Effects on the Human Tear Film of Applying Skin Lipids to the Ocular Surface.

PURPOSE: The effect of skin lipids on the formation and stability of the human tear film was investigated. METHODS: Skin swab substances (SSSs) were applied to the eyes of volunteers and studied using fluorescein or with TearView, which records infrared emissivity showing tear film integrity in real time. Results were compared with similar experiments using castor oil, freshly collected meibum, or acetic acid, which simulated the low pH of the skin. RESULTS: Fluorescein and TearView results were comparable. TearView showed the natural unaltered tear film over the whole eye, instant changes to the tear film, and meibomian gland activity. Minimal amounts of SSS destroyed the integrity of the film and caused pain. Corneal epithelial damage could be detected. TearView showed that SSS stimulated meibomian gland secretion if applied directly to the posterior eyelid margin. Excess meibum had no effect on the tear film spread or integrity. Castor oil formed floating lenses on the tear film which were spread by a blink but then condensed back toward themselves. There was no pain or surface damage with these oils. CONCLUSIONS: SSS contamination of the ocular surface disrupts the tear film, causes stinging, and fluorescein staining of the corneal epithelial cells after a blink. SSS stimulates meibomian gland activity. It is possible that various ocular conditions associated with dry eye, such as blepharitis and ocular rosacea, may compromise a meibomian lipid barrier of the eye lid margin. Skin lipids would then have access to the ocular surface and cause dry eye symptoms.

Comparison of adjuvant mechanisms of medium-chain triacylglycerol in a mouse FITC-induced contact hypersensitivity model.

The number of allergy sufferers has been increasing with the increase in chemicals to which we are potentially exposed. We have discovered that tributyrin, a short-chain triacylglycerol (TAG), enhanced fluorescein isothiocyanate (FITC)-induced contact hypersensitivity in a mouse model. Medium-chain triacylglycerols (MCTs) are used in cosmetics, with which we come into direct contact frequently, to maintain skin conditions and as a thickening agent for cosmetics. In this study, we examined whether MCTs with different side chain lengths enhanced skin sensitization to FITC in the mouse model. During skin sensitization to FITC, the presence of tributyrin (side chain carbon number, 4; C4) as well as that of each MCT, tricaproin (C6), tricaprylin (C8), or tricaprin (C10), resulted in enhanced skin sensitization, whereas that of trilaurin (C12) did not. As to the mechanism underlying the enhanced sensitization, three MCTs (C6, C8 and C10) facilitated migration of FTIC-presenting CD11c+ dendritic cells to draining lymph nodes. These results indicated that not only tributyrin but also MCTs, up to side chain carbon number 10, have an adjuvant effect on FITC-induced skin hypersensitivity in mice.

A general system for evaluating the efficiency of chromophore-assisted light inactivation (CALI) of proteins reveals Ru(II) tris-bipyridyl as an unusually efficient "warhead".

Chromophore-assisted light inactivation (CALI) of proteins is a potentially powerful tool in biological research for the triggered disruption of protein function. It involves the creation of chimeric molecules that can bind specifically to the protein target and can also sensitize the photo-generation of singlet oxygen, which inactivates the target protein. There remains a need for more efficient chromophores for singlet oxygen generation. Here we report a general and convenient system with which to evaluate the efficiency of chromophores in CALI both in crude extracts and in living cells. We employ this system to show that a readily available derivative of ruthenium(II) tris-bipyridyl dication is an unusually efficient "warhead" for CALI, exhibiting a performance markedly superior to the commonly used organic fluorophore, fluorescein.

Hyperbaric oxygen reduces blood-brain barrier damage and edema after transient focal cerebral ischemia.

BACKGROUND AND PURPOSE: Hyperbaric oxygen (HBO) has been shown to protect the brain parenchyma against transient focal cerebral ischemia, but its effects on the ischemic microcirculation are largely unknown. We examined the potential of HBO to reduce postischemic blood-brain barrier (BBB) damage and edema. METHODS: Wistar rats and C57/BL6 mice underwent occlusion of the middle cerebral artery (MCAO) for 2 hours. Forty minutes after filament introduction, animals breathed either 100% O2 at 3.0 atmospheres absolute (ata; HBO group) or at 1.0 ata (control) for 1 hour in an HBO chamber. In rats, MRI was performed 15 minutes after MCAO and after 15 minutes and 3, 6, 24, and 72 hours of reperfusion. In mice, BBB permeability for sodium fluorescein was measured after 24-hour reperfusion. RESULTS: Increased BBB permeability on postcontrast T1-weighted (T1w) images had a biphasic pattern. HBO reduced volumes and intensity of enhancement. Mean abnormal enhancing volumes were 71+/-10 mm3 (control) versus 47+/-10 mm3 (HBO) at 15 minutes; 111+/-21 mm3 versus 69+/-17 mm3 3 hours; 147+/-44 mm3 versus 83+/-21 mm3 6 hours; 150+/-37 mm3 versus 89+/-14 mm3 24 hours; and 322+/-52 mm3 versus 215+/-21 mm3 72 hours (all P<0.05). Interhemispheric quotients of mean gray values on T1w were at 1.73+/-0.11 versus 1.57+/-0.07 15 minutes; 1.74+/-0.07 versus 1.60+/-0.06 at 3 hours; 1.77+/-0.07 versus 1.62+/-0.06 at 6 hours; 1.79+/-0.10 versus 1.60+/-0.05 at 24 hours; and 1.81+/-0.10 versus 1.62+/-0.07 at 72 hours (all P<0.05). HBO-treated mice had significantly lower postischemic BBB permeability than mice treated with either normobaric hyperoxia or room air. Vasogenic edema assessed on T2w images and histologic sections was significantly lower in HBO-treated rats. CONCLUSIONS: Intraischemic HBO therapy reduces early and delayed postischemic BBB damage and edema after focal ischemia in rats and mice.

