Mapping Glucose Uptake, Transport and Metabolism in the Bovine Lens Cortex.

Purpose: To spatially correlate the pattern of glucose uptake to glucose transporter distributions in cultured lenses and map glucose metabolism in different lens regions. Methods: Ex vivo bovine lenses were incubated in artificial aqueous humour containing normoglycaemic stable isotopically-labelled (SIL) glucose (5 mM) for 5 min-20 h. Following incubations, lenses were frozen for subsequent matrix-assisted laser desorption/ionisation (MALDI) imaging mass spectrometry (IMS) analysis using high resolution mass spectrometry. Manually dissected, SIL-incubated lenses were subjected to gas chromatography-mass spectrometry (GC-MS) to verify the identity of metabolites detected by MALDI-IMS. Normal, unincubated lenses were manually dissected into epithelium flat mounts and fibre cell fractions and then subjected to either gel-based proteomic analysis (Gel-LC/MS) to detect facilitative glucose transporters (GLUTs) by liquid chromatography tandem mass spectrometry (LC-MS/MS). Indirect immunofluorescence and confocal microscopy of axial lens sections from unincubated fixed lenses labelled with primary antibodies specific for GLUT 1 or GLUT 3 were utilised for protein localisation. Results: SIL glucose uptake at 5 min was concentrated in the equatorial region of the lens. At later timepoints, glucose gradually distributed throughout the epithelium and the cortical lens fibres, and eventually the deeper lens nucleus. SIL glucose metabolites found in glycolysis, the sorbitol pathway, the pentose phosphate pathway, and UDP-glucose formation were mapped to specific lens regions, with distinct regional signal changes up to 20 h of incubation. Spatial proteomic analysis of the lens epithelium detected GLUT1 and GLUT3. GLUT3 was in higher abundance than GLUT1 throughout the epithelium, while GLUT1 was more abundant in lens fibre cells. Immunohistochemical mapping localised GLUT1 to epithelial and cortical fibre cell membranes. Conclusion: The major uptake site of glucose in the bovine lens has been mapped to the lens equator. SIL glucose is rapidly metabolised in epithelial and fibre cells to many metabolites, which are most abundant in the metabolically more active cortical fibre cells in comparison to central fibres, with low levels of metabolic activity observed in the nucleus.

Cyclic peptides bearing the d-Phe-2-Abz turn motif: Structural characterization and antimicrobial potential.

The effect on secondary structure and antimicrobial activity of introducing different cyclic constraints in linear ß-hairpin antimicrobial peptides has been investigated with the intention of generating cyclic ß sheets as promising antimicrobials with improved therapeutic potential. The linear peptides were cyclized head to tail either directly or after the addition of either a second turn motif or a disulfide bridge. The propensity of these peptides to adopt a cyclic ß-sheet structure has been correlated to their antibacterial activity. All cyclic peptides showed enhanced activity, compared with their linear counterparts against methicillin-resistant Staphylococcus aureus. Scanning electron microscopy and transmission electron microscopy studies showed that this family kills bacteria through membrane lysis. The peptide that showed the best efficacy against all strains (exhibiting nanomolar activity), while retaining low haemolysis, bears two symmetrical, homochiral d-phe-2-Abz-d-ala turns and adopted a flexible structure. Its twin peptide that bears heterochiral turns (one with d-ala and one with L-Ala) showed reduced antibacterial activity and higher percentage of haemolysis. Circular dichroism and nuclear magnetic resonance spectroscopy indicate that heterochirality in the two turns leads to oligomerization of the peptide at higher concentrations, stabilizing the ß-sheet secondary structure. More rigid secondary structure is associated with lower activity against bacteria and loss of selectivity.

An investigation into the effect of ribosomal protein S15 phosphorylation on its intermolecular interactions by using phosphomimetic mutant.

An investigation using recombinant ribosomal proteins and synthetic peptide models was conducted to uncover the effect of the introduction of a negative charge at the C-terminal tail of ribosomal protein S15. Our results help provide a chemical rationale towards understanding how G2019S LRRK2, a common clinical mutation, causes Parkinson's disease.

