When a water droplet spreads on a vertically aligned nanorod surface, the nanorod array will deform into statistically percolated patterns. This nanocarpet effect has been investigated for Si nanorods. Three distinct morphologies were found: a center region due to the water droplet impact, a tilting region due to gravity, and a percolation region due to capillary invasion. The corresponding capillary forces have been estimated through finite element analysis. This effect presents a challenge for nanodevices involving liquid interactions.

The endogenous phospholipid mediator lysophosphatidic acid (LPA) caused growth cone collapse, neurite retraction, and cell flattening in differentiated PC12 cells. Neurite retraction was blocked by cytochalasin B and ADP‐ribosylation of the small‐molecular‐weight G protein Rho by the Clostridium botulinum C‐3 toxin. LPA induced a transient rise in the level of inositol 1,4,5‐trisphosphate, and retraction was blocked by inhibitors of phospholipase β. Repeated application of LPA elicited homologous desensitization of the Ca2+ mobilization response. The activation of the phosphoinositide (PIP)‐Ca2+ second messenger system played a permissive role in the morphoregulatory response. Blockers of protein kinase C—chelerythrine, a myristoylated pseudosubstrate peptide, staurosporine, and depletion of protein kinase C from the cells by long‐term phorbol ester treatment—all diminished neurite retraction by interfering with LPA‐induced Ca2+ mobilization, which was required for the withdrawal of neurites. A brief 15‐min treatment with 4β‐phorbol 12‐myristate 13‐acetate also blocked retraction and Ca2+ mobilization, by inactivating the LPA receptor. Inhibition of protein tyrosine phosphorylation by herbimycin diminished retraction. Although activation of the PIP‐Ca2+ second messenger system appears necessary for the Rho‐mediated rearrangements of the actin cytoskeleton, bradykinin, which activates similar signaling events, failed to cause retraction, indicating that a yet unidentified novel mechanism is also involved in the LPA‐induced morphoregulatory response.

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Previous studies showed that the normal microflora of the large intestine synthesizes biotin and that the colon is capable of absorbing intraluminally introduced free biotin. Nothing, however, is known about the mechanism of biotin absorption in the large intestine and its regulation. To address these issues, we used the human-derived, nontransformed colonic epithelial cell line NCM460. The initial rate of biotin uptake was found to be 1) temperature and energy dependent, 2) Na+ dependent (coupling ratio of 1:1), 3) saturable as a function of concentration [apparent Michaelis constant ( K m) of 19.7 μM], 4) inhibited by structural analogs with a free carboxyl group at the valeric acid moiety, and 5) competitively inhibited by the vitamin pantothenic acid (inhibition constant of 14.4 μM). Pretreatment with the protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl- sn-glycerol significantly inhibited biotin uptake. In contrast, pretreatment with the PKC inhibitors staurosporine and chelerythrine led to a slight, but significant, increase in biotin uptake. The effect of PMA was mediated via a marked decrease in maximal uptake velocity and a slight increase in apparent K m. Pretreatment of cells with modulators of the protein kinase A-mediated pathway, on the other hand, showed no significant effect on biotin uptake. These results demonstrate, for the first time, the functional existence of a Na+-dependent, specialized carrier-mediated system for biotin uptake in colonic epithelial cells. This system is shared with pantothenic acid and appears to be under the regulation of an intracellular PKC-mediated pathway.

