LPS receptor subunits have antagonistic roles in epithelial apoptosis and colonic carcinogenesis.

Colorectal carcinoma (CRC) is characterized by unlimited proliferation and suppression of apoptosis, selective advantages for tumor survival, and chemoresistance. Lipopolysaccharide (LPS) signaling is involved in both epithelial homeostasis and tumorigenesis, but the relative roles had by LPS receptor subunits CD14 and Toll-like receptor 4 (TLR4) are poorly understood. Our study showed that normal human colonocytes were CD14(+)TLR4(-), whereas cancerous tissues were CD14(+)TLR4(+), by immunofluorescent staining. Using a chemical-induced CRC model, increased epithelial apoptosis and decreased tumor multiplicity and sizes were observed in TLR4-mutant mice compared with wild-type (WT) mice with CD14(+)TLR4(+) colonocytes. WT mice intracolonically administered a TLR4 antagonist displayed tumor reduction associated with enhanced apoptosis in cancerous tissues. Mucosa-associated LPS content was elevated in response to CRC induction. Epithelial apoptosis induced by LPS hypersensitivity in TLR4-mutant mice was prevented by intracolonic administration of neutralizing anti-CD14. Moreover, LPS-induced apoptosis was observed in primary colonic organoid cultures derived from TLR4 mutant but not WT murine crypts. Gene silencing of TLR4 increased cell apoptosis in WT organoids, whereas knockdown of CD14 ablated cell death in TLR4-mutant organoids. In vitro studies showed that LPS challenge caused apoptosis in Caco-2 cells (CD14(+)TLR4(-)) in a CD14-, phosphatidylcholine-specific phospholipase C-, sphingomyelinase-, and protein kinase C-ζ-dependent manner. Conversely, expression of functional but not mutant TLR4 (Asp299Gly, Thr399Ile, and Pro714His) rescued cells from LPS/CD14-induced apoptosis. In summary, CD14-mediated lipid signaling induced epithelial apoptosis, whereas TLR4 antagonistically promoted cell survival and cancer development. Our findings indicate that dysfunction in the CD14/TLR4 antagonism may contribute to normal epithelial transition to carcinogenesis, and provide novel strategies for intervention against colorectal cancer.

Single-cell electroporation using proton beam fabricated biochips.

We report the design and fabrication of a novel single cell electroporation biochip featuring high aspect ratio nickel micro-electrodes with smooth side walls between which individual cells are attached. The biochip is fabricated using Proton Beam Writing (PBW), a new direct write lithographic technique capable of fabricating high quality high-aspect-ratio nano and microstructures. By applying electrical impulses across the biochip electrodes, SYTOX® Green nucleic acid stain is incorporated into mouse neuroblastoma (N2a) cells and observed via green fluorescence when the stain binds with DNA inside the cell nucleus. Three parameters; electric field strength, pulse duration, and numbers of pulses have been investigated for the single cell electroporation process. The results indicate high transfection rates as well as cell viability of 82.1 and 86.7% respectively. This single cell electroporation system may represent a promising method for the introduction of a wide variety of fluorophores, nanoparticles, quantum dots, DNAs and proteins into cells.

The antimicrobial activity of heyneanol A extracted from the root of Taiwanese wild grape.

AIMS: To search for antimicrobial compounds against pathogenic bacteria from grape vines (Vitis spp.). To investigate the antimicrobial efficacy of active compounds towards methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: The root extracts of taiwanese wild grape (Vitis thunbergii var. taiwaniana) showed marked activities against Gram-positive bacteria using the disc diffusion method. After purification, the active compound 1 was confirmed as heyneanol A by mass spectroscopy and nuclear magnetic resonance. Heyneanol A showed an minimum inhibitory concentration (MIC) value of 2 microg ml(-1) towards MRSA and a value of 2 to 4 microg ml(-1) for Enterococcus faecium, S. aureus, Streptococcus agalactiae and Streptococcus pyogenes. In addition, the contents of heyneanol A were determined as 36 mg g(-1) in roots of taiwanese wild grape. CONCLUSIONS: The root extracts of grapevines have good antimicrobial activities towards some strains of Gram-positive pathogens. Heyneanol A, the major antimicrobial compound, is especially active towards MRSA. In addition, the abundances of heyneanol A and other stilbenes in the roots of grapevines make it possible to produce natural antimicrobial compounds from this plant species. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports for the first time the antimicrobial compounds in the root extracts of grapevines. The results will have clinical significance owing to their activities against MRSA.

