Cuticular sclerotization in the beetles Pachynoda epphipiata and Tenebrio molitor

The sclerotization of cuticle in two species of beetles, Pachynoda epphipiata and Tenebrio molitor, has been investigated and compared with the sclerotization in the locust, Schistocerca gregaria. Two types of sclerotization, β-sclerotization and quinone tanning, occur in all three species. The main type is β-sclerotization, i.e. cross-linking of proteins by means of N-acetyldopamine which is connected to the proteins through the β-position of its side chain. β-Sclerotization is completed in P. epphipiata when it leaves its cocoon, whereas in adult locusts and in adult Tenebrio β-sclerotization continues for several weeks. The cuticle of all three species contains an insoluble enzyme which activates the β-position of N-acetyldopamine and is presumably responsible for the formation of the cross-links. Locust cuticle contains also small amounts of another enzyme which activates the aromatic ring of N-acetyldopamine, resulting in the formation of an o-quinone, which may be involved in quinone tanning of the cuticle. At emergence adult Tenebrio cuticle is rich in both enzymes, but the quinone-forming enzyme is inactivated after a few days, whereas the β-enzyme first decreases and later increases in activity, so that the β-enzyme is the dominating activity in the cuticle of mature adult Tenebrio. The quinone-forming enzyme is presumably responsible for the formation of the brown colour of Tenebrio exocuticle.The exocuticle of adult beetles contains 3,4-dihydroxyphenylacetic acid, which, although it is not easily extracted from the cuticle, is not covalently bound to cuticular components. In Tenebrio it appears in the cuticle a few days after the final ecdysis.The amino acid compositions of both larval, pupal, and adult cuticle from P. epphipiata have been determined, and they are compared with the composition of the cuticle of the corresponding stages of Tenebrio.     

Differential mechanism of oxidation of N-acetyldopamine and N-acetylnorepinephrine by cuticular phenoloxidase from Sarcophaga bullata

The mechanism of oxidation of two related sclerotizing precursors—N-acetyldopamine and N-acetylnorepinephrine—by the cuticular phenoloxidase from Sarcophaga bullata was studied and compared with mushroom tyrosinase-mediated oxidation. While the fungal enzyme readily generated the quinone products from both of these catecholamine derivatives, sarcophagid enzyme converted N-acetyldopamine to a quinone methide derivative, which was subsequently bound to the cuticle with the regeneration of o-dihydroxy phenolic function as outlined in an earlier publication [Sugumaran: Arch Insect Biochem Physiol, 8, 73 (1988)]. However, it converted N-acetylnorepinephrine to its quinone and not to the quinone methide derivative. Proteolytic digests of N-acetyldopamine-treated cuticle liberated peptides that had covalently bound catechols, while N-acetylnorepinephrine-treated cuticle did not release such peptides. Acid hydrolysis of N-acetyldopamine-treated cuticle, but not N-acetylnorepinephrine-treated cuticle liberated 2-hydroxy-3′,4′-dihydroxyacetophenone and arterenone. These results further confirm the unique conversion of N-acetyldopamine to its corresponding quinone methide derivative and N-acetylnorepinephrine to its quinone derivative by the cuticular phen-oloxidase. Significance of this differential mechanism of oxidation for sclerotization of insect cuticle is discussed.     

Cuticular sclerotization in larval and adult locusts, Schistocerca gregaria

The sclerotization of both larval and adult cuticle from the desert locust, Schistocerca gregaria, has been studied by measuring the incorporation of radioactive dopamine and N-acetyldopamine into the cuticle. The results are compared with the degree of sclerotization of the cuticle and the amount of sclerotizing enzyme present. The various parts of the cuticle differ considerably with respect to the degree of sclerotization: in adult locusts the mandibles and the dorsal mesothoracic cuticle contain about twenty times as much cross-linking material per mg cuticle than is present in the abdominal tergites and sclerites.The degree of sclerotization in the various types of cuticle is apparently not determined by the amounts of sclerotizing enzyme present, and the rate at which radioactive dopamine or N-acetyldopamine is incorporated into the cuticle appears also to be unrelated to the amount of enzyme.The degree of sclerotization of the various parts of the cuticle from fifth instar larvae corresponds with the amounts of labelled dopamine which are incorporated during the first day after ecdysis, whereas there is no correlation between sclerotization and the amounts of labelled dopamine which are incorporated in older larvae. The degree of sclerotization of adult cuticle after 1 day corresponds to the incorporation of dopamine during the first day. When older animals are compared only little correlation is observed. The relative rates of sclerotization in the various parts of the cuticle must therefore change as the adult insect grows older.The changes in the incorporation pattern during the development of the locust are discussed in relation to the physiological control of the sclerotization process.     

