ДАЛИ ЈЕ МЕДИЦИНА ПОСТАЛА БИЗНИС ПРОТИВ ХУМАНОСТИ???!!!! http://news.wustl.edu/news/Pages/21797.aspx
Cancer, Health, Crimes, Alternative Medicine, Treatments| Burton Goldbeg
Cancer and Health Crimes Against Humanity
An Economic Crime Against Humanity
Medicine vs. Business! Which one?(Long read) [Archive] - Bodybuilding.com Forums
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ЗАШТО LINUX НЕМА ВИРУСЕ, А WINDOWS ИХ ИМА У ИЗОБИЉУ http://www.theregister.co.uk/2003/10/06/linux_vs_windows_viruses/WINDOWS
Digital Crime Wave - The Growing Problem
Digital Crime Wave - The Growing Problem
MNOGO VELIKO NEZNANJE U SRPSKOJ INFORMATICI!!!
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У НАШОЈ ЈАДНОЈ СРБИЈИ СВЕ СУ СВЕЛИ НА НОВАЦ, ТАКО ДА СЕ И ОПЕРАТИВНИ СИСТЕМИ ЦЕНЕ НА БАЗИ КОЛИЧИНЕ ПАРА КОЈА СЕ ДАЈЕ ЗА ЊИХ. СОФТВЕР ЈЕ КАО И ЉУДСКА ДУША, А ДАЛИ ДУША ИМА ЦЕНУ??? У СРПСКОЈ МЕДИЦИНИ И НАУЦИ ДУША НЕ ПОСТОЈИ. ЉУДИ СУ САМО МЕСО, КОСТИ И КРВ. ТО ЈЕ САГЛЕДАВАЊЕ НАШЕ НЕРАЗУМНЕ НАУКЕ. ЗАТО ЉУДИ И УМИРУ. НАЈВАЖНИЈА ЈЕ ДУША ТЈ СИСТЕМСКИ СОФТВЕР КОЈИ ПОКРЕЋЕ ТО МЕСО И КОСТИ. ЦЕО ЖИВИ СВЕТ ЈЕ ТАКОЂЕ СВЕДЕН НА ИНФОРМАТИКУ, КОМУНИКАЦИЈА СЕ ОБАВЉА ОД ВИРУСА ПА ДО ЧОВЕКА, КАКО БИ ВИРУСИ ИНАЧЕ ОПСТАЛИ ДА НЕ ПРЕВАРЕ ИМУНИ СИСТЕМ ЧОВЕКА И ЋЕЛИЈУ??? НАШИ ГЛУПИ ЛЕКАРИ И "СТРУЧЊАЦИ" НЕЗНАЈУ НИШТА ИИ КОМУНИКАЦИЈЕ У ОРГАНИЗМИМА !!!
ОБЈАШЊЕЊЕ КОМУНИКАЦИЈЕ !!! http://www.ncbi.nlm.nih.gov/pmc/articles/PMC237095/
У СРБИЈИ СЕ СВЕ СВОДИ НА:
Ljubav do poslednjeg dinara
Press Online :: Zašto političari vole novinarke
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МЕДИЦИНСКИ ЕКСПЕРИМЕНТИ СА ЖИВИМ ЉУДИМА У СВЕТУ,А ВЕРОВАТНО ТО РАДЕ И НАШЕ НАКАЗЕ И УБИЦЕ ЗА ЛОВУ!!!
ЖРТВЕ НЕМОРАЛНЕ СРПСКЕ МЕДИЦИНЕ http://picasaweb.google.com/ralekv/Da?authkey=Gv1sRgCKitlOi-o-rfCQ#5541905902406078546
ДАЛИ ЋЕ НЕЗНАЛИЦЕ ИКАДА СХВТИТИ??? http://www.akademediasrbija.com/index.php?option=com_content&view=article&id=2695:miting-sns-u-beogradu-zavren-mirno&catid=47:ukratko-cat&Itemid=71
МОЈ КОМЕНТАР: КАДА САМ ПРЕ 13 ГОДИНА БИО СА ПОКОЈНОМ СУПРУГОМ КОД МОЈЕГ ПОКОЈНОГ ОЦА У АМЕРИЦИ, МИЛОША ТОДОРОВИЋА, РЕКАО САМ СВОМ ДИВНОМ ОЦУ. "ЋАЛЕ У СРБИЈИ СЕ САДА БОРИМО ЗА ДЕМОКРАТИЈУ". ОН СЕ САМО НАСМЕШИО И РЕКАО МИ, "РАЛЕ, ВИДЕЋЕШ СИНЕ, ШТА ЈЕ ТО ДЕМОКРАТИЈА"!!! И БИО ЈЕ 100% У ПРАВУ.
ВЛАДАВИНА НЕМОРАЛНИХ НЕЗНАЛИЦА И УБИЦА СРБИЈЕ.
