Inhibition of binding of E- and P-selectin to sialyl-Lewis X molecule suppresses the inflammatory response in hypersensitivity pneumonitis in mice.

The carbohydrate structure of sialyl-Lewis X (SLe(x)) can function as a ligand for E- and P-selectin, which play important roles in mediating the initial interactions of leukocytes with the endothelium in inflammatory responses. In this study we evaluated the effects of inhibiting E- and P-selectin function with the SLe(x) molecule on the inflammatory response in an experimental murine model of hypersensitivity pneumonitis (HP). Antigen exposure induced marked interstitial and especially perivascular and peribronchiolar infiltration with lymphocytes and granuloma formation, in murine lung sensitized with Saccaropolyspora rectivirgula. These pathologic changes were significantly suppressed with SLe(x) ganglioside analogues through a reduction in the numbers of lymphocytes in bronchoalveolar lavage fluid, as evidenced by the lung index and histologic scores indicating the severity of the inflammatory response. Using specific antibodies, we also evaluated the immunohistochemical localization of SLe(x) in mononuclear cells in granulomas, and of E- and P-selectin in vascular endothelium. Our findings suggest that the molecular interaction between SLe(x), and E- and P-selectin mediates lymphocyte recruitment into the lung parenchyma, which is critical for the inflammatory response in experimental murine models of HP.

Potassium current suppression in patients with peripheral nerve hyperexcitability.

Acquired neuromyotonia (Isaac's syndrome) is considered to be an autoimmune disease, and the pathomechanism of nerve hyperexcitability in this syndrome is correlated with anti-voltage-gated K(+) channel (VGKC) antibodies. The patch-clamp technique was used to investigate the effects of immunoglobulins from acquired neuromyotonia patients on VGKCs and voltage-gated Na(+) channels in a human neuroblastoma cell line (NB-1). K(+) currents were suppressed in cells that had been co-cultured with acquired neuromyotonia patients' immunoglobulin for 3 days but not for 1 day. The activation and inactivation kinetics of the outward K(+) currents were not altered by these immunoglobulins, nor did the immunoglobulins significantly affect the Na(+) currents. Myokymia or myokymic discharges, with peripheral nerve hyperexcitability, also occur in various neurological disorders such as Guillain-Barré syndrome and idiopathic generalized myokymia without pseudomyotonia. Immuno-globulins from patients with these diseases suppressed K(+) but not Na(+) currents. In addition, in hKv 1.1- and 1.6-transfected CHO (Chinese hamster ovary)-K1 cells, the expressed VGKCs were suppressed by sera from acquired neuromyotonia patients without a change in gating kinetics. Our findings indicate that nerve hyperexcitability is mainly associated with the suppression of voltage-gated K(+) currents with no change in gating kinetics, and that this suppression occurs not only in acquired neuromyotonia but also in Guillain-Barré syndrome and idiopathic generalized myokymia without pseudomyotonia.

Steroidal saponins from the aerial parts of Dracaena draco and their cytostatic activity on HL-60 cells.

Chemical examination of the aerial parts of Dracaena draco has led to the isolation of a total of nine steroidal saponins, including five new ones. The structures of the new saponins were determined by spectral data and a few chemical transformations to be (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta,23,24-tetrol 1-O-{O-(2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl)-(1-->2)-alpha-L -arabinopyranosyl} 24-O-beta-D-fucopyranoside, (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta, 23,24-tetrol 1-O-{O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L -arabinopyranoside}, (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta,23,24-tetrol 1-O-{O-(4-O- acetyl-alpha-L-rhamnopyranosyl)-(1-->2)-alpha-L-arabinopyransoide} , (23S)-spirosta-5,25(27)-diene-1 beta,3 beta,23-triol 1-O-{O-alpha-L- rhamnopyranosyl)-(1-->2)-alpha-L-arabinopyranoside} and (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta,23-triol 1-O-{O-(4-O-acetyl-alpha-L-rhamnopyranosyl)-(1-->2)-alpha-L- arabinopyranoside}. The isolated saponins were evaluated for their cytostatic activity on leukemia HL-60 cells.

