Cytotoxic cardiac glycosides and coumarins from Antiaris toxicaria

Eight new cardiac glycosides/aglycones (antiaritoxiosides A–G, 1–7, and antiarotoxinin B, 8), two new coumarins (anticarins A–B, 41–42), and two new flavanones (antiarones L–K, 43–44) were isolated from trunk bark of Antiaris toxicaria together with 53 known compounds. The new structures were established by extensive analysis of spectroscopic data. Compound 1 (10-carboxy and 3α-hydroxy) and compounds 3–6 (10-hydroxy) contain unique substituents that are rarely found in cardiac glycosides. The cytotoxic effects of isolated compounds against ten human cancer cell lines, KB, KB-VIN, A549, MCF-7, U-87-MG, PC-3, 1A9, CAKI-1, HCT-9 and S-KMEL-2, were tested using the sulforhodamine B assay. Five compounds (12, 16, 20, 22, and 31) showed significant cytotoxicity against all ten cancer cell lines, with notable potency at the ng/mL level against some cell lines, which merits further development as clinical trial candidates.     

Cytotoxic and non-cytotoxic cardiac glycosides isolated from the combined flowers,leaves, and twigs of Streblus asper

A new non-cytotoxic [(+)-17β-hydroxystrebloside (1)] and two known cytotoxic [(+)-3′-de-O-methylkamaloside (2) and (+)-strebloside (3)] cardiac glycosides were isolated and identified from the combined flowers, leaves, and twigs of Streblus asper collected in Vietnam, with the absolute configuration of 1 established from analysis of its ECD and NMR spectroscopic data and confirmed by computational ECD calculations. A new 14,21-epoxycardanolide (3a) was synthesized from 3 that was treated with base. A preliminary structure-activity relationship study indicated that the C-14 hydroxy group and the C-17 lactone unit and the established conformation are important for the mediation of the cytotoxicity of 3. Molecular docking profiles showed that the cytotoxic 3 and its non-cytotoxic analogue 1 bind differentially to Na⁺/K⁺-ATPase. Compound 3 docks deeply in the Na⁺/K⁺-ATPase pocket with a sole pose, and its C-10 formyl and C-5, C-14, and C-4′ hydroxy groups may form hydrogen bonds with the side-chains of Glu111, Glu117, Thr797, and Arg880 of Na⁺/K⁺-ATPase, respectively. However, 1 fits the cation binding sites with at least three different poses, which all depotentiate the binding between 1 and Na⁺/K⁺-ATPase. Thus, 3 was found to inhibit Na⁺/K⁺-ATPase, but 1 did not. In addition, the cytotoxic and Na⁺/K⁺-ATPase inhibitory 3 did not affect glucose uptake in human lung cancer cells, against which it showed potent activity, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na⁺/K⁺-ATPase but not by interacting with glucose transporters.     

Insensitivity of lepidopteran tissues to ouabain: Physiological mechanisms for protection from cardiac glycosides

Neuronal tissues from Manduca sexta, the tobacco hornworm, Hyalophora cecropia, the silkmoth and Danaus plexippus, the Monarch Butterfly, contain Na⁺K⁺-ATPase which is sensitive to cardiac glycoside (ouabain). The K_m for K⁺ stimulation of Na⁺K⁺-ATPase in M. sexta and D. plexippus is 2.2 mM and for Na⁺ stimulation in D. plexippus, 6.0 mM. In vitro ouabain concentrations of 1.0 × 10^?5 M and 5.0 × 10^?5 M in the presence of 7.5 mM K⁺ inhibited Na⁺K⁺-ATPase activity in H. cecropia and M. sexta by 50% respectively. Na⁺K⁺-ATPase from D. plexippus was approximately 300 times less sensitive. High concentrations (10^?3 M in haemolymph) of ouabain had no effect on M. sexta in vivo. This is largely explained by haemolymph K⁺ (>; 30 mM) antagonizing the binding of ouabain to Na⁺K⁺-ATPase. As demonstrated in vitro, 30 mM K⁺ totally protects Na⁺K⁺-ATPase from inhibition by 7.5 × 10^?3 M ouabain in D. plexippus and protects the enzyme by 65% in M. sexta. At least part of the physiological burden incurred in utilization of cardiac glycoside ingestion and storage for protection from predation, however, is probably related to the toxic effects of cardiac glycosides on neuronal Na⁺K⁺-ATPase.     