Oxidative dimerization of ferulic acid by extracts from Sorghum

Extracts from leaves and first internodes of Sorghum bicolor catalyze the conversion of ferulic acid to a β-β-coupled dimer, the dilactone dimer of ferulic acid. The dilactone is then hydrolyzed, probably non-enzymatically, to a blue fluorescing compound, tentatively identified as a β-β-coupled dimer with at least one lactone ring opened to form a carboxylic acid. Both the initial enzymatic and the subsequent non-enzymatic steps are greater at pH 8 than pH 6. The most active preparation is a crude particulate fraction from leaves obtained by centrifugation at 37000 g; white light increases the amount of dimer formed.     

Diphenols in hemolymph and cuticle during development and cuticle tanning of Periplaneta americana (L.) and other cockroach species

Diphenolic compounds in cockroach hemolymph and cuticle were extracted with 1.2 N HCI, partially purified by alumina adsorption, and analyzed by liquid chromatography. Dopamine (DA) is the major catecholamine in hemolymph of Periplaneta americana, Blatta orientalis, Blattella germanica, Gromphadorhina portentosa, and Blaberus craniifer at adult ecdysis, while N-acetyldopamine (NADA) predominates in hemolymph of Leucophaea maderae. In P. americana, NADA is the second most abundant catecholamine, while N-β-alanyldopamine (NBAD), norepinephrine (NE), 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylethanol, 3,4-dihydroxyphenylacetic acid, and 3,4-dihydroxybenzoic acid occur in lesser quantities. Catecholamines occur mainly as acid labile conjugates in hemolymph. Dopamine, conjugated primarily as the 3-sulfate ester, increases in hemolymph from 0.1 to 0.8 mM during the last instar. Concentrations decrease by 75% in pharate adults, partially because of an increase in hemolymph volume. A second smaller peak of DA sulfate occurs after ecdysis followed by a rapid disappearance as the cuticle tans. A conjugate of catechol (o-dihydroxybenzene) is also present in relatively high concentrations at all ages examined. In cuticle, N-β-alanylnorepinephrine accumulates during the early period of adult tanning, while NBAD, NADA, N-acetylnorepinephrine, and DA increase more slowly. The N-β-alanyl and N-acetyl derivatives of DA and NE occure in relatively high concentrations in tanned cutical of P. americana and probably play an important role in the stablization process.     

On the mechanism of formation of arterenone in insect cuticular hydrolyzates

Arterenone (2-amino-3′,4′-dihydroxy acetophenone) is an important hydrolytic product generated from lightly colored sclerotized cuticle that use N-acyldopamine derivatives for crosslinking reactions. It seems to arise from 1,2-dehydro-N-acetyldopamine (dehydro NADA) that has been crosslinked to the cuticular components. However, the mechanism of generation of arterenone, which has two protons on the α-carbon and no proton on the β-carbon atom from dehydro NADA crosslinks that have one proton each on these two side chain carbons, remained elusive and undetermined. To investigate the mechanism of this transformation, we synthesized specifically labeled β-deuterated dehydro NADA and incubated with Sarcophaga bullata cuticle undergoing larval puparial transformation. We also isolated the dimeric products formed during the tyrosinase-mediated oxidation of dehydro NADA. Hydrolysis of both β-deuterated dehydro NADA treated cuticle and β-deuterated dehydro NADA dimer generated arterenone as the major hydrolytic product. Liquid chromatography-mass spectrometric analysis of this arterenone revealed the retention of deuterium from the β-position of dehydro NADA at the α-carbon atom of arterenone. Hydrolysis of β-deuterated dehydro NADA also generated the labeled arterenone under oxidative conditions, but not under anaerobic conditions. These results indicate the unique hydride shift from β-carbon to α-carbon during acid hydrolysis and reveal the mechanism of liberation of arterenone and related compounds from dehydro NADA linked cuticle.     

