Inhibitory action of spermidine on formyl-methionyl-leucyl-phenylalanine stimulated inositol phosphate production in human neutrophils

The effect of the polyamine, spermidine, on formyl-methionyl-leucyl-phenylalanine stimulated hydrolysis of polyphosphoinositides was examined in purified human neutrophils by measurement of inositol phosphate production from radioactively labelled inositol. Spermidine inhibited formyl-methionyl-leucyl-phenylalanine stimulated inositol phosphate production by neutrophil in a dose dependent manner. Inhibition of formyl-methionyl-leucyl-phenylalanine stimulated inositol phosphate accumulation by spermidine was maximal at 10 microM and the IC50 value for this effect was 4.2 microM spermidine. This action of spermidine, thought to be mediated by a membrane component other than phospholipase C, may reflect a control mechanism modulating the response of the polyphosphoinositide system.     

Fluorographic detection of tritiated glycopeptides and oligosaccharides separated on polyacrylamide gels: analysis of glycans from Dictyostelium discoideum glycoproteins

Previous workers have shown that oligosaccharides and glycopeptides can be separated by electrophoresis in buffers containing borate ions. However, normal fluorography of tritium-labeled structures cannot be performed because the glycans are soluble and can diffuse during equilibration with scintillants. This problem has been circumvented by equilibration of the gel with 2,5-diphenyloxazole (PPO) prior to electrophoresis. The presence of PPO in the gel during electrophoresis does not alter mobility of the glycopeptides and oligosaccharides. After electrophoresis, the gel is simply dried and fluorography performed. This allows sensitive and precise comparisons of labeled samples in parallel lanes of a slab gel and, since mobilities are highly reproducible, between different gels. The procedure is preparative in that after fluorography the gel bands can be quantitatively eluted for further study, without any apparent modification by the procedure. In this report, the procedure is illustrated by fractionation of both neutral and anionic glycopeptides produced by the cellular slime mold Dictyostelium discoideum.     

Characterization of the L lambda phase in trehalose-stabilized dry membranes by solid-state NMR and X-ray diffraction

Solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray powder diffraction were used to investigate the mechanism of trehalose (TRE) stabilization of lipid bilayers. Calorimetric investigation of dry TRE-stabilized bilayers reveals a first-order phase transition (L kappa----L lambda) at temperatures similar to the L beta'----(P beta')----L alpha transition of hydrated lipid bilayers. X-ray diffraction studies show that dry mixtures of TRE and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) have a lamellar structure with excess crystalline TRE being present. The L kappa phase shows typical gel-phase X-ray diffraction patterns. In contrast, the L lambda-phase diffraction patterns indicate disordered hydrocarbon chains. 2H NMR of specifically 2H chain-labeled DPPC confirmed that the acyl chains are disordered in the L lambda phase over their entire lengths. 2H spectra of the choline headgroup show hindered molecular motions as compared to dry DPPC alone, and 13C spectra of the sn-2-carbonyl show rigid lattice powder patterns indicating very little motion at the headgroup and interfacial regions. Thus, the sugar interacts extensively with the hydrophilic regions of the lipid, from the choline and the phosphate moieties in the headgroup to the glycerol and carbonyls in the interfacial region. We postulate that the sugar and the lipid form an extensive hydrogen-bonded network with the sugar acting as a spacer to expand the distance between lipids in the bilayer. The fluidity of the hydrophobic region in the L lambda phase together with the bilayer stabilization at the headgroup contributes to membrane viability in anhydrobiotic organisms.     

