Lisa R. Latchney

Purification of F1-ATPase with impaired catalytic activity from partial revertants of Escherichia coliuncA mutant strains

It is shown that F1-ATPase preparations having impaired catalytic rates may be purified from partial revertants of uncA mutant strains of Escherichia coli. Recovery of catalytic activity in the partial revertant F1 was accompanied by recovery of alpha in equilibrium beta intersubunit conformational interaction, supporting the hypothesis that such interaction is required for normal catalysis in F1. The specific ATPase activities of the partial revertant F1 preparations were in the range 1-29% of normal, and some of the preparations showed unusual insensitivity to inhibitors. The properties of a new uncA mutant F1 preparation (uncA498) which has approximately half of normal catalytic rate are also briefly described.

In vivo imaging of retinal pigment epithelium cells in age related macular degeneration.

Morgan and colleagues demonstrated that the RPE cell mosaic can be resolved in the living human eye non-invasively by imaging the short-wavelength autofluorescence using an adaptive optics (AO) ophthalmoscope. This method, based on the assumption that all subjects have the same longitudinal chromatic aberration (LCA) correction, has proved difficult to use in diseased eyes, and in particular those affected by age-related macular degeneration (AMD). In this work, we improve Morgans method by accounting for chromatic aberration variations by optimizing the confocal aperture axial and transverse placement through an automated iterative maximization of image intensity. The increase in image intensity after algorithmic aperture placement varied depending upon patient and aperture position prior to optimization but increases as large as a factor of 10 were observed. When using a confocal aperture of 3.4 Airy disks in diameter, images were obtained using retinal radiant exposures of less than 2.44 J/cm(2), which is ~22 times below the current ANSI maximum permissible exposure. RPE cell morphologies that were strikingly similar to those seen in postmortem histological studies were observed in AMD eyes, even in areas where the pattern of fluorescence appeared normal in commercial fundus autofluorescence (FAF) images. This new method can be used to study RPE morphology in AMD and other diseases, providing a powerful tool for understanding disease pathogenesis and progression, and offering a new means to assess the efficacy of treatments designed to restore RPE health.

Inhibition of Escherichia coli H+-ATPase by venturicidin, oligomycin and ossamycin

The antibiotics venturicidin, oligomycin and ossamycin were investigated as potential inhibitors of the Escherichia coli H+-ATPase. It was found that venturicidin strongly inhibited ATP-driven proton transport and ATP hydrolysis, while oligomycin weakly inhibited these functions. Inhibition of the H+-ATPase by venturicidin and oligomycin was correlated with inhibition of F0-mediate proton transport. Both inhibitors were found to interfere with the covalent reaction between dicyclohexyl[14C]carbodiimide and the F0 subunit c (uncE protein). Ossamycin had no direct inhibitory effect on E. coli F0 or F1; rather, it was found to uncouple ATP hydrolysis from proton transport.

Imaging individual neurons in the retinal ganglion cell layer of the living eye

Significance Retinal ganglion cells are the primary output neurons of the retina that process visual information and transmit it to the brain. We developed a method to reveal these cells in the living eye that does not require the fluorescent labels or high light levels that characterize more invasive methods. The death of these cells causes vision loss in glaucoma, the second leading cause of blindness worldwide. The ability to image these cells in the living eye could accelerate our understanding of their role in normal vision and provide a diagnostic tool for evaluating new therapies for retinal disease. Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain.

Cone and Rod Loss in Stargardt Disease Revealed by Adaptive Optics Scanning Light Ophthalmoscopy

IMPORTANCE Stargardt disease (STGD1) is characterized by macular atrophy and flecks in the retinal pigment epithelium. The causative ABCA4 gene encodes a protein localizing to photoreceptor outer segments. The pathologic steps by which ABCA4 mutations lead to clinically detectable retinal pigment epithelium changes remain unclear. We investigated early STGD1 using adaptive optics scanning light ophthalmoscopy. OBSERVATIONS Adaptive optics scanning light ophthalmoscopy imaging of 2 brothers with early STGD1 and their unaffected parents was compared with conventional imaging. Cone and rod spacing were increased in both patients (P < .001) with a dark cone appearance. No foveal cones were detected in the older brother. In the younger brother, foveal cones were enlarged with low density (peak cone density, 48.3 × 103 cones/mm2). The ratio of cone to rod spacing was increased in both patients, with greater divergence from normal approaching the foveal center, indicating that cone loss predominates centrally and rod loss increases peripherally. Both parents had normal photoreceptor mosaics. Genetic testing revealed 3 disease-causing mutations. CONCLUSIONS AND RELEVANCE This study provides in vivo images of rods and cones in STGD1. Although the primary clinical features of STGD1 are retinal pigment epithelial lesions, adaptive optics scanning light ophthalmoscopy reveals increased cone and rod spacing in areas that appear normal in conventional images, suggesting that photoreceptor loss precedes clinically detectable retinal pigment epithelial disease in STGD1.