Ca2+-dependent and -independent mechanisms of calmodulin nuclear translocation.

Translocation from the cytosol to the nucleus is a major response by calmodulin (CaM) to stimulation of cells by Ca2+. However, the mechanisms involved in this process are still controversial and both passive and facilitated diffusion have been put forward. We tested nuclear translocation mechanisms in electroporated HeLa cells, rat cortical neurons and glial cells using novel calmodulin and inhibitor peptide probes and confocal microscopy. Passive diffusion of calmodulin across the nuclear membrane was measured in conditions in which facilitated transport was blocked and was compared to that of a similarly sized fluorescein-labeled dextran. Wheat germ agglutinin, which blocks facilitated transport but not passive diffusion, inhibited the nuclear entry of both wild-type and Ca2+-binding-deficient mutant calmodulin both in low and elevated [Ca2+]. Ca2+-dependent nuclear translocation was prevented by a membrane-permeant CaM inhibitor, the mTrp peptide, which indicated that it was specific to Ca2+/CaM. Diffusion of free CaM and Ca2+/CaM was considerably slower than the observed nuclear translocation by facilitated transport. Our data show that the majority of CaM nuclear entry occurred by facilitated mechanisms in all cell types examined, in part by a Ca2+-independent and in part by a Ca2+-dependent translocation mechanism.

Suppression of bcr-abl synthesis by siRNAs or tyrosine kinase activity by Glivec alters different oncogenes, apoptotic/antiapoptotic genes and cell proliferation factors (microarray study).

Short 21-mer double-stranded/small-interfering RNAs (ds/siRNAs) were designed to target bcr-abl mRNA in chronic myelogenous leukemia. The ds/siRNAs were transfected into bcr-abl-positive K-562 (derived from blast crisis chronic myelogenous leukemia), using lipofectamine. Penetrating of ds/siRNAs into the cells was detected by fluorescent confocal microscopy, using fluorescein-labeled ds/siRNAs. The cells were treated with mix of three siRNA sequences (3 x 60 nM) during 6 days with three repetitive transfections. The siRNA-treatment was accompanied with significant reduction of bcr-abl mRNA, p210, protein tyrosine kinase activity and cell proliferation index. Treatment of cells with Glivec (during 8 days with four repetitive doses, 180 nM single dose) resulted in analogous reduction of cell proliferation activity, stronger suppression of protein tyrosine kinase activity, and very low reduction of p210. siRNA-mix and Glivec did not affect significantly the viability of normal lymphocytes. Microarray analysis of siRNA- and Glivec-treated K-562 cells demonstrated that both pathways of bcr-abl suppression were accompanied with overexpression and suppression of many different oncogenes, apoptotic/antiapoptotic and cell proliferation factors. The following genes of interest were found to decrease in relatively equal degree in both siRNA- and Glivec-treated cells: Bcd orf1 and orf2 proto-oncogene, chromatin-specific transcription elongation factor FACT 140-kDa subunit mRNA, gene encoding splicing factor SF1, and mRNA for Tec protein tyrosine kinase. siRNA-mix and Glivec provoked overexpression of the following common genes: c-jun proto-oncogene, protein kinase C-alpha, pvt-1 oncogene homologue (myc activator), interleukin-6, 1-8D gene from interferon-inducible gene family, tumor necrosis factor receptor superfamily (10b), and STAT-induced STAT inhibitor.

Folate targeting of haptens to cancer cell surfaces mediates immunotherapy of syngeneic murine tumors.

