Receptor-mediated endocytosis and intracellular trafficking of insulin and low-density lipoprotein by retinal vascular endothelial cells

PURPOSE: The authors investigated the receptor-mediated endocytosis (RME) and intracellular trafficking of insulin and low-density lipoprotein (LDL) in cultured retinal vascular endothelial cells (RVECs). METHODS: Low-density lipoprotein and insulin were conjugated to 10 nm colloidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degrees C and fixed after incubation times between 30 seconds and 1 hour. Control cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control cells were exposed to several concentrations of anti-insulin receptor antibody or a saturating solution of unconjugated insulin before incubation with gold insulin. RESULTS: Using transmission electron microscopy, insulin gold and LDL gold were both observed at various stages of RME. Insulin-gold particles were first seen to bind to the apical plasma membrane (PM) before clustering in clathrin-coated pits and internalization in coated vesicles. Gold was later visualized in uncoated cytoplasmic vesicles, corresponding to early endosomes and multivesicular bodies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of endosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both insulin- and LDL-gold conjugates was seen to accumulate in large lysosome-like compartments. However, a small but significant proportion of the internalized ligands was transcytosed and released as discrete membrane-associated quanta at the basal cell surface. The uptake of LDL gold was greatly increased in highly vacuolated, late-passage RVECs. In controls, anti-insulin receptor antibody and excess unconjugated insulin caused up to 89% inhibition in gold-insulin binding and internalization. CONCLUSION: These results illustrate the internalization and intracellular trafficking by RVECs of insulin and LDL through highly efficient RME, and they provide evidence for at least two possible fates for the endocytosed ligands. This study outlines a route by which vital macromolecules may cross the inner blood-retinal barrier.

Free radical-mediated lipid peroxidation and systemic nitric oxide bioavailability:implications for postexercise hemodynamics

The metabolic vasodilator mediating postexercise hypotension (PEH) is poorly understood. Recent evidence suggests an exercise-induced reliance on pro-oxidant-stimulated vasodilation in normotensive young human subjects, but the role in the prehypertensive state is not known.

Microneedle/nanoencapsulation-mediated transdermal delivery:Mechanistic insights

A systematic study was undertaken to gain more insight into the mechanism of transdermal delivery of nanoencapsulated model dyes across microneedle (MN)-treated skin, a complex process not yet explored. Rhodamine B (Rh B) and fluorescein isothiocyanate (FITC) as model hydrophilic and hydrophobic small/medium-size molecules, respectively, were encapsulated in poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) and delivered through full thickness porcine skin pretreated with MN array. Permeation through MN-treated skin was affected by physicochemical characteristics of NPs and the encapsulated dyes. Dye flux was enhanced by smaller particle size, hydrophilicity, and negative zeta potential of NPs. Regarding encapsulated dyes, solubility at physiological pH and potential interaction with skin proteins proved to outweigh molecular weight as determinants of skin permeation. Data were verified using confocal laser scanning microscopy imaging. Findings coupled with the literature data are supportive of a mechanism involving influx of NPs, particularly of smaller size, deep into MN-created channels, generating depot dye-rich reservoirs. Molecular diffusion of the released dye across viable skin layers proceeds at a rate determined by its molecular characteristics. Data obtained provide mechanistic information of importance to the development of formulation strategies for more effective intradermal and transdermal MN-mediated delivery of nanoencapsulated therapeutic agents.