ГАЛАСАЛИ СМО ЗА ЊИХ И ПОМАГАЛИ, А ОНИ СУ НЕЗНАЊЕМ УБИЛИ ЈАДНУ ГОГУ!!!И САДА НИСУ КРИВИ, ЈЕР У СРБИЈИ НЕМА ПРАВДЕ И ИСТИНЕ http://www.youtube.com/watch?v=qq7DfShRYp0
YouTube - Prkosna pesma
YouTube - Ivana Zigon - Vera nasa
YouTube - Spasi Boze ljude svoje
НАША ИСТИНА !!! http://www.youtube.com/watch?v=JvuhpnpCIOg
YouTube - Frka - Srce (Serbian Rap) (download link!) (tekst / lyrics)
НЕЗНАЛИЦЕ СУ ПРОГЛАСИЛЕ ВИТЕШКИ НАРОД, УБИЦАМА И РОБОВИМА??? http://www.youtube.com/watch?v=L0AGOHuMLyo&feature=related
ДУХОВНО БЛАГО, И ТРАДИЦИЈА. САМО НАС ТО МОЖЕ ИШЧУПАТИ ИЗ ОВОГ БЛАТА. http://www.youtube.com/watch?v=8etjHCqTNVo
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ПЕСМЕ ПУТНИКА??? http://www.youtube.com/watch?v=iek8IwFjEec&feature=related
НИСМО МИ НАРОД ОД ЈУЧЕ http://www.youtube.com/watch?v=Pj_oIBoTqPE&feature=related
ДАЛИ СМО СЕ И МИ У ТО ПРЕТВАРАМО??? БЕЗГРЕШНЕ, БОЛЕСНЕ, НЕМОРАЛНЕ УБИЦЕ. ПРЕТВАРАЊЕ ПЕДИГРЕА У БОЛЕСТ http://www.youtube.com/watch?v=NaZpUMgOetY&playnext=1&list=PL1C95E9B7EC090541
СРУШИЋЕМО И ДРУГЕ, ГЛУПЕ МИТОВЕ!!! http://www.mycity-military.com/Avijacija-i-PVO/Covek-koji-je-oborio-F-117-postao-pekar.html
YouTube - PEKAR, LEKAR, APOTEKAR - RIBLJA ČORBA
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НЕ ЗОВИ МАМА ДОКТОРА, ЊИМА СЕ НЕ МОЖЕ ВИШЕ ВЕРОВАТИ. ОНИ СУ НЕЗНАЛИЦЕ И УБИЦЕ...http://www.youtube.com/watch?v=sOLN5blP4rw&NR=1
ДОКАЗ !!! http://picasaweb.google.com/ralekv/Da?authkey=Gv1sRgCKitlOi-o-rfCQ#slideshow/5541905902406078546
КАКО СЕ ЦЕНИ ЛИЧНОСТ КОЈА ЈЕ НАЈВИШЕ ДОПРИНЕЛА ЧОВЕЧАНСТВУ ОД СВОЈЕГ НАСТАНКА!!! http://www.pula-online.com/article.php?id=2981
ТЕК ПОСЛЕ 150 ГОДИНА
http://www.teslasociety.com/serbia150.htm
А ТЕСЛИНО ВРЕМЕ ТЕК ДОЛАЗИ, ЈОШ СЕ ГРЕЈЕМО НА ДРВА И УГАЉ И ТРОШИМО НАФТУ......ЕНЕРГИЈА ЈЕ СВУД ОКО НАС!!!
ДИВНИ ЉУДИ ЖИВЕ СКРОМНО, И НЕЗАПАЖЕНО, А ЗА СОБОМ ОСТАВЉАЈУ ВЕЛИКА ДЕЛА http://www.youtube.com/watch?v=eoY_7mbm5ng
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ДА СУ КАПИТАЛИСТИ ПОСЛУШАЛИ ТЕСЛУ СТРУЈА БИ СВИМА БИЛА БЕСПЛАТНА, А ПЛАНЕТА ЗЕМЉА БИ БИЛА ЧИСТИЈА И ЗДРАВИЈА, АЛИ ОНИ СУ ВОЛЕЛИ ЛОВУ!!!
САДА СЕ ЈАВЉАЈУ КЛИМАТСКЕ ПРОМЕНЕ, ТОПЕ СЕ ПОЛОВИ, И КО ЗНА ШТА ЋЕ СВЕ БИТИ. ЖИВИ БИЛИ ПА ВИДЕЛИ!!!!
А И САДА САМО КУКАЈУ ЗА ПАРЕ!!!!
ШТА ЈОШ РЕЋИ??? http://www.b92.net/tesla/zivot.php?nav_id=202354
Prevremeni izbori loši za evropske integracije, a naročito za koaliciju na vlasti. Kako će živeti bez vlasti kada je vole??? NAROD MOŽE I DA IZUMRE, ALI MI ŽELIMO U EVROPSKU UNIJU!!!
Politika : Cvetković: Prevremeni izbori loši za evropske integracije : POLITIKA
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У Србији се све изгледа своди на чекање. ТАКО САМ ЈА ЧЕКАО СРПСКЕ "СТРУЧНЕ" ЛЕКАРЕ ДА ИЗЛЕЧЕ МОЈУ ГОГУ А ВРАТИЛИ СУ ЈЕ У САНДУКУ http://bs.wikiquote.org/wiki/%C4%8Cekanje_%28Borges%29
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КАДА СВИ ПРОКОЦКАЈУ НАШЕ ПОВЕРЕЊЕ ТРЕБА СЕ ОСЛОНИТИ НА СЕБЕ. ТАДА СИ БАР САМ КРИВ ЗА СВОЈЕ ГРЕШКЕ. И ОНИ СВИ ГРЕШЕ, А ПРАВЕ СЕ ДА НИСУ КРИВИ ИЛИ ДА ЈЕ ПО ЗАКОНУ. http://www.b92.net/biz/komentari.php?nav_id=476487
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ПОБЕДИЋУ ЗВЕРИ 100% http://sr.wikipedia.org/sr-el/%D0%A1%D0%B0%D0%BC%D0%BE%D0%BF%D0%BE%D1%83%D0%B7%D0%B4%D0%B0%D1%9A%D0%B5
YouTube - How to Build Your Self Confidence
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ДЕБИЛНИ МЕДИЦИНСКИ "СТРУЧЊАЦИ" УБИЈАЈУ НАШЕ НАЈМИЛИЈЕ НЕКАЖЊЕНО, ЗАР НЕ??? http://goran.forumotion.net/t5258-eutanazija-ubistvo-iz-milosra
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KONTROLA BOLA I KOMUNIKACIJA http://www.kewego.fr/video/iLyROoafYv0i.html
SISTEM U HIBERNACIJI!!! Kada će u našoj jadnoj Srbiji, raditi odgovorne poslove ljudi koji su profesionalci, a ne bolesne neznalice koje ubijaju neznanjem. Uvek sam se trudijo da u svom poslu budem vrhunski, nisam lagao, ni varao ljude. Uvek sam im govorio istinu. Srpski lekari nažalost to nisu. Lažu, varaju, pljačkaju, i onda tvrde da su u pravu. Imaju licence, imaju diplome, imaju komoru, koja se brine sama o sebi, ali nemaju znanje i moral. Izmišljamo razne gluopsti insajdere, novinare, proizvodimo tračeve, ali pravog znanja i intelekta nema. Sve se svodi na entuzijazam pojedinaca. Pravde nema, zakona nema, države nema. Sve je slabo, jadno i virtualno. Prosimo po svetu da bi preživeli, a imamo divan, pametan i sposoban narod, koji nema šta da radi. Policija štrajkuje, školstvo štrajkuje, kažu da će i zdravstvo, radnička klasa gladuje, a ne hvataju se ubice, ne razlikuju prirodnu i nasilnu smrt, deca neznaju da misle, lekari ubijaju bezgrešno. Kažnjavaju za pogresno parkiranje i pušenje, teraju na robiju za maltretiranje jadnih, pasa, a naše najmilije ubijaju doktori nakaze i ne može im se ništa.Čemu sluzi takva, glupa, nemoralna, nakazna država. Štaće nam Evropa kad umiru deca i ljudi, i tako hiljade pitanja, a ni jedan odgovor. Postaćemo nebeski narod,ići ćemo kod Manitua u večna lovišta, pa nek vladaju sami sa sobom... Bez svrhe je nauka, tehnologija, znanje, svrha je samo pljačka, pljačka, pljačka...... Tablice, dozvole, porezi, takse, zemljarine, PDV, forma, energija, internet, tv pretplata, .....+beskonačno...... Gde je nestala ljudska svest i moral???
