Chemical constituents of Eclipta alba (L.) Hassk

Ecliptasaponin C (1), a new triterpenoid glucoside, was isolated together with daucosterol (2) and stigmasterol-3-O-glucoside (3) from Eclipta alba. Based on spectral analysis, chemical evidence and hydrolytic results, it's structure was deduced as 3-beta-O-beta-D-glucopyranosyl-19beta-hydroxy olean-12-ene-28-oic acid 28-O-beta-D-glucopyranoside, Compounds 2 and 3 were obtained from Eclipta for the fist time.     

Phlebotomus papatasi saliva inhibits protein phosphatase activity and nitric oxide production by murine macrophages

Leishmania parasites, transmitted by phlebotomine sand flies, are obligate intracellular parasites of macrophages. The sand fly Phlebotomus papatasi is the vector of Leishmania major, a causative agent of cutaneous leishmaniasis in the Old World, and its saliva exacerbates parasite proliferation and lesion growth in experimental cutaneous leishmaniasis. Here we show that P. papatasi saliva contains a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A of murine macrophages. We further demonstrate that P. papatasi saliva down regulates expression of the inducible nitric oxide synthase gene and reduces nitric oxide production in murine macrophages. Partial biochemical characterization of the protein phosphatase and nitric oxide inhibitor indicated that it is a small, ethanol-soluble molecule resistant to boiling, proteolysis, and DNase and RNase treatments. We suggest that the P. papatasi salivary protein phosphatase inhibitor interferes with the ability of activated macrophages to transmit signals to the nucleus, thereby preventing up regulation of the induced nitric oxide synthase gene and inhibiting the production of nitric oxide. Since nitric oxide is toxic to intracellular parasites, the salivary protein phosphatase inhibitor may be the mechanism by which P. papatasi saliva exacerbates cutaneous leishmaniasis.     

Calcineurin protein phosphatase activity in peripheral blood lymphocytes

The protein phosphatase activity of peripheral blood T lymphocytes (PBLs) was examined to quantify the contribution of calcineurin and other members of the family of serine/threonine protein phosphatases. Using selective phosphatase inhibitors, the fractional phosphatase activities of calcineurin, protein phosphatases 1 (PP1), 2A (PP2A), and 2C (PP2C) were determined. Okadaic acid was used to inhibit the activity of both PP1 and PP2A while cyclosporin A/cyclophilin or trifluoperazine were used as a specific inhibitors of the calmodulin-dependent phosphatase calcineurin. Using a [32P]labeled 19-residue phosphopeptide substrate, RII peptide, it was found that PP1 and PP2A comprise the majority of the total phosphatase activity in PBLs with okadaic acid inhibiting 80% of the phosphatase activity. The remaining 20% of the phosphatase activity can be attributed primarily to calcineurin since it was Ca2+ dependent, sensitive to inhibition by the calmodulin antagonist trifluoperazine, and inhibited by the complex of cyclosporin A (CsA) and cyclophilin. These results indicate that PBL extracts contain little PP2C activity. In addition, PBLs treated with CsA had measurably lower calcineurin activity in cell lysates. The measurement of calcineurin activity may provide a useful means of assessing the extent of immunosuppression during drug therapy.     

Ethanol inhibits mitogen activated protein kinase activity and growth of vascular smooth muscle cells in vitro

