Feedback inactivation of D-serine synthesis by NMDA receptor-elicited translocation of serine racemase to the membrane.

D-serine is a physiological coagonist of N-methyl D-aspartate receptors (NMDARs) that plays a major role in several NMDAR-dependent events. In this study we investigate mechanisms regulating D-serine production by the enzyme serine racemase (SR). We now report that NMDAR activation promotes translocation of SR to the plasma membrane, which dramatically reduces the enzyme activity. Membrane-bound SR isolated from rat brain is not extracted from the membrane by high detergent and salt concentration, indicating a strong association. Colocalization studies indicate that most membrane-bound SR is located at the plasma membrane and dendrites, with much less SR observed in other types of membrane. NMDAR activation promotes translocation of the cytosolic SR to the membrane, resulting in reduced D-serine synthesis, and this effect is averted by blockade of NMDARs. In primary neuronal cultures, SR translocation to the membrane is blocked by a palmitoylation inhibitor, indicating that membrane binding is mediated by fatty acid acylation of SR. In agreement, we found that SR is acylated in transfected neuroblastoma cells using [(3)H]palmitate or [(3)H]octanoic acid as precursors. In contrast to classical S-palmitoylation of cysteines, acylation of SR occurs through the formation of an oxyester bond with serine or threonine residues. In addition, we show that phosphorylation of Thr-227 is also required for steady-state binding of SR to the membrane under basal, nonstimulated condition. We propose that the inhibition of D-serine synthesis caused by translocation of SR to the membrane provides a fail-safe mechanism to prevent NMDAR overactivation in vicinal cells or synapses.

Effect of peptides bearing nuclear localization signals on therapeutic ultrasound mediated gene delivery.

BACKGROUND: One of the major limitations of nonviral gene delivery methods is nuclear transport of plasmid DNA (pDNA). Peptides bearing nuclear localization signal (NLS) were shown to mediate nuclear import of macromolecules. We have explored the use of cell-permeable peptides (CPP) bearing NLS sequences to enhance transfection mediated by a nonviral approach: therapeutic ultrasound (TUS). METHODS: Two CPP-NLS peptides which differ in the location of the NLS relative to the CPP were used: S4 13-PV and PV-S4 13. The peptides were attached to pDNA using electrostatic interactions. Gel-electrophoresis and fluorescent assays were performed to evaluate pDNA-peptide interactions and condensation effects. Confocal microscopy was used to evaluate pDNA-peptide interaction inside cells. Transfection studies were conducted with the luciferase gene, using pDNA-peptides alone, or with the application of TUS. RESULTS: Attachment of both peptides to pDNA condensed the pDNA, with higher affinity for the S4(13)-PV peptide. This interaction protected pDNA from endonucleases, but was also reversible. Both peptides mediated pDNA delivery to cell cytoplasm, but less significantly to the nucleus. Thus, both peptides produced transfection in cells, when added after incubation with DNA, with higher transfection-level for PV-S4 13. Application of TUS increased transfection mediated by these peptides, but was not higher compared to transfection using TUS and pDNA alone. CONCLUSIONS: This study suggests that CPP-NLS peptides may be used for condensing pDNA and bringing it into the cell cytoplasm, but their ability to mediate nuclear import of pDNA is insignificant.

Analogous oligo-acyl-lysines with distinct antibacterial mechanisms.

Bactericidal properties were recently shown to emerge from hydrophobicity and charge buildup in oligo-acyl-lysine (OAK) peptide mimetics. Toward understanding the attributes that govern the activity of this novel antimicrobial system, we compared the functional and mechanistic properties of a known octamer and a newly generated hexamer analog. The data provide strong evidence for multiple similarities that included high tissue stability, low hemolysis, large-spectrum antibacterial activity in vitro, and the ability to prevent Escherichia coli-induced mortality in vivo. Despite these similarities, however, the octamer mode of action involved membrane disruption, unlike the hexamer, which acted predominantly through inhibition of DNA functions with characteristically slower bactericidal kinetics. Collectively, the data support the view that the analogous OAKs induced bacterial death by distinct mechanisms and further suggest that relatively minor differences in the sequence of host defense peptides are responsible for selecting one mechanism over another, possibly in conjunction with differential binding affinities to the external and/or cytoplasmic membrane.

Structure-activity relationships of antibacterial acyl-lysine oligomers.

