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Mitochondrial (mt) DNA haplogroups, defined by specific single nucleotide polymorphism (SNP) patterns, represent populations of diverse geographic origins and have been associated with increased risk or protection of many diseases. The H haplogroup is the most common European haplogroup while the K haplogroup is highly associated with the Ashkenazi Jewish population. Transmitochondrial cybrids (cell lines with identical nuclei, but mtDNA from either H (n = 8) or K (n = 8) subjects) were analyzed by the Seahorse flux analyzer, quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry (IHC). Cybrids were treated with amyloid-β peptides and cell viabilities were measured. Other cybrids were demethylated with 5-aza-2′-deoxycytidine (5-aza-dC) and expression levels for APOE and NFkB2 were measured. Results show K cybrids have (a) significantly lower mtDNA copy numbers, (b) higher expression levels for MT-DNA encoded genes critical for oxidative phosphorylation, (c) lower Spare Respiratory Capacity, (d) increased expression of inhibitors of the complement pathway and important inflammasome-related genes; and (e) significantly higher levels of APOE transcription that were independent of methylation status. After exposure to amyloid-β1–42 peptides (active form), H haplogroup cybrids demonstrated decreased cell viability compared to those treated with amyloid-β42–1 (inactive form) (p < 0.0001), while this was not observed in the K cybrids (p = 0.2). K cybrids had significantly higher total global methylation levels and differences in expression levels for two acetylation genes and four methylation genes. Demethylation with 5-aza-dC altered expression levels for NFkB2, while APOE transcription patterns were unchanged. Our findings support the hypothesis that mtDNA-nuclear retrograde signaling may mediate expression levels of APOE, a key factor in many age-related diseases. Future studies will focus on identification of the mitochondrial-nuclear retrograde signaling mechanism(s) contributing to these mtDNA-mediated differences.

Background: Cisplatin, a powerful antitumor agent, causes formation of DNA adducts, and activation of apoptotic pathways. Presently, cisplatin resistance develops in up to 70% of patients but the underlying molecular mechanism(s) are unclear and there are no markers to determine which patients will become resistant. Mitochondria play a significant role not only in energy metabolism but also retrograde signaling (mitochondria to nucleus) that modulates inflammation, complement, and apoptosis pathways. Maternally inherited mitochondrial (mt) DNA can be classified into haplogroups representing different ethnic populations that have diverse susceptibilities to diseases and medications. Methods: Transmitochondrial cybrids, where all cell lines possess identical nuclear genomes but either the H (Southern European) or J (Northern European) mtDNA haplogroups, were treated with cisplatin and analyzed for differential responses related to viability, oxidative stress, and expression levels of genes associated with cancer, cisplatin-induced nephrotoxicity and resistance, apoptosis and signaling pathways. Results: The cisplatin-treated-J cybrids showed greater loss of cell viability along with lower levels of reactive oxygen species and mitochondrial membrane potential compared to cisplatin-treated-H cybrids. After cisplatin treatment, J cybrids showed increased gene expression of BAX, CASP3, and CYP51A, but lower levels of SFRP1 compared to untreated-J cybrids. The cisplatin-treated-H cybrids had elevated expression of CDKN1A/P21, which has a role in cisplatin toxicity, compared to untreated-H cybrids. The cisplatin-treated H had higher transcription levels of ABCC1, DHRS2/HEP27, and EFEMP1 compared to cisplatin-treated-J cybrids. Conclusions: Cybrid cell lines that contain identical nuclei but either H mtDNA mitochondria or J mtDNA mitochondria respond differently to cisplatin treatments suggesting involvement of the retrograde signaling (from mitochondria to nucleus) in the drug-induced cell death. Varying toxicities and transcription levels of the H vs. J cybrids after cisplatin treatment support the hypothesis that mtDNA variants play a role in the expression of genes affecting resistance and side effects of cisplatin.

Mitochondrial (mt) DNA haplogroups, defined by specific single nucleotide polymorphism (SNPs) patterns, represent populations of diverse geographic origins and may play a role in disparate disease susceptibilities found in different ethnic/racial populations. The most common European haplogroup is H, while the K haplogroup is highly associated with Ashkenazi Jewish populations. Studies using transmitochondrial cybrids (cell lines with identical nuclei but mitochondria from either H or K haplogroup subjects) demonstrated significant molecular and biological differences but mechanisms for these disparities are unclear. In this study, we hypothesized that there is differential retrograde signaling occurring between the Stimulator of Interferon Genes (STING) pathway and H versus K mtDNA haplogroups. Results showed that K cybrids exhibit increased levels of cytoplasmic mtDNA fragments. After STING Knock-Down, H cybrids had lower expression levels for EGFR, BRCA1, DNMT3A, DNMT3B, HDAC1, and IFNα genes, but upregulated DNMT3A compared to control H cybrids. The STING-KD K cybrids showed downregulation of EGFR, DNMT3A, HDAC1, HCAD9, CFH, and CHI, along with upregulation of DNMT1 and IL-6 compared to control K cybrids. Since all cybrids have identical nuclei, the STING DNA sensor system interacts differently with K haplogroup mtDNA compared to H mtDNA for genes related to cancer (EGFR, BRCA1), methylation (DNMT1, DNMT3A, DNMT3B), acetylation (HDAC1, HDCA9), complement (CFH, CHI) and inflammation (IFNα, IL-6). In summary, in non-pathologic conditions, (a) STING is an important retrograde signaling mechanism(s) and (b) cybrids possessing Ashkenazi Jewish mtDNA (K haplogroup) interact with the STING complex differently compared to H cybrids which affects various disease-related pathways.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

In a volcano monitoring application, various geophysical and geochemical sensors generate continuous high-fidelity data, and there is a compelling need for real-time raw data for volcano eruption prediction research. It requires the network to support network synchronized sampling, online configurable sensing and situation awareness, which pose significant challenges on sensing component design. Ideally, the resource usages shall be driven by the environment and node situations, and the data quality is optimized under resource constraints. In this paper, we present our smart sensing component design, including hybrid time synchronization, configurable sensing, and situation awareness. Both design details and evaluation results are presented to show their efficiency. Although the presented design is for a volcano monitoring application, its design philosophy and framework can also apply to other similar applications and platforms.

Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, and expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and -J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2'-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases.