Immunohistochemical assessment of fractalkine, inflammatory cells, and human herpesvirus 7 in human salivary glands.

Human fractalkine (CX3CL1), a 8-chemokine, is implicated in the mediation of multiple cell functions. In addition to serving as a chemotactic factor for mononuclear cell subtypes, membrane-bound fractalkine may promote viral infection by interacting with virions that encode putative fractalkine-binding proteins. Fractalkine expression in normal epithelial tissues studied to date is either constitutive or is upregulated with inflammation. In salivary glands, the expression of fractalkine is unknown. Moreover, salivary glands are a major site for the persistent and productive infection by human herpesvirus (HHV)-7, which encodes two putative fractalkine-binding gene products, U12 and U51. Surprisingly, the cellular distribution of HHV-7 in major salivary glands has not been explored. We therefore determined by immunohistochemistry the cellular localization of fractalkine in three different salivary glands: parotid, submandibular, and labial glands. Fractalkine expression was highly variable, ranging from high to undetectable levels. We further examined the association of fractalkine with inflammatory cell infiltration or HHV-7 infection of salivary epithelial cells. Inflammatory cells were always adjacent to epithelial cells expressing fractalkine, consistent with a function of fractalkine in inflammatory cell recruitment and/or retention in salivary glands. In contrast, HHV-7-infected epithelial cells did not always express fractalkine, suggesting that fractalkine may not be an absolute requirement for viral entry.

Biochemical and immunological studies and assay of rat sublingual mucins.

Original studies of rat sublingual mucins raised questions as to the existence of a second mucin species as distinguished by binding to hydroxyapatite. The existence of multiple mucin species is of concern in pharmacological studies of mucous-cell secretion as each species could represent distinct mucous-cell populations that respond differently to secretagogues. Thus a separate hydroxyapatite-bound mucin pool expressed in rat sublingual glands was isolated and characterized. Biochemical comparison of hydroxyapatite-bound mucins to total and hydroxyapatite-unbound sublingual mucins demonstrated no substantial differences in either amino acid and carbohydrate contents or in size distributions. In addition, a radioimmunoassay was developed using antisera prepared previously against unbound mucins. The three mucin pools exhibited equal specificities in displacement of radiolabelled unbound mucin tracer in the radioimmunoassay. Thus, bound and unbound mucins are indistinguishable, both immunologically and in biochemical composition. The radioimmunoassay was then evaluated for use in pharmacological studies of acinar mucous-cell secretion. Measurement by radioimmunoassay of secretion from isolated acini in response to carbachol was concentration-dependent (EC50 approx. 0.3 microM and maximal stimulation at 1 microM carbachol). In immunolocalization studies the antiserum was highly selective for mucous cells, recognized all mucous cells within histological sections, and was localized subcellularly to mucous-cell secretion granules and trans-Golgi, further validating the radioimmunoassay as a method to detect exocrine secretion from the entire pool of acinar mucous cells. Moreover, the radioimmunoassay was compared and found equivalent to an acid-precipitation method to assess relative secretion, suggesting the acid-precipitation method is also valid for pharmacological studies of isolated acini.

Drusen in patient-derived hiPSC-RPE models of macular dystrophies

Significance Age-related macular degeneration (AMD) and related macular dystrophies (MDs) are a major cause of vision loss. However, pharmacological treatments in these diseases are limited due to the lack of knowledge of underlying disease mechanisms, partly because appropriate human models to study AMD and related MDs are lacking. Furthermore, in the living human eye, the entire retina acts as a functional unit, making it difficult to investigate the specific contribution of a particular retinal cell type in the disease. Here, we established human models of multiple MDs, which demonstrated similar molecular and phenotypic manifestations within these diseases. Furthermore, we showed that dysfunction of an individual cell type, retinal pigment epithelium cells in the retina, is sufficient for the development of key pathological features in these MDs. Age-related macular degeneration (AMD) and related macular dystrophies (MDs) are a major cause of vision loss. However, the mechanisms underlying their progression remain ill-defined. This is partly due to the lack of disease models recapitulating the human pathology. Furthermore, in vivo studies have yielded limited understanding of the role of specific cell types in the eye vs. systemic influences (e.g., serum) on the disease pathology. Here, we use human induced pluripotent stem cell-retinal pigment epithelium (hiPSC-RPE) derived from patients with three dominant MDs, Sorsby’s fundus dystrophy (SFD), Doyne honeycomb retinal dystrophy/malattia Leventinese (DHRD), and autosomal dominant radial drusen (ADRD), and demonstrate that dysfunction of RPE cells alone is sufficient for the initiation of sub-RPE lipoproteinaceous deposit (drusen) formation and extracellular matrix (ECM) alteration in these diseases. Consistent with clinical studies, sub-RPE basal deposits were present beneath both control (unaffected) and patient hiPSC-RPE cells. Importantly basal deposits in patient hiPSC-RPE cultures were more abundant and displayed a lipid- and protein-rich “drusen-like” composition. Furthermore, increased accumulation of COL4 was observed in ECM isolated from control vs. patient hiPSC-RPE cultures. Interestingly, RPE-specific up-regulation in the expression of several complement genes was also seen in patient hiPSC-RPE cultures of all three MDs (SFD, DHRD, and ADRD). Finally, although serum exposure was not necessary for drusen formation, COL4 accumulation in ECM, and complement pathway gene alteration, it impacted the composition of drusen-like deposits in patient hiPSC-RPE cultures. Together, the drusen model(s) of MDs described here provide fundamental insights into the unique biology of maculopathies affecting the RPE–ECM interface.