New insights into image processing of cortical blood flow monitors using laser speckle imaging.

Laser speckle imaging has increasingly become a viable technique for real-time medical imaging. However, the computational intricacies and the viewing experience involved limit its usefulness for real-time monitors such as those intended for neurosurgical applications. In this paper, we propose a new technique, tLASCA, which processes statistics primarily in the temporal direction using the laser speckle contrast analysis (LASCA) equation, proposed by Briers and Webster. This technique is thoroughly compared with the existing techniques for signal processing of laser speckle images, including, the spatial-based sLASCA and the temporal-based modified laser speckle imaging (mLSI) techniques. sLASCA is an improvement of the basic LASCA technique. In sLASCA, the derived contrasts are further averaged over a predetermined number of raw speckle images. mLSI, on the other hand, is the technique in which temporal statistics are processed using the equation described by Ohtsubo and Asakura. tLASCA preserves the original image resolution similar to mLSI. tLASCA outperforms sLASCA (window size M = 5) with faster convergence of K values (5.32 versus 20.56 s), shorter per-frame processing time (0.34 versus 2.51 s), and better subjective and objective quality evaluations of contrast images. tLASCA also outperforms mLSI with faster convergence of K values (5.32 s) compared to N values (10.44 s), shorter per-frame processing time (0.34 versus 0.91 s), smaller intensity fluctuations among frames (8%-10% versus 15%-35%), and better subjective and objective quality evaluations of contrast images. As laser speckle imaging becomes an important tool for real-time monitoring of blood flows and vascular perfusion, tLASCA is proven to be the technique of choice.

Isolation and characterization of antioxidant compounds from Aspergillus candidus broth filtrate.

The objectives of this study were to isolate the antioxidative components in the broth filtrate of Aspergillus candidus (CCRC 31543), to characterize their antioxidative properties, and to evaluate their safety. Three major compounds were isolated and identified as 3,3' '-dihydroxyterphenyllin, 3-hydroxyterphenyllin, and candidusin B. In the linoleic acid peroxidation system, the inhibition of peroxidation in these three compounds was greater than 95% and was significantly higher than that of alpha-tocopherol but equal to that of BHA at 12.5-200 microg/mL. As measured using the Rancimat method in lard, 3,3' '-di-OH-terphenyllin exhibited a protection factor value of 7.82, which was substantially higher than those of BHA (5.58) and alpha-tocopherol (4.29) at 200 microg/mL. 3,3' '-di-OH-terphenyllin and 3-OH-terphenyllin also exhibited marked scavenging effects on the alpha,alpha-diphenyl-beta-picrylhydrazyl radicals (94.7 and 96.0%, respectively), which were similar to those of BHA and alpha-tocopherol. Safety studies showed that these three compounds were neither cyto- nor geno-toxic toward human intestine 407 (INT 407) cells, nor mutagenic toward Salmonella typhimurium TA98 and TA100.

Suppression effect of soy isoflavones on nitric oxide production in RAW 264.7 macrophages.

Genistein, daidzein, and glycitein, as primary isoflavones in soybeans, are reported to have beneficial effects on atherosclerosis, chronic inflammatory diseases, and cancers that are conducted by nitric oxide (NO) injury. The objectives of this study were to investigate the effects and mechanisms of these soy isoflavones on the inducible nitric oxide synthase (iNOS) system in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Genistein, daidzein, and glycitein dose-dependently suppress NO production (IC(50) = 50 microM) in supernatants of LPS-activated macrophages as measured on the basis of nitrite accumulation. In addition, direct inhibition of iNOS activity, determined by means of the conversion of L-[(3)H]arginine to L-[(3)H]citrulline, and markedly reduced iNOS protein and mRNA levels, evaluated by means of Western blot and RT-PCR, respectively, were found in homogenates of LPS-activated cells treated with each isoflavone. Moreover, genistein was found to have a greater inhibitory effect on NO production but no significant effect on iNOS activity or protein and gene expression to daidzein and glycitein. These observations reveal that the suppression of NO production by genistein, daidzein, and glycitein might be due to the inhibition of both the activity and expression of iNOS in LPS-activated macrophages. The result suggests that soy isoflavones might attenuate excessive NO generation at inflammatory sites.