The yeast Saccharomyces cerevisiae has a finite replicative life span. Yeasts possess two prohibitins, Phb1p and Phb2p, in similarity to mammalian cells. These proteins are located in the inner mitochondrial membrane, where they are involved in the processing of newly-synthesized membrane proteins. We demonstrate that the elimination of one or both of the prohibitin genes in yeast markedly diminished the replicative life span of cells that lack fully-functional mitochondria, while having no effect on cells with functioning mitochondria. This deleterious effect was suppressed by the deletion of the RAS2 gene. The expression of PHB1 and PHB2 declined gradually up to 5-fold during the life span. Cells in which PHB1 was deleted in conjunction with the absence of a mitochondrial genome displayed remarkable changes in mitochondrial morphology, distribution, and inheritance. This loss of mitochondrial integrity was not seen in cells devoid of PHB1 but possessing an intact mitochondrial genome. In a subset of the cells, the changes in mitochondrial integrity were associated with increased production of reactive oxygen species, which co-localized with the altered mitochondria. The mitochondrial deficits described above were all suppressed by deletion of RAS2. Our data, together with published information, are interpreted to provide a unified view of the role of the prohibitins in yeast aging. This model posits that the key initiating event is a decline in mitochondrial function, which leads to progressive oxidative damage that is exacerbated in the absence of the prohibitins. This aggravation of the initial damage is ameliorated by the suppression of the production of mitochondrial proteins in the absence of Ras2p signaling of mitochondrial biogenesis.

Human Molecular Genetics, 2014, Vol. 23, No. 13 doi:10.1093/hmg/ddu065 Advance Access published on February 28, 2014 Inherited mitochondrial DNA variants can affect complement, inflammation and apoptosis pathways: insights into mitochondrial –nuclear interactions M. Cristina Kenney 1,2, ∗ , Marilyn Chwa 1 , Shari R. Atilano 1 , Payam Falatoonzadeh 1 , Claudio Ramirez 1 , Deepika Malik 1 , Mohamed Tarek 1 , Javier Ca´ceres-del-Carpio 1 , Anthony B. Nesburn 1,4 , David S. Boyer 5 , Baruch D. Kuppermann 1 , Marquis Vawter 3 , S. Michal Jazwinski 6 , Michael Miceli 6 , Douglas C. Wallace 7 and Nitin Udar 1 Gavin Herbert Eye Institute, 2 Department of Pathology and Laboratory Medicine, 3 Functional Genomics Laboratory, Department of Psychiatry and Human Behavior, University of California Irvine, Irvine, CA, USA, 4 Cedars-Sinai Medical Center, Los Angeles, CA, USA, 5 Retina-Vitreous Associates Medical Group, Beverly Hills, CA, USA, 6 Tulane Center for Aging, Tulane University, New Orleans, LA, USA and 7 Children’s Hospital of Philadelphia, Center for Mitochondrial and Epigenomic Medicine, Philadelphia, PA, USA Received December 17, 2013; Revised February 3, 2014; Accepted February 10, 2014 Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries. While linked to genetic polymorphisms in the complement pathway, there are many individuals with high risk alleles that do not develop AMD, suggesting that other ‘modifiers’ may be involved. Mitochondrial (mt) haplogroups, defined by accu- mulations of specific mtDNA single nucleotide polymorphisms (SNPs) which represent population origins, may be one such modifier. J haplogroup has been associated with high risk for AMD while the H haplogroup is protective. It has been difficult to assign biological consequences for haplogroups so we created human ARPE-19 cybrids (cyto- plasmic hybrids), which have identical nuclei but mitochondria of either J or H haplogroups, to investigate their effects upon bioenergetics and molecular pathways. J cybrids have altered bioenergetic profiles compared with H cybrids. Q-PCR analyses show significantly lower expression levels for seven respiratory complex genes encoded by mtDNA. J and H cybrids have significantly altered expression of eight nuclear genes of the alternative complement, inflammation and apoptosis pathways. Sequencing of the entire mtDNA was carried out for all the cybrids to identify haplogroup and non-haplogroup defining SNPs. mtDNA can mediate cellular bioenergetics and expression levels of nuclear genes related to complement, inflammation and apoptosis. Sequencing data sug- gest that observed effects are not due to rare mtDNA variants but rather the combination of SNPs representing the J versus H haplogroups. These findings represent a paradigm shift in our concepts of mt–nuclear interactions. INTRODUCTION Mitochondria (mt) are unique organelles that have their own DNA. Human mtDNA is maternally inherited and forms a circle of double-stranded DNA with 16 569 nucleotide pairs. The non-coding mt-Dloop (also called the control region) con- tains 1121 nucleotides important for replication and transcrip- tion. The coding region of mtDNA encodes for 37 genes, including 13 protein subunits essential for oxidative phosphorylation (OXPHOS), 2 ribosomal RNAs and 22 transfer RNAs (1 – 3). The mtDNA can be categorized into haplogroups that are defined by a group of single nucleotide polymorphism (SNP) variants that have accumulated over tens of thousands of years and correspond to various geographic populations. While some studies have shown that certain mtDNA hap- logroups can be either protective or increase risk for human dis- eases (4–10), others report no haplogroup association with diseases (11– 13). However, many of these studies identified To whom correspondence should be addressed at: Gavin Herbert Eye Institute, Discovery Center for Eye Research, University of California Irvine, Hewitt Hall, Room 2028, 843 Health Science Road, Irvine, CA 92697, USA. Tel: +1 9498247603; Fax: +1 9498249626; Email: mkenney@uci.edu # The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Purpose. Due to a possible link in humans between atherosclerosis, a high-fat diet, and the development of age-related retinal degenerations, we investigated retinal changes with age and diet in high-fat-fed C57BL/6 mice, a mouse model of human atherosclerosis. Methods. We fed C57BL/6J mice either a normal chow diet or an atherogenic diet containing 15% total fat for either 15, 30 or 45 weeks. We sacrificed the animals and examined the eyes using fluorescence microscopy, light microscopy and transmission electron microscopy. Results. At 15 weeks, in high-fat-fed mice, there was an increase in the number of autofluorescent granules in the retinal pigment epithelium (RPE) compared to chow-fed mice. By light microscopy, we noted no remarkable differences in the inner retinal layers between mice fed either diet for 15 or 30 weeks. In contrast, we noted major changes in transmission electron micrographs from the 30 week high-fat group. In the RPE, these included: (1) an increase in the number and size of autop...

