A protein tyrosine kinase associated with the ATP-dependent inactivation of adipose diacylglycerol acyltransferase

The activity that has been previously reported to reversibly inactivate adipose glycerolphosphate acyltransferase (GPAT) and diacylglycerol acyltransferase (DGAT)in vitro in the presence of ATP is shown here to be partially purified from adipose tissue with an apparent molecular weight of 68 kDa. The activity responsible for inactivating DGAT is associated with a kinase activity as determined by phosphate incorporation both into microsomal proteins and into a synthetic tyrosine-containing peptide as substrate for protein tyrosine kinase. Two microsomal polypeptides of 53 and 69 kDa are major substrates of this kinase. Both DGAT inactivating and kinase activities assayed from the purified sample have been found to be insensitive to the Ser/Thr kinase inhibitor H-7 while being sensitive to genistein and tyrphostin-25. A crude protein phosphatase preparation from liver was capable of reversing the effects of both activities. The purified sample was also shown to inactivate GPAT in the presence of ATP. These results suggest that a protein tyrosine kinase, in concert with a protein tyrosine phosphatase, may regulate the activities of DGAT and GPAT by a phosphorylation-dephosphorylation mechanism.     

Activation of rat liver cholesterol ester hydrolase by cAMP-dependent protein kinase and protein kinase C

Short term regulation of hepatic cholesterol ester hydrolase by reversible phosphorylation is described. Two different kinase systems seem to be involved in this regulation. The addition of ATP, cyclic AMP and Mg²⁺ to rat liver 104,000× g supernatant (S104) produced a 100–140% increase in cholesterol ester hydrolase activity. This stimulation was abolished when protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and Mg²⁺. Cholesterol ester hydrolase activity was also stimulated when calcium ions, phosphatidylserine, and diolein were added to S104 along with ATP and Mg²⁺. Diolein in this reaction could be substituted by phorbol 12-myristate 13-acetate. Preincubation of S104 with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. The addition of increasing concentrations of Mg²⁺ to S104 produced increasing inhibition of cholesterol ester hydrolase activity, and this effect was blocked by NaF. It is suggested that rat liver cholesterol ester hydrolase is activated by cyclic AMP dependent protein kinase and protein kinase C. Deactivation is accomplished by dephosphorylation catalyzed by a phosphoprotein phosphatase, dependent on Mg²⁺. This work was presented at the Twenty-Third Southeastern Regional Lipid Conference, held October 26–28, in Cashiers, North Carolina.     

Properties of diacylglycerol kinase purified from bovine brain

A nearly homogeneous but somewhat unstable diacylglycerol kinase (ca. MW 72,000 daltons) was purified from bovine brain by modification of the procedure of Kanoh et al. (Kanoh, H., Kondoh, H., and Ono, T. [1983]J. Biol. Chem. 258, 1767–1774). The purification consisted of four steps (brain cytosol isolation and successive chromatography on DEAE-cellulose, Sephadex G-25 for desalting and ATP-agarose) carried out in buffers stabilized with EDTA, ATP and dithiothreitol (DTT). Specific activities, determined within 4 hr of purification, ranged from 908–1857 nmol ATP incorporated/min/mg protein, with the variation reflecting the instability. Optimal activities required deoxycholate (0.1%), one of the phosphoglycerides [phosphatidylcholine (PC), phosphatidylethanolamine (PE) or phosphatidylserine (PS)] (0.025–0.25 mM), ATP (5 mM, apparent K_m=0.57 mM), 1,2-dioleoyl-rac-glycerol (5 mM, apparent K_m=1 mM) and Mg²⁺ (10 mM, apparent K_m=2.2 mM). Phosphatidylinositol (PI) was slightly less effective than PC, PE or PS and noninhibitory in combination with PC, PE or PS. Relative to PC phosphatidic acid (PA) (52%), sphingomyelin (48%), lyso-PC (1.5%) and lyso-PI (28.6%) were less effective activators. The sulfhydryl reagents,p-chloromercuribenzoic acid (PCMB) (1.0 mM),N-ethylmaleimide (NEM) (1.0 and 2.0 mM) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) (1.0 mM), showed strong inhibition of activity which was prevented by 0.5 mM DTT. In contrast to other reports, this purified enzyme showed no monoacylglycerol kinase activity. Comparison of diacylglycerols of varying fatty acid composition indicated that the enzyme showed a preference for substrates with at least one unsaturated fatty acid, particularly in the 2-position. With saturated fatty acids the order of preference was C₁₀ and C₁₂>C₁₄>C₁₆>C₁₈. Such a pattern indicates that the enzyme shows little selectivity that favors the generation of particular molecular species of PA.     

