Actin redistribution in mosquito malpighian tubules after a blood meal and cyclic AMP stimulation

Fluid secretion by mosquito Malpighian tubules is critical to maintaining fluid and electrolyte balance after a blood meal. Endogenous cAMP levels increase in Malpighian tubules after a blood meal. Here, we determined if corresponding changes in intracellular actin distribution occur after a blood meal or dibutyryl-cAMP (db-cAMP) stimulation and whether altering actin turnover inhibits secretion. In untreated Malpighian tubules, beta-actin immunostaining was more intense in the apical region of adult Malpighian tubules than in the cytoplasm. Stimulation by a blood meal or db-cAMP significantly decreased beta-actin immunostaining in the non-apical region of the cell. Db-cAMP had similar effects in larvae and pupae Malpighian tubules. In contrast, no detectable shift in F-actin distribution was detected; however, F-actin bundles within the cytoplasm increased in size after treatment with db-cAMP. Pretreatment of Malpighian tubules with agents perturbing actin fiber assembly and disassembly decreased basal secretion rates and inhibited the stimulatory effects of db-cAMP. Our results show (1) beta-actin redistributes toward the apical membrane after a blood meal and this correlates temporally with increase urine flow rate and intracellular cAMP levels, (2) Malpighian tubules from all developmental stages exhibit this same response to db-cAMP-stimulation, and (3) dynamic assembly and disassembly of beta-actin is required for db-cAMP-stimulated secretion.     

A novel mass spectrometric assay for the cerebroside sulfate activator protein (saposin B) and arylsulfatase A

A mass spectrometric method is described for monitoring cerebrosides in the presence of excess concentrations of alkali metal salts. This method has been adapted for use in the assay of arylsulfatase A (ASA) and the cerebroside sulfate activator protein (CSAct or saposin B). Detection of the neutral glycosphingolipid cerebroside product was achieved via enhancement of ionization efficiency in the presence of lithium ions. Assay samples were extracted into the chloroform phase as for the existing assays, dried, and diluted in methanol-chloroform-containing lithium chloride. Samples were analyzed by electrospray ionization mass spectrometry with a triple quadrupole mass spectrometer in the multiple reaction monitoring tandem mass spectrometric mode. The assay has been used to demonstrate several previously unknown or ambiguous aspects of the coupled ASA/CSAct reaction, including an absolute in vitro preference for CSAct over the other saposins (A, C, and D) and a preference for the non-hydroxylated species of the sulfatide substrate over the corresponding hydroxylated species. The modified assay for the coupled ASA/CSAct reaction could find applicability in settings in which the assay could not be performed previously because of the need for radiolabeled substrate, which is now not required.     

Alkaloidal and other constituents of Uncaria elliptica and canthium dicoccum

A new alkaloid roxburghine X, along with roxburghine D, formosanine and mitraphylline, has been isolated from the bark of Uncaria elliptica. Sitosterol, quinovaic acid, acetylquinovaic acid and scopoletin were isolated from the bark of Canthium dicoccum.     

D-VITAMINS AND THEIR PRECURSORS AS GROWTH REGULATORS IN AXENICALLY CULTIVATED MARINE MACROALGAE1

Light petroleum extracts from thalli of Fucus vesiculosus (L.) and Laminaria digitata (Huds.) Lamour. stimulate growth of axenically cultured Fucus spiralis L. and Enteromorpha compressa (L.) Grev. in amounts per litre culture medium corresponding to one to 10 mg wet weight of the brown algae. This indicates the presence of a lipophilic, very active growth regulator in the blades. Extracts from the meristematic zone of Laminaria are much less active. Vitamins D₃, D₂ and 1,25-(OH)₂-D₃ at 1·10^?8– 1·10^?7 M increase the fresh weight of Enteromorpha compressa and Fucus spiralis up to 100% or more. The dry weight of Nemalion helminthoides (Veil, in With.) Batt. is also increased under the same conditions. Ergosterol and cholesterol are also growth-stimulating but higher concentrations (e.g. 1·10^?6 M) are necessary. The effects of the D vitamins manifest themselves without any auxins being added to the medium.     

Sphingomyelin synthase as a potential target for D609-induced apoptosis in U937 human monocytic leukemia cells

Tricyclodecan-9-yl-xanthogenate (D609) is a selective tumor cytotoxic agent. However, the mechanisms of action of D609 against tumor cells have not been well established. Using U937 human monocytic leukemia cells, we examined the ability of D609 to inhibit sphingomyelin synthase (SMS), since inhibition of SMS may contribute to D609-induced tumor cell cytotoxicity via modulating the cellular levels of ceramide and diacylglycerol (DAG). The results showed that D609 is capable of inducing U937 cell death by apoptosis in a dose- and time-dependent manner. The induction of U937 cell apoptosis was associated with an inhibition of SMS activity and a significant increase in the intracellular level of ceramide and decrease in that of sphingomyelin (SM) and DAG, which resulted in an elevation of the ratio between ceramide and DAG favoring the induction of apoptosis. In addition, incubation of U937 cells with C(6)-ceramide and/or H7 (a selective PKC inhibitor) reduced U937 cell viability; whereas pretreatment of the cells with a PKC activator, PMA or 1-oleoyl-2-acetylglycerol (OAG), attenuated D609-induced U937 cell apoptosis. These results suggest that SMS is a potential target of D609 and inhibition of SMS may contribute to D609-induced tumor cell death via modulation of the cellular levels of ceramide and DAG.     