Dynamic soliton-like modes.

Incoherent optical spatial solitons require noninstantaneous nonlinearity, i.e., the local intensity fluctuation of the solitons must be faster than the medium can respond. Observing partially incoherent bicomponent solitons, we find that there exists a threshold speed. When the fluctuation of the soliton intensity, resulting from the time-varying interference of its constituent modes, is below the threshold, the soliton beam and its induced waveguide oscillate violently. Just above the threshold, the soliton-induced waveguide is observed to be dragged by the soliton beam.

Mutagenicity and identification of mutagenic compounds of fumes obtained from heating peanut oil.

Since the fume of cooking oil has been reported to increase the risk of lung cancer, the objectives of this study were to evaluate the mutagenicity and to find the mutagens in the fumes of peanut oil heated to the smoke point. Peanut oil prepared from roasted peanut kernel showed a lower smoke point, less unsaturated fatty acids, more fume formation, and stronger mutagenicity than that from unroasted kernel. Further investigation of mutagenic compounds was performed by the Ames test and gas chromatography/mass spectrometry analysis. Among the 12 compounds identified from the neutral fraction of methanol extract, four compounds at a dose of 10 microg per plate were mutagenic to Salmonella Typhimurium TA98 and TA100 in the order of trans-trans-2,4-decadienal > trans-trans-2,4-nonadienal > trans-2-decenal > trans-2-undecenal. Results report the enal compounds formed as the mutagens in the fumes of peanut oil and indicate that inhaling cooking fumes might cause carcinogenic risk.

Generation of IgM anti-platelet autoantibody in dengue patients.

Dengue virus infection causes a wide range of diseases from dengue fever to life-threatening dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). The mechanisms involved in DHF/DSS pathogenesis remain unclear. Patient sera collected from an outbreak in southern Taiwan from November 1998 to January 1999 were studied. The presence of antibodies which cross-reacted with platelets could be detected in patient sera, and the isotype of these autoantibodies was IgM. The anti-platelet IgM levels were higher in DHF/DSS than in dengue fever patient sera in disease acute phase. These autoantibodies were still detectable in convalescent stage (1-3 weeks after acute phase) and even eight to nine months after illness. The platelet binding activity was not observed in other virus-infected patient sera tested. Further investigation showed that dengue patient sera caused platelet lysis in the presence of complement. The platelet cytotoxicity induced by DHF/DSS patient sera was higher than that by dengue fever sera. Dengue patient sera also inhibited platelet aggregation which, however, appeared to be not related to DHF/DSS development.

Simultaneous determination of sweeteners and preservatives in preserved fruits by micellar electrokinetic capillary chromatography.

A micellar electrokinetic capillary method for the simultaneous determination of the sweeteners dulcin, aspartame, saccharin, and acesulfame-K and the preservatives sorbic acid; benzoic acid; sodium dehydroacetate; and methyl-, ethyl-, propyl-, isopropyl-, butyl-, and isobutyl-p-hydroxybenzoate in preserved fruits is developed. These additives are ion-paired and extracted using sonication followed by solid-phase extraction from the sample. Separation is achieved using a 57-cm fused-silica capillary with a buffer comprised of 0.05 M sodium deoxycholate, 0.02 M borate-phosphate buffer (pH 8.6), and 5% acetonitrile, and the wavelength for detection is 214 nm. The average recovery rate for all sweeteners and preservatives is approximately 90% with good reproducibility, and the detection limits range from 10 to 25 microg/g. Fifty preserved fruit samples are analyzed for the content of sweeteners and preservatives. The sweeteners found in 28 samples was aspartame (0.17-11.59 g/kg) or saccharin (0.09-5.64 g/kg). Benzoic acid (0.02-1.72 g/kg) and sorbic acid (0.27-1.15 g/kg) were found as preservatives in 29 samples.