We have previously shown that an experimental, low-zinc environment decreased catalase activity in cultured human fetal retinal pigment epithelial (RPE) cells. The purpose of this study was to investigate the effect of zinc supplementation on catalase expression in cultured human fetal RPE cells.Confluent fetal RPE cells incubated in Coon's modified Ham's F12 (CMF-12) were treated (18 h) with zinc chloride (ZnCl2) (15, 30, or 100 microM) to assess changes in catalase enzyme activity or for 6 h to assess the induction of catalase mRNA by Northern analysis and in situ hybridization. RPE cells were also treated with 30 microM ZnCl2 for 2, 6, 24, 48 and 72 h to assess the time course of changes in catalase enzyme activity, changes in mRNA levels and status of the Sp1 transcription factor.Catalase activity was increased above control by the addition of 15, 30 and 100 microM ZnCl2. Catalase gene expression was induced by 30 microM zinc in 6 h, but decreased to non-treated control levels by 24 h. The transcription factor Sp1 was also activated by zinc treatment (30 microM) which peaked at 2 h and declined to non-treated control levels by 24 h. Catalase enzyme activity peaked at 24 h and decreased to control levels by 72 h.Our results demonstrate that zinc treatment of RPE cells increases catalase expression and activates the transcription factor Sp1. The results suggest zinc may play a role in the transcriptional regulation of catalase in RPE cells.

Mitochondrial DNA Variants Mediate Energy Production and Expression Levels for CFH, C3 and EFEMP1 Genes: Implications for Age-Related Macular Degeneration M. Cristina Kenney 1 *, Marilyn Chwa 1 , Shari R. Atilano 1 , Janelle M. Pavlis 1 , Payam Falatoonzadeh 1 , Claudio Ramirez 1 , Deepika Malik 1 , Tiffany Hsu 1 , Grace Woo 1 , Kyaw Soe 1 , Anthony B. Nesburn 1,2 , David S. Boyer 3 , Baruch D. Kuppermann 1 , S. Michal Jazwinski 4 , Michael V. Miceli 4 , Douglas C. Wallace 5 , Nitin Udar 1 1 Gavin Herbert Eye Institute, University of California Irvine, Irvine, California, United States of America, 2 Cedars-Sinai Medical Center, Los Angeles, California, United States of America, 3 Retina-Vitreous Associates Medical Group, Beverly Hills, California, United States of America, 4 Tulane Center for Aging and Department of Medicine, Tulane University, New Orleans, Louisiana, United States of America, 5 Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania, United States of America Abstract Background: Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD). Methodology/Principal Findings: Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism. Conclusion/Significance: Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD. Citation: Kenney MC, Chwa M, Atilano SR, Pavlis JM, Falatoonzadeh P, et al. (2013) Mitochondrial DNA Variants Mediate Energy Production and Expression Levels for CFH, C3 and EFEMP1 Genes: Implications for Age-Related Macular Degeneration. PLoS ONE 8(1): e54339. doi:10.1371/journal.pone.0054339 Editor: Walter Lukiw, Louisiana State University Health Sciences Center, United States of America Received October 1, 2012; Accepted December 10, 2012; Published January 24, 2013 Copyright: s 2013 Kenney et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding provided by The Discovery Eye Foundation www.discoveryeye.org, Lincy Foundation lincyinstitute.unlv.edu/lincy.html, Beckman Macular Research Initiative www.beckmanmacular.org, The Henry Guenther Foundation, Polly and Michael Smith Foundation, Research to Prevent Blindness Foundation www.rpbusa.org, and the National Institute on Aging (AG006168). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mkenney@uci.edu specific SNP variants that have accumulated over tens of thousands of years and correspond to different geographic populations of the world. The H haplogroup is the most common European haplogroup, while the J haplogroup originates from the Northern European region and is defined by SNP variants that are associated with heat production as an adaptation to colder climates [4]. The mtDNA plays an important role in aging and diseases [4– 6]. Specific haplogroups are associated with a variety of eye diseases including age-related macular degeneration (AMD) [7– 10], diabetic retinopathy [11], pseudoexfoliation glaucoma [12,13], primary open-angle glaucoma [14], keratoconus [15], multiple sclerosis-related optic neuritis [16,17], and Leber Introduction Mitochondria provide critical cellular energy using the tricar- boxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and beta-oxidation of fatty acids for metabolism, cell division, production of reactive oxygen species (ROS), and apoptosis. Human mitochondrial (mt) DNA forms a circle of double stranded DNA with 16,569 nucleotide pairs. The non-coding mtDNA Dloop contains 1121 nucleotides and is important for replication and transcription. The coding region of mtDNA encodes for 37 genes including 13 protein subunits essential for OXPHOS, 2 ribosomal RNAs, and 22 transfer RNAs [1–3]. The mtDNA can be categorized into haplogroups that are defined by a set of PLOS ONE | www.plosone.org January 2013 | Volume 8 | Issue 1 | e54339

We previously reported an age-related decline of catalase activity and metallothionein (MT) in human retinal pigment epithelium (RPE) isolated from donor eyes. MT content and catalase activity also were shown to decrease in RPE cells cultured in zinc-deficient medium. We now sought to determine whether zinc content and superoxide dismutase activity change with age and signs of macular degenerative disease in isolated human RPE. Eyes from 57 donors were graded with respect to disease by the presence or absence of visible drusen and macular pigment changes. RPE was collected from macular and peripheral regions. Tissues from 39 donors were separated into soluble and pigment granule fractions and zinc content determined by flame atomic absorption spectroscopy. RPE from 18 separate donors was used for SOD activity analysis. Total RPE zinc was 9% lower in eyes from donors >70 years of age compared to that from donors age 70 and showed a greater decline in the soluble fraction of macular RPE in eyes with signs of macular disease. Total SOD activity increased significantly (P < .05) with donor age. We conclude that zinc content decreased dramatically in the soluble fraction of macular RPE with age and signs of macular degeneration. Total SOD activity correlated negatively with changes in zinc content. It remains to be determined whether the age-related decline in macular zinc is causal with respect to previously observed decreases in cellular MT and catalase. © 1996 Wiley-Liss, Inc.