The intrathecal (i.t.) injection of endothelins to conscious rats was found to cause respiratory arrest. To gain some insights into this central phenomenon, peripheral vascular permeability and lung oedema were measured after i.t. and i.v. injections of these peptides. When injected at T-8 spinal cord level, endothelin-1 (65 and 650 pmol) and endothelin-3 (650 pmol) enhanced vascular permeability in the lungs by 22-fold and 7-fold, respectively, and caused sudden death at the highest dose. Less prominent increases (between 1.4- and 2.2-fold) of vascular permeability were observed in other tissues (trachea, kidney, ears, skin of hind paws and back skin) with endothelin-1. Endothelin-1 (650 pmol) caused a similar increase (27-fold) in lung vascular permeability when injected at T-2, although the response was significantly less (P < 0.05) if injected at the L-4 (15-fold) spinal cord level. Only endothelin-1 produced lung oedema when injected at the T-2 or T-8 level. In contrast, intravenous injection of endothelins-1 and -3 (650 pmol) did not produce lung oedema and the lung vascular permeability was increased by only 1.4-1.6-fold and all rats survived. The prior i.t. injection of 6.5 nmol BQ-123 (cyclo[D-Trp, D-Asp, L-Pro, D-Val, L-Leu]), a selective endothelin ET(A) receptor antagonist, prevented the increases of lung vascular permeability and oedema and the mortality induced by i.t. endothelin-1 (650 pmol). Whereas i.v. treatment with phentolamine (2 mg/kg) or pentolinium (25 mg/kg + 50 mg/kg per h x 15 min) abolished the lung vascular permeability changes evoked by endothelin-1 (650) pmol), atropine (1 mg/kg), NG-nitro-L-arginine (50 mg/kg) or indomethacin (5 mg/kg) had no effect. Moreover, the effects of endothelin-1 were attenuated in capsaicin pretreated rats (125 mg/kg, 10 days earlier) and almost abolished in rats subjected to sympathectomy with 6-hydroxydopamine (100 mg/kg, 24-48 h earlier). All these treatments except atropine and NG-nitro-L-arginine prevented the endothelin-1-induced lung oedema and reduced the lethality by around 50%. These results suggest that the increases of pulmonary vascular permeability and oedema induced by i.t. endothelin-1 are due to an intense pulmonary vasoconstriction mediated by alpha-adrenoceptors following the release of catecholamines in response to the activation of endothelin ET(A) receptor in the spinal cord. This central phenomenon seems to be reflexogenic, including the involvement of primary afferent C-fibers and spinal cord ascending fibers to the brain. Thus, endothelin-1 could play a role in neurogenic pulmonary oedema through a central mechanism.     

Gallium nitrate inhibits alkaline phosphatase activity in a differentiating mesenchymal cell culture

The effect of gallium nitrate on alkaline phosphatase activity in a differentiating chick limb-bud mesenchymal cell culture was monitored in order to gain insight into the observation that rachitic rats treated with gallium nitrate failed to show the expected increase in serum alkaline phosphatase activity. Cultures maintained in media containing 15 microM gallium nitrate showed drastically decreased alkaline phosphatase activities in the absence of significant alterations in total protein synthesis and DNA content. However, addition of 15 microM gallium nitrate to cultures 18 h before assay for alkaline phosphatase activity had little effect. At the light microscopic and electron microscopic level, gallium-treated cultures differed morphologically from gallium-free cultures: with gallium present, there were fewer hypertrophic chondrocytes and cartilage nodules were flatter and further apart. Because of altered morphology, staining with an antibody against chick cartilage alkaline phosphatase appeared less extensive; however, all nodules stained equivalently relative to gallium-free controls. Histochemical staining for alkaline phosphatase activity was negative in gallium-treated cultures, demonstrating that the alkaline phosphatase protein present was not active. The defective alkaline phosphatase activity in cultures maintained in the presence of gallium was also evidenced when cultures were supplemented with the alkaline phosphatase substrate, beta-glycerophosphate (beta GP). The data presented suggest that gallium inhibits alkaline phosphatase activity in this culture system and that gallium causes alterations in the differentiation of mesenchymal cells into hypertrophic chondrocytes.     

Comparative study of androgen uptake and metabolism in domestic and wild mallard drakes (Anas platyrhynchos L.)

