Vacuolar proton-translocating ATPases (V-ATPases) 2 are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. They reside on intracellular membranes of all eukaryotic cells and function to acidify a variety of intracellular compartments, including secretory vesicles, endosomes, lysosomes, the trans-Golgi network, and the central vacuole of yeast (1-4). Acidification of the vacuolar compartments plays an important role in a number of cellular processes, including receptor-mediated endocytosis, intracellular targeting of newly synthesized lysosomal enzymes, macromolecular processing and degradation, and coupled transport of small molecules. In mammals, V-ATPases also reside at high levels on the plasma membrane of some specialized cells such as kidney epithelial cells and osteoclasts, where they are responsible for transepithelial or cellular proton transport required for normal acid-base homeostasis of the body or bone remodeling (5). Mutations in V-ATPase subunits have been shown to cause renal tubular acidosis (6, 7) and osteopetrosis (8).Although V-ATPases play important physiological roles in a variety of cellular processes, how the V-ATPase-directed proton transport is coupled to cellular metabolism remains poorly understood. Disassembly of V-ATPase in Saccharomyces cerevisiae was first reported to occur in the absence of extracellular glucose (9). Glucose-dependent assembly and regulation of V-ATPase was also observed in mammalian cells (10). However, V-ATPase was found to assemble normally in a panel of yeast mutants with deficiencies in the known glucose-sensing pathways, including the Ras-cAMP, Snf1p, protein kinase C, and Rts1p pathways (11). The glucose-induced assembly of V-ATPase is, therefore, independent of these pathways. In a recent report, the glycolytic enzyme phosphofructokinase has been shown to interact with the "a" subunit of human V-ATPase (12). It remains to be determined whether the interaction between phosphofructose kinase and V-ATPase is physiologically relevant to V-ATPase regulation in either mammalian or yeast cells.In a search for protein partners that interact with V-ATPase, we discovered that the glycolytic enzyme aldolase physically associated with three distinct subunits of V-ATPase (13,14). We examined the growth pattern of yeast mutant cells deficient in aldolase and observed a growth phenotype similar to that previously reported in the V-ATPase subunit deletion mutant cells (14). Our data suggest that aldolase deficiency in yeast cells results in V-ATPase malfunction by disrupting the physical association between aldolase and V-ATPase. Furthermore, when the wild-type aldolase gene was introduced and expressed in aldolase deletion mutant cells, abnormalities in V-ATPase assembly and protein expression were restored to normal levels (14). As a result, ATP hydrolysis and proton transport activities of V-ATPase were also restored to normal levels by aldolase complementation (14). Taken together, these findings ...

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Bevacizumab repackaged in plastic syringes could contain protein aggregates and is contaminated by silicone oil microdroplets. Freeze-thawing or other mishandling can further increase levels of particle contaminants.

INTRODUCTION|: To investigate potentially adverse effects of different topical glaucoma medications and preservatives on cultured ocular epithelial cells. METHODS|: Confluent cultures of human corneal (10.014 pRSV-T) and conjunctival cells (1-5c-4) were assayed with 100 μL of different glaucoma medications for 25 minutes at 37°C and 5% CO₂. We also tested the preservative sofZia® (Alcon Laboratories, Fort Worth, TX, USA), as well as a range of concentrations of the preservative benzalkonium chloride (BAK; 0.001% to 0.050%). Balanced salt solution was used as the "live" control and a solution containing 70% methanol and 0.2% saponin was used as a "dead" control. The LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Carlsbad, CA, USA) was used to determine the percentage of dead and live cells via ethidium homodimer and calcein fluorescence, respectively. RESULTS|: The toxicity of the prostaglandin analogs latanoprost, tafluprost and travoprost preserved with BAK was similar to the toxicity observed in their respective BAK concentrations. The prostaglandin analog travoprost (0.004%) preserved with the oxidizing preservative sofZia had much greater corneal and conjunctival cell survival than travoprost preserved with BAK. Travoprost (0.004%) containing polyquad also performed statistically better than its BAK-preserved formulation. CONCLUSION|: Ocular surface side effects have previously been demonstrated with chronic, long-term exposure to intraocular pressure-lowering medications containing the common preservative BAK. BAK alone has significant in-vitro cytotoxicity to cultured ocular epithelial cells. Substitution of BAK with polyquad or sofZia resulted in significantly higher percentages of live conjunctival and corneal cells. Further studies are needed to understand the- clinical implications of these findings.