The goal of this study was to determine the effect of extracellular matrix components on the phagocytic function of the retinal pigment epithelial (RPE) cell. Cultured human fetal RPE cells were established in culture and plated on three commercially-prepared substrates: collagen IV, fibronectin and laminin and on three native matrices: bovine corneal endothelial cell matrix (BCEM) denuded bovine Bruch's membrane and denuded human Bruch's membrane. Cultured cells were allowed to become confluent and maintained for an additional two weeks before uptake of fluorescent bovine retinal outer segments (ROS) was measured by flow cytometry. Morphology by phase contrast microscopy and melanization was also determined as measures of differentiation. The results showed that morphology, melanization and ROS uptake by cells on collagen IV, laminin and fibronectin were not different from control cells plated on tissue culture plastic. However, ROS uptake by cells plated on BCEM was significantly less than that of cells cultured on plastic and melanization was greater. ROS uptake by cells plated on both types of Bruch's membrane was also significantly less than control cells. Treatment of cells plated on tissue culture plastic with 44 mM NaHCO3, which increased melanization, also reduced ROS uptake. We conclude that native matrices seem to contain components that significantly depress ROS uptake in culture. The inhibition is not mimicked by collagen, laminin or fibronectin coated wells. The ECM may play a significant role in controlling phagocytosis of ROS either by determining morphology, increasing differentiation or by directly influencing intracellular metabolism, and thus serve as another level of control for this RPE function which may not occur in cells plated on tissue culture plastic. These results may also have implications for the effects of aging or disease in which there are changes in the ECM.

An investigation was undertaken to determine the toxicity of an intravitreal injection of a novel peptide drug, Shiva-1, in rabbits. The drug, a synthetic peptide modeled after lytic peptides secreted by certain insects, has antiproliferative and antibacterial properties. Initial in vitro experiments showed that the drug, at a concentration of 100 microM, was toxic to both Y-79 retinoblastoma cells and human retinal pigment epithelial cells. A wide range of doses (6-1200 micrograms) was injected into the rabbit vitreous in an attempt to determine the maximum tolerated dose. Retinal toxicity was evaluated clinically, by electroretinography, and by light microscopy. Some localized toxicity was evident at 200 micrograms; all doses of 240 micrograms and above were toxic. While the drug appears to exhibit a narrow range between effective and toxic doses, the results suggest that this and other peptides of similar design merit further investigation for the treatment of proliferative and infectious diseases of the eye.

This study investigates the mechanism by which insulin stimulates phagocytosis in cultured human retinal pigment epithelium, using a flow cytometric assay of retinal outer segment uptake. RPE cells were isolated from adult human donors and grown in culture. Retinal outer segments were isolated from fresh bovine eyes and covalently labeled with the fluorescent dye carboxy-SNAFL-2. Retinal outer segment binding and uptake was quantified by direct visualization and the increase in cellular fluorescence measured using a flow cytometer and the methods compared. Uptake measured by flow cytometry was further characterized by concentration, time, temperature and serum stimulation, and shown to be comparable to other published methods. Uptake of retinal outer segments was acutely stimulated by insulin in the absence or presence of serum with half maximal stimulation occurring between 0.1 and 1.0 microgram ml-1. Theophylline, forskolin and cholera toxin all reduced retinal outer segment uptake by RPE cells, but had no effect upon insulin stimulation. Measurements of cAMP showed that insulin did not change intracellular cAMP concentration compared to controls in the absence or presence of added retinal outer segments. One hundred nanomolar okadaic acid also inhibited retinal outer segment uptake but not insulin-stimulated uptake, nor did 100 microM genistein have an effect upon insulin-stimulated uptake. Preincubation of RPE cells with 25 microM ZnCl2 overnight stimulated retinal outer segment uptake and appeared to inhibit the insulin stimulation. Preincubation of the cells in 25 mM glucose overnight also increased the uptake of retinal outer segments over control and reduced the effect of insulin. We conclude that insulin stimulates retinal outer segment phagocytosis by an as yet unknown process which may involve specific tyrosine phosphatases.

To investigate the nature of the oxidative event that occurs during phagocytosis of retinal outer segments (ROS) by cultured human retinal pigment epithelial (RPE) cells, cells were incubated with isolated bovine ROS labeled with either the fluorescence probe carboxy-SNAFL-2 or the nonfluorescent, oxidizable probe 2',7'-dichlorodihydrofluorescein (H2DCF). The increase in fluorescence following phagocytosis was measured by a flow cytometer. Other measurements included: oxygen consumption using a Clark-type oxygen electrode, extracellular superoxide release by superoxide dismutase inhibitable lucigenin chemiluminescence, intracellular hydrogen peroxide (H2O2) production, and the effect of catalase inhibition on cellular thiobarbituric acid-reactive substances (TBARS) caused by phagocytosis. The activities of the enzymes NADPH oxidase and palmitoyl-CoA oxidase were also measured. H2DCF attached to bovine ROS was oxidized during phagocytosis with a time course suggesting oxidation subsequent to ROS uptake. Measurements of oxygen consumption showed a time-dependent increase of 10%, 4 h after ROS feeding, attributable to a doubling of the cyanide-resistant oxygen consumption. Intracellular H2O2 production also doubled 4 h after ROS phagocytosis. ROS uptake by RPE cells produced no significant extracellular superoxide, while extracellular superoxide production was readily demonstrated in a control macrophage cell line. Enzyme activity measurements showed that incubation of RPE cells with ROS doubled catalase activity without affecting superoxide dismutase or glutathione peroxidase activities. Inhibition of catalase during ROS uptake increased TBARS by 66%. Other enzyme activity measurements showed that human RPE cells possess both NADPH oxidase and palmitoyl-CoA oxidase activities. We conclude that ROS phagocytosis subjects RPE cells to an oxidative event on the same order of magnitude as measured in a macrophage. The event is not an extracellular macrophage-type respiratory burst and may be due to intracellular H2O2 resulting from an NADPH oxidase in the phagosome or from beta-oxidation of ROS lipids in peroxisomes. Irrespective of case, the enzyme catalase appears to be essential in protecting the RPE cell against reactive oxygen species produced during phagocytosis.

Grids, clouds and cloud-like infrastructures are capable of supporting a broad range of data-intensive applications. There are interesting and unique performance issues that appear as the volume of data and degree of distribution increases. New scalable data-placement and management techniques, as well as novel approaches to determine the relative placement of data and computational workload, are required. We develop and study a genome sequence matching application that is simple to control and deploy, yet serves as a prototype of a data-intensive application. The application uses a SAGA-based implementation of the All-Pairs pattern. This paper aims to understand some of the factors that influence the performance of this application and the interplay of those factors. We also demonstrate how the SAGA approach can enable data-intensive applications to be extensible and interoperable over a range of infrastructure. This capability enables us to compare and contrast two different approaches for executing distributed data-intensive applications—simple application-level data-placement heuristics versus distributed file systems.