Electrophysiological and immunocytochemical characterization of DRG neurons on an organosilane surface in serum-free medium

We are attempting to recreate a stretch reflex circuit on a patterned Bio-MEMS (bio-microelectromechanical systems) chip with deflecting micro-cantilevers. The first steps to recreate this system is to be able to grow individual components of the circuit (sensory neuron, motoneuron, skeletal muscle, and muscle spindle) on a patternable, synthetic substrate coating the MEMS device. Sensory neurons represent the afferent portion of the stretch reflex arc and also play a significant role in transmitting the signal from the muscle spindle to the spinal cord motoneurons. We have utilized a synthetic silane substrate N-1[3-(trimethoxysilyl) propyl) diethylenetriamine (DETA) on which to grow and pattern the cells. DETA forms a self-assembled monolayer on a variety of silicon substrates, including glass, and can be patterned using photolithography. In this paper, we have evaluated the growth of sensory neurons on this synthetic silane substrate. We have investigated the immunocytochemical and electrophysiological properties of the sensory neurons on DETA and compared the resultant properties with a biological control substrate (ornithine/laminin). Immunocytochemical studies revealed the survival and growth of all three subtypes of sensory neurons: trkA, trkB, and trkC on both surfaces. Furthermore, whole-cell patch clamp recordings were used to study the electrophysiological properties of the sensory neurons on the two surfaces. There were no significant differences in the electrical properties of the neurons grown on either surface. This is the first study analyzing the immunocytochemical and electrophysiological properties of sensory neurons grown long-term in a completely defined environment and on a nonbiological substrate.     

Deregulation of Beclin 1 in patients with tobacco-related oral squamous cell carcinoma

Autophagy is a physiologically regulated and evolutionary conserved process that plays a critical role in degradation of cytoplasmic proteins and other macromolecules within the lysosomes. Beclin-1, the mammalian orthologue of yeast Atg6, is an important mediator of autophagy that has been studied in many human cancers. However, the expression of Beclin-1 has not yet been investigated in oral cancer. We for the first time investigated the expression of Beclin-1 in serum and tissues and correlated it with the clinic-pathological features of oral cancer patients. m-RNA expression of Beclin-1 was evaluated in tumor and normal areas of surgical specimens from 10 oral cancer patients by real-time PCR. Approximately, 8-fold lower expression (p<0.001) of Beclin-1 mRNA was observed in tumor tissue as compared to the normal tissue. Serum levels of Beclin-1 were evaluated by SPR and ELISA. No significant difference was observed in serum Beclin-1 levels in patients as compared to healthy subjects, similarly no correlation was found between serum levels and clinic-pathological parameters such as stage, lymph node involvement and tumor size. Our results demonstrate that down-regulation of Beclin-1 may play an important role in the development and progression of oral cancer possibly by dysregulation of autophagy in tumor cells.     

Sequence and structure space of RNA-binding peptides

Studies of RNA-binding peptides, and recent combinatorial library experiments in particular, have demonstrated that diverse peptide sequences and structures can be used to recognize specific RNA sites. The identification of large numbers of sequences capable of binding to a particular site has provided extensive phylogenetic information used to deduce basic principles of recognition. The high frequency at which RNA-binding peptides are found in large sequence libraries suggests plausible routes to evolve sequence-specific binders, facilitating the design of new binding molecules and perhaps reflecting characteristics of natural evolution.     

Recent Advances in Lipid Nanoparticle Formulations with Solid Matrix for Oral Drug Delivery

Lipid nanoparticles based on solid matrix have emerged as potential drug carriers to improve gastrointestinal (GI) absorption and oral bioavailability of several drugs, especially lipophilic compounds. These formulations may also be used for sustained drug release. Solid lipid nanoparticle (SLN) and the newer generation lipid nanoparticle, nanostructured lipid carrier (NLC), have been studied for their capability as oral drug carriers. Biodegradable, biocompatible, and physiological lipids are generally used to prepare these nanoparticles. Hence, toxicity problems related with the polymeric nanoparticles can be minimized. Furthermore, stability of the formulations might increase than other liquid nano-carriers due to the solid matrix of these lipid nanoparticles. These nanoparticles can be produced by different formulation techniques. Scaling up of the production process from lab scale to industrial scale can be easily achieved. Reasonably high drug encapsulation efficiency of the nanoparticles was documented. Oral absorption and bioavailability of several drugs were improved after oral administration of the drug-loaded SLNs or NLCs. In this review, pros and cons, different formulation and characterization techniques, drug incorporation models, GI absorption and oral bioavailability enhancement mechanisms, stability and storage condition of the formulations, and recent advances in oral delivery of the lipid nanoparticles based on solid matrix will be discussed.