The effect of proteoglycans of cartilage and over-sulphated polysaccharides on the development of calcium-hydroxy-apatite (CHA) crystal formation in vitro

A coulometric model system is described which facilitates the quantitative study of the kinetics of transformation of amorphous calcium phosphate (ACP) into calcium-hydroxy-apatite (CHA) crystals. Proteoglycans of high molecular weight and over-sulphated polysaccharides (Arteparon, dextran sulphate) delayed CHA crystal formation. The results have enabled us to characterize the structure activity relationship of inhibitors of CHA formation, and to postulate a general structural requirement for molecules with inhibitory effect. As working mechanism, binding of calcium ions by sulphate groups of polyanions was supposed, which might reversibly impair "the critical nuclei formation", and/or further deposition of calcium ions in the CHA crystals. The clinical, therapeutical significance of the determination of the threshold concentration of different compounds is discussed.     

Extraction of mitochondrial protein-lipid complexes into organic solvents: an approach to study the interaction between the ATPase and the mitochondrial ATPase-inhibitor protein

Protein-lipid complexes were transferred directly from mitochondria and submitochondrial particles into hexane and ether. The protein-lipid residue left after solvent removal from these extracts was used to form liposomes which display low-temperature-resistant ATPase activity. Centrifugation experiments indicate that the ATPase activity is associated to the vesicles. Most of the F1-ATPases appear to be accessible to the external water phase of the liposomes. The ATPase activity of these particles was insensitive to dicyclohexylcarbodiimide and oligomycin. Incubation of these vesicles at room temperature activated (4--10-fold) the ATPase through a process that is partially sensitive to phenylmethylsulfonyl fluoride. The results with purified ATPase-inhibitor protein and (F1--ATPase)-inhibitor complex indicate that the activation process in the liposomes is due to the abolition of the inhibitory action of the inhibitor protein bound to a large fraction of the extracted ATPases. Liposomes prepared from hexane extracts obtained from submitochondrial particles having different levels of ATPase activity displayed an activation ratio which correlated with the number of ATPases that are inhibited by the inhibitor protein in the submitochondrial particles. The extraction of mitochondrial ATPase and its incorporation into liposomes followed by activity measurements may be used to judge the number of ATPases that in a given preparation contain the inhibitor protein in its inhibiting site.     

Structure and expression of the gene encoding cystatin D, a novel human cysteine proteinase inhibitor

A new member of the human cystatin multigene family has been cloned from a genomic library using a cystatin C cDNA probe. The complete nucleotide sequence of a 4.3-kilobase DNA segment, containing a complete gene with structure very similar to those of known Family 2 cystatin genes, was determined. The novel gene, called CST4, is composed of three exons and two introns. It contains the coding information for a protein of 142 amino acid residues, which has been tentatively called cystatin D. The deduced amino acid sequence includes a putative signal peptide and presents 51-55% identical residues with the sequences of either cystatin C or the secretory gland cystatins S, SN, or SA. The cystatin D sequence contains all regions of relevance for cysteine proteinase inhibitory activity and also the 4 cysteine residues that form disulfide bridges in the other members of cystatin Family 2. Northern blot analysis revealed that the cystatin D gene is expressed in parotid gland but not in seminal vesicle, prostate, epididymis, testis, ovary, placenta, thyroid, gastric corpus, small intestine, liver, or gall-bladder tissue. This tissue-restricted expression is in marked contrast with the wider distribution of all the other Family 2 cystatins, since cystatin C is expressed in all these tissues and the secretory gland cystatins are present in saliva, seminal plasma, and tears. Cystatin D, being the first described member of a third subfamily within the cystatin Family 2, thus appears to have a distinct function in the body in contrast to other cystatins.     

Mechanism of the HDL2 stimulation of progesterone secretion in cultured placental trophoblast