MapReduce has emerged as an important data-parallel programming model for data-intensive computing – for Clouds and Grids. However most if not all implementations of MapReduce are coupled to a specific infrastructure. SAGA is a high-level programming interface which provides the ability to create distributed applications in an infrastructure independent way. In this paper, we show how MapReduce has been implemented using SAGA and demonstrate its interoperability across different distributed platforms – Grids, Cloud-like infrastructure and Clouds. We discuss the advantages of programmatically developing MapReduce using SAGA, by demonstrating that the SAGA-based implementation is infrastructure independent whilst still providing control over the deployment, distribution and runtime decomposition. The ability to control the distribution and placement of the computation units (workers) is critical in order to implement the ability to move computational work to the data. This is required to keep data network transfer low and in the case of commercial Clouds the monetary cost of computing the solution low. Using data-sets of size up to 10GB, and upto 10 workers, we provide detailed performance analysis of the SAGA-MapReduce implementation, and show how controllingthe distribution of computation and the payload per worker helps enhance performance.

1 Abstract Previously the cost and extremely limited capabilities of sensors prohibited Quality of Service (QoS) implementations in wireless sensor networks. With advances in technology, sensors are becoming significantly less expensive and the increases in computational and storage capabilities are opening the door for new, sophisticated algorithms to be implemented. Newer sensor network applications require higher data rates with more stringent priority requirements. We introduce a dynamic scheduling algorithm to improve bandwidth for high priority data in sensor networks, called Tiny-DWFQ. Our Tiny-Dynamic Weighted Fair Queuing scheduling algorithm allows for dynamic QoS for prioritized communications by continually adjusting the treatment of communication packages according to their priorities and the current level of network congestion. For performance evaluation, we tested Tiny-DWFQ, Tiny-WFQ (traditional WFQ algorithm implemented in TinyOS), and FIFO queues on an Imote2-based wireless sensor network and report their throughput and packet loss. Our results show that Tiny-DWFQ performs better in all test cases.

Summary Yeast cells become older with each division, but their daughters are born young. Mutational analysis shows that maintenance of this age asymmetry requires segregation of a complement of active mitochondria to daughters and that this process breaks down in older mother cells. This decline has implications for stem cell aging in higher organisms. PEX6, a peroxisome biogenesis gene, has been isolated as a multicopy suppressor of an atp2 age asymmetry mutant. Suppression depended on the presence of particular amino acid residues in Atp2p, and required adenosine triphosphate (ATP) binding and/or ATP hydrolysis activity of Pex6p. Extra copies of PEX6 corrected the deficit in Atp2p in mitochondria in the mutant by improving its import kinetics, resulting in near normal mitochondrial inheritance by daughter cells. The novel function of Pex6p described here may provide insights into peroxisomal and mitochondrial disorders and into metabolic diseases in general.

Purpose To measure changes in nuclear gene expression resulting from mitochondrial dysfunction in retinal pigment epithelial cells. Methods ARPE-19 retinal pigment epithelial cells were depleted of their mitochondrial (mt)DNA by passaging in a low concentration of ethidium bromide. Loss of mitochondrial DNA was determined by uridine auxotrophy and quantitative real-time polymerase chain reaction of isolated DNA. Loss of mitochondrial membrane potential was estimated by uptake of JC-1. Changes in nuclear gene expression were determined by quantitative real-time reverse transcription-polymerase chain reaction of isolated total RNA from ethidium-bromide-treated and untreated cells. Morphologic and phenotypic changes were determined by phase-contrast microscopy, sensitivity to the oxidant tert-butyl hydroperoxide (tBH), and invasion assay. Results ARPE-19 cells became auxotrophic for growth on uridine after eight passages in 50 ng/mL ethidium bromide. Quantitative PCR revealed almost complete loss of mitochondrial DNA (rho(0) cells). Uptake of JC-1 was reduced in the rho(0) cells, indicating reduction of mitochondrial membrane potential. Quantitative RT-PCR measured increased expression of genes coding for drusen components, lipid transport, extracellular matrix components, and responses to inflammation in the rho(0) cells. The rho(0) cells also exhibited an increased sensitivity to killing by tBH and increased migration and invasion through solubulized basement membrane-coated tissue culture inserts. Conclusions ARPE-19 cells respond to loss of mitochondrial function by changes in nuclear gene expression that resemble changes observed in age-related macular degeneration. The results lead to the hypothesis that loss of mitochondrial function with age and resultant changes in nuclear gene expression may explain some of the changes in the macula that are associated with the known clinical manifestations of age-related macular degeneration.

The yeast Saccharomyces cerevisiae has a finite replicative life span. Yeasts possess two prohibitins, Phb1p and Phb2p, in similarity to mammalian cells. These proteins are located in the inner mitochondrial membrane, where they are involved in the processing of newly-synthesized membrane proteins. We demonstrate that the elimination of one or both of the prohibitin genes in yeast markedly diminished the replicative life span of cells that lack fully-functional mitochondria, while having no effect on cells with functioning mitochondria. This deleterious effect was suppressed by the deletion of the RAS2 gene. The expression of PHB1 and PHB2 declined gradually up to 5-fold during the life span. Cells in which PHB1 was deleted in conjunction with the absence of a mitochondrial genome displayed remarkable changes in mitochondrial morphology, distribution, and inheritance. This loss of mitochondrial integrity was not seen in cells devoid of PHB1 but possessing an intact mitochondrial genome. In a subset of the cells, the changes in mitochondrial integrity were associated with increased production of reactive oxygen species, which co-localized with the altered mitochondria. The mitochondrial deficits described above were all suppressed by deletion of RAS2. Our data, together with published information, are interpreted to provide a unified view of the role of the prohibitins in yeast aging. This model posits that the key initiating event is a decline in mitochondrial function, which leads to progressive oxidative damage that is exacerbated in the absence of the prohibitins. This aggravation of the initial damage is ameliorated by the suppression of the production of mitochondrial proteins in the absence of Ras2p signaling of mitochondrial biogenesis.