Two chromone-secoiridoid glycosides and three indole alkaloid glycosides from Neonauclea sessilifolia

From the dried roots of Neonauclea sessilifolia, two new chromone-secoiridoid glycosides, sessilifoside and 7"-O-beta-D-glucopyranosylsessilifoside, and three novel indole alkaloid glycosides, neonaucleosides A, B, and C, were isolated along with the main known glycosides, 5-hydroxy-2-methylchromone-7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside, sweroside, loganin, grandifloroside, and quinovic acid 3 beta-O-beta-D-quinovopyranoside-28-O-beta-D-glucopyranoside. The structures of these new glycosides were determined by spectroscopic and chemical means. Neonaucleoside A and its C-3 epimer were prepared from secologanin and tryptamine.     

三种植物源杀虫活性成分对东方粘虫中肠细胞的毒力测定

采用MTT([3-(4,5-二甲基噻唑-2)-2,5-二苯基]四氮唑溴盐)比色法、中性红摄取法和台盼蓝拒染法,分别测定植物源杀虫活性成分苦皮藤素Ⅴ、白鲜碱和梣皮酮对中肠细胞的毒力。结果表明:急性分离的东方粘虫中肠细胞能在Grace’s昆虫细胞培养基中维持生长;3种供试药剂对中肠细胞均有明显的细胞毒性。MTT比色法、中性红摄取法和台盼蓝拒染法测得苦皮藤素Ⅴ对中肠细胞的LC50依次为9.0,7.79和10.94μg/mL;白鲜碱为27.85,31.77和36.42μg/mL;梣皮酮为186.66,164.00和192.34μg/mL。     

The piRNA response to BmNPV infection in the silkworm fat body and midgut

Bombyx mori nucleopolyhedrovirus (BmNPV) is a DNA virus that causes huge losses to the silkworm industry but the piRNA responses during BmNPV infection in the silkworm remain uninvestigated. Here, silkworm piRNA profiles of uninfected and BmNPV-infected fat body and midgut were determined by high-through sequencing in the early stages of BmNPV infection. A total of 2675 and 3396 genome-derived piRNAs were identified from fat body and midgut, respectively. These genome-derived piRNAs mainly originated from unannotated instead of transposon regions in the silkworm genome. In total, 572 piRNAs were associated with 280 putative target genes in fat body and 805 piRNAs with 380 target genes in midgut. Compared to uninfected tissues, 322 and 129 piRNAs were significantly upregulated in BmNPV-infected fat body and midgut, respectively. In addition, 276 and 117 piRNAs were significantly downregulated. Moreover, differentially expressed (DE) piRNAs during BmNPV infection differed significantly between fat body and midgut. Putative DE piRNA–targeted genes were associated with “response to stimulus” and “environmental information processing” in fat body after infection with BmNPV, which may indicate an active piRNA response to BmNPV infection in fat body. This study may lay the foundation for future research of the potential roles of the piRNA pathway and specific piRNAs in BmNPV pathogenesis.     

The fine structural morphology of the midgut of adult Drosophila: A morphometric analysis

The midgut of one day old Drosophilia was examined morphometrically at the electron microscopic level. Results suggest that parenchymal cells, with the exception of basal cells, possess identical functions. Drosophilia midgut cells are smaller than those of other insects studied, and the surface densities of the rER was less, indicating that its protein synthetic activity is also less than that of other insects.     

Implications for the functions of the four known midgut differentiation factors: An immunohistologic study of Heliothis virescens midgut

Antibodies to the peptides that induce differentiation of midgut larval stem cells, the midgut differentiating factors MDF-2, MDF-3, and MDF-4, bind to columnar cells in midgut cultures and in intact midgut of Heliothis virescens, in manners similar to the binding of anti- MDF-1 to those tissues. Antibodies to MDF-2 and MDF-3 also stained droplets in the midgut lumen, suggesting that columnar cells may also release MDF-2- and MDF-3-like cytokines to the lumen. Antibody to MDF-4 exhibited similar staining patterns but also recognized stem and differentiating cells, the presumed targets of peptides that regulate stem cell differentiation. Antibody to MDF-4 also bound to one type of endocrine cell in midgut cultures and in sections of midgut, as well as to the endocrine secretion released both to the midgut lumen and the hemolymph. Antibodies to the MDFs 1, 2, and 3, incubated with cultures of midgut cells, did not appear to prevent differentiation of the stem cells in the cultures but affected viability of mature cells, reflected in increased apoptosis and doubling of the number of differentiating cells compared to controls. Only antibody to MDF-4 induced temporary necrosis and inhibition of population recovery, indicating that MDF4 may be the true differentiation factor. The other MDFs may have additional functions beyond regulation of midgut stem cell differentiation in vivo.     

Thiol addition to protected allyl glycosides: An improved method for the preparation of spacer-arm glycosides

A useful method for the preparation of differently functionalized sulfide spacer-arm glycosides is presented. Several protected allyl glycosides were variously elongated via a radical addition reaction with pentanethiol, methyl 3-mercaptopropionate, or 2-mercaptoethanol. The hydroxyl function of protected 3-(2-hydroxyethylthio)propyl glycosides was subsequently transformed into an azide function.     