Synthetic diacylglycerols (DAG) and DAG-lactones as activators of protein kinase C (PK-C)

The central role of protein kinase C (PK-C) in cellular signal transduction has established it as an important therapeutic target for cancer and other diseases. We have developed a series of 4,4-disubstituted-gamma-butyrolactones, which contain a constrained glycerol backbone (DAG-lactones) and behave as potent and selective activating ligands of PK-C with affinities that approach those of the structurally complex natural product agonists, such as the phorbol esters. This Account traces the design and construction of these molecules. Initially, we examined the consequences of reducing the entropic penalty associated with the transformation of a DAG into a DAG-lactone. Then, using molecular modeling to extend insights arising from the newly solved crystal structure of a C1 domain complexed with phorbol ester, we incorporated amino acid-specific branched hydrophobic chains to provide a new generation of DAG-lactones that have the capacity to bind to PK-C with low nanomolar affinity. Depending on the specific pattern of hydrophobic substitution, some DAG-lactones are able to induce selective translocation of individual PK-C isozymes to different cellular compartments, and since the specific nature of these hydrophobic interactions influences biological outcome, some of these compounds exhibit cell-specific antitumor activity. The ability to direct specific PK-C isozyme translocation with sets of structurally simple, yet highly potent molecules provides a powerful tool for engineering a plethora of molecules with novel biological functions.     

Substrate selectivity of diacylglycerol kinase in guinea pig brain

The present study was conducted to test the selectivity of the microsomal diacylglycerol kinase (ATP:1,2-diacyl-sn-glycerol phosphotransferase) from guinea pig brain towards different 1,2-diacyl-sn-glycerols. The molecular species added to the incubation medium as substrates were a mixture of the 1-[³H]palmitoyl plus 1-[¹⁴C]stearoyl homologs of either the monoenoic (2-oleoyl), dienoic (2-linoleoyl), tetraenoic (2-arachidonoyl), or hexaenoic (2-docosahexaenoyl) diacylglycerols. Rates of phosphatidic acid synthesis (1-palmitoy plus 1-stearoyl homologs) with each of the four classes of unsaturated diacylglycerols were not greatly different, although they were moderately higher in the case of the monoenes. No marked enzyme selectivity for either the 1-palmitoyl or 1-stearoyl homolog of the various 1-saturated 2-unsaturated diacylglycerols was apparent. Generally similar results were obtained with the diacylglycerol kinase in rat brain microsomes. Presented in part at the AOCS Annual Meeting, St. Louis, May 1978.     