Further studies on the mechanism of oxidation of N-acetyldopamine by the cuticular enzymes from Sarcophaga bullata and other insects

In accordance with our earlier results, quinone methide formation was confirmed to be the major pathway for the oxidation of N-acetyldopamine (NADA) by cuticle-bound enzymes from Sarcophaga bullata larvae. In addition, with the use of a newly developed HPLC separation condition and cuticle prepared by gentle procedures, it could be demonstrated that 1, 2-dehydro-NADA and its dimeric oxidation products are also generated in the reaction mixture containing a high concentration of NADA albeit at a much lower amount than the NADA quinone methide water adduct, viz., N-acetylnorepinephrine (NANE). By using different buffers, it was also possible to establish the accumulation of NADA quinone in reaction mixtures containing NADA and cuticle. That the 1,2-dehydro-NADA formation is due to the action of a NADA desaturase system was established by pH and temperature studies and by differential inhibition of NANE production. Of the various cuticle examined, adult cuticle of Locusta migratoria, presclerotized cuticle of Periplaneta americana, and white puparial cases of Drosophila melanogaster exhibited more NADA desaturase activity than NANE generating activity, while the reverse was observed with the larval cuticle of Tenebrio molitor and pharate pupal cuticle of Manduca sexta. These studies indicate that both NADA quinone methide and 1, 2-dehydro NADA are formed during enzymatic activation of NADA in insect cuticle. Based on these results, a unified mechanism for β-sclerotization involving quinone methides as the reactive species is presented.     

Quinone methides—and not dehydrodopamine derivatives—as reactive intermediates of β-sclerotization in the puparia of flesh fly Sarcophaga bullata

Cuticular phenoloxidase(s) from Sarcophaga bullata larvae oxidized a variety of o-diphenolic compounds. While catechol, 3,4-dihydroxybenzoic acid, dopa, dopamine, and norepinephrine were converted to their corresponding quinone derivatives, other catechols such as 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenethyl alcohol, 3,4-dihydroxyphenyl glycol, 3,4-dihy-droxymandelic acid, and N-acetyldopamine were oxidized to their side-chain oxygenated products. In addition, the enzyme-catalyzed oxidation of the latter group of compounds accompanied the formation of colorless catecholcuticle adducts consistent with the operation of β-sclerotization. Radioactive trapping experiments failed to support the participation of 1,2-dehydro-N-acetyldopamine as a freely formed intermediate during phenoloxidase-mediated oxidation of N-acetyldopamine. When specifically tritiated substrates were provided, cuticular enzyme selectively removed tritium from [7-³H]N-acetyldopamine and not from either [8-³H] or [ring-³H]N-acetyldopamine during the initial phase of oxidation. The above results are consistent with the generation and subsequent reactions of quinone methides as the initial products of enzyme-catalyzed N-acetyldopamine oxidation and confirm our hypothesis that quinone methides and not 1,2-dehydro-N-acetyldopamine are the reactive intermediate of β-sclerotization of sarcophagid cuticle. Quinone methide sclerotization resolves a number of conflicting observations made by previous workers in this field.     

Investigation of an Ortho-quinone isomerase from larval cuticle of the American silkmoth,Hyalophora cecropia

Insect cuticles catalyze the formation of N-acetylnorepinephrine (NANE) and N-β-alanylnorepinephrine (NBANE) from N-acetyldopamine (NADA) and N-β-alanyldopamine (NBAD), respectively. An enzyme, involved in the reaction, has now been isolated from fifth stage larval cuticle of Hyalophora cecropia and partially characterized. The enzyme alone has hardly any activity towards NADA, but together with diphenoloxidases [catechol oxidases (EC 1.10.3.1) or laccases (EC 1.10.3.2)] it will produce NANE as the main product from NADA, indicating that NADA-quinone is the actual substrate for the enzyme. The enzyme is presumably an ortho-quinone para-quinone methide isomerase, and formation of NANE is due to non-enzymatic addition of water to the quinone methide. The enzyme combination mushroom tyrosinase-cuticular isomerase has pH optimum at 5.5, and the optimal substrate concentration is about 10 mM NADA.Together with the endogenous cuticular diphenoloxidases the isomerase can account for the formation of NANE observed when pieces of intact cuticle are incubated with NADA, and for the presence of NANE and NBANE in sclerotized cuticle.The possible roles of the enzyme in sclerotization and defense reactions in insects are briefly discussed.     