Calcium and mitochondria

The literature suggests that the physiological functions for which mitochondria sequester Ca(2+) are (1). to stimulate and control the rate of oxidative phosphorylation, (2). to induce the mitochondrial permeability transition (MPT) and perhaps apoptotic cell death, and (3). to modify the shape of cytosolic Ca(2+) pulses or transients. There is strong evidence that intramitochondrial Ca(2+) controls both the rate of ATP production by oxidative phosphorylation and induction of the MPT. Since the results of these processes are so divergent, the signals inducing them must not be ambiguous. Furthermore, as pointed out by Balaban [J. Mol. Cell. Cardiol. 34 (2002 ) 11259-11271], for any repetitive physiological process dependent on intramitochondrial free Ca(2+) concentration ([Ca(2+)](m)), a kind of intramitochondrial homeostasis must exist so that Ca(2+) influx during the pulse is matched by Ca(2+) efflux during the period between pulses to avoid either Ca(2+) buildup or depletion. In addition, mitochondrial Ca(2+) transport modifies both spatial and temporal aspects of cytosolic Ca(2+) signaling. Here, we look at the amounts of Ca(2+) necessary to mediate the functions of mitochondrial Ca(2+) transport and at the mechanisms of transport themselves in order to set up a hypothesis about how the mechanisms carry out their roles. The emphasis here is on isolated mitochondria and on general mitochondrial properties in order to focus on how mitochondria alone may function to fulfill their physiological roles even though the interactions of mitochondria with other organelles, particularly with endoplasmic and sarcoplasmic reticulum [Sci. STKE re1 (2004) 1-9], may also influence this story.     

Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells

A methodology for simple convenient preparation of bright, negatively or positively charged, water-soluble CdSe/ZnS core/shell nanocrystals (NCs) and their stabilization in aqueous solution is described. Single NCs can be detected using a standard epifluorescent microscope, ensuring a detection limit of one molecule coupled with an NC. NCs solubilized in water by DL-Cys were stabilized, to avoid aggregation, by poly(allylamine) and conjugated with polyclonal anti-mouse antibodies (Abs). NC-Abs conjugates were tested in dot-blots and exhibited retention of binding capacity within several nanograms of antigen detected. We further demonstrated the advantages of NC-Abs conjugates in the immunofluorescent detection and three-dimensional (3D) confocal analysis of p-glycoprotein (p-gp), one of the main mediators of the MDR phenotype, overexpressed in the membrane of MCF7r breast adenocarcinoma cells. Immunolabeling of p-gp with NC-Abs conjugates was 4200-, 2600-, and 420-fold more resistant to photobleaching than its labeling with fluorescein isothiocyanate-Abs, R-phycoerythrin-Abs, and AlexaFluor488-Abs, respectively. The labeling of p-gp with NC-Abs conjugates was highly specific, and the data were used for confocal reconstruction of 3D images of the p-gp distribution in the MCF7r cell membrane. Finally, we demonstrated the applicability of NC-Abs conjugates obtained by the method described to specific detection of antigens in paraffin-embedded formaldehyde-fixed cancer tissue specimens, using immunostaining of cytokeratin in skin basal carcinoma as an example. We conclude that the NC-Abs conjugates may serve as easy-to-do, highly sensitive, photostable labels for immunofluorescent analysis, immunohistochemical detection, and 3D confocal studies of membrane proteins and cells.     

Gangliosides modulate the activity of the plasma membrane Ca(2+)-ATPase from porcine brain synaptosomes

We systematically examined the effects of gangliosides on the plasma membrane Ca(2+)-ATPase (PMCA) from porcine brain synaptosomes. Our results showed that GD1b (two sialic acid residues) stimulated the activity, GM1 (one sialic acid residue) slightly reduced the activity, while asialo-GM1 (no sialic acid residue) markedly inhibited it, suggesting that sialic acid residues of gangliosides are important in the modulation of the PMCA. We also examined the oligosaccharide effects by using GM1, GM2, and GM3 whose only difference was in the length of their oligosaccharide chain. GM1, GM2, and GM3 reduced the enzyme activities, whereas GM2 and GM3 were potent inhibitors. Gangliosides affect both affinity for Ca(2+) and the Vmax of enzyme. It was observed that GD1b and GM2 increased the affinity of the enzyme for Ca(2+). GD1b, GM2 affected the Vmax with an increase of GD1b, but decreases of GM2. The study of the affinity for ATP and the Vmax of enzyme in the presence of gangliosides showed that GD1b and GM2 had little effect on the ATP binding to the enzyme, but the Vmax was apparently changed. Moreover, the effects of gangliosides are additive to that of calmodulin, suggesting that the modulation of PMCA by gangliosides should be through a different mechanism. The conformational changes induced by gangliosides were probed by fluorescence quenching. We found that fluorescent quenchers (I(-) and Cs(+)) with opposite charges had different accessibility to the IAEDANS binding to the PMCA in the presence of gangliosides. An apparent red shift (25nm) with increased maximum of fluorescence spectrum was also observed in the presence of GD1b.     

Cloning, characterization, and structural analysis of a C-type lectin from Bothrops insularis (BiL) venom

Lectins are carbohydrate-binding molecules that mediate a variety of biological processes. In this work, we identify and characterize a lectin from Bothrops insularis venom, with respect to its biochemical properties and theoretical structure. Initially, from a venom gland cDNA library, we cloned and sequenced a cDNA encoding a protein with high identity to snake venom lectins. A lectin molecule was purified to homogeneity from the venom by affinity column and gel filtration. This protein named BiL displayed hemagglutinating activity that was inhibited by galactose, lactose, and EDTA. Mass spectrometry analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that BiL is a disulfide-linked dimeric protein consisting of monomers with 16,206 m/z. The amino acid sequence, deduced from its cDNA sequence, was confirmed by Edman sequencing and by peptide mass fingerprint analysis. BiL shows similarity to other C-type lectin family members. Modeling studies provide insights into BiL dimeric structure and its structural determinants for carbohydrate and calcium binding.