Trypsin cleavage of the α-subunit of beef heart F1-ATPase abolishes ATP synthesis and ATP-driven energy-transduction capabilities

Previous work has shown that mild trypsin treatment eliminates energy-transduction capability and tight (non-exchangeable)nucleotide binding in beef heart mitochondrial F1-ATPase (Leimgruber, R.M. and Senior, A.E. (1976) J. Biol. Chem. 251, 7103-7109). The structural change brought about by trypsin was, however, too subtle to be identified by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, and was not defined. In this work we have applied two-dimensional electrophoresis (isoelectric focussing then sodium dodecyl sulfate polyacrylamide gradient electrophoresis) to the problem, and have determined that the alpha-subunit of F1 is altered by the mild trypsin treatment, whereas no change was detected in beta-, gamma-, delta- or epsilon-subunits. Binding of ADP to the trypsin-treated F1 was compared to binding to control enzyme over a range of 0-40 muM ADP in a 30 min incubation period. There was no difference between the two enzymes, KADPd in Mg2+ -containing buffer was about 2 muM in each. Since the tight (nonexchangeable)sites are abolished in trypsin-treated F1, this shows that tight exchangeable ADP-binding sites are different from the tight nonexchangeable ADP-binding sites. There was no effect of trypsin cleavage of the alpha-subunit on beta-subunit conformation as judged by aurovertin fluorescence studies. The cleavage of the alpha-subunit which occurred was judged to occur very close to the C- or N-terminus of the subunit and constitutes therefore a small and specific chemical modification which abolishes overall function in F1 but leaves partial functions intact.

Fluorescence Adaptive Optics Scanning Laser Ophthalmoscope for Detection of Reduced Cones and Hypoautofluorescent Spots in Fundus Albipunctatus

IMPORTANCE Fundus albipunctatus (FA) is a form of congenital stationary night blindness characterized by yellow-white spots, which were classically described as subretinal. Although night blindness and delayed dark adaptation are hallmarks of this condition, recent studies have described a macular phenotype, particularly among older patients. Using a fluorescence adaptive optics scanning laser ophthalmoscope (FAOSLO), this study provides in vivo morphologic data at the cellular level in FA. OBJECTIVE To study the cone photoreceptors and the albipunctate spots in FA at single-cell resolution. DESIGN, SETTING, AND PARTICIPANT A woman in her 30s with FA underwent a complete ophthalmic examination, including conventional imaging tests, at the University of Rochester. A FAOSLO was used to obtain infrared reflectance images of the cone mosaic at the central fovea and along the superior and temporal meridians to 10° eccentricity. Cone density was measured at the foveal center, and cone spacing was calculated in sampling windows eccentrically. In the area of the albipunctate spots, autofluorescence FAOSLO images (excitation, 561 nm; emission, 624 Δ 40 nm) were simultaneously obtained. MAIN OUTCOMES AND MEASURES Structural appearance of cones, cone density and spacing, and reflectance and autofluorescence of albipunctate spots. RESULTS Cone density was reduced to 70% of the lower limit of the normal range at the foveal center (78.7 × 10(3) cones/mm(2); mean [SD] reference range, 199 [87] × 10(3) cones/mm(2)), and cone spacing was increased eccentrically to 10° (sign test, P = .045). Individual cone central core reflectances appeared dim, suggesting loss of photoreceptor outer segments. The albipunctate spots were hypoautofluorescent. No photoreceptors or retinal pigment epithelium cells were identified at the locations of the albipunctate spots. CONCLUSIONS AND RELEVANCE Although the predominant clinical symptom of night blindness and the electroretinography results suggest a primary rod dysfunction, examination with a FAOSLO demonstrates that cone density is also reduced. This finding may represent an early sign of progression to macular phenotype in FA. The hypoautofluorescence suggests that the albipunctate spots do not represent lipofuscin.