A cytoplasmic factor, calpastatin and ATP together reverse run-down of Ca2+ channel activity in guinea-pig heart.

1. The cytoplasmic extract of bovine heart was separated into four fractions by gel filtration: H (molecular mass > 300 kDa), P (250-300 kDa), L1 (180-250 kDa) and L2 (< 180 kDa). The effects of these fractions on the run-down of L-type Ca2+ channel activity were investigated in guinea-pig ventricular myocytes. 2. After run-down induced by inside-out patch formation, Ca2+ channel activity was restored by P or H (+ 3 mM ATP) to 7.5 and 5.8 % of that in the cell-attached mode, respectively, but to as high as 86 % by P + H + ATP. 3. The reversal of run-down brought about by the P fraction was mimicked by calpastatin. 4. The restorative effect of calpastatin + ATP showed a biphasic time course: 38 % in the early transient phase and 11 % in the late phase. However, calpastatin + H + ATP showed a sustained effect: 66 % in the early transient phase, and 87 % in the late phase. 5. The effective component of the H fraction showed a protein-like nature: heat and trypsin sensitivity. 6. The activities of cAMP-dependent protein kinase, casein kinase I, casein kinase II, protein tyrosine kinase, protein serine/threonine or tyrosine phosphatases were measured. However, these kinases and phosphatases were not confirmed as the effective component of cytoplasm or the H fraction. 7. Run-down was not prevented by 2 microM phalloidin or 2 microM paclitaxel, suggesting that neither actin filaments nor microtubules are directly involved in the run-down. 8. Our results support the view that the basal activity of the Ca2+ channel is maintained by at least three factors: a protein-like factor in the H fraction, calpastatin, and ATP.

Aculeoside B, a new bisdesmosidic spirostanol saponin from the underground parts of Ruscus aculeatus.

From the underground parts of Ruscus aculeatus, a new bisdesmosidic spirostanol saponin named aculeosides B (2) was isolated, and its structure was determined on the basis of spectroscopic analysis, including 2D NMR techniques. Aculeoside A (1), which was previously isolated from the same plant source, exhibited inhibitory activity on cell growth of leukemia HL-60 cells with an IC50 value of 0.48 microgram mL(-1), while aculeoside B (2) was inactive.

Steroidal saponins from the rhizomes of Hosta sieboldii and their cytostatic activity on HL-60 cells.

A total of eighteen steroidal saponins were isolated from the rhizomes of Hosta sieboldii, one of which appeared to be the first isolation from a plant source and six to be new compounds. The structures of the new saponins were determined by spectral data and a few chemical transformations to be (25R)-2 alpha, 3 beta-dihydroxy-5 alpha-spirostan-12-one (manogenin) 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-beta-D-glucopyranosyl -(1-->4)-beta-D-galactopyranoside¿, (25R)-2 alpha,3 beta-dihydroxy-5 alpha-spirost-9-en-12-one (9,11-dehydromanogenin) 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-beta-D- glucopyranosyl-(1-->4)-beta-D-galactopyranoside¿, 9,11-dehydromanogenin 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-[O-alpha-L- rhamnopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->3)]-O-beta-D- glucopyranosyl-(1-->4)-beta-D-galactopyranoside¿, (25R)-2 alpha,3 beta-dihydroxy-26-beta-D-glucopyranosyloxy-22-methoxy-5 alpha-furostan-12-one 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-[beta-D-xylopyranosyl-(1-->3)]-O- beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranoside¿, (25R)-2 alpha, 3 beta-dihydroxy-26-beta-D-glucopyranosyloxy-22-methoxy-5 alpha-furost-9-en-12-one 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-[beta-D-xylopyranosyl-(1-->3)]-O- beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranoside¿ and (25R)-5 alpha-spirostan-2 alpha,3 beta,12 beta-triol 3-O-¿O-alpha-L- rhamnopyranosyl-(1-->2)-beta-D-galactopyranoside¿, respectively. Cytostatic activity of the isolated saponins on leukaemia HL-60 cells was examined.