Oxidative modification of neurogranin by nitric oxide: an amperometric study.

Neurogranin (Ng) is a neuron-specific protein kinase C (PKC) substrate, which contains four cysteine (Cys) residues. Recently, it has been shown that Ng is a redox-sensitive protein and is a likely target of nitric oxide (NO) and other oxidants [F.-S. Sheu, C.W. Mahoney, K. Seki, K.-P. Huang, Nitric oxide modification of rat brain neurogranin affects its phosphorylation by protein kinase C and affinity for calmodulin, J. Biol. Chem. 271 (1996) 22407-22413: J. Li, J.H. Pak, F.L. Huang, K.-P. Huang, N-methyl-D-aspartate induces neurogranin/RC3 oxidation in rat brain slices, J. Biol. Chem. 274 (1999) 1294-1300]. In this study, we directly examine the redox reactions between dissolved NO and Cys as well as between NO and bacterial expressed Ng in its reduced form, at concentrations approximate to the physiological levels in phosphate buffer solution (PBS) under aerobic conditions. The reaction kinetics are measured directly by our newly developed electrochemical sensor. Our sensor is based on the chemical modification of electrode with immobilized nanoparticles of transition metal palladium (Pd) which serves as catalytic centers for the electrochemical oxidation of thiol and NO selectively and quantitatively at different potentials. It detects Cys and Ng in a linear range from nano to micromolar concentration at + 450 mV, vs. a saturated calomel reference electrode (SCE), while the detection of NO at the sensor can be optimally achieved at + 700 mV (vs. SCE) with a linear current-to-concentration range of nM to microM. It thus provides a selective control to monitor two reactants independently. With this sensor as a detector, we found that (1) the oxidation of either Cys or Ng by NO is a fast reaction which reaches a near completion within 1-2 min at its physiological concentration; and (2) after the completion of reaction, NO is mostly, if not all, regenerated, an observation consistent with the reaction mechanism involving the formation of S-nitrosothiol as an intermediate. The reaction kinetics of both NO to Cys and NO to Ng implies that NO can achieve local action on cellular proteins in addition to its effect on targets located in neighboring cells via concentration-gradient-dependent diffusion.

Direct observation of trapping and release of nitric oxide by glutathione and cysteine with electron paramagnetic resonance spectroscopy.

While the biosynthesis of nitric oxide (NO) is well established, one of the key issues that remains to be solved is whether NO participates in the biological responses right after generation through biosynthesis or there is a "secret passage" via which NO itself is trapped, transported, and released to exert its functions. It has been shown that NO reacts with thiol-containing biomolecules (RSH), like cysteine (Cys), glutathione (GSH), etc., to form S-nitrosothiols (RSNOs), which then release nitrogen compounds, including NO. The direct observation of trapping of NO and its release by RSNO has not been well documented, as most of the detection techniques measure the content of NO as well as nitrite and nitrate. Here we use spin-trapping electron paramagnetic resonance (EPR) technique to measure NO content directly in the reaction time course of samples of GSH and Cys ( approximately mM) mixed with NO ( approximately microM) in the presence of metal ion chelator, which pertains to physiological conditions. We demonstrate that NO is readily trapped by these thiols in less than 10 min and approximately 70-90% is released afterward. These data imply that approximately 10-30% of the reaction product of NO does not exist in the free radical form. The NO release versus time curves are slightly pH dependent in the presence of metal ion chelator. Because GSH and Cys exist in high molar concentrations in blood and in mammalian cells, the trapping and release passage of NO by these thiols may provide a mechanism for temporal and spatial sequestration of NO to overcome its concentration gradient-dependent diffusion, so as to exert its multiple biological effects by reacting with various targets through regeneration.

