Oral Delivery of Peptide Formulations and Their Cellular Evaluation

International Journal of Peptide Research and Therapeutics - The development of peptide-based formulations presents numerous challenges to the formulator due to their complexity, delicate structure...     

Effect of functional electrical stimulation on the effort and walking speed,surface electromyography activity,and metabolic responses in stroke subjects

ObjectiveTo investigate the effects of functional electrical stimulation (FES) combined with conventional rehabilitation program on the effort and speed of walking, the surface electromyographic (sEMG) activity and metabolic responses in the management of drop foot in stroke subjects.MethodsFifteen patients with a drop foot resulting from stroke at least 3 months prior to the start of the trial took part in this study. All subjects were treated 1 h a day, 5 days a week, for 12 weeks, including conventional stroke rehabilitation program and received 30 min of FES to the tibialis anterior (TA) muscle of the paretic leg in clinical settings. Baseline and post-treatment measurements were made for temporal and spectral EMG parameters of TA muscle, walking speed, the effort of walking as measured by physiological cost index (PCI) and metabolic responses.ResultsThe experimental results showed a significant improvement in mean-absolute-value (21.7%), root-mean-square (66.3%) and median frequency (10.6%) of TA muscle EMG signal, which reflects increased muscle strength. Mean increase in walking speed was 38.7%, and a reduction in PCI of 34.6% between the beginning and at end of the trial. Improvements were also found in cardiorespiratory responses with reduction in oxygen consumption (24.3%), carbon dioxide production (19.9%), heart rate (7.8%) and energy cost (22.5%) while walking with FES device.ConclusionsThe results indicate that the FES may be a useful therapeutic tool combined with conventional rehabilitation program to improve the muscle strength, walking ability and metabolic responses in the management of drop foot with stroke patients.     

Antimicrobial Protegrin-1 Forms Ion Channels: Molecular Dynamic Simulation, Atomic Force Microscopy, and Electrical Conductance Studies

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among β-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood. Using complementary approaches, including molecular dynamics simulations, atomic force microscopy (AFM) imaging, and planar lipid bilayer reconstitution, we provide computational and experimental evidence that PG-1, a β-hairpin peptide, forms ion channels. Simulations indicate that PG-1 forms channel-like structures with loosely attached subunits when reconstituted in anionic lipid bilayers. AFM images show the presence of channel-like structures when PG-1 is reconstituted in dioleoylphosphatidylserine/palmitoyloleoyl phosphatidylethanolamine bilayers or added to preformed bilayers. Planar lipid bilayer electrical recordings show multiple single channel conductances that are consistent with the heterogeneous oligomeric channel structures seen in AFM images. PG-1 channel formation seems to be lipid-dependent: PG-1 does not easily show ion channel electrical activity in phosphatidylcholine membranes, but readily shows channel activity in membranes rich in phosphatidylethanolamine or phosphatidylserine. The combined results support a model wherein the β-hairpin PG-1 peptide acts as an antibiotic by altering cell ionic homeostasis through ion channel formation in cell membranes.     

Deletions in chromosome 4 differentially associated with the development of cervical cancer: evidence of slit2 as a candidate tumor suppressor gene

The aim of this study was to locate the candidate tumor suppressor genes (TSGs) loci in the chromosomal 4p15-16, 4q22-23 and 4q34-35 regions associated with the development of uterine cervical carcinoma (CA-CX). Deletion mapping of the regions by microsatellite markers identified six discrete areas with high frequency of deletions, viz. 4p16.2 (D1: 40%), 4p15.31 (D2: 35–38%), 4p15.2 (D3: 37–40%), 4q22.2 (D4: 34%), 4q34.2-34.3 (D5: 37–59%) and 4q35.1 (D6: 40–50%). Significant correlation was noted among the deleted regions D1, D2 and D3. The deletions in D1, D2, D5 and D6 regions are suggested to be associated with the cervical intraepithelial neoplasia (CIN), and deletions in the D2, D3, D5 and D6 regions seems to be associated with progression of CA-CX. The deletions in the D2 and D6 regions showed significant prognostic implications (P = 0.001; 0.02). The expression of the candidate TSG SLIT2 mapped to D2 region gradually reduced from normal cervix uteri →CIN → CA-CX. SLIT2 promoter hypermethylation was seen in 28% CIN samples and significantly increased with tumor progression (P = 0.04). Significant correlation was seen between SLIT2 deletion and its promoter methylation (P = 0.001), indicating that both these phenomena could occur simultaneously to inactivate this gene. Immunohistochemical analysis showed reduced expression of SLIT2 in cervical lesions and CA-CX cell lines. Although no mutation was detected in the SLIT2 promoter region (−432 to + 55 bp), CC and AA haplotypes were seen in −227 and −195 positions, respectively. Thus, it indicates that inactivation of SLIT2-ROBO1 signaling pathway may have an important role in CA-CX development.     