Production and protein kinase C activation of diacylglycerols containing polymethylene-interrupted PUFA

Sciadonic acid (20∶3, Δ-5c,11c,14c) is a polymethylene-interrupted PUFA (PMI-PUFA) that is present in conifer seeds and known to be incorporated into animal cells and to accumulate in membrane PI as a substitute for arachidonate. In this study, we investigated whether PI having sciadonate could serve as source of DAG that could activate protein kinase C (PKC). When Swiss 3T3 cells cultured with sciadonic acid were stimulated with 100 nM of bombesin, 1-stearoyl-2-sciadonoyl-glycerol (G) and 1-stearoyl-2-arachidonoyl-G were produced. The net increments of these two molecular species of DAG reflected the levels of the two molecular species in the PI in the cells. When cells cultured with juniperonic acid (20∶4, Δ-5c,11c,14c,17c) were stimulated 1-stearoyl-2-juniperonoyl-G was produced in proportion to the level of this molecular species in PI in the cells. We also examined PKC activation by synthetic DAG using a partially purified PKC fraction from rat brain and found that both 1-stearoyl-2-sciadonoyl-G and 1-stearoyl-2-juniperonoyl-G could activate PKC comparably to 1-stearoyl-2-arachidonoyl-G. These results indicate that 1-stearoyl-PI having these C₂₀ PMI-PUFA residues can serve as sources of potential signaling molecules.     

喹啉类蛋白酪氨酸激酶抑制剂的研究进展

蛋白酪氨酸激酶的过度表达与多种肿瘤的发生、发展及转移相关,抑制酪氨酸激酶活性可有效抑制肿瘤。目前已有11个小分子蛋白酪氨酸激酶抑制剂作为抗肿瘤药物上市,1个由美国FDA批准接受预登记的药物,超过100个候选药物处于临床试验阶段。其中喹唑啉结构的酪氨酸激酶抑制剂已成为临床上最常见的抗肿瘤化疗药物类别之一。综述了近年来与喹唑啉结构相似的具有喹啉骨架的酪氨酸激酶抑制剂研究进展。     

Synthetic caged DAG-lactones for photochemically controlled activation of protein kinase C

Switching on kinases: Synthetic caged DAG-lactones have been developed and showed decreases of two orders of magnitude, relative to the corresponding parent compounds, in their binding affinities towards PKC. The caged compounds had no effect on the translocation of PKC until after photoactivation. This approach is a potentially powerful tool for probing the PKC signaling cascade.     

Effects of hexadecylphosphocholine on protein kinase C and TPA-induced differentiation of HL60 cells

Several structural analogs of alkylphosphocholine (APC) were studied for their effects on protein kinase C (PKC) and 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited biochemical and cellular events in HL60 cells. Hexadecylphosphocholine (He-PC₂), the APC prototype, inhibited PKC competitively with respect to phosphatidylserine and noncompetitively with respect to CaCl₂, both with an apparent K_i of about 15 μM. Inhibition of PKC by He-PC₂ was selective, since cyclic AMP dependent protein kinase and Ca²⁺/calmodulin dependent protein kinase II were relatively unaffected. He-PC₂ inhibited TPA-induced depletion of PKC and TPA-stimulated phosphorylation of cellular proteins in HL60 cells. TPA-induced differentiation of HL60 cells was also inhibited by He-PC₂, and this inhibition was synergistic or additive to the effects of 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), a PKC inhibitor. The present findings are consistent with the hypothesis that inhibition of PKC might be related, in part, to the antineoplastic effect of He-PC₂ and ether lipid analogs such as ET-18-OCH₃ (1-octadecyl-2-methyl-glycero-3-phosphocholine).     

Effect of dietary fat on colonic protein kinase C and induction of aberrant crypt foci

