholipase C

NAD+ glycohydrolase (NADase) present on the surface of rabbit erythrocytes is a membrane-bound ectoenzyme that can be solubilized by phosphatidylinositol-specific phospholipase C (PIPLC). As much as 70% of the cell-associated NADase was made soluble by treatment with PIPLC. The portion of NADase that remained cell-associated after an initial PIPLC treatment proved to be resistant to subsequent solubilization attempts. Further analysis showed that release of NADase from erythrocytes could not be attributed to the action of proteinases or phospholipase C. Erythrocytes obtained from other mammals were analyzed and found to have variable amounts of PIPLC-susceptible NADase. Practically, this finding can be used to easily solubilize membrane-bound NADase as a first step in its purification.     

Inactivation of Acinetobacter calcoaceticus acetate kinase by diethylpyrocarbonate

Acetate kinase purified from Acinetobacter calcoaceticus was inhibited by diethylpyrocarbonate with a second-order rate constant of 620 M-1.min-1 at pH 7.4 at 30 degrees C and showed a concomitant increase in absorbance at 240 nm due to the formation of N-carbethoxyhistidyl derivative. Activity could be restored by hydroxylamine and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.64. Complete inactivation of acetate kinase required the modification of seven residues per molecule of enzyme. Statistical analysis showed that among the seven modifiable residues, only one is essential for activity. 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuryphenylsulfonate, N-ethylmaleimide and phenylglyoxal did not affect the enzyme activity. These results suggest that the inactivation is due to the modification of one histidine residue. The substrates, acetate and ATP, protected the enzyme against inactivation, indicating that the modified histidine residue is located at or near the active site.     

Digitalis-like pregnanes. Cardiac and renal effects of a glycoside of 14 beta-hydroxyprogesterone

The synthesis of the glucoside, 3 beta-[(beta-D-glucopyranosyl)oxy]-14-hydroxy-14 beta-pregn-4-en-20-one, a 14 beta-hydroxyprogesterone glucoside (14 beta-OHP-glu), is described. This compound has an IC50 of 1 microM in a [3H]ouabain binding assay, and is about 10 times more potent than the aglycone. Like 14 beta-hydroxyprogesterone, the glucoside enhances contractility of isolated cardiac muscle. 14 beta-OHP-glu or ouabain, when infused at comparable doses into the renal artery of the anesthetized rat, markedly increases urine volume. Whereas ouabain significantly enhances urinary potassium excretion with little or no effect on sodium excretion, 14 beta-OHP-glu promotes a marked natriuresis with no significant effect on potassium excretion.     

Homogeneous, structurally defined heparin-oligosaccharides with low anticoagulant activity inhibit the generation of the amplification pathway C3 convertase in vitro

This paper demonstrates that heparin-oligosaccharides with low anticoagulant activity have a high capacity to inhibit activation of the amplification pathway of complement in vitro. We prepared heparin-oligosaccharides by partial depolymerization of heparin using purified flavobacterial heparinase. The resulting oligosaccharide mixture was then fractionated using strong anion exchange-high pressure liquid chromatography to produce individual oligosaccharide components of this mixture, with degree of polymerization ranging from 2 to 16. These heparin-oligosaccharides were examined for both their anticoagulant activity and capacity to inhibit activation of the amplification pathway of complement. Although there was little difference among commercial heparins, a correlation between molecular weight and activity to inhibit convertase generation was clearly established for heparin-oligosaccharides between degree of polymerization 2 through 16. Heparin-oligosaccharides of degree of polymerization 10-16 (Mr 3888-5320) demonstrated up to 54% of heparin's activity on a molar basis (and up to 163% of heparin's activity on a weight basis) in inhibiting the amplification pathway of complement in vitro while showing almost no anticoagulant activity. These studies, for the first time, completely separate heparin's ability to inhibit complement activation from its anticoagulant activity.     

Production of penicillin in a fluidized-bed bioreactor using a carrier-supported mycelial growth

A carrier-supported mycelial growth of Penicillium chrysogenum was applied to penicillin fermentation system using celite as a support material. Hyphal growth through the pore matrices of the material showed strong anchorages and provided highly stable biofilm growth. With bioparticles developed in such a manner, both cell growth and penicillin production were observed to increase significantly compared to the conventional dispersed filamentous cultures. Maximum values of specific penicillin production rate were found to be constant regardless of the growth form. A three-phase fluidized-bed fermentor was designed and tested for penicillin production using the bioparticles. Two modes of operation, semicontinuous and repeated fed batch, of the fermentor were tried. It was noted that the overgrowth of free mycelia and the development of fluffy loose bioparticles caused poor mixing and made the fermentor operation quite difficult. Control of the bioparticle size and the extension of production phase were therefore considered important to maintain the reactor productivity at a desired level. From the results of repeated fed-batch operation it was found that the control of bioparticle size could be successfully achieved by phosphate-limiting culture condition. Penicillin production under this condition was also observed to be maintained at a high level (about 80% of the maximum) for at least 1 month.     