ДАЛИ СРПСКИ МЕДИЦИНСКИ РАДНИЦИ, ПРАВНИЦИ, СУДИЈЕ, ПОЛИТИЧАРИ ИМАЈУ РАЗУМ??? http://sr.wikipedia.org/sr-el/%D0%A0%D0%B0%D0%B7%D1%83%D0%BC
ИСТИНА http://sr.wikipedia.org/sr-el/%D0%98%D1%81%D1%82%D0%B8%D0%BD%D0%B0
СТАЛНО СЕ НАМЕЋЕ ЈЕДНО ПИТАЊЕ. ЗАШТО СЕ НЕЗНАЛИЦЕ БАВЕ МЕДИЦИНОМ??? http://www.google.com/images?hl=en&q=zasto%20se%20neznalice%20bave%20medicinom&psj=1&um=1&ie=UTF-8&source=og&sa=N&tab=wi&biw=1280&bih=753
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ПРОФЕСОРИ УНИВЕРЗИТЕТА НЕЗНАЛИЦЕ И УБИЦЕ НАШИХ НАЈМИЛИЈИХ!!!
ОВЕ ЗВЕРИ ВАСПИТАВАЈУ БУДУЋЕ "МОРАЛНЕ" ЛЕКАРЕ!!!
ОНДА НАША НАЈБОЉА ПРИЈАТЕЉИЦА КАЖЕ:"НИШТА ЈОЈ НИЈЕ ИШЛО НА РУКУ" И ШТА ОНДА РЕЋИ???
Dodato posle 29 minuta:
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ДА НЕЗНАЛИЦЕ НАУЧЕ ЦЕО УНИВЕРЗУМ ЈЕ КАО ВЕЛИКИ ИНФОРМАЦИОНИ СИСТЕМ, ПА И ВИРУСИ И БАКТЕРИЈЕ КОМУНИЦИРАЈУ СА ОРГАНИЗМОМ УЗ ПОМОЋ ЕЛЕТРОМАГНЕТНИХ ТАЛАСА!!!
JOURNAL OF VIROLOGY, Oct. 1994,p. 6745-6750 Vol. 68, No. 10
0022-538X/94/$04.00+0
Copyright C 1994, American Society for Microbiology NOTES
Feline Immunodeficiency Virus Decreases Cell-Cell Communication
and Mitochondrial Membrane Potential INDRANI R. DANAVE,1 EVELYN TIFFANY-CASTIGLIONI,2 ELISABETH ZENGER,2 ROLA BARHOUMI,2
ROBERT C. BURGHARDT,2 AND ELLEN W. COLLISSON3*
Departments of Biology,1 Veterinary Anatomy and Public Health, 2 and Veterinary Pathobiology,3
Texas A&M University, College Station, Texas 77843
Received 25 April 1994/Accepted 6 July 1994
The in vitro effects of viral replication on mitochondrial membrane potential (MMP) and gap junctional intercellular communication (GJIC) were evaluated as two parameters of potential cellular injury. Two distinct cell types were infected with the Petaluma strain of feline immunodeficiency virus (FIV). Primary astroglia supported acute FIV infection, resulting in syncytia within 3 days of infection, whereas immortalized Crandell feline kidney (CRFK) cells of epithelial origin supported persistent FIV infection in the absence of an obvious
cytopathic elect. An examination of cells under conditions that included an infection rate of more than 90% for either population revealed that the astroglia produced about four times more virus than the CRFK cells.
The mitochondrial uptake of the cationic fluorescent dye rhodamine 123 in infected astroglia was less than 45% of that of normal control cells, whereas the MMP of the CRFK cells, which produced about one-fourth as much virus, was 80.8% of that of the normal cells. Cell-cell communication between adjacent cells was determined by the recovery of fluorescence following photobleaching of a single cell. In spite of the lower level of innate cell-cell communication among cultured CRFK cells than among astroglia, viral replication resulted in a 30%Yo
decrease in the GJIC of both astroglia and CRFK cells. These studies indicate that cell injury, as defined by an inhibition of MMP and GJIC, can occur as a result of persistent and acute infection with the Petaluma strain of FIV.
