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.

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.

Selective decline in protein F1 phosphorylation in hippocampus of senescent rats.

Certain forms of neuronal plasticity have been found to be expressed through alterations in brain protein phosphorylation, and its regulation by protein kinase activity. Of interest in this regard is the possibility that the decline in neuronal plasticity and cognitive function that occurs in advanced age may result in part from altered phosphorylation of specific proteins. As a first attempt to identify age-related changes in phosphoproteins, we assayed in vitro phosphorylation of proteins in hippocampus, cerebellum, entorhinal cortex, and frontal cortex from Fischer-344 rats of 5 months, 11 months, and 25 months of age. Compared to the middle-aged animals, the aged rats showed a selective 46% decline in phosphorylation of the 47 kDa protein (F1) in hippocampus, with no change in the phosphorylation of other proteins measured in this structure. Aged animals also showed decreased phosphorylation relative to young animals. No age-related change was observed in any protein band for the other brain areas examined. Since protein F1 is phosphorylated by protein kinase C (PKC), the cytosolic and membrane distribution of this enzyme was compared across age groups. The activity of PKC in hippocampus did not change across age. The explanation of this age-related decline in protein F1 phosphorylation is likely to be a decline in the substrate protein itself. The results are discussed in terms of protein F1's possible role in age-related decline of hippocampal synaptic plasticity.

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.

Dose-dependent phorbol ester facilitation or blockade of hippocampal long-term potentiation: relation to membrane/cytosol distribution of protein kinase C activity.

We have proposed that the translocation/activation of protein kinase C (PKC) in synergism with a Ca2+-mediated event plays an essential role in hippocampal long-term potentiation (LTP). In a previous study, we saw no effect of PKC-activating phorbol esters alone on baseline responses, although it has been reported by others to enhance synaptic transmission. To resolve this discrepancy, we investigated the dose-response to phorbol esters of both baseline and potentiated granule cell responses elicited with perforant path stimulation. It was confirmed that iontophoretic ejection of phorbol ester to the dentate hilus, which alone had no effect on baseline responses, prolonged the persistence of potentiation produced by 2 trains of 400 Hz stimulation. These data support the proposed synergistic model in which the effects of phorbol ester and high frequency stimulation together produce a long-lasting potentiation of synaptic activation. A similar synergism was observed with ejection of a lower dose of phorbol ester into the perforant path synaptic zone in the molecular layer. Higher doses delivered to the synaptic zone without 400 Hz stimulation were sufficient to enhance baseline synaptic responses, but these doses inhibited the initial potentiation induced with 2 trains of 400 Hz stimulation delivered immediately after ejection. There was at times a slowly developing enhancement observed after the initial blockade. Thus, induction of a persistent synaptic enhancement was observed without initial potentiation. Measurement of PKC activity in membrane and cytosol indicated that PKC activation is only associated with the persistence phase of LTP. In contrast, there was no change in PKC subcellular distribution associated with the blockade of initial potentiation by higher doses of PDBu.

Protein kinase C activity and substrate (F1/GAP-43) phosphorylation in developing cat visual cortex.

Protein kinase C (PKC) and substrate proteins such as F1/GAP-43 have been previously implicated in the synaptic plasticity of long-term potentiation (LTP). As a first step in determining whether they participate in the plasticity observed during the critical period of visual cortex development, we have studied cytosol and membrane PKC activity as well as the endogenous phosphorylation of visual cortical proteins in cat cortical areas 17, 18 at postnatal days 1 and 3, weeks 1, 3, 5, 7, 9, 13, 28 and 51, and adult year 5. There was an 8.4 to 10.9 fold increase in cytosolic PKC activity relative to day 1 level during the critical period of synaptic plasticity (weeks 3-13) which then dramatically decreased back to 2.5-fold of day 1 level by week 51. This was near the adult level of cytosolic PKC. Since there was an increase of 1.8- to 2.1-fold in membrane PKC activity during the critical period, this argues against a PKC translocation event and for an increase in enzyme synthesis. Endogenous phosphorylation in the same visual cortex tissue revealed an increase in protein F1 phosphorylation during the critical period. This level of PKC substrate activity was maintained in the adult providing a mechanism for plasticity in adult cat visual cortex.

NMDA receptor blockade prevents the increase in protein kinase C substrate (protein F1) phosphorylation produced by long-term potentiation.

Recent evidence has implicated activation of the N-methyl-D-aspartate (NMDA) class of glutamate receptor in the initiation of hippocampal long-term potentiation (LTP), an electrophysiological model of information storage in the brain. A separate line of evidence has suggested that activation of protein kinase C (PKC) and the consequent phosphorylation of its substrates is necessary for the maintenance of the LTP response. To determine if PKC activation is a consequence of NMDA receptor activation during LTP, we applied the NMDA receptor antagonist drug, DL-aminophosphonovalerate (APV) both immediately prior to and following high frequency stimulation, resulting in successful and unsuccessful blockade of LTP initiation, respectively. We then measured the phosphorylation of a PKC substrate (protein F1) in hippocampal tissue dissected from these animals. Only successful blockade of LTP initiation by prior application of APV was seen to block the LTP-associated increase in protein F1 phosphorylation measured in vitro (P less than 0.001 by ANOVA). This suggests that NMDA receptor-mediated initiation triggers maintenance processes that are, at least in part, mediated by protein F1 phosphorylation. These data provide the first evidence linking two mechanisms associated with LTP, NMDA receptor activation and PKC substrate phosphorylation.

Phorbol ester promotes growth of synaptic plasticity.

Iontophoretic application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to the intact rat hippocampus enhances potentiation produced by subsequent high frequency stimulation of the perforant path. The decay of the enhanced population spike amplitude recorded in the hilar dentate gyrus was prevented in animals receiving ejections of TPA, as compared to controls which decayed to baseline values within 2 h following high frequency stimulation. In fact, growth of the potentiated response was observed beginning at 45 min. Similar results were observed with the population excitatory postsynaptic potential slope, a measure of the synaptic response. Since tumour-promoting phorbol esters are known to translocate and activate protein kinase C (PKC) and PKC is translocated to the membrane following hippocampal potentiation, a role for membrane-associated PKC in the regulation of synaptic plasticity is suggested.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.