A Novel Synthesis of Poly(ester-alt-sulfide)s by the Ring-Opening Alternating Copolymerization of Oxiranes with gamma-Thiobutyrolactone Using Quaternary Onium Salts or Crown Ether Complexes as Catalysts.

Poly(ester-alt-sulfide) (polymer 1) was synthesized by the alternating copolymerization of glycidyl phenyl ether (GPE) with gamma-thiobutyrolactone (TBL) catalyzed by either quaternary onium salts or crown ether complexes. The copolymerization proceeded to produce polymer 1 with good yields in neat or in various organic solvents at 30-120 degreesC, in which quaternary onium salts having Cl- as a counteranion such as tetrabutylammonium chloride (TBAC) had higher activity than quaternary onium salts such as tetrabutylammonium bromide having Br- as a counteranion. It was also found that the alternate copolymer (polymer 1) of GPE with TBL was obtained selectively under different feed ratios of GPE and TBL, although ring-opening homopolymerizations of GPE and TBL did not proceed. Copolymerizations of various oxiranes such as butyl glycidyl ether, styrene oxide, and 1,2-hexene oxide with TBL catalyzed by TBAC also proceeded, and the corresponding poly(ester-alt-sulfide)s (polymers 2, 3, and 4) were obtained under the same conditions as for the synthesis of polymer 1.

Steroidal saponins from the underground parts of Ruscus aculeatus and their cytostatic activity on HL-60 cells.

Phytochemical examination of the underground parts of Ruscus aculeatus has been undertaken as part of systematic study of plants of the Liliaceae. Six new spirostanol saponins and five new furostanol saponins were isolated, and their structures were assigned on the basis of spectroscopic analysis, including two-dimensional NMR techniques, and hydrolysis. Ruscogenin diglycoside with three acetyl groups attached to the inner galactosyl moiety and its corresponding 26-glucosyloxyfurostanol saponin showed cytostatic activity on leukemia HL-60 cells.

Run-down of L-type Ca2+ channels occurs on the alpha 1 subunit.

Run-down of L-type Ca2+ channels in CHO cells stably expressing alpha 1c, alpha 1c beta 1a, or alpha 1c beta 1a alpha 2 delta gamma subunits was studied using the patch-clamp technique (single channel recording). The channel activity (NPo) of alpha 1c channels was increased 4- and 8-fold by coexpression with beta 1a and beta 1a alpha 2 delta gamma, respectively. When membranes containing channels composed of different subunits were excised into basic internal solution, the channel activity exhibited run-down, the time-course of which was independent of the subunit composition. The run-down was restored by the application of calpastatin (or calpastatin contained in cytoplasmic P-fraction) + H-fraction (a high molecular mass fraction of bovine cardiac cytoplasm) + 3 mM ATP, which has been shown to reverse the run-down in native Ca2+ channels in the guinea-pig heart. The restoration level was 64.7, 63.5, and 66.4% for channels composed of alpha 1c, alpha 1c beta 1a, and alpha 1c beta 1a alpha 2 delta gamma, respectively, and was thus also independent of the subunit composition. We conclude that run-down of L-type Ca2+ channels occurs via the alpha 1 subunit and that the cytoplasmic factors maintaining Ca2+ channel activity act on the alpha 1 subunit.

New steroidal constituents of the underground parts of Ruscus aculeatus and their cytostatic activity on HL-60 cells.

Phytochemical examination of the underground parts of Ruscus aculeatus has led to the isolation of a total of twelve steroidal saponins, including seven new ones. The structures of the new saponins were determined by spectroscopic analysis and chemical evidence. The furostanol saponin, having a diglycoside moiety modified with a (2S,3S)-2-hydroxy-3-methylpentanoic acid group and an acetic acid group, and its corresponding spirostanol saponin exhibited cytostatic activity on leukemia HL-60 cells.