Characterization of quinone tautomerase activity in the hemolymph of Sarcophaga bullata larvae

The hemolymph of Sarcophaga bullata larvae was activated with either zymosan or proteolytic enzymes such as chymotrypsin or subtilisin and assayed for phenoloxidase activity by two different assays. While oxygen uptake studies readily attested to the wide specificty of activated phenoloxidase, visible spectral studies failed to confirm the accumulation of quinone products in the case of 4-alkyl substituted catechols such as N-acetyldopamine and N-β-alanyldopamine. Sepharose 6B column chromatography of the activated hemolymph resolved phenoloxidase activity into two fractions, designated as A and B. Peak A possessed typical o-diphenoloxidase (o-diphenol, oxygen oxidoreductase EC 1.10.3.1) activity, while peak B oxidized physiologically important catecholamine derivatives such as N-acetyldopamine, N-acetylnorepinephrine, and N-β-alanyldopamine into N-acetylnorepinephrine, N-acetylarterenone, and N-β-alanylnorepinephrine, respectively, and converted 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylglycol into 3,4-dihydroxymandelic acid, 3,4-dihydroxybenzaldehyde, and 2-hydroxy-3′,4′-dihydroxyacetophenone, respectively. These transformations are consistent with the conversion of phenoloxidase-generated quinones to quinone methides and subsequent non-enzymatic transformations of quinone methides. Accordingly, Peak B contained both o-diphenoloxidase activity and quinone tautomerase activity. Sepharose 6B column chromatography of unactivated hemolymph resulted in the separation of quinone tautomerase from prophenoloxidase. The tautomerase rapidly converted both chemically made and mushroom tyrosinase-generated quinones to quinone methides. Thus the failure to observe the accumulation of quinones with N-acyl derivatives of dopamine and related compounds in the whole hemolymph is due to the rapid conversion of these long lived toxic quinones to short lived quinone methides. The latter, being unstable, undergo rapid non-enzymatic transformations to form side-chain-oxygenated products that are non-toxic. The possible roles of quinone isomerase and its reaction products—quinone methides—as essential components of sclerotization of cuticle and defense reaction of Sarcophaga bullata are discussed.     

Cuticle laccase of the silkworm,Bombyx mori: Purification,gene identification and presence of its inactive precursor in the cuticle.

Laccase is a multi-copper enzyme found in variety of organisms including plants, fungi and bacteria. In insects, laccase is thought to play an important role in cuticle sclerotization with its ability to catalyze the oxidation of phenolic compounds to their corresponding quinones. From the newly ecdysed pupae of the silkworm, Bombyx mori, we purified a dimer form of cuticular laccase with 70-kDa polypeptides. Mass spectrometric analysis of the tryptic fragments and cDNA sequence analysis revealed that the gene for the purified laccase (BmLaccase2) is an ortholog of laccase2, one of the multiple laccase genes found in insect genomes. BmLaccase2 is highly expressed in the epidermis prior to ecdysis, suggesting that the BmLaccase2 protein accumulates before ecdysis. However, the cuticle of newly ecdysed pupa does not have laccase activity, and the activity only becomes detectable several hours after ecdysis. These data suggest that cuticle laccase is synthesized as an inactive precursor, which is later activated after ecdysis. We also found that urea-solubilized cuticle protein extract contains an inactive form of laccase that can be activated by trypsin treatment.     

Procyanidin xylosides from the bark of Betula pendula

A procyanidin dimer xyloside, catechin-(4α → 8)-7-O-β-xylopyranosyl-catechin, was isolated from the inner bark of Betula pendula and its structure was determined using 1D and 2D NMR, CD and high-resolution ESIMS. Interestingly, the 7-O-β-xylopyranose unit was found to be present in the lower terminal unit of the dimer. In addition to this procyanidin dimer xyloside, an entire series of oligomeric and polymeric procyanidin xylosides was detected. Their structures were investigated by hydrophilic interaction HPLC–HRESIMS. Procyanidin glycosides are still rarely found in nature.     

Catecholamines and related o-diphenols in the hemolymph and cuticle of the cockroach Leucophaea maderae (F.) during sclerotization and pigmentation