Lipoprotein cholesterol (C) supports the high rate of progesterone production by the human placenta as endogenous cholesterol synthesis is low. To study underlying mechanisms whereby lipoproteins, including high density lipoprotein-2 (HDL2), stimulate progesterone secretion, trophoblast cells were isolated from human term placentas and maintained in primary tissue culture. Lipoproteins were added at several concentrations and medium progesterone secretion was determined. HDL2 (d 1.063-1.125 g/ml) as well as low density lipoproteins (LDL) (d 1.019-1.063 g/ml) but not HDL3 (d 1.125-1.21 g/ml) stimulated progesterone secretion in a dose-dependent manner, with HDL2 cholesterol entering the cell and serving as substrate for progesterone synthesis. Conversely, LDL and HDL2 produced a significant decrease in [2-14C]acetate incorporation into cell cholesterol. Cholesterol-depleted lipoproteins did not stimulate progesterone secretion. The stimulating effect of LDL was abolished by apolipoprotein modification by cyclohexanedione or reductive methylation and by the addition of anti-LDL receptor antibody or 10 microM chloroquine to the medium. [14C]acetate conversion into cholesterol was accelerated by these procedures. However, HDL2 stimulation of progesterone secretion and reduction of [14C]acetate incorporation into cholesterol was not blocked by chemical modification of apolipoproteins, anti-LDL receptor antibody, or chloroquine. Treatment of HDL2 with tetranitromethane or dimethylsuberimidate also did not block the stimulation of progesterone. To determine whether the capacity of HDL2 to deliver cholesterol to the trophoblast cells was restricted to subfractions differing in apoE content, HDL2 was chromatographed on heparin-Sepharose and three fractions (A, B, and C) were obtained. Fraction A was poorest in apoE and free cholesterol, fraction B contained the majority of cholesterol, and fraction C was the richest in apoE and free cholesterol. When added to trophoblast cells, fraction A stimulated little progesterone secretion, fraction B stimulated moderately, and fraction C did so greatly. Modification of these subfractions with cyclohexanedione or reductive methylation did not inhibit these effects. In conclusion, HDL2 stimulated progesterone secretion in human trophoblast cell culture. Contrary to LDL, the HDL effect was not mediated by apolipoproteins or the LDL receptor pathway. The ability of HDL2 to stimulate progesterone secretion is consistent with the passive transfer of free cholesterol to the cell membrane from a physicochemically specific subfraction of HDL. This mechanism may be an auxiliary source of cholesterol for human steroidogenic cells.     

Enhancement by Cytidine of Membrane Phospholipid Synthesis

Cytidine, as cytidine 5'-diphosphate choline, is a major precursor in the synthesis of phosphatidylcholine in cell membranes. In the present study, we examined the relationships between extracellular levels of cytidine, the conversion of [14C]choline to [14C]phosphatidylcholine, and the net syntheses of phosphatidylcholine and phosphatidylethanolamine by PC12 cells. The rate at which cytidine (as [3H]cytidine) was incorporated into the PC12 cells followed normal Michaelis-Menten kinetics (Km = 5 microM; Vmax = 12 x 10(-3) mmol/mg of protein/min) when the cytidine concentrations in the medium were below 50 microM; at higher concentrations, intracellular [3H]cytidine nucleotide levels increased linearly. Once inside the cell, cytidine was converted mainly into cytidine triphosphate. In pulse-chase experiments, addition of cytidine to the medium caused a time- and dose-dependent increase (by up to 30%) in the incorporation of [14C]choline into membrane [14C]-phosphatidylcholine. When the PC12 cells were supplemented with both cytidine and choline for 14 h, small but significant elevations (p less than 0.05) were observed in their absolute contents of membrane phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, all increasing by 10-15% relative to their levels in cells incubated with choline alone. Exogenous cytidine, acting via cytidine triphosphate, can thus affect the synthesis and levels of cell membrane phospholipids.     

Kinetics of a model of autocatalysis, coupling of a reaction in which the enzyme acts on one of its substrates.

A global kinetic analysis is presented of a model of an enzyme autocatalytic process, to which a reaction is coupled, in which the enzyme acts upon one of its substrates. The kinetic equations of both the transient phase and the steady state are derived for this mechanism. In addition, we determine the corresponding kinetic equations for several particular cases which are characterized by certain relations between the rate constants. Finally, a kinetic data analysis is proposed for one of these particular cases. It can easily be extended to any of the other cases.     

Bretylium-induced voltage-gated sodium current in human lymphocytes.

Using the whole-cell variation of the patch-clamp technique it has been determined that 0.25-3 mM bretylium tosylate (BT) exerts a repolarizing effect on partially depolarized human lymphocytes. The repolarizing effect was ouabain (40 microM)-sensitive, and was inhibited by the removal of external Na+ or by the Na(+)-channel-blocker amiloride (10-44 microM), but K(+)-channel-blockers 4-aminopyridine (0.1-5 mM) and quinine (100 microM) had no effect. The drug induced a sodium dependent, amiloride-sensitive transient inward current reaching its maximum value approx. 20-30 s after the administration of BT and lasting for 6-10 min. This current was activated by depolarization within 25 ms at around -42 mV, its inactivation took about 2 s and its reversal potential was +24 +/- 5 mV. An increase in the intracellular sodium concentration (1.8-3.2 mM) has been observed upon the addition of BT by monitoring the SBFI fluorescence of the dye-loaded cells. It has been shown that whole-cell K+ currents are significantly decreased by BT. The existence of voltage and ligand (BT)-gated sodium channels has been postulated in human lymphocytes. These channels are thought to participate in the initiation of membrane repolarization in human lymphocytes, and thereby influence mitogenic or antigen-induced cell-activation processes.     