Feline immunodeficiency virus (FIV) is a common pathogen of domestic cats throughout the world (24, 25, 37). Clinical signs of FIV infection, such as wasting, diarrhea, lymphoid atrophy, and opportunistic infections, resemble those attribut-able to human immunodeficiency virus (HIV) infection in humans (24, 37, 38, 42, 46). FIV, however, apparently has a broader range of tropism than HIV, with certain strains readily infecting cells of epithelial origin (such as Crandell feline kidney [CRFK] cells) as well as CD8+ and CD4+ T lymphocytes, macrophages, and astroglia (2, 3, 15, 37). FIV and HIV-1 have also been associated with similar neurologic signs and
central nervous system lesions (6, 11, 13-15, 43). FIV has been isolated from both cerebrospinal fluid and brain tissues of naturally and experimentally infected cats (45, 46).
Like HIV, FIV replicates in vitro in astroglia and microglia of the central nervous system but apparently not in oligodendrocytes or neurons (12, 13, 15). Neurologic disease could be the result of FIV- or HIV-induced alterations in astroglial or microglial function. Astroglia, the most abundant cells of the central nervous system, are macroglia that maintain ion concentrations, take up and metabolize neurotransmitters, regulate the differentiation and function of myelinated axons, and aid in the formation of tight junctions between endothelial cells in the blood-brain barrier (21, 27-29, 41). They are also a source for cytokines that are involved in cellular signaling,
inflammatory responses, and antigen presentation (19, 20, 22, 34).
The cellular mechanisms for lentivirus-induced lesions are not well studied, although these viruses often induce cell-cell
fusion, resulting in multinucleated cells and limited viability in susceptible target cells. In addition, there is evidence suggest-ing that HIV infection alters membrane integrity and that membrane-HIV interactions play a critical role in the pathogenesis of AIDS (7, 17, 23, 33). Membrane interactions of the host cell with HIV or viral proteins are postulated to cause permeability changes in the membrane, interference with the normal homeostasis of the cell, alteration of ion fluxes, elevation of intracellular Ca2+ concentrations, and an increase in membrane unsaturated oleic acid (7, 8, 17, 23, 30, 33, 36).
Virally induced effects of gap junctional intercellular communication (GJIC) and the dissipation of mitochondrial membrane potential (MMP) are two critical end points of cellular injury which are very sensitive to changes in intracellular Ca2+ levels. MMP is essential in the generation of energy for cellular homeostasis. Whereas the outer mitochondrial membrane is permeable to most ions, the inner membrane houses the electron transport chain-driven proton pump. The resulting electrochemical gradient facilitates a continuous cycling of H+,
allowing the transport of certain ions, such as Ca2' and K+, and high-energy phosphates between the cytosol and the mitochondria (31).
Direct intercellular communication occurs through gap junctions within the cytoplasmic membrane. These junctions are aggregates of cell-cell channels which join adjacent cells and mediate bidirectional diffusion of inorganic ions and small molecules (<1 kDa) between cells but do not mediate the passage of macromolecules (32). Individual channels between adjacent cells are formed by the alignment of two hexameric
*
Corresponding author. Phone: (409) 845-4122. Fax: (409) 845-
9972. Electronic mail address: EWC0087@Venus.TAMU.EDU.
6745
6746
NOTES
aggregates of proteins called connexons which form a hydro-
pore about 1 nm wide. GJIC is involved in the mainte-
philic
nance of cellular homeostasis, growth control, cell repair, and
neoplastic transformation (18).
In order to evaluate potential molecular targets of FIV-
induced cellular injury, fluorescent probes for specific cellular
functions were used to examine the in situ effects of viral
replication on MMP and GJIC. The Petaluma strain of FIV
acutely infects primary astroglia, producing obvious syncytia
and cell death, whereas this strain chronically infects the
immortalized CRFK cells in the absence of a cytopathic effect
(9, 10, 14, 15, 44).
FIV infection of CRFK cells and astroglia. MMP and GJIC
in astroglia acutely infected and CRFK cells persistently
infected with the Petaluma strain of FIV were measured.
Cultured CRFK cells maintained in Dulbecco's modified Ea-
gle's medium with 10% calf serum, lx minimal essential
medium (Gibco, Gaithersburg, Md.), and 1 mM sodium pyru-
vate with penicillin and streptomycin (Sigma, St. Louis, Mo.)
had been persistently infected for more than 6 months with the
Petaluma strain of FIV (NIH AIDS Repository). Astroglia
from fetal domestic cats at 50 to 60 days of gestation were
prepared as described by Zenger et al. (47) and maintained in
Dulbecco's modified Eagle's medium F12 (1:1, vol/vol) with
15% fetal bovine serum. A glial fibrillary acidic protein assay
with rabbit anti-bovine glial fibrillary acidic protein antibody
(Dako, Carpinteria, Calif.) was used to confirm the astroglial
nature of the cultured central nervous system cells (47).
Astroglia were inoculated by incubation for 1 h at 37°C with
viral stock from supernatant fluid of Petaluma strain-infected
CRFK cells having a reverse transcriptase activity of 7.8 x 106
cpm/ml. The astroglia in a 75-cm flask were infected at
approximately 50% confluency with 1 ml of the stock virus or
with medium. After incubating at 37°C for 1 h, the cells were
given fresh medium and observed daily for the presence of
syncytia. Uninfected cells were added two or three times per
week to maintain viability. Infected astroglia were analyzed at
15 to 20 days postinfection. Both cell types were subcultured at
a density of 2 x 105 viable cells in 35-mm-diameter plastic
tissue culture dishes 48 h prior to evaluation of MMP or GJIC.
An ACAS 570 interactive laser cytometer (Meridian Instru-
ments, Okemos, Mich.) permitted evaluations of these param-
eters in individual cells as well as in populations of cells.