A major objective of the present study was to determine whether a high-fat diet affects early events during colon carcinogenesis. Female Sprague-Dawley rats were injected with saline or azoxymethane (20 mg/kg) and fed either a normal (5% corn oil w/w) or a high (5% corn oil and 15% beef tallow w/w) fat diet. To assess the effect of a known tumor-promoting diet on the early events of neoplastic transformation, Study 1 examined the induction and growth of aberrant crypt foci (ACF) as well as of proliferative indices. The total number of ACF were similar in both groups even after 8 wk of dietary treatment; however, ACF with accelerated growth characteristics (≥4 crypts/focal lesions) were more prevalent (P≤0.05) in the colons of animals fed the high-fat diet. Metaphase arrest cells and 5′-bromo-2′-deoxyuridine labelled cells showed no appreciable response to dietary changes. To determine whether changes in colonic signal transduction pathways represent an early response to dietary modification, Study 2 evaluated the activity of protein kinase C (PKC), proliferative indices and changes in phospholipid fatty acid profiles. In comparison to the normal fat group, the colons of high-fat fed animals exhibited higher (P≤0.05) membranes and lower soluble PKC activity; however, proliferation patterns of these colons were not altered. Changes in the membrane lipid composition were minor; however, an increase in the phosphatidylcholine/phosphatidylethanolamine ratio and in 20∶4n−6 was noted. Our results demonstrate that in comparison to a normal-fat diet, a high-fat diet stimulated the growth of a population of ACF, i.e., preneoplastic lesions leading to advanced growth characteristics. In addition, a high-fat diet exerted a marked influence on total, cytosolic and membrane associated PKC activities. The findings suggest that modulation of PKC may play a critical role at the early stages of colon carcinogenesis. This work was presented by L.M.Z. Lafave as a recipient of the Honored Student Award at the American Oil Chemists' Society annual meeting, Anaheim, California, 1993. L.M.Z. Lafave is the recipient of a Natural Sciences and Engineering Research Council Postgraduate Scholarship.     

C60高分子衍生物的制备与应用

从合成方法的角度，详细的介绍了C60的高分子衍生物的制备方法。包括Fridel—Crafts烷基化反应、形成桥键的加成反应、Diels-Alder环加成反应、胺加成反应、自由基加成反应(一般自由基加成反应和活性自由基聚合)、离子聚合及配位聚合等。同时也介绍了在光学、电学、色谱学、生物医学及摩擦学等方面的应用。     

Dimeric C34 Derivatives Linked through Disulfide Bridges as New HIV-1 Fusion Inhibitors

C34, a 34-mer fragment peptide, is contained in the HIV-1 envelope protein gp41. A dimeric derivative of C34 linked through a disulfide bridge at its C terminus was synthesized and found to display potent anti-HIV activity, comparable with that of a previously reported PEGylated dimer of C34REG. The reduction in the size of the linker moiety for dimerization was thus successful, and this result might shed some light on the mechanism of the suppression of six-helix bundle formation by these C34 dimeric derivatives. Addition of a Gly-Cys(CH₂CONH₂)-Gly-Gly motif at the N-terminal position of a C34 monomeric derivative significantly increased the anti-HIV-1 activity. This moiety functions as a new pharmacophore, and this might provide a useful insight into the design of potent HIV-1 fusion inhibitors.     

Modulation by sphingosine of substrate phosphorylation by protein kinase C in bovine mammary gland

The effect of sphingosine on the phosphorylation of endogenous proteins by protein kinase C (PKC) was investigated in bovine mammary gland. Several proteins were shown to be substrates for PKC in both cytosolic and total particulate fractions by phosphorylation in the absence or presence of 1-oleoy-2-acetyl-sn-glycerol, phosphatidylserine (PS) and Ca²⁺. At concentrations of 83 μM or less, sphingosine inhibited phosphorylation of several substrates for PKC in both fractions. Phosphorylation of cytosolic 36 kDa, 21 kDa and particulate 36 kDa proteins was particularly sensitive to sphingosine. Cytosolic 97 kDa phosphorylation (which was enhanced by Ca²⁺ alone) was also sensitive to sphingosine. The inhibition was reversed by excess addition of lipid cofactors, particularly PS, but not by Ca²⁺. At higher concentrations (167 and 417 μM), in addition to the inhibition seen at lower concentrations, sphingosine stimulated phosphorylation of several proteins, including cytosolic 19 kDa and particulate 53 kDa, which were not detected in the absence of sphingosine. The sphingosine-induced phosphorylation disappeared with excess addition of PS, but not with addition of Ca²⁺. The results point toward the importance of the interaction of sphingosine with membrane phospholipids in the signal transduction pathway mediated by PKC-dependent phosphorylation in bovine mammary gland.     