To determine the relative expression of metallothionein isoforms and their differential induction by oxidative stress in cultured RPE cells and to localize the isoforms in the human chorioretinal complex.Total RNA was isolated from cultured human retinal pigment epithelial cells using TRI-Reagent. An "anchor-oligo-dT primer" was used for the synthesis of cDNA, reverse transcribed using avian reverse transcriptase and subsequently subjected to PCR analysis using oligonucleotides specific for metallothionein (MT) I, MT II, and MT III. The selected transcripts were then used to assess the expression of the above elements in fixed tissue sections by in situ hybridization. Cultured RPE cells were allowed to phagocytose bovine photoreceptor outer segments (ROS) or were treated with H(2)O(2) for 6 hours and then analyzed by RT-PCR or in situ hybridization to ascertain the effect of oxidative stress on metallothionein mRNA isoform expression.Relative density analysis of amplified products demonstrate the presence of MT I, MT II and MT III in RPE cells, with an apparent relative expression MT IIMT IMT III [corrected]. Expression of MT I and MT II mRNA was increased by both phagocytosis and hydrogen peroxide, however MT III was not induced by either stress. In situ hybridization corroborated the findings of the RT-PCR analysis and showed that MTs were mainly localized in the RPE and the photoreceptor layer of the retina.The localization of MT and the response of MT to oxidative stress are consistent with a role for MT as an antioxidant in the RPE and retina. Studies are ongoing to determine the specific mechanisms of action of these antioxidants in RPE cells.

This study was undertaken to determine whether bioavailable zinc can influence the effects of oxidative stress on cultured human retinal pigment epithelial (RPE) cells. RPE cells were maintained for 7 d in culture medium containing 14 microM total zinc, or in medium containing 0.55 microM total zinc. After 1 week, MTT assays were performed to determine the relative cytotoxicity of H2O2 or paraquat on RPE cells. Conjugated dienes and thiobarbituric acid reactive substances (TBARS) were measured in RPE cells treated with 0, 0.5 mM H2O2, 10 microM FeSO4 + 0.5 mM H2O2 or 10 microM FeSO4 + xanthine/xanthine oxidase for 24 h or paraquat for 7 d. Oxidized proteins were determined by the formation of carbonyl residues. The antioxidants metallothionein, catalase, superoxide dismutase, and glutathione peroxidase were also measured. The MTT assays showed that zinc protected cultured RPE from the toxicity of H2O2 and paraquat. RPE cells in 0.55 microM zinc medium contained higher levels of TBARS, conjugated dienes and protein carbonyls due to the oxidative stresses, compared to cells in 14 microM zinc. Catalase and MT content were reduced in cells cultured in 0.55 microM zinc medium and were reduced additionally when treated with above stresses. Superoxide dismutase activity increased in 0.55 microM zinc medium in response to these stresses. Our results show RPE cells cultured in zinc-reduced medium are more susceptible to oxidative insult.

Numerous reports have shown metallothionein protects tissue from reactive oxygen intermediates, considered to be a factor in aging. Because zinc stimulates MT induction and because loss of metal increases proteolysis of the apoprotein, zinc may have a protective role and may indirectly protect against aging. Our laboratory has shown age-related decreases in catalase, zinc and metallothionein in human retinal pigment epithelium. This study was undertaken to investigate the role of bioavailable zinc on MT content in RPE cells subjected to an oxidative stress.

To examine the internalization of liposomes containing various phospholipids by human retinal pigment epithelial (RPE) cells in culture. The internalization was compared to that exhibited by macrophages and fibroblast cells.The uptake into cells was monitored by using a non-degradable, radioactive cholesterol-hexadecyl ether derivative and a pH-sensitive, water-soluble fluorescent dye. Variables tested to determine their effect on uptake included time, phospholipid concentration, the presence or absence of serum in medium and the presence or absence of cholesterol in the liposome bilayer. The most avidly ingested liposome was tested to determine its ability to deliver a liposome-dependent drug.Liposome uptake was time and concentration dependent. Uptake for RPE was maximal in serum-free media, while the opposite was true for the other cells tested. Cell survival following exposure to fluoroorotic acid-containing liposomes correlated with the radioactive cholesterol-hexadecyl ether and fluorescent dye uptake data.No single liposome carrier or set of conditions tested appeared to be optimum for the delivery of a cytotoxic agent to these three cell types. This bears consideration when designing strategies for the prevention and treatment of intraocular proliferative diseases such as proliferative vitreoretinopathy (PVR).

Zinc is a necessary micronutrient, usually abundant in human RPE. Our study was undertaken to determine the effects of short-term, zinc deficiency on human retinal pigment epithelium (RPE) using a culture model of fetal human RPE cells. Human fetal RPE cells were isolated and cultured in Coon's modified Ham's F-12 medium. For zinc depletion studies, cells were cultured for 1 week in Chelex-treated Dulbecco's modified Eagle's medium containing low (0.25 microM) or physiologic (11 microM) total zinc concentrations as determined by flame atomic absorption spectroscopy. Protein synthesis was determined by incorporation of 35S-cysteine/methionine and labeled proteins analysed by polyacrylamide gel electrophoresis. Several cell parameters and enzymes were significantly reduced below control when cultured in low zinc: zinc content (40%), proliferation (63%), protein/well (50%), catalase activity (68%), alkaline phosphatase activity (61%), alpha-mannosidase activity (68%), and metallothionein (82%). No statistically significant decline was seen in acid phosphatase activity, superoxide dismutase activity, glutathione peroxidase activity and dexamethasone induction of metallothionein. Zinc repletion (100 microM, 1 h) increased catalase and alpha-mannosidase activities from 32% and 33% of control to 75% and 73%, respectively. Cycloheximide did not inhibit this short-term zinc-induced repletion of catalase or alpha-mannosidase. Protein synthesis in low zinc medium was depressed, but not significantly, as shown by incorporation of radiolabeled 35S-cysteine/methionine into newly synthesized proteins. The effects of zinc deficiency in cultured human RPE are selective. Adequate intracellular zinc was required for maximal activity of some enzymes. The dependence of catalase activity on zinc was not predicted and may help explain the observed decline in catalase activity seen with age in RPE. Our model of zinc deficiency should prove useful in elucidating the complex effects of zinc deficiency and repletion in human RPE.