Therefore, conditions were selected so that a high percentage
of cells in the sample population were supporting viral repli-
cation. Unlike infected CRFK cells and normal control cells,
astroglia infected with the Petaluma strain of FIV began
forming syncytia 3 days after subculturing with added viable,
uninfected cells. The cultured CRFK cells and astroglia were
examined 48 h after subculturing in order to avoid measuring
changes that might reflect membrane fusion in the latter cells.
At this time, evaluation of the infected cells by indirect
immunofluorescence assay indicated that 95% of the CRFK
cells and 91% of the astrocytes were infected with FIV (40)
and microscopic examination indicated that the cells were well
attached and were not showing signs of a cytopathic effect.
Although similar percentages of astroglia and CRFK cells were
infected, the amount of virus in the supernatants as measured
by the reverse transcriptase assay (2) was more than four times
greater in the infected astroglia (15.1 x 105 cpm/106 cells) than
in the CRFK cells (3.49 x 105 cpm/106 cells). The intensity of
FIV-specific immunofluorescence was also consistently greater
in the infected astroglia (data not shown). Therefore, the
amount of virus produced by the astroglia was considerably
greater than the amount produced by the persistently infected
CRFK cells. For each experiment, the cell cultures were
J. VIROL.
divided into one control and at least two treated groups, with
three to six culture dishes per group. Means were compared by
Duncan's multiple range test for variables and the General
Linear Models procedure. The P values of <0.05 were consid-
ered significant.
Effects of viral replication on MMP. The mitochondrial
incorporation of the cationic fluorescent dye rhodamine 123 is
dependent on the presence of an intact electrochemical gradi-
ent across the mitochondrial inner membrane. Therefore, a
decrease in fluorescence values indicates a dissipation of MMP
(4, 16). The cationic fluorescent dye rhodamine 123, prepared
as a 2-mg/ml stock in ethanol, was used as a probe for
examining the dissipation of MMP (4, 16). After overnight
incubation at 37°C, the infected and uninfected control cul-
tured cells were washed four times with Dulbecco's phosphate-
buffered saline (PBS) and incubated for 30 min at 37°C with
rhodamine 123 diluted from the stock solution to 5 ,ug/ml in
serum-free and phenol red-free medium. The cells were sub-
sequently washed four times in serum-free medium without
phenol red, and the fluorescence intensity was immediately
evaluated with the ACAS 570 laser cytometer at an excitation
wavelength of 488 nm. Eight areas per dish in three dishes per
experimental group were examined in experiments from two
different days. The mean fluorescence intensity was deter-
mined for 50 to 60 cells per treatment group to provide a
relative measure of MMP based on the Nermstian distribution
of the dye (16).
A significant difference (P < 0.05) in the incorporation of
rhodamine 123 in the infected CRFK cells and astroglia
compared with the uninfected cells was observed (Fig. 1). The
mean fluorescence intensity in the infected astroglia was 44.9%
of that of the uninfected controls, and the intensity in the
infected CRFK cells was 80.8% of that of the uninfected
controls (Fig. 2). While the MMPs of normal astroglia and
CRFK cells were comparable, the incorporation of rhodamine
123 in infected astroglia was consistently less than that in the
infected CRFK cells. In addition, although equivalent numbers
of cells were infected, the amount of virus produced as
measured by reverse transcriptase activity was about four times
greater in the acutely infected astroglia. MMP may be quan-
titatively affected by the amount of replication because infec-
tion of astroglia which produced more virus than the CRFK
cells resulted in nearly twice the decrease in MMP compared
with that in corresponding uninfected control cells. Alterna-
tively, the greater impact of infection on the astroglia could be
the result of fundamental differences in the two cell types. The
inhibition of MMP may not, however, be a requirement for a
productive FIV infection because under conditions in which
the MMP in infected cells could be maintained at a normal
level (with camptothecin treatment), the amount of virus
produced was equivalent to that in infected CRFK cells with
the dissipation of MMP (unpublished data).
Elects of viral replication on GJIC. Fluorescence recovery
in photobleached cells is dependent on the ability of non-
membrane-permeating carboxyfluorescein to flow to bleached
cells from the neighboring cells through gap junctions. GJIC
was evaluated with the ACAS 570 by an assay of fluorescence
recovery after photobleaching (1, 44). The cells were washed
four times with PBS and stained for 15 min at 37°C with
5-carboxyfluorescein diacetate diluted to 10 ,ug/ml (from a
2-mg/ml stock solution in dimethyl sulfoxide in serumless
Dulbecco's modified Eagle's medium without phenol red. The
5-carboxyfluorescein diacetate is taken up by cells and con-
verted to carboxyfluorescein, which is able to diffuse through
gap junctions but not through nonjunctional membrane. The
cultures were rinsed four times and maintained in serumless,
NOTES
VOL. 68, 1994
6747
FIG. 1. Decreased rhodamine 123 uptake in FIV-infected astroglia and CRFK cells indicates a decrease in MMP. The relative fluorescence
intensity scale is shown on the left. Fluorescence from rhodamine 123 is shown in a field of uninfected (a) and infected (b) astroglia and uninfected
(c) and infected (d) CRFK cells. Bar, 50 ,um.
phenol red-free medium before microscopic fields containing
aggregates of cells were selected. Single cells, when present,
served as photobleached negative controls. Single cells or small
isolated groups of nonphotobleached cells were used to mon-
itor potential background photobleaching resulting from image
scans and/or the presence of uncleaved dye. Two to three
abutting cells were selected from each field to monitor fluo-
rescence transfer at an excitation wavelength of 488 nm. One
or two cells from each field were photobleached while a single
cell or small group of cells were left unbleached and demar-
cated as positive controls. Photobleaching was done by deliv-
ering a high-intensity laser beam, which reduces the carboxy-
fluorescein due photochemically in the selected cells and
results in a reduction of fluorescence (20 to 40% of the original
value, depending on the cell type). Sufficient bleaching of
2-
0
0
Astroglia CRFK Cells
FIG. 2. The effects of FIV infection on the mean uptake of
rhodamine 123. The fluorescence intensity means and standard errors
for 68 uninfected astroglia and 232 uninfected CRFK cells are
represented by the solid bars, and those for 57 infected astroglia and
222 infected CRFK cells are represented by the hatched bars.
fluorescence to measure recovery without causing visible cell
damage at the light-microscopic level was the criterion used to
determine laser strength, number of bleaches per cell, and scan
and bleach intensities. A series of five postbleach image scans
were generated. One scan was taken immediately postbleach,
and the subsequent four scans were taken at 1-min intervals
to measure the redistribution of intracellular fluorescence
through gap junctions. At least three analyses of cells from
each of six dishes (15 to 20 cells) per treatment group were
conducted in two separate experiments. Fluorescence levels
were quantified by computer-assisted evaluation of data.