Effects of the cyclic AMP second messenger system were studied on the retraction of neurites elicited by the phospholipid mediator lysophosphatidic acid (LPA) in PC12 cells. LPA stimulation inhibited adenylyl cyclase, indicating that the LPA receptor couples to the heterotrimeric G, proteins. However, pertussis toxin or expression of dominant negative Ras did not prevent neurite retraction. In contrast, cholera toxin, forskolin, and application of dibutyryl-cyclic AMP prevented neurite retraction. The neurite-protective effect of forskolin was blocked by Rp-adenosine 3',5'-phosphorothioate. Forskolin and dibutyryl-cyclic AMP both failed to protect neurites in Al 26-1 B2 and 123.7 cells, which lack cyclic AMP-activated protein kinase. Data indicate that elevation of cyclic AMP levels triggers a cyclic AMP-activated protein kinase-dependent mechanism that opposes the functioning of the morphoregulatory signaling activated by LPA. ADP-ribosylation of Rho by the Clostridium botulinum C-3 toxin in 123.7 cells caused neuronal differentiation, indicated by neurite extension, and blocked LPAinduced neurite retraction. LPA activates G 0-and G~linked signaling in parallel; therefore, a morphoregulatory signaling network hypothesis is proposed versus the simplistic approach of a signaling pathway. The signaling network integrates the receptor-activated individual, sequential, and parallel signaling events into an interactive network whose individual components may fulfill required and permissive functions encoding the cellular response. Key Words: Lipid mediator-Signal transduction -Adenylyl cyclase-Forskol in -Receptor-operated Ca2+ influx-Rp -Adenosine 3 ',S' -phosphorothioate-Cyclic AM P-dependent protein kinase-C-3 ADP-ribosyltransferase-Rho.

Lysophosphatidic acid (LPA) induces mitogenic responses in cultured fibroblasts through a pertussis toin-sensitive signaling pathway. In contrast, we have shown that LPA inhibits the proliferation of Sp2/0-Agl4 myeloma cells. To resolve this apparent controversy, LPA-elicited responses in cell proliferation and the underlying second messenger mecanisms were compared in Sp2/0-Ag14 myeloma and NIH 3T3 fibroblast cells.

Blood sera from many vertebrate species elicit large oscillatory chloride currents in oocytes from the frog Xenopus laevis. Rabbit serum was active at dilutions as great as one part in 10 million. Intracellularly applied serum was ineffective, and externally applied serum failed to trigger oscillatory currents when the intracellular level of ionized calcium was prevented from rising by loading the oocyte with EGTA. The serum also caused an increase of inositol 1,4,5-trisphosphate in the oocyte. We conclude that serum contains a factor which activates a membrane receptor that is coupled to the phosphatidylinositol second messenger system. The active factor is a protein with an apparent molecular mass of 60-70 kDa in gel permeation chromatography. Although the normal function of the serum factor is still unknown, it may have far-reaching implications, because it acts on the multifunctional phosphatidylinositol phosphate signaling system. Also, because of its great potency the serum factor and Xenopus oocytes are very useful for probing the operation of the phosphatidylinositol system.

Although the mechanism of folate intestinal transport has been the subject of intensive studies, very little is known about the molecular identity of the transport system(s) involved. In this investigation, we screened a mouse intestinal cDNA library using as probe the cDNA clone of a reduced folate carrier (RFC1) of mouse leukemia L1210 cells, and identified a positive clone, IFC1(RFC1). The cloned cDNA consisted of 2274 base pairs with an open reading frame that encodes a putative polypeptide of 512 amino acids with a predicted molecular mass of 58,112 daltons and 12 putative transmembrane domains. The polypeptide appears to carry a net positive charge (pI = 8.6) which may be important for its interaction with the negatively charged substrate. Functional identity of the IFC1(RFC1) clone was established by expression in Xenopus oocytes. An 11-fold increase in 5-methyltetrahydrofolate (5-MTHF) uptake was observed in oocytes injected with 10 ng IFC1(RFC1) cRNA compared to water-injected controls. The expressed folate uptake in the cRNA injected oocyte was (1) 4,4'-diisothiocyanatosilbene-2,2'-disulfonic acid (DIDS)-sensitive; and (2) saturable with an apparent Km of 1.99 +/- 0.32 micrometers and a V(max) of 3782 +/- 188 fmol/oocyte per h. The distribution of mRNA species complementary to IFC1(RFC1) in different mouse tissues was examined by Northern blot analysis. In addition to the small intestine, expression of such mRNA species were also found in the kidney, large intestine, brain, heart and liver. Furthermore, mRNA species complementary to IFC1(RFC1) were also detected by Northern blot analysis in the small intestine of human and other animal species (rat and rabbit). Expression of mRNA complementary to IFC1(RFC1) was markedly higher in rat intestinal villus cells than in crypt cells. These results represent the first identification of a folate transporter in mammalian intestine.