Changes in blood flow and blood redistribution were measured by impedance plethysmography in the pelvic and leg regions of six male and four female subjects during three 5-min exposures to -20, -40, and -60 mm Hg lower body negative pressure (LBNP). Female subjects demonstrated significantly higher mean heart rate and lower leg blood flow indices than the male subjects during the recumbent control periods. Men had slightly higher mean resting systolic and diastolic blood pressures and higher mean control pelvic blood flow indices. Women demonstrated significantly less blood pooling in the legs and slightly less in the pelvic region than the men. All of the 18 tests with male subjects at -60 mm Hg were completed without initial signs of syncope, while only two of the tests with women were completed successfully without the subject exhibiting presyncopal conditions. The results of this study indicate that impedance plethysmography can be used to measure segmental cardiovascular responses during LBNP and that females may be less tolerant to -60 mm Hg LBNP than males.     

Ion uptake and growth of wheat seedlings treated with nitrite at different pH values

Increasing concentrations of NaNO2 caused decreases in the uptake of K+, Na+ and SO4(2-) by wheat roots at pH 4. At pH 6 or 7, the detrimental effect was much less. The translocation of nutrients to the shoots was also impaired. The root growth was strongly inhibited by increasing NaNO2 concentrations, but the shoot development was little affected. At low pH, however, the inhibitory effect was much higher.     

Okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells

Alkaline phosphatase activity is regulated by various hormones and growth factors at least in part through the phosphorylation of target proteins during the bone cell differentiation. To investigate the role of protein phosphorylation in alkaline phosphatase activity in MC3T3-E1 osteoblast, we used okadaic acid which is a potent specific inhibitor of serine/threonine protein phosphatases to type 1 and 2A. Alkaline phosphatase activity in cellular layer was measured by spectrophotometer using p-nitrophenyl phosphate as substrate and data were expressed as p-nitrophenyl of nmol/min/mg of protein. Okadaic acid (1-50 ng/ml) caused the inhibition of alkaline phosphatase activity in MC3TC-E1 cells. At 50 ng/ml of okadaic acid showed the maximal inhibitory effect on alkaline phosphatase activity. Okadaic acid (50 ng/ml) also inhibited alkaline phosphatase activity in all differentiation stages. These results indicate that okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells.     

An anti-okadaic acid-anti-idiotypic antibody bearing an internal image of okadaic acid inhibits protein phosphatase PP1 and PP2A catalytic activity

Okadaic acid (OA), produced by marine phytoplankton, is the parent compound of a family of marine toxins responsible for diarrheic shellfish poisoning (DSP). A monoclonal antibody to OA (6/50) (Ab1) has been raised and in turn used for immunization of syngeneic animals. Mice inoculated with the 6/50 idiotype produced both anti-idiotypic antibodies (Ab2) and OA binding antibodies (Ab3). The selected anti-idiotypic antibody 1/59 bound to the immunizing 6/50 idiotype but not to F(ab')2 fragments of pooled normal mouse Ig. It inhibited the binding of OA to solid-phase attached F(ab')2 of 6/50 IgG as well as the binding of 6/50 IgG to a solid-phase bound OA. Like OA, 1/59 anti-idiotypic antibody inhibited protein phosphatase 1 and 2A catalytic subunits in a 32P-phosphorylase a phosphatase radioassay. Thus, 1/59 IgG is a novel internal image anti-idiotypic antibody (Ab2 beta) and can serve as a surrogate of OA in biological assays.     

Cerium toxicity, uptake and translocation in Arabidopsis thaliana seedlings

Arabidopsis thaliana seedlings were cultivated in 0-500μmol/L of extraneous cerium(Ce)for 7 d to investigate the toxicity,uptake and translocation of rare earth elements(REEs).The results showed that Ce could be largely absorbed by the roots of A.thaliana and translocated to the shoots.But the uptake rates of Ce by the roots were much higher than the translocation rates from roots to shoots.Ultra-structural analysis revealed that Ce was mainly distributed on the cell wall.At higher concentration,Ce could also enter cell,destroy the ultra-structure of cells and disturb the intrinsic balance of nutrient elements of A.thaliana.Addition of Ce(50-500μmol/L)to the culture medium significantly inhibited the elongation of primary roots,decreased chlorophyll content,rosette diameter and fresh mass of plants.The damage increased with the increase of Ce concentration in culture medium,although primary root elongation,chlorophyll content,and rosette diameter were stimulated by relatively low concentration(0.5μmol/L)of Ce.Thus,it is speculated that REEs may become a new type contamination if we don’t well control the release of REEs into the environment.     