Estimation of a rate constant (k) for fluorescence recovery
was performed by fitting the percent fluorescence intensity at a
given time, F(t), to the following equation: F(t) = Feq(l-e-kt)
+ F(O), where Feq represents the percent fluorescence recov-
ery of the bleached cell at equilibrium, and e-k' is the estimate
of the rate constant at a given time, and F(O) is the percent
fluorescence intensity immediately following photobleaching
(1). The value of Feq depends on the number of contacting
cells and the initial level of bleaching. Data from at least 30
cells from each treatment group were pooled to obtain the
mean k values by curve fitting regression analysis, which
permits extrapolation of fluorescence recovery versus time.
The GJIC was monitored in both uninfected and infected
CRFK cells and astroglia. The cells were examined 24 h before
any syncytia could be detected in the cultured astroglia. Figure
3a and b represents a field of uninfected and of infected
astroglia, respectively, before and immediately after photo-
bleaching and at 4 min after photobleaching, showing the
recovery of fluorescence. The cell labelled 3 in each field was
left unbleached as a positive control, and those labelled 1 and
2 were bleached. The GJIC was lower in the infected astroglia
(cells 1 and 2 in Fig. 3b) than in the uninfected astroglia (cells
1 and 2 in Fig. 3a). Cell 2 of the infected population nearly
ceased to communicate with adjacent cells.
At any time up to 4 min after photobleaching of a population
of either cell type, the mean fluorescent recovery was less in
the infected cells than in the corresponding uninfected cells
6748
J. VIROL.
NOTES
b
FIG. 3. Effects of FIV infection on GJIC in astroglia as measured by the recovery of fluorescence after photobleaching of individual uninfected
(a) and infected (b) astroglia. A single field is shown before (prebleach), immediately after (O min), and at 4 min following bleaching of cells
labelled 1 and 2. Cell 3, which was not bleached, was used as a reference control. The graphs represent the recovery of fluorescence from each
cell. Green line, cell 1; red line, cell 2; yellow line, cell 3.
(Fig. 4a). The mean communication rates values (Fig. 4b)
indicated that the GJIC between infected cells was significantly
less than that between the corresponding uninfected cells (P,
<0.03 for CRFK cells and <0.02 for astroglia). The rates of
recovery for the infected CRFK cells and infected astroglia
were 71.5 and 70.1%, respectively, of the rates for the corre-
sponding uninfected cells.
Although the magnitude of virally induced reduction in both
cell types was similar, GJIC in infected and uninfected astro-
glia was greater than that in the CRFK cells examined. The
mean percent recovery of astroglia ranged from about 37% at
zero time to between 67 and 77% at 4 min after bleaching
compared with less than 20% to between 35 and 45% in the
CRFK cells. The difference in the communication rates of the
two types of cells was statistically significant (P < 0.0001).
It has been suggested that the closing of gap junctions is a
mechanism for isolating injured cells (32). Although the GJIC
of infected cells within tissues is not known, a possible expla-
nation for the GJIC decrease in infected cell culture could be
that cells tend to seal themselves off as a response to infection,
in general, rather than as a function of the presence of a virus
or viral replication in the cell.
Digital imaging procedures that have proven valuable in
examining mechanisms of mycotoxin and heavy metal cytotox-
icity have been exploited to examine the effects of viral
replication on critical cellular functions (5, 39). These sensitive
Minutes
4
Astroglia
CRFK Cells
FIG. 4. Effects of FIV infection on the GJIC of a population of cells. (a) Mean percent fluorescence recoveries following photobleaching of
32 uninfected (closed circles) and 29 infected (open circles) astroglia and 26 uninfected (closed squares) and 27 infected (open squares) CRFK
cells. (b) Mean communication rates (per minute) calculated from panel a, with standard errors. Uninfected cells are represented by the solid bars;
infected cells are represented by the hatched bars.
molecular procedures utilize vital fluorescent probes to deter-
mine virally induced alterations in cellular homeostasis. Rep-
lication of the Petaluma strain of FIV was consistently associ-
ated with quantitative decreases in the MMP and GJIC in
cultured cells. It appears that these alterations are general
consequences of infection with this virus because decreases
were observed in two cell types which differ in origin, function,
and susceptibility to FIV replication. The astroglia were pri-
mary cell cultures that supported an acute infection, producing
large multinucleated cells, and the CRFK were immortalized
cultured cells supporting persistent FIV infection in the ab-
sence of syncytia or other signs of a cytopathic effect. Since an
ultrastructural analysis of gap junctions was not done, it is not
possible to determine whether reduced GJIC was due to
disruption of gap junctions or to reduced permeability of
channels due to other causes, such as cytoplasmic acidification
or altered phosphorylation of channel proteins.
The contributions of these alterations to a cytopathic effect
are not known. Membrane-associated changes could be related
to cell-cell fusion and syncytium formation. Viral replication
may directly alter GJIC and MMP or, alternatively, alter the
homeostasis of interacting end points. Zenger et al. (47) have
found other indicators of cell injury in astroglia; e.g., FIV
infection of astroglia decreased cytoplasmic glutathione levels
and altered Ca2' homeostasis (47). Because glutathione plays
a crucial role in protecting cells against free radicals and
electrophiles and in maintaining membrane integrity (26, 35),
its reduced activity could contribute to the observed injury im
mitochondrial membranes. Differences in intracellular Ca2+
stores were detected following ionophore-induced Ca2+ fluxes.