Objective: Hyaluronic acid (HA) has a key role in the structure and organization of the extracellular matrix. We sought to identify the distribution of HA in human eye tissue with regard to age using a biotinylated HA-binding protein. Methods: Fetal and adult (from donors ranging from 28 to 94 years of age) eye tissues were fixed less than 24 hours post mortem and embedded in JB-4 medium (Polysciences, Warrington, Pa). Sections of 2-μm thickness were used. Control sections were pretreated either with Streptomyces hyaluronidase or HA-binding protein inactivated by HA. The binding of the protein to HA was detected with avidinbiotin alkaline phosphatase and developed by incubation with naphthol as-mx phosphate and Texas Red Salt (Pierce, Rockford, Ill). Results: Specific staining for HA was observed in fetal eyes in the choroid, Bruch's membrane, sclera, retinal pigment epithelium, and developing retina from the vitreoretinal interface to the inner plexiform layer. Specific staining decreased with age in the choroid, retinal pigment epithelium, and Bruch's membrane. Hyaluronic acid—specific staining was undetectable in tissues from donors over 50 years of age. Conclusions: The localization of HA in the chorioretinal complex and its disappearance after the fifth decade of life may play a role in aging and age-related retinal disorders.

We studied zinc uptake by nonhuman primate retinal pigment epithelium (RPE) and choroid, using 65Zn as a probe. With intravenously administered 65ZnCl2, virtually all detectable tracer was lost from the plasma after 20 hours but the pigment epithelium-choroid showed prominent uptake and retention. Plasma concentrations of oral 65ZnO remained high 20 hours after feeding. Uptake and retention of orally administered 65Zn as 65ZnO from the bloodstream by the RPE/choroid was avid in both young and old animals. Excretion in urine and feces was minimal. All pigmented ocular tissues took up and retained 65Zn. A survey of total zinc content of human and nonhuman primate ocular tissues showed that the pigmented tissues had consistently higher concentrations of zinc. Our results demonstrate for the first time direct uptake and retention of zinc from the blood by primate RPE and other ocular tissues.

We investigated the inhibition of proliferation of human retinal pigment epithelial cells in vitro by the 4-quinolone, ciprofloxacin, and the steroid, dexamethasone. The concentration of ciprofloxacin that inhibited growth by 50% (IC50) was found to be 14.1 micrograms/mL. Growth was 100% inhibited at 83 micrograms/mL. At 166 micrograms/mL, all the cells became completely detached and appeared dead at the end of seven days. The IC50 for dexamethasone in RPE cells was found to be 141 micrograms/mL. A dexamethasone concentration of 1.3 mg/mL inhibited proliferation 100% after five days. When the two drugs were combined, the inhibitory effect was found to be additive; i.e., the IC50 dose of the two drugs in combination inhibited RPE cell proliferation by 75%. A combination of the two drugs was also tested for retinal toxicity in rabbit eyes. An examination of histological sections and electroretinograms showed that a dose of 100 micrograms of ciprofloxacin, alone or in combination with 200 micrograms of dexamethasone in saline, was not toxic to the rabbit retina. These studies indicate that a combination of ciprofloxacin and dexamethasone has the potential for reducing the risk of PVR formation and aiding in the prevention of endophthalmitis.

The acute effects of doxorubicin on coronary perfusion and left ventricular pressures and intracellular phosphate metabolite levels, the latter obtained by 31P nuclear magnetic resonance, were measured simultaneously in isolated, isovolumic rat hearts (Langendorf preparation) perfused at constant flow. Nineteen experimental hearts were perfused for 70 min with oxygenated HEPES-buffered solution containing 6 mg/L doxorubicin. These were compared with 18 control hearts (C), perfused under identical conditions but without doxorubicin, by repeated measures analysis of variance. In the experimental group, coronary perfusion pressure steadily increased to 226.3 +/- 13.8% (mean +/- SEM) of initial levels (p less than 0.0001 vs. C). Because flow was constant, the increase in coronary perfusion pressure in experimental hearts indicates a greater than twofold increase in coronary resistance. Intracellular phosphocreatine and ATP decreased to 80.3 +/- 3.9% (p less than 0.005 vs. C) and 82.1 +/- 6.4% (p less than 0.05 vs. C), whereas inorganic phosphate increased to 149.7 +/- 19.1% (p less than 0.05 vs. C) of initial levels, respectively. Accompanying these changes, diastolic pressure steadily increased to 521.7 +/- 91.4% of initial levels (p less than 0.0001 vs. C). Developed pressure initially increased to 107.1 +/- 4.5% at 30 min, and thereafter decreased to 76.2 +/- 6.3% at 70 min (p less than 0.05 vs. C). Typical structural alterations in myocyte nuclei were noted. Cellular calcium was not increased in doxorubicin-exposed hearts. Thus, acute doxorubicin cardiotoxicity is characterized by an increase in coronary resistance and is closely correlated with alterations in ventricular function and a decrease in intracellular high-energy phosphate content.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated adult rat hearts perfused in an isovolumic mode were used to study the effects of sodium-potassium pump inhibition and sodium-calcium exchange alterations on the tissue content of adenosine triphosphate, phosphocreatine, inorganic phosphate, and intracellular pH, all measured by phosphorus-31 nuclear magnetic resonance spectroscopy. Rates of oxygen consumption, contractile function, and the cell contents of calcium, sodium, and potassium also were determined. The inhibition of sodium-potassium adenosine triphosphatase, either by the reduction in perfusate potassium from 5.9 to 1 millimolar or less, or by the addition of 10(-4) molar ouabain, transiently increased systolic pressure. This was followed by a decrease in systolic pressure, an increase in diastolic pressure, and eventual inexcitability. This contractile profile was accompanied by a persistent increase in oxygen consumption, a monotonic decline in cellular adenosine triphosphate and phosphocreatine content, the development of marked intracellular acidosis, a gain in cell sodium and calcium content, and a reduction in cell potassium. Quite similar metabolic changes were also observed when cell calcium was increased after a reduction in perfusate sodium. These metabolic and contractile effects could be prevented or reversed by decreasing perfusate calcium. The results emphasize the profound role of calcium in modulating cell oxygen consumption, energy balance, pH, excitability, and force production. These data are discussed in light of changes in the myocardial energy supply/demand balance, as well as from the viewpoint of the known competition between mechanisms for mitochondrial calcium transport vs. high-energy phosphate production.