Over-expression of protein tyrosine phosphatase 1 (PTP1) alters IL-3-dependent growth and tyrosine phosphorylation

Interleukin-3 (IL-3) is a hematopoietic growth factor receptor which stimulates the proliferation of multilineage progenitor cells. It is known that IL-3 stimulates tyrosine phosphorylation while transducing a mitogenic signal. The signal transduction pathways activated by the IL-3 receptor, however, are not fully understood. In this study a protein tyrosine phosphatase has been over-expressed in the IL-3 dependent, murine myeloid progenitor cell line, 32D cl3 in order to test whether altering the levels of tyrosine phosphorylation would change IL-3 stimulated proliferation. These cells were transfected with a metal-inducible expression vector containing a rat cDNA encoding PTP1. A low basal level of rat PTP1 message and protein was detected in cells transfected with the PTP1 vector, and zinc treatment resulted in a three- to fourfold increase in the amount of PTP1 message, protein and catalytic activity. Over-expression of PTP1 resulted in a two- to threefold decrease in IL-3 stimulated proliferation. Cells over-expressing PTP1 also exhibited decreased levels of tyrosine phosphorylation; phosphorylation of the IL-3 receptor beta subunit and the Shc protein were both dramatically decreased. Thus, PTP1 over-expression negatively modulated IL-3 signal transduction. To identify potential substrates of PTP1, 32D cl3 cells were transfected with a catalytically inactive PTP1 mutant, PTP1(C/S). Three tyrosine-phosphorylated proteins of MW 140, 79 and 69 k coprecipitated with PTP1(C/S). We believe that the 140 kDa protein represents the beta subunit of the IL-3 receptor. In addition, a GST-fusion protein containing active PTP1 dephosphorylated the beta-subunit in an in vitro assay. By immunofluorescent microscopy over-expressed PTP1(C/S) co-localized largely with calnexin, an endoplasmic reticulum-associated protein. Immunofluorescent microscopy also indicated that PTP1(C/S) and the beta subunit co-localized at discrete sites at the plasma membrane and around a cytoplasmic organelle where most of the beta subunit was located. These observations suggest PTP1 over-expression may down-regulate the growth response to IL-3 through dephosphorylation of the IL-3 receptor, perhaps in an intracellular compartment, thereby inhibiting propagation of the IL-3 mitogenic signal.     