Synthesis and Antibacterial Analysis of Analogues of the Marine Alkaloid Pseudoceratidine.

In an effort to gain more understanding on the structure activity relationship of pseudoceratidine 1, a di-bromo pyrrole spermidine alkaloid derived from the marine sponge Pseudoceratina purpurea that has been shown to exhibit potent biofouling, anti-fungal, antibacterial, and anti-malarial activities, a large series of 65 compounds that incorporated several aspects of structural variation has been synthesised through an efficient, divergent method that allowed for a number of analogues to be generated from common precursors. Subsequently, all analogues were assessed for their antibacterial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. Overall, several compounds exhibited comparable or better activity than that of pseudoceratidine 1, and it was found that this class of compounds is generally more effective against Gram-positive than Gram-negative bacteria. Furthermore, altering several structural features allowed for the establishment of a comprehensive structure activity relationship (SAR), where it was concluded that several structural features are critical for potent anti-bacterial activity, including di-halogenation (preferable bromine, but chlorine is also effective) on the pyrrole ring, two pyrrolic units in the structure and with one or more secondary amines in the chain adjoining these units, with longer chains giving rise to better activities.

Cyclic Tetrapeptides from Nature and Design: A Review of Synthetic Methodologies, Structure, and Function.

Small cyclic peptides possess a wide range of biological properties and unique structures that make them attractive to scientists working in a range of areas from medicinal to materials chemistry. However, cyclic tetrapeptides (CTPs), which are important members of this family, are notoriously difficult to synthesize. Various synthetic methodologies have been developed that enable access to natural product CTPs and their rationally designed synthetic analogues having novel molecular structures. These methodologies include the use of reversible protecting groups such as pseudoprolines that restrict conformational freedom, ring contraction strategies, on-resin cyclization approaches, and optimization of coupling reagents and reaction conditions such as temperature and dilution factors. Several fundamental studies have documented the impacts of amino acid configurations, N-alkylation, and steric bulk on both synthetic success and ensuing conformations. Carefully executed retrosynthetic ring dissection and the unique structural features of the linear precursor sequences that result from the ring dissection are crucial for the success of the cyclization step. Other factors that influence the outcome of the cyclization step include reaction temperature, solvent, reagents used as well as dilution levels. The purpose of this review is to highlight the current state of affairs on naturally occurring and rationally designed cyclic tetrapeptides, including strategies investigated for their syntheses in the literature, the conformations adopted by these molecules, and specific examples of their function. Using selected examples from the literature, an in-depth discussion of the synthetic techniques and reaction parameters applied for the successful syntheses of 12-, 13-, and 14-membered natural product CTPs and their novel analogues are presented, with particular focus on the cyclization step. Selected examples of the three-dimensional structures of cyclic tetrapeptides studied by NMR, and X-ray crystallography are also included.

Making Solid-Phase Peptide Synthesis Greener: A Review of the Literature.

To date, the synthesis of peptides is concurrent with the production of enormous amounts of toxic waste. DMF, CH2 Cl2 , and NMP are three of the most toxic organic solvents used in chemical synthesis and are the most common solvents used for peptide synthesis. Additionally, concerns about the hepatotoxicity caused by exposure to DMF and from the toxic and allergenic nature of additives used in peptide synthesis necessitates the need for a green, environmentally friendly, and safer protocol for peptide synthesis. This review summarizes the current literature on green solid-phase peptide synthesis successes and challenges encountered. The review concludes with suggestions for future research towards a simple and efficient green peptide synthesis protocol.

Acyclic peptides incorporating the d-Phe-2-Abz turn motif: Investigations on antimicrobial activity and propensity to adopt ß-hairpin conformations.