Effects of point substitutions on the structure of toxic Alzheimer's β-amyloid channels: atomic force microscopy and molecular dynamics simulations

Alzheimer's disease (AD) is a misfolded protein disease characterized by the accumulation of β-amyloid (Aβ) peptide as senile plaques, progressive neurodegeneration, and memory loss. Recent evidence suggests that AD pathology is linked to the destabilization of cellular ionic homeostasis mediated by toxic pores made of Aβ peptides. Understanding the exact nature by which these pores conduct electrical and molecular signals could aid in identifying potential therapeutic targets for the prevention and treatment of AD. Here using atomic force microscopy (AFM) and molecular dynamics (MD) simulations, we compared the imaged pore structures with models to predict channel conformations as a function of amino acid sequence. Site-specific amino acid (AA) substitutions in the wild-type Aβ(1-42) peptide yield information regarding the location and significance of individual AA residues to its characteristic structure-activity relationship. We selected two AAs that our MD simulation predicted to inhibit or permit pore conductance. The substitution of Phe19 with Pro has previously been shown to eliminate conductance in the planar lipid bilayer system. Our MD simulations predict a channel-like shape with a collapsed pore, which is supported by the AFM channel images. We suggest that proline, a known β-sheet breaker, creates a kink in the center of the pore and prevents conductance via blockage. This residue may be a viable target for drug development studies aiming to inhibit Aβ from inducing ionic destabilization toxicity. The substitution of Phe20 with Cys exhibits pore structures indistinguishable from the wild type in AFM images. MD simulations predict site 20 to face the solvated pore. Overall, the mutations support the previously predicted β-sheet-based channel structure.     

Models of toxic beta-sheet channels of protegrin-1 suggest a common subunit organization motif shared with toxic alzheimer beta-amyloid ion channels

Antimicrobial peptides (AMPs) induce cytotoxicity by altering membrane permeability. The electrical properties of membrane-associated AMPs as well as their cellular effects have been extensively documented; however their three-dimensional structure is poorly understood. Gaining insight into channel structures is important to the understanding of the protegrin-1 (PG-1) and other AMP cytolytic mechanisms, and to antibiotics design. We studied the β-sheet channels morphology using molecular dynamics simulations. We modeled PG-1 channels as intrinsic barrel-stave and toroidal membrane pores, and simulated them in zwitterionic and anionic lipid bilayers. PG-1 channels consist of eight β-hairpins in a consecutive NCCN (N and C represent the β-hairpin's N- and C-termini) packing organization yielding antiparallel and parallel β-sheet channels. Both channels preserve the toroidal, but not the barrel-stave pores. The two lipid leaflets of the bilayer bend toward each other at the channels’ edges, producing a semitoroidal pore with the outward-pointing hydrophobic residues preventing the polar lipid headgroups from moving to the bilayer center. In all simulated lipid environments, PG-1 channels divide into four or five β-sheet subunits consisting of single or dimeric β-hairpins. The channel morphology with subunit organization is consistent with the four to five subunits observed by NMR in the POPE/POPG bilayer. Remarkably, a β-sheet subunit channel motif is in agreement with Alzheimer ion channels modeled using the universal U-shape β-strand-turn-β-strand structure, as well as with high resolution atomic force microscopy images of β-amyloid channels with four to six subunits. Consistent with the toxic β-amyloid channels that are ion-conducting, the PG-1 channels permeate anions.     

New structures help the modeling of toxic amyloidbeta ion channels

The mechanism of amyloid toxicity is poorly understood and there are two schools of thought in this hotly debated field: the first favors membrane destabilization by intermediate-to-large amyloid oligomers, with consequent thinning and non-specific ion leakage; the second favors ion-specific permeable channels lined by small amyloid oligomers. Published results currently support both mechanisms. However, the amyloidbeta (Abeta) peptide has recently been shown to form a U-shaped 'beta-strand-turn-beta-strand' structure. This structure and the available physiological data present a challenge for computational biology--to provide candidate models consistent with the experimental data. Modeling based on small Abeta oligomers containing extramembranous N-termini predicts channels with shapes and dimensions consistent with experimentally derived channel structures. These results support the hypothesis that small Abeta oligomers can form ion channels. Molecular dynamics modeling can provide blueprints of 3D structural conformations for many other amyloids whose membrane association is key to their toxicity.     

ClpB/Hsp100 proteins and heat stress tolerance in plants

High-temperature stress can disrupt cellular proteostasis, resulting in the accumulation of insoluble protein aggregates. For survival under stressful conditions, it is important for cells to maintain a pool of native soluble proteins by preventing and/or dissociating these aggregates. Chaperones such as GroEL/GroES (Hsp60/Hsp10) and DnaK/DnaJ/GrpE (Hsp70/Hsp40/nucleotide exchange factor) help cells minimize protein aggregation. Protein disaggregation is accomplished by chaperones belonging to the Caseinolytic Protease (Clp) family of proteins. ClpB/Hsp100 proteins are strikingly ubiquitous and are found in bacteria, yeast and multi-cellular plants. The expression of these proteins is regulated by heat stress (HS) and developmental cues. Bacteria and yeast contain one and two forms of ClpB proteins, respectively. Plants possess multiple forms of these proteins that are localized to different cellular compartments (i.e. cytoplasm/nucleus, chloroplast or mitochondria). Overwhelming evidence suggests that ClpB/Hsp100 proteins play decisive roles in cell adaptation to HS. Mutant bacteria and yeast cells lacking active ClpB/Hsp100 proteins are critically sensitive to high-temperature stress. Likewise, Arabidopsis, maize and rice mutants lacking cytoplasmic ClpB proteins are very sensitive to heat. In this study, we present the structural and functional attributes of plant ClpB forms.