Developmental profiles of catecholamines and related o-diphenols in the hemolymph and cuticle of Leucophaea maderae were determined during sclerotization and pigmentation of last instar nymphs and adults. N-Acetyldopamine (NADA) and dopamine (DA) were the major o-diphenols in hemolymph, whereas 3,4-dihydroxyphenylketoethanol (DOPKET), N-β-alanyldopamine (NBAD), norepinephrine, 3,4-dihydroxyphenylethanol, 3,4-dihydroxyphenylacetic acid, and 3,4-dihydroxyphenylalanine were detected at lower concentrations. The o-diphenols occurred primarily as acid-labile conjugates in hemolymph. Dopamine, conjugated as the 3-O-sulfate ester, and a NADA conjugate(s) were equal in concentration (0.06 mM) in nymphs shortly before adult apolysis. However, NADA increased after adult ecdysis to a peak at 6 h (0.18 mM), while its precursor DA decreased, suggesting N-acetylation of the latter or its metabolism to melanin pigments in the cuticle. In cuticle, NADA, N-acetylnorepinephrine (NANE), DOPKET, and N-β-alanylnorepinephrine (NBANE) accumulated during the early period of adult cuticle sclerotization. DOPKET and NADA (0.4 μmol g⁻¹ each), and NANE (0.2 μmole g⁻¹) occurred at the highest concentrations in tanned adult cuticle. Large amounts of DOPKET conjugates extracted by cold 1.2 M HCl from tanned cuticle which released DOPKET upon hydrolysis at 100°C for 10 min. DA and NBANE (0.2 μmole g⁻¹ each) predominated in tanned nymphal cuticle. Therefore, sclerotization of nymphal cuticle may require more of the N-β-alanyl catecholamines, whereas the adult cuticle contains larger quantities of the N-acetyl derivatives and ketocatechol (DOPKET) metabolites. Black pigmentation of nymphal and adult cuticle occurs during the first few hours after ecdysis, which correlates with relatively high levels of dopamine.     

Properties of an adrenal cytochrome P-450 (P-45011β) for the hydroxylations of corticosteroids

A highly purified preparation of cytochrome P-450, designated as P-450_11β, has been obtained from bovine adrenal cortex mitochondria. The P-450_11β exhibits remarkably high steroid hydroxylase activity in the reconstituted adrenal electron-donating system from NADPH via NADPH:adrenal ferredoxin oxidoreductase (EC 1.6.7.1) and adrenal ferredoxin. The turnover numbers (moles of hydroxylated product formed per minute per mole of P-450-heme) are 110 and 18 for respective 11β- and 18-hydroxylase activity when deoxycorticosterone is the substrate. The apparent K_m value is 6 μm for both reactions. The ratio, about 6:1 between the two activities, is constant under various experimental conditions including those in the presence of competitive inhibitors of hydroxylation. In addition to deoxycorticosterone, other steroids such as 11-deoxycortisol, 4-androstene-3,17-dione and testosterone are the hydroxylatable substrates. In cases in which 4-androstene-3,17-dione, a C₁₉-steroid, is the substrate, the hydroxylatable sites appear to be its respective 11β- and 19-position. The ratio between the two activities is about 4:1. In view of these results, it is concluded that one hemoprotein species, the P-450_11β, is responsible for the hydroxylase reactions of various Corticosteroids. 2-Methyl-1,2-di-3-pyridyl-1-propanone (metyrapone) inhibits the P-450_11β-catalyzed steroid hydroxylase reactions of either deoxycorticosterone at 11β- and 18-position or 4-androstene-3,17-dione at 11β- and 19-position (K_i = 0.1-0.2 μM). The P-450_scc-catalyzed cholesterol desmolase reaction is also inhibited, although weakly (K_i = 160 μM). In addition, both adrenal cytochromes appeared to differ from each other in spectral response to metyrapone.     

Enzymatic activities involved in incorporation of N-acetyldopamine into insect cuticle during sclerotization

During sclerotization of insect cuticle, N-acetyldopamine (NADA) is enzymatically oxidized before reaction with cuticular proteins. Not all oxidized NADA reacts with cuticular structural materials, a small fraction reacts with water or other available low molecular weight compounds to give soluble products. Various types of cuticle were incubated with excess NADA and the products studied by reversed phase high performance liquid chromatography (RP-HPLC) to obtain information on the enzymatic activities in the cuticle. The occurrence of at least two enzymes competing for NADA and present in different proportions in the various types of cuticle can explain the results. NADA may be incorporated into cuticle via α,β-dehydro-NADA (β-sclerotization) or via quinone methides and o-quinones, and the actual course of sclerotization will depend upon the relative activities of the enzymes involved. The various pathways may all be used simultaneously.     

Cholesterol Modulates the Dimer Interface of the β2-Adrenergic Receptor via Cholesterol Occupancy Sites

The β₂-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β₂-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β₂-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.     