Predator cue studies reveal strong trait-mediated effects in communities despite variation in experimental designs

Nonconsumptive or trait-mediated effects of predators on their prey often outweigh density-mediated interactions where predators consume prey. For instance, predator presence can alter prey behaviour, physiology, morphology and/or development. Despite a burgeoning literature, our ability to identify general patterns in prey behavioural responses may be influenced by the inconsistent methodologies of predator cue experiments used to assess trait-mediated effects. We therefore conducted a meta-analysis to highlight variables (e.g. water type, predator husbandry, exposure time) that may influence invertebrate prey's behavioural responses to fish predator cues. This revealed that changes in prey activity and refuge use were remarkably consistent overall, despite wide differences in experimental methodologies. Our meta-analysis shows that invertebrates altered their behaviour to predator cues of both fish that were fed the focal invertebrate and those that were fed other prey types, which suggests that invertebrates were not responding to specific diet information in the fish cues. Invertebrates also altered their behaviour regardless of predator cue addition regimes and fish satiation levels. Cue intensity and exposure time did not have significant effects on invertebrate behaviour. We also highlight that potentially confounding factors, such as parasitism, were rarely recorded in sufficient detail to assess the magnitude of their effects. By examining the likelihood of detecting trait-mediated effects under large variations in experimental design, our study demonstrates that trait-mediated effects are likely to have pervasive and powerful influences in nature.

NaH Mediated Isomerisation-Allylation Reaction of 1,3-Substituted Propenols

A base mediated isomerisation-allylation protocol of 1,3- disubstituted propenols has been established. The use of diaryl and aryl-silyl substrates is reported alongside the use of substituted allyl bromides. Mechanistic experiments have also been conducted to elucidate the reaction pathway.

Base-Mediated Cascade Rearrangements of Aryl-Substituted Diallyl Ethers

Two base-mediated cascade rearrangement reactions of diallyl ethers were developed leading to selective [2,3]-Wittig–oxy-Cope and isomerization–Claisen rearrangements. Both diaryl and arylsilyl-substituted 1,3-substituted propenyl substrates were examined, and each exhibits unique reactivity and different reaction pathways. Detailed mechanistic and computational analysis was conducted, which demonstrated that the role of the base and solvent was key to the reactivity and selectivity observed. Crossover experiments also suggest that these reactions proceed with a certain degree of dissociation, and the mechanistic pathway is highly complex with multiple competing routes.

Histamine H3 receptor-mediated inhibition of sympathetically evoked mydriasis in rats

This study was designed to determine if the histamine H3 receptor agonist R-alpha-methylhistamine would play a role in modulation of sympathetically evoked mydriasis in anesthetized rats, and if so, to ascertain the specific receptor subtype(s) involved. Reproducible frequency-response curves of pupillary dilation were generated by stimulation of the cervical preganglionic sympathetic nerve (1-32 Hz). Systemic administration of R-alpha-methylhistamine (0.3-3.0 mg kg(-1)) produced a dose-related inhibition of the evoked mydriasis. The greatest inhibition was seen at lower frequency levels, with about 43% depression observed at 2 Hz. The specific histamine H3 receptor antagonist, clobenpropit (3.0 mg kg(-1), i.v.), blocked the inhibitory effect of R-alpha-methylhistamine, whereas neither the histamine H2 receptor antagonist, cimetidine (5.0 mg kg(-1), i.v.), nor the histamine H1 receptor antagonist, chlorpheniramine (0.5 mg kg(-1), i.v.), was effective. The histamine H2 receptor agonist, dimaprit (10 mg kg(-1), i.v.), was also without effect on the evoked mydriasis. R-alpha-methylhistamine (3.0 mg kg(-1)) did not inhibit phenylephrine-induced mydriasis. These results support the conclusion that R-alpha-methylhistamine produces inhibition of sympathetically evoked mydriasis via histamine H3 receptor stimulation, presumably by an action on presynaptic histamine H3 receptors.