Phorbol ester stimulates PAF synthesisvia the activation of protein kinase C in rat leukocytes

When rat pleural mononuclear leukocytes were stimulated with 1 μM phorbol myristate acetate (PMA), platelet-activating factor (PAF)-like activity was detected in the supernatant and the cellular fractions of the incubation mixture, as measured by rabbit platelet aggregation. C₁₆PAF activity peaked at 30 min in both fractions. Acetyltransferase activity in the microsomal fraction of the stimulated cells also increased rapidly and showed a peak at 10 min. A protein kinase C inhibitor, staurosporine, and an inhibitor of phospholipase A₂,p-bromophenacylbromide, inhibited stimulated PAF formation in both fractions. Staurosporine also inhibited PMA induced acetyltransferase activity. The data suggest that PMA stimulates PAF synthesis by the remodeling pathway in rat pleural cells through activation of both phospholipase A₂ and acetyltransferase, and that the acetyltransferase, in turn, may be activated through activation of protein kinase C. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

In vitro activation of mouse skin protein kinase C by fatty acids and their hydroxylated metabolites

To understand how dietary fatty acids differentially modulate mouse skin tumorigenesis, the ability of specific fatty acids and their derivatives to activate murine epidermal protein kinase C (PKC)in vitro was investigated. Total PKC from untreated female SSIN mouse skin was partially purified and incubated with specific fatty acids at concentrations up to 300 μM in the presence of Ca²⁺ and phosphatidylserine. Thecis-unsaturated fatty acids tested, ranging from 16∶1 to 22∶6, stimulated PKC activity in a similar dose-dependent manner with an approximate threefold maximum increase over control. Neither the number ofcis-double bonds nor the chainlength of these fatty acids affected their relative ability to activate PKC.trans-Fatty acids, with the exception of linoelaidic acid (t,t-18∶2n−6), exhibited about half of the potency of their correspondingcis-isomers in stimulating PKC at the plateau concentration (200 μM) or lower. Substitutions close to the double bond oncis-fatty acids abolished their ability to activate PKC. The hydroxylated metabolites of arachidonic acid (20∶4n−6) and linoleic acid (c,c-18∶2n−6), i.e., the hydroxyeicosatetraenoic acids (HETE) and hydroxyoctadecadienoic acids (HODE), also activated mouse skin PKCin vitro, but only about half as effectively as did the respective parent fatty acids. The results suggest that both hydroxyl substitution andtrans-configuration of HETE and HODE are responsible for their reduced ability to activate PKC. Overall the data suggests that the reduced skin tumor yield observed in mice fed diets high inc,c-18∶2n−6 is not likely to be due to differences in the ability ofc,c-18∶2n−6 or 20∶4n−6, or their matabolites, to activate PKC.     

Substituted thiobisphenols as antioxidants: Correlation between antioxidant activities and13C chemical shifts

The antioxidant activities of ten thiobisphenols were evaluated by an oxygen-absorption method at 60°C in tetralin and determination of peroxide values at 160°C in paraffin. For the 4,4′-thiobisphenols, alkyl groupsortho to the phenolic hydroxyl groups increased the induction period compared to that of other thiobisphenols for both oxidation of tetralin and paraffin. The data on electrochemical oxidation potentials that were determined by linear-sweep voltammetry and the¹³C nuclear magnetic resonance chemical shifts (δ) of these compounds were associated with antioxidant activities. In particular, the activities exhibited during the induction period closely correlated with the¹³C chemical shifts of ipso-carbon of the OH substituent.     

维生素C及其衍生物的制备工艺和应用

介绍了维生素C的结构、理化性质、用途及制备方法的沿革,综述了7种维生素C衍生物的制备工艺和用途。