Three linear peptides incorporating d-Phe-2-Abz as the turn motif are reported. Peptide 1, a hydrophobic ß-hairpin, served as a proof of principle for the design strategy with both NMR and CD spectra strongly suggesting a ß-hairpin conformation. Peptides 2 and 3, designed as amphipathic antimicrobials, exhibited broad spectrum antimicrobial activity, with potency in the nanomolar range against Staphylococcus aureus. Both compounds possess a high degree of selectivity, proving non-haemolytic at concentrations 500 to 800 times higher than their respective minimal inhibitory concentrations (MICs) against S. aureus. Peptide 2 induced cell membrane and cell wall disintegration in both S. aureus and Pseudomonas aeruginosa as observed by transmission electron microscopy. Peptide 2 also demonstrated moderate antifungal activity against Candida albicans with an MIC of 50 µM. Synergism was observed with sub-MIC levels of amphotericin B (AmB), leading to nanomolar MICs against C. albicans for peptide 2. Based on circular dichroism spectra, both peptides 2 and 3 appear to exist as a mixture of conformers with the ß-hairpin as a minor conformer in aqueous solution, and a slight increase in hairpin population in 50% trifluoroethanol, which was more pronounced for peptide 3. NMR spectra of peptide 2 in a 1:1 CD3 CN/H2 O mixture and 30 mM deuterated sodium dodecyl sulfate showed evidence of an extended backbone conformation of the ß-strand residues. However, inter-strand rotating frame Overhauser effects (ROE) could not be detected and a loosely defined divergent hairpin structure resulted from ROE structure calculation in CD3 CN/H2 O. The loosely defined hairpin conformation is most likely a result of the electrostatic repulsions between cationic strand residues which also probably contribute towards maintaining low haemolytic activity.

Molecular Weight and Charge Density Effects of Guanidinylated Biodegradable Polycarbonates on Antimicrobial Activity and Selectivity.

Six guanidine functionalized aliphatic biodegradable polycarbonates with varying molecular weights and charge densities were synthesized via postsynthesis modification of alkyne containing polycarbonates using Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry. The concept of passive diluting group was to modify the cationic charge density of the polycarbonate without changing its hydrophilicity. Within the molecular weight range from 8000 to 30000 g mol-1, these guanidine polycarbonates exhibited broad-spectrum biocidal activity with low toxicity to red blood cells (RBCs). The lowest molecular weight homopolymer sample (PG-8k-100) showed the best antimicrobial activity (MIC = 40 µg/mL against Escherichia coli and MIC = 20 µg/mL against Staphylococcus epidermidis) and least RBC toxicity (0.6% hemolysis at MIC). Within the three guanidine charge densities from 20% to 70%, the low to medium dilution samples (PG-8k-7030 and PG-8k-5050) had no obvious loss in antimicrobial activities compared to the nondiluted control sample PG-8k-100. However, upon further dilution, PG-8k-2080 gave the lowest antimicrobial activity.

A review on comparative mechanistic studies of antimicrobial peptides against archaea.

Archaea was until recently considered as a third domain of life in addition to bacteria and eukarya but recent studies support the existence of only two superphyla (bacteria and archaea). The fundamental differences between archaeal, bacterial, and eukaryal cells are probably the main reasons for the comparatively lower susceptibility of archaeal strains to current antimicrobial agents. The possible emerging pathogenicity of archaea and the role of archaeal methanogens in methane emissions, a potent greenhouse gas, has led many researchers to examine the sensitivity patterns of archaea and make attempts to find agents that have significant anti-archaeal activity. Even though antimicrobial peptides (AMPs) are well known with several published reviews concerning their mode of action against bacteria and eukarya, to our knowledge, to date no reviews are available that focus on the action of these peptides against archaea. Herein, we present a review on all the peptides that have been tested against archaea. In addition, in an attempt to shed more light on possible future work that needs to be performed we have included a brief overview of the chemical characteristics, spectrum of activity, and the known mechanism of action of each of these peptides against bacteria and/or fungi. We also discuss the nature of and key physiological differences between Archaea, Bacteria, and Eukarya that are relevant to the development of anti-archaeal peptides. Despite our relatively limited knowledge about archaea, available data suggest that AMPs have an even broader spectrum of activity than currently recognized.