Perfluorophenanthrene, a liquid fluorocarbon with a specific gravity approximately twice that of water, potentially offers certain advantages as a vitreous substitute in vitreoretinal surgery. To determine its efficacy and safety we first purified it by chemical methods used in the preparation of experimental blood substitutes to a level at which it was not at all or only minimally toxic to culture-grown retinoblastoma cells. Nineteen of 22 vitrectomized eyes of white New Zealand rabbits injected with this purified perfluorophenanthrene showed satisfactory clinical tolerance. Light and electron microscopy showed minimal or no toxic effects in the 19 eyes, although uptake of perfluorophenanthrene by some preretinal cells was observed 28 days after implantation. Postoperative light-adapted electroretinography recordings of eight of the injected eyes showed no significant change. Perfluorophenanthrene injected into the anterior chamber of the rabbit eyes had toxic effects on the cornea. If further experimentations confirm our findings, perfluorophenanthrene may be a suitable transparent high-density liquid for temporary use in surgery to repair retinal detachments.

Fluorosilicone oil, a high specific gravity fluorinated siticene oil, has been recently reevaluated for temporary use in surgery to repair complicated retinal detachments. We evaluated the toxicfty of a low-viscosity 300 centistokes) fluorosilicone oil as a vitreous substitute in vitrectomized eyes of albino rabbits. No toxicrties could be demonstrated by histopathologic and electroretinographic examinations 6 weeks following surgery. In vitro experiments also showed no toxic effects on cultured retinoblastoma cells. When fluorosilicone oil was injected into the anterior chamber, endothelial cell damage could be seen 2 weeks following injection. Low-viscosity fluorosilicone oil appears to be a safe vitreous substitute for temporary use; it is easily injected and removed, and it maintains adequate surface tension for intraocular tamponade. Because of its relative low viscosity, 300-cst fluorosilicone oil (FS) may be a better vitreous substitute than perfluorocarbon liquids for both intraoperative manipulation of the retina and short-term intraocular tamponade for complicated retinal detachments.

1. 1. In the adult pulmonate snail Helisoma sp., ca 29% of the total phosphorus is present as alkylphosphonate, principally 2-aminoethylphosphonic acid (AEP), while in freshly-laid eggs, 98% is present as alkylphosphonate. 2. 2. 31P NMR analysis showed major differences in the phosphonate region of the spectrum of hot water extracts from eggs, compared to snails. 3. 3.Hot water extractable material from eggs had a mol. wt of greater than 1.5 × 106 by gel filtration and contained 2.5% AEP, 48% hexose and less than 1% amino acids. 4. 4.|AEP appears to be attached to the high mol. wt material by a phosphonomonoester bond.

In freshly laid egg masses of Helisoma sp., more than 95 percent of the phosphorus is found in alkylphosphonic acids, as determined by phosphorus-31 nuclear magnetic resonance spectroscopy. These compounds are metabolized during embryonic development, as shown by differential acid hydrolysis and experiments with phosphorus-33-labeled phosphoric acid. Further, nuclear magnetic resonance spectroscopy indicates phosphonic acid involvement in related snail families, including the schistosomal vector Biomphalaria glabrata.

The 31 P nuclear magnetic resonance spectrum of cultured human Y-79 retinoblastoma cells was obtained at 121 MHz on intact cells trapped in agarose threads. The spectrum was dominated by monoester peaks, which varied in relative concentration from preparation to preparation. Resonances from phosphocreatine, phosphodiesters and diphosphodiesters also exhibited variability relative to ATP. The main monoester was identified as phosphorylcholine by 31 P-NMR of perchloric acid extracts. It was determined that the changes in monoester concentration correlated with feeding pattern. Phosphorus spectra of cells 1,2 and 3 days post feeding showed a 40% decrese in the relative concentration of phosphorylcholine concentration over the 3 day period. Phosphocreatine, phosphodiesters and diphosphodiesters increased relative to ATP during the same period. Growth curve experiments and oxygen consumption measurements indicated that the decrease in phosphorylcholine correlated with a decrease in cellular growth and oxygen consumption. We conclude that monoester concentration may be a useful indicator of nutritional status in these cells and possibly in intact tumors.

31P NMR was applied to an examination of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis. Resonances from sugar phosphates, inorganic phosphate, adenylates and arginine phosphate were identified. Two peaks of Pi were identified corresponding to intracellular and extracellular Pi. Anoxia produced an expected decrease in peak intensities of ATP and arginine phosphate while the peak of intracellular Pi was enhanced and shifted to indicate intracellular acidification during anoxia. Spectra of whole larvae were monitored over a temperature range from -30 degrees to +25 degrees C. No abrupt alterations in the spectra were seen at the point of extracellular freezing which occurs at about -8 degrees C but temperature had dramatic effects upon the peak intensities of ATP and arginine phosphate. A reversible increase/decrease in peak intensities, relative to Pi, was observed as temperature was raised/lowered. At 15 degrees and -20 degrees C, the beta peak of ATP was 64% and 2% of the peak intensity of Pi while that of arginine phosphate was 78% and 11%, respectively. This temperature effect was not an artifact of instrumentation (as model solutions containing Pi, ATP and arginine phosphate did not show this effect) or a result of changes in the total amounts of these compounds in the cell with temperature. Rather it is apparent that these molecules become restricted in their rotational movement as temperature is lowered perhaps via binding to subcellular components. Changes in the amounts of freely soluble ATP and arginine phosphate with temperature could have important implications for metabolism and its control. Analysis of the effect of temperature on the chemical shift of Pi was also used to determine pH in the intracellular and extracellular compartments. Temperature change had no effect on extracellular (hemolymph) pH which remained constant at 6.1-6.3. Intracellular pH varied with temperature, however, from pH 6.8 at 15 degrees C to pH 7.3 at -12 degrees C with a change, delta pH/delta 0, of -0.0185 degrees C consistent with alphastat regulation.

Retinal pigment epithelium (RPE) was isolated from the globe of a donor positive for human immunodeficiency virus (HIV) who had cytomegalovirus (CMV) retinitis secondary to acquired immunodeficiency syndrome (AIDS). In culture, the cells exhibited normal epithelioid morphology by phase contrast microscopy. After two weeks the cells developed cytomegaly and dense intranuclear and cytoplasmic inclusions and, eventually, died. Transmission electron microscopy (EM) demonstrated intranuclear nonenveloped virus particles 80-120 nm in diameter consistent with a herpes type infection. Immunofluorescence staining demonstrated the presence of CMV antigens. Conditioned medium from the infected cells caused infection in RPE cells isolated from normal donors. Hybridization assay demonstrated the presence of CMV DNA and indicated that the time course of the infection was similar, but not identical to infection in MRC-5 and HEL cells. We conclude that cultured human RPE is a permissive host for CMV.