Effect of sublethal Bacillus thuringiensis crystal endotoxin treatment on the larval midgut of a moth, Manduca: SEM study

The effect of a single, sublethal dose of B. thuringiensis crystal endotoxin on the midgut of the moth Manduca sexta larvae was monitored during acute and recovery stages. Initially both goblet and columnar cells swelled. Many columnar cells produced membrane extrusions. In some cases the affected cells ruptured, extruding cellular debris into the midgut lumen. Following the acute stage, the midgut tissue recovered, the damaged cells being extruded into the midgut lumen apparently as newly regenerated cells rose to take their place. The insects appeared to recover completely and continue normal development.     

The role of stem cells in midgut growth and regeneration

Summary TheManduca sexta (L.) [Lepidoptera: Sphingidae] andHeliothis virescens (F.) [Lepidoptera: Noctuidae] midguts consist of a pseudostratified epithelium surrounded by striated muscle and tracheae. This epithelium contains goblet, columnar, and basal stem cells. The stem cells are critically important in that they are capable of massive proliferation and differentiation. This growth results in a fourfold enlargement of the midgut at each larval molt. The stem cells are also responsible for limited cell replacement during repair. While the characteristics of the stem cell population vary over the course of an instar, stem cells collected early in an instar and those collected late can start in vitro cultures. Cultures of larval stem, goblet, and columnar cells survive in vitro for several mo through proliferation and differentiation of the stem cells. One of the two polypeptide differentiation factors which have been identified and characterized from the culture medium has now been shown to be present in midgut in vivo. Thus the ability to examine lepidopteran midgut stem cell growth in vitro and in vivo is proving to be effective in determining the basic features of stem cell action and regulation. Mention of any product in this publication does not imply endorsement by the USDA.     

Subchronic treatment with vanadate does not potentiate the toxicity of cardiac glycosides

Since it has been claimed that vanadate is an endogenous regulator of Na/K-ATPase activity and that it potentiates the toxicity of cardiac glycosides, we were alarmed to discover that certain Finnish physicians were prescribing vanadate in combination with other trace minerals to elderly patients for many different chronic diseases (e.g., cancer, rheumatism). To study the interaction of vanadate and cardiac glycosides, we fed vanadate in the drinking water (25 μg/mL) to guinea pigs for 20 d, and studied either their sensitivity to the acute toxicity of the cardiac glycoside ouabain or whether the vanadate would influence the subacute toxicity of ouabain. Vanadate had no influence on the toxicity of ouabain either acute or subchronically administered, nor was there any sign of inhibition of Na/K-ATPase activity as measured by⁸⁶Rb-uptake into intact erythrocytes (RBCs), RBC content of sodium or potassium or Na/K-ATPase activity in RBC membranes prepared from the vanadate-treated guinea pigs. Vanadate had been absorbed in substantial quantities from the gastrointestinal tract, since serum, heart, liver, and especially kidney contained measurable amounts of vanadium in contrast to controls, but it is concluded that this vanadate is not in a biologically active form.     

The occurrence of acylated flavonol glycosides in the cruciferae

Nine acylated glycosides of kaempferol or quercetin were identified in Sisymbrium gilliesii, and in three Crambe spp. They were usually present together with the related unacylated glycosides. Acylation is a very common characteristic of the four crucifer species studied.     

Compartmentalization of proteinases and amylases in Nauphoeta cinerea midgut.

Compartmentalization of proteinases, amylases, and pH in the midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was studied in order to understand the organization of protein and starch digestion. Total proteolytic activity measured with azocasein was maximal at pH 11.5 both in anterior (AM) and posterior (PM) halves of the midgut, but the bulk of activity (67%) was found in PM. Total AM and PM preparations were fractionated on a Sephadex G-50 column and further analysed by means of activity electrophoresis and specific inhibitors and activators. The major activity in PM was classified as an unusual SH-dependent proteinase with M(r) 24,000 and pH optimum with synthetic substrate BApNA at 10.0. The enzyme was 43-fold activated in the presence of 1 mM DTT, insensitive to synthetic inhibitors of serine (PMSF, TLCK, TPCK) and cysteine (IAA, E-64) proteinases, strongly inhibited by STI, and displayed four active bands on zymograms. In PM, activities of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine proteinases were observed. Aspartic and metalloproteinases were not detected. In AM, activity of unusual SH-dependent proteinase also dominated and activity of chymotrypsin-like proteinase was observed, but their levels were much lower than in PM. Distribution of amylase activity, exhibiting an optimum at pH 6.0, was quite the opposite. The major part of it (67%) was located in AM. Treatment of amylase preparation with proteinases from AM and PM reduced amylase activity twofold. pH of the midgut contents was 6.0-7.2 in AM, 6.4-7.6 in the first and 8.8-9.3 in the second halves of PM. Thus, pH in AM is in good agreement with the optimal pH of amylase, located in this compartment, but the activity of proteinases, including the ability to degrade amylase, in such an environment is low. Active proteolysis takes place in the second half of PM, where pH of the gut is close to the optimal pH of proteinases.     