Characterization and partial purification of the cytoplasmic factor that maintains cardiac Ca2+ channel activity.

Using the patch clamp method we attempted to characterize the cytoplasmic factor in guinea-pig cardiac myocytes which restores L-type Ca2+ channel activity after run-down. The factor was eluted from a diethylaminoethyl (DEAE) sepharose column by KCl at 100-360 mM. On gel filtration the factor had an apparent molecular mass (Mr) of 250-300 kDa. Two-dimensional electrophoresis of the partially purified factor showed at least nine spots, of which the major spot had a Mr of about 100 kDa and an isoelectric point of 4.8, suggesting that the physicochemical properties of the factor resemble those of calpastatin, an endogenous inhibitor of Ca2+-activated protease, calpain. Calpastatin activity was increased in the partially purified cytoplasm and an antibody raised against calpastatin recognized the major band. Reduction of calpastatin in the cytoplasm decreased the potency of Ca2+ channel activation. These results suggest that calpastatin might interact with the Ca2+ channel and maintain channel activity.

Run-down of the cardiac L-type Ca2+ channel: partial restoration of channel activity in cell-free patches by calpastatin.

We have found previously that run-down of cardiac Ca2+ channels in cell-free patches is reversed by cytoplasm plus adenosine triphosphate (ATP). Characterization of the factor in cytoplasm revealed that it is likely to be calpastatin (CS), an endogenous inhibitor of calpain (Ca2+-activated neutral protease). We therefore investigated the possible restoring effect of CS obtained from various tissues (activity 1.3-23 U/ml) on Ca2+ channel activity after run-down in inside-out patches. Although CS from porcine erythrocytes (plus 3 mM ATP) had only a minimal effect in restoring channel activity (to 4% of the control level recorded before the run-down), CS from porcine heart restored channel activity to 19% of control. The product of recombinant complementary deoxyribonucleic acid (cDNA) of human heart CS, a membrane-bound CS partially purified from bovine heart and CS from rabbit skeletal muscle (Sigma) restored channel activity to 28%, 23% and 10% of control levels, respectively. These results suggest that tissue-type CS, but not erythrocyte-type (truncated) CS, seems to have an effect on the cardiac Ca2+ channel to maintain its activity. Purified CS had relatively small effects compared to that of crude cytoplasm, implying that some other factor(s) might contribute also to the regulation of Ca2+ channel activity.

Run-down of the cardiac Ca2+ channel: characterization and restoration of channel activity by cytoplasmic factors.

Possible mechanisms for run-down in the Ca2+ channel, such as proteolysis or dephosphorylation of the channel, were examined in guinea-pig ventricular myocytes. The Ca2+ channel current, recorded in inside-out patches using a pipette solution containing 50 mM Ba2+ and 3 microM Bay K 8644, ran down with a mean survival time of 2.35 min. The survival time was not significantly affected by adenosine triphosphate (ATP) (3 mM), 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA) (2 mM), isoprenaline (l-5 microM), phosphate (l20 mM) and leupeptin (l0 microM). Stimulation of guanosine triphosphate (GTP)-binding proteins was also ineffective. The catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA, 0.5-2 microM) slightly and transiently increased channel activity, but had minimal effects on the channel when applied after complete run-down. On the other hand, cytoplasm from the heart, skeletal muscle, brain and liver, but not kidney, induced channel activity. There was a positive correlation between NPo (the product of the number of channels N and the open probability Po) value before run-down and that after the application of cytoplasm, suggesting that the activity of once-active channels was restored ba the exogenous cytoplasm. The potency of cytoplasm in tissues in inducing channel activity was not related to PKA activity nor to the number of dihydropyridine binding sites. These results suggest that the run-down of the cardiac Ca2+ channel is not mediated by dephosphorylation or proteolysis of the channel, but involves other factor(s), possibly interaction of the channel protein with a cytoplasmic regulatory protein.