Decreases in MMP could, therefore be due to mitochondrial
uptake of Ca2' and/or changes in plasma membrane function.
These differences in the sequestering of calcium could be
associated with a calcium-induced loss of MMP (47).
These studies examined intracellular molecular alterations
induced by the replication of the Petaluma strain of FIV.
Similar studies with other strains of FIV will determine
whether these cellular changes are a common property of FIV
or a peculiarity of this strain. Such indicators of cell injury
could provide the tools to characterize the mechanisms for
acute and persistent infections and discriminate the vital
functions that determine virus-induced injury and cell death.
This work was funded by National Institute of Allergy and Infectious
Diseases grants AI 32360-01 and AI 90221-OlAl.
REFERENCES
1. Barhoumi, R., J. A. Bowen, L S. Stein, J. Echol, and R. C.
Burghardt. 1993. Concurrent analysis of intracellular glutathione
content and gap junctional intercellular communication. Cytom-
etry 14:747-756.
2. Brown, W. C., L. Bissey, K. S. Logan, N. C. Pedersen, J. H. Elder,
and E. W. Collisson. 1991. Feline immunodeficiency virus infects
both CD4+ and CD8+ T lymphocytes. J. Virol. 65:3359-3364.
3. Brunner, D., and N. C. Pedersen. 1989. Infection of peritoneal
macrophages in vitro and in vivo with feline immunodeficiency
virus. J. Virol. 63:5483-5488.
4. Bunting, J. R., T. V. Phan, E. Kamali, and R. M. Dowben. 1989.
Fluorescent cationic probes of mitochondria: metrics and mecha-
nism of interaction. Biophys. J. 56:979-993.
5. Burghardt, R. C., R. Barhoumi, E. H. Lewis, R. H. Bailey, K. A.
Pyle, B. A. Clement, and T. D. Phillips. 1992. Patulin-induced
cellular-cytotoxicity: a vital fluorescence study. Toxicol. Appl.
Pharmacol. 112:235-244.
6. Cheng-Mayer, C., J. T. Rutka, M. L Rosenblum, T. McHugh, T. P.
Stites, and J. A. Levy. 1987. Human immunodeficiency virus can
productively infect cultured human glial cells. Proc. Natl. Acad.
Sci. USA 84:3526-3530.
7. Cloyd, M. W., and W. S. Lynn. 1991. Perturbation of host cell
membrane is a primary mechanism of HIV cytopathology. Virol-
ogy 181:500-511.
8. Cloyd, M. W., W. S. Lynn, K. Ramsey, and S. Baron. 1989.
Inhibition of human immunodeficiency virus (HIV-1) by diphenyl-
hydantion (dilantin) implicates the role of cellular calcium in the
viral life-cycle. Virology 173:581-590.
9. Crandell, R. A., and E. Q. Despeau. 1959. Cytopathology of feline
viral rhinotracheitis virus in tissue cultures of feline renal cells.
Proc. Soc. Exp. Biol. Med. 154:1403-1418.
10. Crandell, R A., C. G. Fabricant, and W. A. Nelson-Rees. 1973.
Developmental, characterization and viral susceptibility of Felis
catus renal cell line (CRFK). In Vitro (Rockville) 9:176.
11. Dewhurst, S., J. Bresser, M. Stevenson, K. Sakai, M. J. Evinger-
Hodges, and D. J. Volsky. 1987. Susceptibility of human glial cells
to human immunodeficiency virus (HIV). FEBS Lett. 213:138-143.
6750
J. VIROL.
NOTES
12. Dickson, D. W., L. A. Mattiace, K. Kure, K. Hutchins, W. D.
Lyman, and C. F. Brosana. 1991. Microglia in human disease, with
emphasis on the human immune deficiency syndrome. Lab. Invest.
64:135-156.
13. Dow, S. W. 1989. Retrovirus infection of the central nervous
system. Proceedings of the 7th American College Veterinary
Internal Medicine Forum, San Diego, Calif.
14. Dow, S. W., M. J. Dreitz, and E. A. Hoover. 1992. Feline immu-
nodeficiency virus neurotropism: evidence that astrocytes and
microglia are the primary target cells. Vet. Immunol. Immuno-
pathol. 35:23-35.
15. Dow, S. W., M. L. Poss, and E. A. Hoover. 1990. Feline immuno-
deficiency virus: a neurotropic lentivirus. J. Acquired Immune
Defic. Syndr. 3:658-668.
16. Ehrenberg, B., V. Montana, M. D. Wei, J. P. Wuskell, and L. M.
Loew. 1988. Membrane potential can be determined from individ-
ual cells from the Nernstian distribution of cationic dyes. Biophys.
J. 53:785-794.
17. Fermin, C. D., and R. F. Garry. 1992. Membrane alterations linked
to early alterations of HIV with the cell surface. Virology 191:941-
946.
18. Fletcher, W. H., C. V. Byus, and D. A. Walsh. 1987. Receptor
mediated action without receptor occupancy: a function for cell-
cell communication in ovarian follicles, p. 299-323. In V. B.
Mahesh, D. S. Dhindsa, E. Anderson, and S. P. Kalra (ed.),
Regulation of ovarian and testicular function. Plenum Publishing
Corp., New York.
19. Fontana, A., F. Kristensen, R Dubs, D. Gemsa, and E. Weber.
1982. Production of prostaglandin E and an interleukin I like
factor by cultured astrocytes and C6 glioma cells. J. Immunol.
129:2413-2419.
20. Frei, K., S. Bodmer, C. Schwerdel, and A. Fontana. 1986. Astro-
cyte derived interieukin 3 as a growth factor for microglia cells and
peritoneal macrophages. J. Immunol. 137:3521-3527.