The proteases and the protease inhibitor in the midgut of Leucophaea maderae

The caseinolytic enzymes of the midgut lumina and epithelia of Leucophaea were purified through precipitation by 60% saturated (NH₄)₂SO₄, followed by gel permeation on Sephadex G-200 and subsequent DEAE anionexchange chromatography. At least four peaks with enzyme activity were eluted from anionexchange chromatography columns. Gregarines of the midgut lumen apparently do not contribute to the caseinolytic activity within the midgut. Elution profiles of lumen and epithelial enzymes were nearly identical. The same enzymes were identified in the lumina of epithelial microsomal vesicles. This allows the conclusion that these enzymes are produced by the midgut epithelia.Practically all protease activity of the midgut was found in the posterior half, both in the lumen and epithelium. Feeding stimulated protease production primarily in the posterior midgut. The pH optimum of the proteases lay between 9.0 and 9.5 which was closely matched by the observed pH of the posterior midgut where most of the activity is seen. The anterior midgut pH was determined to be around 8.0.The anterior midgut of Leucophaea contained a heatstable protease inhibitor with characteristics of a competitive inhibitor. This inhibitor was precipitable by 60% saturated (NH₄)₂SO₄ and eluted from a Sephadex G-200 column more or less together with the proteases. From a DEAE anionexchange column it was eluted by 0.8 M NaCl, i.e. after the main portion of the proteases. The biological significance of the protease inhibitor in the anterior portion of the midgut is obscure.     

Fine structure of the larval midgut of the fly Rhynchosciara and its physiological implications

The midgut of Rhynchosciara americana larvae consists of a cylindrical ventriculus from which protrudes two gastric caeca formed by polyhedral cells with microvilli covering their apical faces. The basal plasma membrane of these cells is infolded and displays associated mitochondria which are, nevertheless, more conspicuous in the apical cytoplasm. The anterior ventricular cells possess elaborate mitochondria-associated basal plasma membrane infoldings extending almost to the tips of the cells, and small microvilli disposed in the cell apexes. Distal posterior ventricular cells with long apical microvilli are grouped into major epithelial foldings forming multicellular crypts. In these cells the majority of the mitochondria are dispersed in the apical cytoplasm, minor amounts being associated with moderately-developed basal plasma membrane infoldings. The proximal posterior ventriculus represents a transition region between the anterior ventriculus and the distal posterior ventriculus. The resemblance between the gastric caeca and distal posterior ventricular cells is stressed by the finding that their microvilli preparations display similar alkaline phosphatase-specific activities. The results lend support to the proposal, based mainly on previous data on enzyme excretion rates, that the endo-ectoperitrophic circulation of digestive enzymes is a consequence of fluid fluxes caused by the transport of water into the first two thirds of midgut lumen, and its transference back to the haemolymph in the gastric caeca and in the distal posterior ventriculus.     

A simple mechanical method to isolate the basal lamina of insect midgut epithelial cells

In mealworms (Tenebrio molitor) the midgut epithelium is surrounded by a 1.6mum thick basal lamina of low electron density with a framework of high electron density imbedded in a part of it. The lamina can be isolated by ultrasonication followed by repeated filtrations and high-speed centrifugations, making large-scale preparation of the lamina for further analyses possible. The isolation of the basal lamina is confirmed by electron microscopy.     

Protective effect of total phenylethanoid glycosides from Monochasma savatieri Franch on myocardial ischemia injury

The present study was designed to investigate the cardioprotective effect of total phenylethanoid glycosides from Monochasma savatieri Franch (TPG). The data showed that there were mainly four phenylethanoid glycosides isolated and identified from TPG. TPG significantly increased cells viability and inhibited morphological changes on H9c2 cardiomyocytes induced by H₂O₂ or Na₂S₂O₄. In addition, TPG significantly decreased T-wave elevation and histopathological changes of heart tissues in myocardial infracted rats induced by isoproterenol. It also significantly reduced the infarct size induced by ligating the coronary artery in rats, increased the activities of antioxidative enzymes superoxide dismutase (SOD), the content of glutathione (GSH), and decreased the leakage of lactic dehydrogenase (LDH), the activities of creatine kinase (CK) and the content of maleic dialdehyde (MDA). In conclusion, these results suggested that TPG from Monochasma savatieri Franch might be developed as new natural medicine or food additives with effects of prevention of coronary artery disease due to its significant antioxidant activity.