ATP regulates cardiac Ca2+ channel activity via a mechanism independent of protein phosphorylation.

The role of adenosine triphosphate (ATP) in the regulation of L-type Ca2+ channel activity was investigated in inside-out patches from guinea-pig ventricular cells, in which the Ca2+ channel activity had been reprimed by application of cytoplasm from bovine heart. Passing the cytoplasm through a diethylaminoethyl (DEAE)-sepharose column or heating at 60 degrees C for 20 min attenuated the induction Ca2+ channel activity to 6-13% of that in the preceding cell-attached patch. Addition of 10 mM MgATP to the cytoplasm greatly improved the potency of cytoplasm in restoring Ca2+ channel activity (to 83 +/- 22%, mean +/- SE). This effect of MgATP was also produced, although with lower potency, by K2ATP (61 +/- 20%) or 5'-adenylylimidodiphosphate (AMP-PNP, 39 +/- 7%), a non-hydrolyzable ATP analogue, suggesting that hydrolysis of ATP is not required for the stimulatory effect on channel activity. A non-specific protein kinase inhibitor H8 (50-100 microM) did not inhibit the effect of cytoplasm + MgATP on channel activity, suggesting the involvement of a pathway independent of phosphorylation. We conclude that ATP regulates Ca2+ channel activity in dual pathways: one with, and the other without, protein phosphorylation.

Steroidal glycosides from the underground parts of Hosta plantaginea var. japonica and their cytostatic activity on leukaemia HL-60 cells.

A new C22-steroid glycoside was isolated from the underground parts of Hosta plantaginea var. japonica, together with a known furostanol saponin and three known spirostanol saponins. The structure of the new steroid glycoside was characterized by spectroscopic analysis and acid-catalysed hydrolysis as 2 alpha, 3 beta, 16 beta-trihydroxy-5 alpha-pregn-20(21)-ene-carboxylic acid gamma-lactone 3-O-¿O-beta-D-glucopyranosyl-(1-->2)-O-beta-D-glucopyranosyl-(1--> 4)-beta-D-galactopyranoside¿. The isolated compounds were assayed for their cytostatic activity on leukaemia HL-60 cells. The spirostanol saponins showed cytostatic activity in a dose-dependent manner with the IC50 values ranging between 1 and 3 micrograms ml-1.

Cyclic AMP-dependent protein kinase but not protein kinase C regulates the cardiac Ca2+ channel through phosphorylation of its alpha 1 subunit.

The voltage-dependent L-type Ca2+ channel in the heart is regulated by cAMP-dependent protein kinase (PKA) and possibly by protein kinase C (PKC). We have investigated the channel modulation through phosphorylation by these protein kinases, using liposomes into which Ca2+ channels from bovine heart were reconstituted. Phosphorylation of the proteoliposomes with PKA increased the dihydropyridine-sensitive Ca2+ efflux from them by about 70%. PKA rapidly phosphorylated membrane proteins of 210 and 170 kDa. A dihydropyridine-class Ca2+ channel blocker, [3H]azidopine, specifically photo-labeled a protein of 210 kDa, suggesting that the 210-kDa phosphoprotein might be the alpha 1 subunit of the Ca2+ channel. In contrast, phosphorylation of the proteoliposomes with PKC failed to modulate the Ca2+ efflux. Although PKC catalyzed the phosphorylation of membrane proteins of 150, 130, 95, 67, and 62 kDa, the 210- and 170-kDa proteins were not phosphorylated by this kinase. These results suggest that phosphorylation of the 210-kDa protein in the cardiac sarcolemma by PKA may be responsible for modulation of the channel function, whereas modulation of the channel by PKC, if it occurs, must be the result of an indirect mechanism, e.g. phosphorylation of a cytoplasmic protein or an associated channel polypeptide, that cannot function in the reconstituted system.