Analysis of patulin in apple juice by diphasic dialysis extraction with in situ acylation and mass spectrometric determination.

A procedure combining diphasic dialysis extraction with in situ acylation and gas chromatography/mass spectrometry (GC/MS) determination was developed for detection and quantification of the mycotoxin patulin in apple juice. Apple juice samples spiked with 4-N,N-dimethylaminopyridine were dialyzed using methane chloride and acetic anhydride inside dialysis tubing. Patulin was derivatized into its acetate and collected in the tubing after diphasic dialysis and was directly determined using GC/MS with the selective ion monitoring mode without further concentration and cleanup steps. Quantification was carried out by a calibration curve with an internal standard of correlation. The appropriate parameters of both dialysis and derivatization were examined. The linear range of the calibration curve was found to be 10-250 microg/L for patulin, and the limit of quantification was 10 microg/L. Levels of patulin ranging from 0 to 107.2 microg/L with 77-109% recovery were found in 10 apple samples. The technique combining diphasic dialysis extraction and acylation was demonstrated and showed potential for other applications.

Photorefractive polymeric optical spatial solitons.

We predict the formation of optical spatial solitons in photorefractive polymers. The orientational enhancement from the doped chromophores and the dependency of the quantum efficiency of generating mobile holes on the electric field make the polymeric solitons behave differently from other photorefractive solitons.

Microscopic observations of the different morphological changes caused by anti-bacterial peptides on Klebsiella pneumoniae and HL-60 leukemia cells.

Natural anti-bacterial peptides cecropin B (CB) and its analogs cecropin B-1 (CB-1), cecropin B-2 (CB-2) and cecropin B-3 (CB-3) were prepared. The different characteristics of these peptides, with amphipathic/hydrophobic alpha-helices for CB, amphipathic/amphipathic alpha-helices for CB-1/CB-2, and hydrophobic/hydrophobic alpha-helices for CB-3, were used to study the morphological changes in the bacterial cell, Klebsiella pneumoniae and the leukemia cancer cell, HL-60, by scanning and transmission electron microscopies. The natural and analog peptides have comparable secondary structures as shown by circular dichroism measurements. This indicates that the potency of the peptides on cell membranes is dependent of the helical characteristics rather than the helical strength. The microscopic results show that the morphological changes of the cells treated with CB are distinguishably different from those treated with CB-1/CB-2, which are designed to have enhanced anti-cancer properties by having an extra amphipathic alpha-helix. The morphological differences may be due to their different modes of action on the cell membranes resulting in the different potencies with lower lethal concentration and higher concentration of 50% inhibition (IC50) of CB on bacterium and cancer cell, respectively, as compared with CB-1/:CB-2 (Chen et al. 1997. Biochim. Biophys. Acta 1336, 171-179). In contrast, CB-3 has little effect on either the bacterium or the cancer cell. These results provide microscopic evidence that different killing pathways are involved with the peptides.

Nitric oxide modification of rat brain neurogranin affects its phosphorylation by protein kinase C and affinity for calmodulin.

Neurogranin (Ng) is a prominent protein kinase C (PKC) substrate which binds calmodulin (CaM) in the absence of Ca2+. Rat brain Ng contains four cysteine residues that were readily oxidized by nitric oxide (NO) donors, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEANO) and sodium nitroprusside, and by oxidants, H2O2 and o-iodosobenzoic acid. NO oxidation of Ng resulted in a conformational change detectable by increased electrophoretic mobility upon SDS-polyacrylamide gel electrophoresis. The NO-mediated mobility shift was reversed by treatment with dithiothreitol and was blocked by modification of Ng sulfhydryl groups with 4-vinylpyridine. Both the nonphosphorylated and PKC-phosphorylated Ng were susceptible to NO oxidation. Modification of Ng by DEANO was blocked by CaM in the absence of Ca2+; while in the presence of Ca2+, CaM did not protect Ng from oxidation by DEANO. CaM also failed to protect DEANO-mediated oxidation of PKC-phosphorylated Ng with or without Ca2+. Oxidation of Ng by the various oxidants apparently resulted in the formation of intramolecular disulfide bond(s) as judged by a reduction of apparent Mr on SDS-polyacrylamide gel electrophoresis; this oxidized form, unlike the reduced form, did not bind to CaM-affinity column. The oxidized Ng was also a poorer substrate for PKC; both the reduced and oxidized forms had similar Km values, but the Vmax of the oxidized form was about one-fourth of the reduced one. When comparing the rate of DEANO-mediated nitrosation of Ng with other sulfhydryl-containing compounds, it became evident that Ng ranked as one of the best NO acceptors among those tested, including serum albumin, glutathione, and dithiothreitol. Ng present in the rat brain synaptosomal preparations was also oxidized by DEANO in a dose-dependent manner when analyzed by immunoblot with a polyclonal antibody against this protein. These results suggest that Ng is a likely target of NO and other oxidants and that oxidation/reduction may serve as a mechanism for controlling both the PKC phosphorylation and the CaM-binding affinity of this protein.