Tyrosine and catecholamine levels in the hemolymph of tobacco hornworm larvae,Manduca sexta,parasitized by the braconid wasp,Cotesia congregata,and in the developing parasitoids

Parasitism of fifth instar Manduca sexta larvae by the gregarious parasitoid Cotesia congregata prevented normal storage of tyrosine in the hemolymph, whereas total tyrosine levels increased over eight times in the hemolymph of unparasitized larvae by day 4. Tyrosine glucoside, the hemolymph storage form of tyrosine and the precursor for pupal cuticle sclerotizing agents, was found only in trace amounts in parasitized larvae at the time of parasitoid emergence, but had increased to over 6 mM in hemolymph of unparasitized larvae. Concentrations of dopamine and N-β-alanyldopamine (NBAD), precursors for melanization and sclerotization of cuticle, respectively, had approximately doubled in the hemolymph of parasitized larvae by the day of parasitoid emergence, but not in unparasitized larvae. Catecholamine biosynthesis may be transiently stimulated for wound-healing, as black melanic pigmentation appeared around the wasp emergence holes in the host integument. C. congregata larvae accumulate tyrosine, dopamine, and NBAD by the time of emergence and cocoon spinning, either by direct uptake or by synthesis from precursors obtained from the host. NBAD increased in parasitoid larvae close to pupation, suggesting it functions as the main precursor for pupal cuticle tanning. Both dopamine and NBAD increased dramatically in pharate adult wasps just before eclosion and N-acetyldopamine (NADA) appeared for the first time. Dopamine was highest in concentration and total amount, and it can serve both as a precursor for black melanic pigmentation of adult wasp cuticle and for synthesis of NADA and NBAD, the precursors for cuticle sclerotization. Arch. Insect Biochem. Physiol. 38:193–201, 1998. © 1998 Wiley-Liss, Inc.     

Dimeric antioxidant and cytotoxic triterpenoid saponins from Terminalia ivorensis A. Chev.

Three saponins, including two dimeric triterpenoid glucosides possessing an unusual skeleton, ivorenosides A and B, and a monomeric triterpenoid saponin (ivorenoside C), together with the known sericoside, were isolated from the bark of Terminalia ivorensis. Their structures were established on the basis of 1D and 2D NMR data, chemical methods and tandem MS–MS spectrometry as a dimer of β-d-glucopyranosyl-18,19-seco-2α,3β,19,19,24-pentahydroxyolean-12-en-28-oate and β-d-glucopyranosyl-2α,3β,19α,24-tetrahydroxyolean-12-en-28-oate (ivorenoside A, 1), a dimer of β-d-glucopyranosyl-18,19-seco-24-carboxyl-2α,3β,19,19-tetrahydroxyolean-12-en-28-oate and β-d-glucopyranosyl-2α,3β,19α,24-tetrahydroxyolean-12-en-28-oate (ivorenoside B, 2) and β-d-glucopyranosyl-2α,3β,19β,24-tetrahydroxyolean-11-oxo-olean-12-en-28-oate (ivorenoside C, 3). Ivorenosides A and B are the first examples in nature of dimeric triterpenoid saponins with a 18,19-seco E ring of one of the two units. These isolated compounds were evaluated for their antioxidant properties and further for their cytotoxic activity against four human cancer cell lines. Ivorenoside B and C exhibited scavenging activity against DPPH and ABTS⁺ radicals with IC₅₀ values comparable with that of the standard drug Trolox and ivorenoside A showed antiproliferative activity against MDA-MB-231 and HCT116 human cancer cell lines with IC₅₀ values of 3.96 and 3.43 μM, respectively.     

Purification of deoxycytidine kinases from two P815 murine neoplasms and their separation from deoxyguanosine kinase

Deoxycytidine kinase, which catalyzes the phosphorylation of deoxycytidine and 1-βd-arabinofuranosylcytosine (Ara-C) at the 5′-position, has been extracted and extensively purified from a murine neoplasm P815, either sensitive (P815) or resistant (P815/ Ara-C) to 1-β-d-arabinofuranosylcytosine. Gel filtration and ion-exchange chromatography were used to accomplish the purification. The purified enzyme exhibited a single band upon disc electrophoresis. During the extraction procedure an enzyme catalyzing the phosphorylation of deoxyguanosine and deoxyadenosine was successfully separated for the first time from deoxycytidine kinase. The K_m values and turnover numbers with deoxycytidine as phosphate acceptor for the kinase from P815 cells sensitive to 1-β-darabinofuranosylcytosine and that from P815 cells resistant to the drug are 9.3 μm, 4.7 × 10⁶/min and 15.4 μm, 8.0 × 10⁴/min, respectively.