A new cytotoxic cholestane bisdesmoside from Ornithogalum saundersiae bulbs.

Bioassay-guided fractionation of the MeOH extract of Ornithogalum saundersiae bulbs led to the isolation of a new cholestane bisdesmoside with potent cytotoxic activities toward leukemia HL-60 and MOLT-4 cells. The structure was deduced mainly from spectroscopic information.

Total synthesis of VIM-2 ganglioside isolated from human chronic myelogenous leukemia cells.

A total synthesis of the tumor-associated glycolipid antigen, VIM-2, is described [2]. Phenyl 2,3,4-tri-O-benzoyl-6-O-benzyl-beta-D-galactopyranosyl-(1-->4)-6-O-benzy l-2- deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (7), a key intermediate prepared by condensation of phenyl 6-O-benzyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (6) and 2,3,4-tri-O-benzoyl-6-O-benzyl-alpha-D-galactopyranosyl bromide (5), was glycosylated with methyl 2,3,4-tri-O-benzyl-1-thio-beta-L-fucopyranoside (8) to give the trisaccharide donor 9, which, on coupling with 2-(trimethylsilyl)ethyl 2,4,6-tri-O-benzyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-be ta-D- glucopyranoside (10), afforded the pentasaccharide 11. The regioselective glycosylation of 12 (derived by O-debenzoylation of 11) with 7 gave the heptasaccharide 13, which was converted by treatment with hydrazine monohydrate and subsequent N-acetylation into the hexasaccharide acceptor 14. The stereo- and regio-selective glycosylation of 14 with methyl (phenyl 5-acetamido-4,7,8,9-O-benzoyl-3,5-dideoxy-2-thio-D-glycero-beta-D-galact o-2- nonulopyranosid)onate (16) gave the desired octasaccharide 18. Hydrogenolytic removal of the benzyl groups in 18 and successive O-acetylation, removal of the 2-(trimethylsilyl)ethyl group, and treatment with trichloroacetonitrile gave the alpha-trichloro-acetimidate 21, which was then coupled with (2S,3R,4E)-2-azido-3-O-(tert-butyldiphenylsilyl)-4-octade cene-1,3-diol (22) to give 23. Compound 23 was transformed, via selective reduction of the azido group, N-introduction of octadecanoic acid, O-desilylation, O-deacylation, and saponification of the methyl ester group, into the title VIM-2 ganglioside 26.

Steroidal saponins from Allium chinense and their inhibitory activities on cyclic AMP phosphodiesterase and Na+/K+ ATPase.

The saponin fraction prepared from the methanolic extract of Allium chinense bulbs exhibited inhibitory activities on cyclic AMP phosphodiesterase (cAMP PDE) (43.5%) and Na+/K+ ATPase (59.3%) at a sample concentration of 100 micrograms ml-1, respectively. Attempted purification of the active fraction through column chromatography on silica gel and ODS silica gel resulted in the isolation of six steroidal saponins, one of which appeared to be a new compound and one to be the first isolation from a natural source. (25R,S)-5 alpha-Spirostan-3 beta-ol tetrasaccharide showed inhibitory activities on both cAMP PDE and Na+/K+ ATPase, while (25R)-3 beta-hydroxy-5 alpha-spirostan-6-one di- and tri-saccharides inhibited only cAMP PDE.

A novel hexahydrodibenzofuran derivative with potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells from Lindera umbellata bark.

A novel cinnamoyl-hexahydrodibenzofuran derivative (1) was isolated from the bark of Lindera umbellata. The structure was determined by extensive spectroscopic analysis to be (5aR*,6R*,9R*,9aS*)-4-cinnamoyl-3,6-dihydroxy-1-methoxy-6-me thyl- 9-(1-methylethyl)-5a,6,7,8,9,9a-hexahydrodibenzofuran. Compound 1 showed potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells without causing any cytotoxicity in the cultured cells or skin irritation in guinea pig.