This retrospective case series reports sustained elevation of intraocular pressure (IOP) after single or repeated intravitreal injections of bevacizumab (Avastin; Genentech, San Francisco, CA) for wet age-related macular degeneration (AMD). All six cases experienced significant and sustained elevation in IOP after single or repeated intravitreal injections of bevacizumab. Initiation or advancement of IOP-lowering therapy was required in all cases. The results support the need for further studies investigating the incidence of this potential side effect and the need for close long-term surveillance of IOP after injection of bevacizumab, particularly in patients with glaucoma or suspected glaucoma. Future in vitro and in vivo studies are needed to better understand the reasons for this observed phenomenon.

There are significant differences in IgG concentration measured from repackaged bevacizumab syringes. A trend exists for an increase in micron-sized protein aggregates with the decrease in IgG concentration. Large particulate matter within some samples may lead to obstruction of aqueous outflow and subsequent elevation in intraocular pressure. Additional studies are warranted to explore these findings.

-ATPase translocation from cytoplasmic tubulovesicles to apical plasma membrane in parietal cells, resulting in HCl secretion. We studied the mechanisms involved in tubulovesicle translocation with a permeabilized gland system. Streptolysin O (SLO)-treated glands were permeabilized such that exogenous fluorescently labeled actin incorporated into cytoskeleton in a pattern mimicking endogenous F-actin. As shown by accumulation of the weak base aminopyrine (AP), SLO-permeabilized glands are stimulated to secrete acid by addition of cAMP and ATP and inhibited by proton pump inhibitors. Direct visualization with the fluorescent pH probe Lysosensor showed acid accumulation in glandular lumen and parietal cell canaliculi. ME-3407, an antiulcer drug with inhibitory action implicated to involve ezrin, inhibited AP uptake in and effectively released ezrin from intact and SLOpermeabilized glands. In contrast, wortmannin, an effective secretion inhibitor in intact glands, had minimal effects on ezrin or AP accumulation in SLO-permeabilized glands. The finding that SNARE protein syntaxin 3 is associated with H ϩ ,K ϩ -ATPase-containing tubulovesicles suggested that it is involved in membrane fusion. Addition of recombinant syntaxin 3, but not syntaxin 5 or heat-denatured syntaxin 3, dose-dependently inhibited acid secretion. Our studies are consistent with a membrane recycling hypothesis that activation of protein kinase cascades leads to SNARE-mediated fusion of H ϩ ,K ϩ -ATPase-containing tubulovesicles to apical plasma membrane. SNARE hypothesis; membrane fusion; ezrin; wortmannin; hydrogen,potassium-adenosinetriphosphatase THE DEVELOPMENT OF PERMEABILIZED cell preparations has provided a valuable experimental tool linking in vitro studies that use subcellular fractions with direct assays of cellular function. In parietal cell physiology, a number of important functions have been elucidated through the study of gastric glands permeabilized with detergents (reviewed in Ref. 13). One of the major drawbacks with many detergent-like molecules (digitonin or saponin) was that the treated glands were unable to transform from the resting to the stimulated state, although preparations stimulated before treatment could be maintained to secrete acid (10, 15). It was then discovered that pore-forming bacterial toxins permeabilize gastric glands while allowing the transition of resting to a stimulated state (22). These toxins have been valuable in generating permeable cell systems that facilitate better understanding of the parietal cell activation process (19,21,26). Depending on the size of the plasma membrane pore, one can introduce a variety of molecules into the cell to facilitate or interfere with normal functional activity. In previous work, we found ␣-toxin to be a superior permeabilization agent over digitonin and other pore-forming bacterial toxins. We determined (19) that stimulation was dependent on addition of cAMP, that addition of ATP led to synthesis of cAMP and therefore could trigger stimulation, and that addition o...

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