We describe structure-activity relationships that emerged from biophysical data obtained with a library of antimicrobial peptide mimetics composed of 103 oligoacyllysines (OAKs) designed to pin down the importance of hydrophobicity (H) and charge (Q). Based on results obtained with OAKs displaying minimal inhibitory concentration < or = 3 microM, the data indicate that potent inhibitory activity of the gram-negative Escherichia coli and the gram-positive Staphylococcus aureus required a relatively narrow yet distinct window of HQ values where the acyl length played multiple and critical roles, both in molecular organization and in selective activity. Thus, incorporation of long-but not short-acyl chains within a peptide backbone is shown to lead to rigid supramolecular organization responsible for poor antibacterial activity and enhanced hemolytic activity. However, sequence manipulations, including introduction of a tandem lysine motif into the oligomer backbone, enabled disassembly of aggregated OAKs and subsequently revealed tiny, nonhemolytic, yet potent antibacterial derivatives.

In vivo fate and therapeutic efficacy of PF-4/CTF microspheres in an orthotopic human glioblastoma model.

The correlation between glioma grade and angiogenesis suggests that antiangiogenic therapies are potentially therapeutically effective for these tumors. However, to achieve tumor suppression, antiangiogenic therapies need to be administered daily using high systemic quantities. We designed a biodegradable polymeric device that overcomes those barriers by providing sustained local delivery of a C-terminal fragment of platelet factor 4 (PF-4/CTF), an antiangiogenic agent. Fluorescent-labeled microspheres composed of poly lactic-coglycolic acid (PLGA) were loaded with rhodamine-labeled PF-4/CTF and formulated to release their contents over time. Fluorescent labeling enabled the correlation between the in vitro to the in vivo kinetic and release studies. PF-4/CTF microspheres were injected into established intracranial human glioma tumors in nude mice. Noninvasive magnetic resonance imaging (MRI) was used to assess the therapeutic response. Tumor size, microvessel density, proliferation, and apoptosis rate were measured by histological analysis. Intracranially, the microspheres were located throughout the tumor bed and continuously released PF-4/CTF during the entire experimental period. MRI and histological studies showed that a single injection of microspheres containing PF-4/CTF caused a 65.2% and 72% reduction in tumor volume, respectively, with a significant decrease in angiogenesis and an increase in apoptosis. Our data demonstrate that polymeric microspheres are an effective therapeutic approach for delivering antiangiogenic agents that result in the inhibition of glioma tumor growth.

Improved antimicrobial peptides based on acyl-lysine oligomers.

We describe peptidomimetic oligomers that show rapid, nonhemolytic, broad-spectrum bactericidal properties in mice and do not induce the emergence of resistance. The oligomers contain acyl chains, which prevent the formation of stable secondary structure. This design appears advantageous over conventional antimicrobial peptides with respect to in vivo efficacy and safety, and may provide a convenient platform for the development of peptide antibiotics.

Effects of acyl versus aminoacyl conjugation on the properties of antimicrobial peptides.

To investigate the importance of increased hydrophobicity at the amino end of antimicrobial peptides, a dermaseptin derivative was used as a template for a systematic acylation study. Through a gradual increase of the acyl moiety chain length, hydrophobicity was monitored and further modulated by acyl conversion to aminoacyl. The chain lengths of the acyl derivatives correlated with a gradual increase in the peptide's global hydrophobicity and stabilization of its helical structure. The effect on cytolytic properties, however, fluctuated for different cells. Whereas acylation gradually enhanced hemolysis of human red blood cells and antiprotozoan activity against Leishmania major, bacteria displayed a more complex behavior. The gram-positive organism Staphylococcus aureus was most sensitive to intermediate acyl chains, while longer acyls gradually led to a total loss of activity. All acyl derivatives were detrimental to activity against Escherichia coli, namely, but not solely, because of peptide aggregation. Although aminoacyl derivatives behaved essentially similarly to the nonaminated acyls, they displayed reduced hydrophobicity, and consequently, the long-chain acyls enhanced activity against all microorganisms (e.g., by up to 12-fold for the aminolauryl derivative) but were significantly less hemolytic than their acyl counterparts. Acylation also enhanced bactericidal kinetics and peptide resistance to plasma proteases. The similarities and differences upon acylation of MSI-78 and LL37 are presented and discussed. Overall, the data suggest an approach that can be used to enhance the potencies of acylated short antimicrobial peptides by preventing hydrophobic interactions that lead to self-assembly in solution and, thus, to inefficacy against cell wall-containing target cells.