Increased protein kinase C activity in myotrophin-induced myocyte growth

Myotrophin, a novel protein that has been shown to stimulate myocyte growth, has been isolated, purified, and sequenced from the hearts of spontaneously hypertensive rats and dilated cardiomyopathic human tissue. Recently, the cDNA clones encoding myotrophin have been isolated and expressed in Escherichia coli, and the recombinant myotrophin was found to be as biologically and immunologically active as natural myotrophin. The mechanism by which myotrophin stimulates protein synthesis and initiates myocardial hypertrophy is not known. To evaluate the involvement of protein kinase C (PKC) in myotrophin-induced hypertrophy, PKC activity and its distribution in the subcellular fraction were determined in cultured neonatal and adult myocytes. PKC activity was determined by measuring the incorporation of 32P into histone type III-S and PKCepsilon substrate peptide (epsilon(pep)) from [gamma-32P]ATP in neonatal myocytes. Myotrophin significantly stimulated PKC activity in neonatal myocytes and was associated with a significant increase in protein synthesis. The effect of myotrophin on the stimulation of PKC activity and [3H]leucine incorporation was abolished by pretreatment with either staurosporine or H-7, two selective, pharmacological PKC inhibitors. Pretreatment of myocytes with staurosporine also reduced the myotrophin-induced mRNA levels of c-fos and beta-myosin heavy chain. To evaluate the subcellular events whose occurrence was due to myotrophin and translocation of PKC, we studied the effect of genistein, a tyrosine kinase inhibitor, on myotrophin-induced neonatal myocyte growth. Genistein attenuated the [3H]leucine incorporation induced by myotrophin. To define the specificity of the PKC isoform(s) involved in myotrophin-stimulated myocyte growth, both neonatal and adult myocytes were treated with myotrophin, and Western blot analyses were performed by using the antibodies of different PKC isoforms. Results showed that both PKCalpha and PKCepsilon isoforms participated in the myotrophin-induced neonatal myocyte growth, whereas only the PKCepsilon isoform was involved in myotrophin-induced adult myocyte hypertrophy. PKCdelta and PKCzeta do not seem to participate in either neonatal or adult myocyte growth induced by myotrophin. Treatment with antisense oligonucleotides specific for PKCalpha and PKCepsilon isoforms further supported this result. PKCalpha is the major PKC isoform in neonatal myocytes and needs Ca2+ and phospholipids for its activation, and PKCepsilon (the Ca2+-independent PKC isoform) is present in both neonatal and adult myocytes; the 15-mer antisense oligodeoxynucleotides of each were used for this study. Treatment of neonatal myocytes with the PKCalpha and PKCepsilon antisense oligodeoxynucleotides for 5 days significantly reduced Ca2+-dependent and Ca2+-independent PKC activity, respectively, as well as the [3H]leucine incorporation induced by myotrophin. Furthermore, myotrophin-induced PKC activity was primarily located in the particulate fraction and did not result in a concomitant decrease in the cytosolic fraction. Myotrophin does not change PKC isoform expression (both Ca2+ dependent and independent PKC isoforms used in this study) in rat neonatal cardiac fibroblasts. Our data suggest that myotrophin exerts its action on protein synthesis, possibly through a tyrosine kinase-coupled pathway and translocation of PKC from the cytosol to the cell membrane.     

Platelet factor 4 modulates fibroblast growth factor 2 (FGF-2) activity and inhibits FGF-2 dimerization

Platelet factor 4 (PF-4) inhibits angiogenesis in vitro and in vivo. The mechanism of inhibition is poorly understood. We have investigated the mechanism of inhibition by examining the interaction of PF-4 and the fibroblast growth factor-2 (FGF-2)/fibroblast growth factor receptor (FGFR) system. PF-4 inhibited the binding of FGF-2 to high-affinity and low-affinity binding sites in murine microvascular endothelial cells (LEII cells) and proliferation. Maximum inhibition of binding to endothelial FGF receptors was observed at PF-4 concentrations between 5 and 10 microg/mL (half maximum inhibition at 0.6 micro/mL), and proliferation was completely inhibited at 2 microg/mL. At this concentration, PF-4 reduced internalization of 125I-FGF-2 by threefold and delayed degradation. To gain insight into the mechanism of inhibition, we have analyzed the interaction of PF-4 with FGF-2/FGFR by using mutant heparan sulfate-deficient Chinese hamster ovary (CHO) cells transfected with the FGFR-1 cDNA (CHOm-FGFR-1) and by examining the direct interaction with FGF-2. In the absence of heparin, PF-4 inhibited binding of 125I-FGF-2 to CHOm-FGFR-1 cells in a concentration-dependent manner, although not completely. In the presence of heparin, PF-4 abolished totally the stimulatory effect of heparin. Furthermore, PF-4 complexed to FGF-2 and inhibited endogenous or heparin-induced FGF-2 dimerization. These results indicate that PF-4 interacts with FGF-2 by complex formation, inhibiting FGF-2 dimerization, binding to FGF receptors, and internalization. This mechanism most likely contributes to the antiangiogenic properties of PF-4.     