Regulation of the final phase of mammalian melanogenesis. The role of dopachrome tautomerase and the ratio between 5,6-dihydroxyindole-2-carboxylic acid and 5,6-dihydroxyindole.

The regulation of the final steps of the melanogenesis pathway, after L-2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) formation, is studied. It is shown that both tyrosinase and dopachrome tautomerase are involved in the process. In vivo, it seems that tyrosinase is involved in the regulation of the amount of melanin formed, whereas dopachrome tautomerase is mainly involved in the size, structure and composition of melanin, by regulating to the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the polymer. Moreover, using L-3,4-dihydroxyphenylalanine (dopa) and related compounds, it was shown that the presence of dopachrome tautomerase mediates an initial acceleration of melanogenesis since L-dopachrome is rapidly transformed to DHICA, but that melanin formation is inhibited because of the stability of this carboxylated indole compared to 5,6-dihydroxyindole (DHI), its decarboxylated counterpart obtained by spontaneous decarboxylation of L-dopachrome. Using L-dopa methyl ester as a precursor of melanogenesis, it is shown that this carboxylated indole does not polymerize in the absence of DHI, even in the presence of tyrosinase. However, it is incorporated into the polymer in the presence of both tyrosinase and DHI. Thus, this study suggests that DHI is essential for melanin formation, and the rate of polymerization depends on the ratio between DHICA and DHI in the medium. In the melanosome, this ratio should be regulated by the ratio between the activities of dopachrome tautomerase and tyrosinase.     

Specificity of Zea mays histone deacetylase is regulated by phosphorylation.

Mono Q ion exchange high performance liquid chromatography (HPLC) reveals that the main histone deacetylase activity (HD1) of germinating Zea mays embryos consists of multiple enzyme forms. Chromatography of HD1 after treatment with alkaline phosphatase yields two distinct histone deacetylase forms (HD1-A, HD1-B). The same is true for chromatography after phosphatase treatment of a total cell extract. One of these enzyme forms (HD1-A) is subject to phosphorylation, which causes a change in the substrate specificity of the enzyme, as shown with HPLC-purified individual core histone species; the substrate specificity for H2A increases more than 2-fold after phosphorylation, whereas the specificity for H3 decreases to about 60%. The total histone deacetylase activity is quantitatively released from isolated nuclei after extraction with moderate ionic strength buffers; no significant residual enzyme activity could be detected in the nuclear matrix.     

Further characterization of alpha N-acetyl beta-endorphin-(1-31) regulatory activity, I: Effect on opioid- and alpha 2-mediated supraspinal antinociception in mice.

Picomol doses of the acetylated derivative of beta-endorphin-(1-31), injected intracerebroventricularly (icv) in mice, reduced the analgesic activity of morphine, etorphine and beta-endorphin-(1-31), while the efficiency of DAGO and DADLE in producing analgesia was enhanced. The effects of the delta agonists DPDPE and [D-Ala2]-Deltorphin II were not altered by this treatment. After alpha N-acetyl beta-endorphin-(1-31) injection, morphine antagonized the analgesia of DAGO. The regulatory effect of alpha N-acetyl beta-endorphin-(1-31) was exhibited when giving the peptide both before (up to 24 h) and after the opioids. Naloxone did not prevent or reverse that modulatory activity; moreover, pretreatment with the acetylated peptide did not change the pA2 value displayed by the antagonist at the mu receptor. The antinociceptive activity of the alpha 2-adrenoceptor agonist clonidine was also increased in mice treated with alpha N-acetyl beta-endorphin-(1-31). The reducing activity of alpha N-acetyl beta-endorphin-(1-31) upon morphine- and beta-endorphin-induced analgesia was not exhibited in mice undergoing treatment with pertussis toxin or N-ethylmaleimide, agents known to impair the function of Gi/Go transducer proteins. However, the enhancing activity displayed by this peptide upon DAGO- DADLE and clonidine-evoked antinociception was still manifested. These results confirm and strengthen the idea of alpha N-acetyl beta-endorphin-(1-31) acting as a non-competitive regulator of mu opioid- and alpha 2-adrenoceptor-mediated supraspinal antinociception. A neural substrate acted on by both receptors (likely Gi/Go transducer proteins) appears to be involved in the effects of that neuropeptide.