Retroviral-mediated gene transfer of the human erythropoietin gene into a factor-dependent cell line, TF-1

The factor-dependent cell line, TF-1, established from a patient with erythroleukaemia, shows characteristics of immature erythroblasts. Addition of granulocyte-macrophage colony stimulating factor (GM-CSF) to the culture medium is required for long-term growth of the cells. Erythropoietin (Epo) can also be used to sustain TF-1 cells but for only limited periods (approximately a week). Low levels of both growth factors can act synergistically to maintain proliferation for a longer period of time than Epo alone. To eliminate the requirement of exogenous Epo for growth, TF-1 cells were co-cultured with a retroviral secreting cell line containing the human erythropoietin (hEpo) gene and a neomycin (neo) selectable marker. TF-1 cells which exhibited neo resistance (indicating infection by the retrovirus) were then grown in low concentrations of GM-CSF without the addition of Epo. Under these conditions growth of normal TF-1 cells was not sustained. The neo-resistant cells survived for more than 14 days indicating synergy between GM-CSF and the Epo synthesised by the co-cultured TF-1 cells. Radioimmunoassays performed on growth media detected concentrations up to 1 mU/ml of Epo, implying that stable integration of the retroviral vector and expression of the hEpo gene have been achieved.

Macrophages Control Vascular Stem/Progenitor Cell Plasticity Through Tumor Necrosis Factor-α-Mediated Nuclear Factor-κB Activation

Objective: Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process.

Approach and Results: We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-α (TNF-α) via TNF-α receptor 1 and canonical nuclear factor-κB activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-α receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-α. Furthermore, TNF-α–mediated EC differentiation was driven by direct binding of nuclear factor-κB (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-α. The lack of TNF-α in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation.

Conclusions: Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α–mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases such as vein graft.

Dorsal and ventral stream mediated visual processing in genetic subtypes of Prader-Willi syndrome

Previous work has suggested that there are specific deficits in dorsal stream processing in a variety of developmental disorders. Prader-Willi syndrome (PWS) is associated with two main genetic subtypes, deletion and disomy. Relative strengths in visual processing are shown in PWS, although these strengths may be specific to the deletion subtype. We investigated visual processing in PWS using an adapted Simon task which contrasted location (dorsal stream) and shape identity (ventral stream) tasks. Compared to a group of typically developing children, children with PWS deletion showed a greater degree of impairment in the dorsal stream task than in the ventral stream task, a pattern similar to that shown in a group of boys with Fragile-X syndrome. When matched on a measure of non-verbal ability, children with PWS disomy showed the opposite pattern with better performance in the location compared to the shape task, although these task performance asymmetries may have been linked to executive control processes. It is proposed that children with PWS deletion show a relative strength in visual processing in the ventral stream along with a specific deficit in dorsal stream processing. In contrast, children with PWS disomy show neither effect. (C) 2009 Published by Elsevier Ltd.

Water-mediated electrochemical nano-writing on thin ceria films

Bias dependent mechanisms of irreversible cathodic and anodic processes on a pure CeO2 film are studied using modified atomic force microscopy (AFM). For a moderate positive bias applied to the AFM tip an irreversible electrochemical reduction reaction is found, associated with significant local volume expansion. By changing the experimental conditions we are able to deduce the possible role of water in this process. Simultaneous detection of tip height and current allows the onset of conductivity and the electrochemical charge transfer process to be separated, further elucidating the reaction mechanism. The standard anodic/cathodic behavior is recovered in the high bias regime, where a sizable transport current flows between the tip and the film. These studies give insight into the mechanisms of the tip-induced electrochemical reactions as mediated by electronic currents, and into the role of water in these processes, as well as providing a different approach for electrochemical nano-writing.

Microneedle-Mediated Minimally Invasive Patient Monitoring

Background: The emerging field of microneedle-based minimally invasive patient monitoring and diagnosis is reviewed. Microneedle arrays consist of rows of micron-scale projections attached to a solid support. They have been widely investigated for transdermal drug and vaccine delivery applications since the late 1990s. However, researchers and clinicians have recently realized the great potential of microneedles for extraction of skin interstitial fluid and, less commonly, blood, for enhanced monitoring of patient health.

Methods: We reviewed the journal and patent literature, and summarized the findings and provided technical insights and critical analysis.

Results: We describe the basic concepts in detail and extensively review the work performed to date.

Conclusions: It is our view that microneedles will have an important role to play in clinical management of patients and will ultimately improve therapeutic outcomes for people worldwide.