Binding of myristoylated alanine-rich protein kinase C substrate to phosphoinositides attenuates the phosphorylation by protein kinase C.

The myristoylated aline-rich protein kinase C substrate (MARCKS) is a peripheral membrane protein that undergoes phosphorylation-dependent translocation between membrane and cytosol. MARCKS binds to acidic phospholipids with high affinity (Kd less than 0.5 microM) but binds poorly to neutral phospholipids. Although interaction of MARCKS with acidic phospholipids lacks specificity when determined by binding assay, these phospholipids exert distinctive effects on the phosphorylation of this protein by protein kinase C (PKC). Preincubation of MARCKS with phosphatidylserine (PS) or phosphatidylglycerol enhanced the phosphorylation; whereas with phosphatidic acid, phosphatidylinositol (PI), phosphatidylinositol-4-phosphate, or phosphatidylinositol-4,5-biphosphate inhibited the phosphorylation of this substrate by PKC. Phosphoinositide inhibition of MARCKS phosphorylation was apparently directed at the substrate rather than at the kinase as the phosphorylation of two other phospholipid-binding PKC substrates, neuromodulin and neurogranin, exhibited different responses from those of MARCKS. Furthermore, the inhibition of phosphoinositides on MARCKS phosphorylation was seen with PKC isozymes alpha, beta, gamma, and delta and with the catalytic fragment of PKC, protein kinase M. A 25-amino-acid synthetic peptide corresponding to the phosphorylation site domain (PSD) of MARCKS, but not to the myristoylated N-terminal peptide, competed equally effectively with MARCKS in binding to either PS- or PI-containing vesicles, suggesting that both phospholipids bind to the PSD of MARCKS. Binding of PI to MARCKS inhibited PKC phosphorylation of all three phosphorylation sites. These results suggest that phosphoinositides and PS bind at different residues within the MARCKS PSD, so that the resulting phospholipid/MARCKS complexes are differentially phosphorylated by PKC.

Differential responses of protein kinase C substrates (MARCKS, neuromodulin, and neurogranin) phosphorylation to calmodulin and S100.

Phosphorylation of three physiological substrates of protein kinase C (PKC), MARCKS, neuromodulin (Nm), and neurogranin (Ng), was analyzed to determine their relative efficacy as substrates of PKC alpha, beta, and gamma and sensitivities to inhibition by calmodulin (CaM) and S100. Comparison of the Vmax/Km of the phosphorylation of each individual substrate indicated the order of efficacy as PKC substrate was MARCKS > Nm > Ng. Phosphorylation of these proteins in a mixture by PKC beta and gamma was indistinguishable from that when each individual substrate was phosphorylated by these two isozymes. In contrast, the rates of PKC alpha-catalyzed phosphorylation of Nm and Ng in a mixture also containing MARCKS were significantly reduced as compared to that when Nm or Ng was individually phosphorylated by this isozyme. When these substrates were present in a mixture, both CaM and S100 inhibited the PKC-catalyzed phosphorylation of MARCKS to a higher degree than that of Nm or Ng. Protease-activated catalytic fragment of PKC (PKM) was used to determine the effects of Ca2+ and phospholipid on the CaM and S100-mediated inhibition of PKC substrate phosphorylation. CaM and S100 inhibited the PKM-catalyzed phosphorylation of MARCKS only in the presence of Ca2+ and addition of phosphatidylserine (PS)/dioleoylglycerol (DG) did not influence the inhibitory effect. Phosphorylation of Nm or Ng by PKM was inhibited by CaM to a higher degree in the absence than in the presence of Ca2+. S100 was ineffective in inhibiting the phosphorylation of Nm and Ng without Ca2+ and only poorly effective in the presence of Ca2+. The CaM-mediated inhibition of Nm or Ng phosphorylation by PKM was also not affected by PS/DG either with or without Ca2+. The results presented here demonstrate that MARCKS is a preferred substrate of PKC and its phosphorylation by PKC is most sensitive to inhibition by regulatory proteins such as CaM and S100.