A variety of human cancers overexpress a cell surface receptor with high affinity for the vitamin, folic acid ( K(d) approximately 10(-10)M). Covalent attachment of therapeutic agents to folic acid has been shown to allow efficient targeting of the folate-drug conjugates to folate receptor-expressing cancer cells, with little or no uptake by normal tissues except the kidneys. We report here the use of folate's ability to deliver attached molecules specifically to cancer cells to convert poorly immunogenic tumors into highly immunogenic tissue targets. By linking folic acid to a model hapten, we have been able to decorate folate receptor-expressing cancer cell surfaces with >10(6) haptens/cell in vivo. Following marking of such cells with haptens, the cells are observed to become opsonized with autologous anti-hapten antibodies, which is presumed to mediate cell removal via antibody-dependent cellular cytotoxicity (ADCC). Supplemental administration of low levels of ADCC-activating cytokines [e.g. interleukin-2 (IL-2) and interferon-alpha (IFN-alpha)] has been shown to synergize with the folate-targeted immunotherapy. Thus, using M109 syngeneic lung cancer cells injected intraperitoneally into Balb/c mice that were previously immunized against fluorescein, a significant extension of life span is observed following treatment with folate-fluorescein conjugates, and complete cures are observed upon supplementation with moderate levels of IL-2 and IFN-alpha. Because control tumor-bearing mice treated with the same cytokines but with non-targeted fluorescein show no extension of life span, we conclude that tumor-specific opsonization is an essential step in this immunotherapy. Finally, because the anti-fluorescein antibodies are unable to access the folate receptors on the apical membranes of the kidney proximal tubules, no kidney or other normal tissue cytotoxicity is observed. These data suggest that retargeting of haptens to folate receptor-expressing cancers might constitute a method for mobilizing the immune system specifically against poorly immunogenic tumors.

Efficacy and residues of phloxine B and uranine for the suppression of Mediterranean fruit fly in coffee fields.

The field efficacy of a bait containing phloxine B, uranine and Provesta 621 protein was tested against Mediterranean fruit fly (Ceratitis capitata; Medfly) by aerial and ground spraying in about 84 ha of coffee fields in Kauai, Hawaii, USA. Concurrently, soil and crop samples were collected from the aerially sprayed field and its unsprayed control field for residue studies. Efficacy of the sprays was assessed through trapping with both protein-baited and trimedlure-baited traps and through the infestation level of coffee cherries collected at least three-quarters ripe. The C capitata population was low at the start of the aerial and ground spray studies, but dramatically increased in the control fields. This increase coincided with initial ripening of coffee cherries. During times of peak population levels, C capitata populations were reduced by more than 91% in the ground-sprayed field and 99% in the aerial-sprayed field, relative to the populations in their respective control fields and based on protein-baited trap catches. Results of residue analyses indicated that uranine dissipated quickly compared with phloxine B on coffee and soil. Coffee samples collected at pre-spray periods had phloxine B residues of 7.2-25.5 ng g-1 on berries. Phloxine B concentrations were much higher on coffee leaves (163-1120 ng g-1). Lower concentrations of the dye were found from coffee samples collected during rainy days. Average phloxine B concentrations immediately after spraying were 56 and 2840 ng g-1 in coffee berries and leaves, respectively. Dissipation of phloxine B on berries was fast, with a half-life (t1/2) of 3 days. Dissipation of phloxine B on leaves was fitted to two linear phases: the initial (0-4 days) with a shorter t1/2 of 3 days and the later phase (4-28 days) with a longer t1/2 of 15 days. Average concentrations of phloxine B in the top soil ranged from 50 to 590 ng g-1 at pre-spray. Phloxine B initial concentration (770 ng g-1) reached a plateau immediately after the last spraying, but showed a steady decline over time with t1/2 of 16 days. Fast dissipation of the dyes in the field indicates that these chemicals may be environmentally compatible and therefore a promising alternative for fruit fly control.

Calmodulin regulates the disassembly of cortical F-actin in mast cells but is not required for secretion.

Secretion is dependent on a rise in cytosolic Ca(2+)concentration and is associated with dramatic changes in actin organization. The actin cortex may act as a barrier between secretory vesicles and plasma membrane. Thus, disassembly of this cortex should precede late steps of exocytosis. Here we investigate regulation of both the actin cytoskeleton and secretion by calmodulin. Ca(2+), together with ATP, induces cortical F-actin disassembly in permeabilized rat peritoneal mast cells. This effect is strongly inhibited by removing endogenous calmodulin (using calmodulin inhibitory peptides), and increased by exogenous calmodulin. Neither treatment, however, affects secretion. Low concentrations ( approximately 1 microM) of a specific inhibitor of myosin light chain kinase, ML-7, prevent F-actin disassembly, but not secretion. In contrast, a myosin inhibitor affecting both conventional and unconventional myosins, BDM, decreases cortical disassembly as well as secretion. Observations of fluorescein-calmodulin, introduced into permeabilized cells, confirmed a strong (Ca(2+)-independent) association of calmodulin with the actin cortex. In addition, fluorescein-calmodulin enters the nuclei in a Ca(2+)-dependent manner. In conclusion, calmodulin promotes myosin II-based contraction of the membrane cytoskeleton, which is a prerequisite for its disassembly. The late steps of exocytosis, however, require neither calmodulin nor cortical F-actin disassembly, but may be modulated by unconventional myosin(s).