Retinoic acid modifies positional memory in the anteroposterior axis of regenerating axolotl limbs

The effects of retinoic acid (RA) on anteroposterior (AP) positional memory of regenerating axolotl limbs were tested after removing the anterior or posterior half from the zeugopodium (lower arm or leg). RA (150 micrograms/g body wt) was injected into groups of animals bearing the following types of limbs: (1) anterior and posterior half zeugopodia grafted to the eyesocket and amputated distally 7 days later; (2) unamputated anterior and posterior half zeugopodia in situ; (3) double anterior and double posterior half zeugopodia amputated distally 7 days after their construction; (4) sham-operated zeugopodia amputated distally 7 days after operation. Controls consisted of these four groups injected with the retinoid solvent, dimethyl sulfoxide, or not injected. Control half zeugopodia grafted to the eyesocket regenerated no more than one or two digits. Control unamputated half zeugopodia in situ underwent partial or complete regeneration of the missing half from the proximal and midline wound surfaces exposed during construction of the half zeugopodia. Control double anterior and posterior zeugopodia both regenerated symmetrical, hypomorphic regenerates with 1-3 digits in the double anteriors and 1-6 digits in the double posteriors. Sham-operated controls regenerated normally. Regenerating anterior and posterior halves responded differently to RA. RA-treated anterior half zeugopodia in the eyesocket, and anterior half stumps adjacent to the unamputated posterior half zeugopodia in situ both produced regenerates that duplicated stump structures in the proximodistal axis and formed a complete and normal AP pattern. RA-treated double anterior zeugopodia regenerated proximodistal-duplicated pairs of mirror-imaged limbs, each with a complete and normal AP pattern. In contrast, half posterior zeugopodia in the eyesocket, the posterior half stumps of unamputated half anterior zeugopodia in situ, and double posterior zeugopodia all failed to regenerate. These results suggest that RA modifies positional memory in only one direction in the AP axis, posterior.     

Glycerol stress and platelet integrity

Platelet response to glycerol gradient was studied using a few in vitro parameters. These were platelet count, mean platelet size, platelet response to hypotonic stress (PHRS), and collagen-induced platelet aggregation. An equal volume of 1-10% (w/v) glycerol in plasma was added at once to the platelet concentrate resulting in 0.5-5% (w/v) final glycerol concentration. The concentrate was kept at 22 degrees C for 60 min. Platelets were then separated by one centrifugation and resuspended in glycerol-free plasma. A loss in platelet count was observed when the gradient of glycerol was more than 3%. This was associated with an increase in mean cell size and a reduction in aggregability. With 5% glycerol stress, a loss of 30% in cell count, an increase in 18% in cell size, and a 78% loss in aggregability was observed. Declining of PRHS was shown already with a 1% glycerol gradient and 69% of this function was suppressed by 5% glycerol stress. In other experiments, 5% glycerol was first added, them removed in 5 steps with a gradient of 1% each. When time interval between each step was less than 0.5 min, platelet loss and PRHS reduction were 17 and 47% respectively. These values were gradually improved to 4% and 11-20%, respectively, as increasing time interval up to 15 min. It was concluded that a gradient of 1% glycerol and a 15-min interval for each step minimizes the detrimental osmotic stress on platelets while glycerol is added or removed. Our findings may lead up to devising an improved protocol for platelet cryopreservation with glycerol.     

Studies on naturally occurring proteinous inhibitor for transmethylation reactions

An inhibitor for S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases has been purified from rat liver particulate fraction to apparent homogeneity, as judged by high-performance liquid chromatography, two-dimensional paper electrophoresis and isoelectric focusing chromatography. This inhibitor molecule, which is composed of 27 amino acid residues with an additional fluorescent chromophore, is rich in glycine, contains no basic amino acid, and has an isoelectric point (pI) of 3.70. A single absorption peak was observed at 248 nm in acidic as well as in neutral media, while two peaks were detected in alkaline medium at 206 nm and 248 nm. The former peak was found to be quite labile. The fluorescent spectra with excitation peak at 285 nm and emission peak at 358 nm are greatly influenced by the pH, being the highest in alkaline medium. The purified inhibitor inhibits all the AdoMet-dependent methyltransferases examined.     

Effects of prostaglandins E2 and F2 alpha on progesterone metabolism by rat granulosa cells

Rat granulosa cells were cultured with or without PGE2 and/or PGF2 alpha. Accumulation of endogenous progesterone and 20 alpha-hydroxy-4-pregnen-3-one was determined. Additionally, [4-14C]progesterone metabolism was assessed. PGE2 increased progesterone accumulation, in part, by decreasing progesterone catabolism to 20 alpha-reduced progestins. In contrast, PGF2 alpha stimulated 20 alpha-hydroxysteroid dehydrogenase activity, thus increasing progesterone catabolism. Combined treatment with PGE2 and PGF2 alpha augmented progesterone accumulation to levels above controls but below those attained with PGE2 alone. These data indicate that PGE2 and PGF2 alpha exert opposite effects on progesterone production and catabolism and that the ratio of PGE2 to PGF2 alpha in the local granulosa cell milieu may be of importance in determining overall progesterone output.     

Expression of c-myc oncogene in human fibroblasts during in vitro senescence

Expression of the c-myc oncogene was determined in pre-confluent early and late passage human (IMR-90) fibroblast by dot blot analysis of cellular mRNA. Significant decreases in c-myc levels were found in late passage when compared to levels found in early passage cells. Cells restimulated with serum after serum restriction also showed reduced levels of c-myc in late passage. Confluent cells expressed levels of c-myc similar to that of pre-confluent cells, when serum stimulation was the same in both cases. Southern blots of Eco R1 digested DNA showed 2 fragments of similar size hybridizing to c-myc sequences in both early and late passage cells.