Glial-derived S100b protein selectively inhibits recombinant beta protein kinase C (PKC) phosphorylation of neuron-specific protein F1/GAP43.

Protein F1/GAP43 is neuron-specific, associated with neurite outgrowth during development and a substrate for PKC. This protein is present in high levels in serotonergic neurons which in culture sprout in response to the glial-derived S100b, the beta-beta homodimer. As an initial step in determining whether S100b acts on F1/GAP43 we studied the regulation by S100b of PKC phosphorylation of F1/GAP43. Either the S100b or a mixture of S100a and S100b, both from a brain glial cell source, inhibited in vitro phosphorylation of purified F1/GAP43 by purified PKC in a dose-dependent manner. Using recombinant PKC subtypes, purified S100b preferentially inhibited the F1/GAP43 phosphorylation by the beta subtype. The IC50 of S100b for beta I and beta II PKC was 8 microM while for alpha and gamma PKC it was 64 microM. S100b inhibition was thus subtype-selective. Histone III-S phosphorylation by the four PKC subtypes was not inhibited by S100b. S100b inhibition was thus substrate-selective. Moreover, the effect of S100b on phosphorylation could not be explained by a direct inhibition of kinase activity. Together with earlier studies implicating a role for S100 in synaptic plasticity and neurite outgrowth, the present results suggest that S100b may regulate such functions through its inhibition of neuron-specific PKC substrate (F1/GAP43) phosphorylation. The regulation of this neuron-specific substrate phosphorylation by glial S100 suggests the potential for a novel neuro-glial interaction. Finally, the location of S100 gene on chromosome 21, trisomic in Down's syndrome, and over-expressed in this disorder, as well as in Alzheimer's disease, suggests a link to cognitive impairments in human.

Learning selectively increases protein kinase C substrate phosphorylation in specific regions of the chick brain.

The effect of imprinting, an early form of exposure learning, on the phosphorylation state of the protein kinase C substrates myristoylated alanine-rich C-kinase substrate (MARCKS) and protein F1/43-kDa growth-associated protein (F1/GAP-43) was studied in two regions of the chick forebrain. One region, the intermediate and medial part of the hyperstriatum ventrale (IMHV), is probably a site of long-term memory; the other, the wulst, contains somatic sensory and visual projection areas. After imprinting, a significant increase in MARCKS protein phosphorylation was observed in the left IMHV but not the right IMHV. No significant alteration in F1/GAP-43 was observed in IMHV. MARCKS was resolved into two acidic components of pI approximately 5.0 and approximately 4.0. Phosphorylation of the pI approximately 5.0 MARCKS but not the pI approximately 4.0 MARCKS was significantly altered by imprinting. The partial correlation between preference score (an index of learning) and phosphorylation, holding constant the effect of approach activity during training, was significant only for the pI approximately 5.0 MARCKS in the left IMHV. A significant negative partial correlation between preference score and F1/GAP-43 phosphorylation in the right wulst was observed. Because the imprinting-induced alteration in MARCKS is selective with respect to phosphoprotein moiety, hemispheric location, and brain region, we propose that these alterations may be central to the learning process.