Transient and persistent increases in protein phosphatase activity during long-term depression in the adult hippocampus in vivo

In a 34-month prospective study to determine the prevalence of Staphylococcus aureus small colony variants (SCVs) in cystic fibrosis (CF) patients, S. aureus SCVs or SCVs plus normal S. aureus were recovered from 26 of 78 patients; 27 patients harbored only normal S. aureus. By pulsed-field gel electrophoresis, clonal identity was demonstrated of SCV and normal strains isolated at the same time and of multiple S. aureus SCV and normal strains in consecutive specimens from individual patients. All S. aureus SCVs were resistant to antifolate antibiotics, while the corresponding parent strains were susceptible, and in 11 of 12 SCV/normal pairs, gentamicin was less active against S. aureus with the SCV phenotype than against the normal isolate. Analysis of the underlying auxotrophism of SCVs revealed hemin, thymidine, and/or menadione dependencies. Thus, S. aureus SCVs are highly prevalent in respiratory secretions of CF patients, persist over extended periods, and may contribute to S. aureus persistence in CF patients.     

DNA binding by the KP repressor protein inhibits P-element transposase activity in vitro

P elements are a family of mobile DNA elements found in Drosophila. P-element transposition is tightly regulated, and P-element-encoded repressor proteins are responsible for inhibiting transposition in vivo. To investigate the molecular mechanisms by which one of these repressors, the KP protein, inhibits transposition, a variety of mutant KP proteins were prepared and tested for their biochemical activities. The repressor activities of the wild-type and mutant KP proteins were tested in vitro using several different assays for P-element transposase activity. These studies indicate that the site-specific DNA-binding activity of the KP protein is essential for repressing transposase activity. The DNA-binding domain of the KP repressor protein is also shared with the transposase protein and resides in the N-terminal 88 amino acids. Within this region, there is a C2HC putative metal-binding motif that is required for site-specific DNA binding. In vitro the KP protein inhibits transposition by competing with the transposase enzyme for DNA-binding sites near the P-element termini.     

A protease-resistant form of insulin-like growth factor (IGF) binding protein 4 inhibits IGF-1 actions

Smooth muscle cells (SMC) secrete a serine protease that cleaves insulin-like growth factor (IGF) binding protein (IGFBP)-4 into fragments that have low affinity for IGF-1. When IGFBP-4 is added to monolayer cultures of cell types that do not secrete this protease, IGF-1 stimulation of DNA synthesis is significantly inhibited. In contrast, if cell types that secrete this protease are used, IGFBP-4 is a much less potent inhibitor. These studies were conducted to determine whether proteolysis of IGFBP-4 accounted for its reduced capacity to inhibit IGF-1-stimulated DNA synthesis. The cleavage site in IGFBP-4 that the SMC protease uses was determined to be lysine120, histidine121. A protease-resistant mutant form of IGFBP-4 was prepared, expressed, purified, and tested for biologic activity using porcine SMC cultures. Addition of the protease-resistant mutant resulted in inhibition of DNA and cell migration responses to IGF-1. The inhibition was concentration dependent and was maximal when 500 ng/ml (20 nM) of the mutant was added with 20 ng/ml (2.8 nM) of IGF-1. When the mutant was added in the absence of IGF-1, it had no activity. The results show that cleavage of IGFBP-4 at lysine120, histidine121 results in inactivation of the ability of IGFBP-4 to bind to IGF-1. Creation of a mutant form of IGFBP-4 that was not cleaved by the protease resulted in inhibition of IGF-1-stimulated actions. The results suggest that IGFBP-4 can act as a potent inhibitor of the anabolic effects of IGF-1 and that the variables that regulate protease activity may indirectly regulate IGF-1 actions.