21. Frohman, E. M., S. Van Der Noort, and S. Gupta. 1989. Astrocytes
and intracerebral immune responses. J. Clin. Immunol. 9:1-9.
22. Giulian, D., J. Woodward, D. G. Young, J. F. Krebs, and L. B.
Lachman. 1988. Interleukin 1 injected into mammalian brain
stimulates astrogliosis and neovascularization. J. Neurosci.
8:2485-2490.
23. Grewe, C., A. Beck, and H. R Gelderblom. 1990. HIV: early
virus-cell interactions. J. Acquired Immune Defic. Syndr. 10:965-
974.
24. Harbour, D. A., P. D. Williams, T. J. Gruffydd-Jones, J. Burbridge,
and G. R Pearson. 1988. Isolation of a T lymphotropic lentivirus
from a persistently leucopenic domestic cat. Vet. Rec. 122:84-
86.
25. Ishida, T., T. Washizu, K. Toriyabe, S. Motoyoshi, L. Tomoda, and
N. C. Pedersen. 1989. Feline immunodeficiency virus in cats of
Japan. JAMA 194:221-225.
26. Jain, A., J. Martensson, E. Stole, P. A. M. Auld, and A. Meister.
1991. Glutathione deficiency leads to mitochondrial damage in the
brain. Proc. Natl. Acad. Sci. USA 88:1913-1917.
27. Janzer, R. C., and M. C. Raff. 1987. Astrocytes induce blood brain
properties in endothelial cells. Nature (London) 325:253-257.
28. Kimelberg, H. K. 1986. Occurrence and functional significance of
serotonin and catecholamine uptake by astrocytes. Biochem.
Pharmacol. 35:2273-2281.
29. Kimelberg, M. R., and B. R Ransom. 1986. Physiological and
pathological aspects of astrocyte swelling, p. 129-158. In S. Fed-
eroff and A. Vernadis (ed.), Astrocyte cell biology and pathology
of astrocytes, vol. 3. Academic Press, New York.
30. Kinchington, D., W. Barker, S. Galpin, and K. Apostolov. 1992.
Temperature enhancement of syncytium formation by HIV and
Sendai virus. J. Med. Virol. 36:44-48.
Lehninger, A. L. 1984. Principles of Biochemistry, p. 491-492.
Worth Publishers Inc., New York.
Loewenstein, W. R 1966. Permeability of membrane junctions.
Ann. N. Y. Acad. Sci. 137:441-471.
Lynn, W. S., A. Tweedale, and M. W. Cloyd. 1988. Human
immunodeficiency virus (HIV-1): perturbation of the cell mem-
brane and depression of phospholipid synthesis. Virology 163:43-
51.
Malipiero, U. V., K. Frei, and A. Fontana. 1990. Production of
hemopoietic and colony stimulating factors by astrocytes. J. Im-
munol. 144:3816-3821.
Meister, A., and M. E. Anderson. 1983. Glutathione. Annu. Rev.
Biochem. 52:711-760.
Miller, M. A., R. F. Garry, J. M. Jaynes, and R. C. Montelaro.
1991. A structural correlation between lentivirus transmembrane
proteins and natural cytolytic peptides. AIDS Res. Hum. Retro-
viruses 7:511-519.
Pedersen, N. C., E. W. Ho, M. L. Brown, and J. K. Yamamoto.
1987. Isolation of a T lymphotropic virus from domestic cats with
immunodeficiency-like syndrome. Science 235:790-793.
Pumarola-Sune, T., B. A. Navia, C. Cordon-Cardo, E. S. Cho, and
R W. Price. 1987. HIV antigens in the brain of patients with AIDS
dementia complex. Ann. Neurol. 21:490-496.
Rahn, C. A., D. W. Bombick, and D. J. Doolittle. 1991. Assessment
of mitochondrial membrane potential as an indicator of cytotox-
icity. Fundam. Appl. Toxicol. 16:435-448.
Rey, M. A., B. Spire, D. Dormont, F. Barre-Sinoussi, L. Montag-
nier, and J. C. Chermann. 1984. Characterization of the RNA
dependent DNA polymerase of a new human T lymphotropic
retrovirus (lymphadenopathy associated virus). Biochem. Biophys.
Res. Commun. 121:126-133.
Rodriguez, M., M. L. Pierce, and E. L. Howie. 1987. Immune
response gene products (Ia antigens) on glial and endothelial cells
in virus induced demyelination. J. Immunol. 138:3438-3442.
Sparger, E. E., P. A. Luciw, J. H. Elder, et al. 1989. Feline
immunodeficiency virus is a lentivirus associated with an AIDS-
like disease in cats. J. Acquired Immune Defic. Syndr. 3(1):43-45.
Stavrou, D., P. Mehraein, W. Mellert, K. Bise, K. Schmidtke, E.
Rothemunds, I. Funke, U. Stocher, L. Babaryka, C. Zietz, V. Frydl,
F. Prantl, and V. Erfle. 1989. Evaluation of intracerebral lesions in
patients with acquired immunodeficiency syndrome, neuropatho-
logical findings and experimental data. Neuropathol. Appl. Neu-
robiol. 15:207-222.
Wade, M. H., J. E. Trosko, and M. Schindler. 1986. A fluorescence
photobleaching assay of gap junction mediated communication
between human cells. Science 232:525-528.
Yamamoto, J. K., H. Hansen, E. W. Ho, T. Y. Morishita, T. Okuda,
T. R. Sawa, R. M. Nakamura, W. P. Kau, and N. C. Pedersen.
1989. Epidemiologic and clinical aspects of feline immunodefi-
ciency virus infection of cats from the continental United States
and Canada and possible modes of transmission. J. Am. Vet. Med.
Assoc. 194:213-220.
Yamamoto, J. K., E. Sparger, E. W. Ho, P. R. Anderson, T. P.
O'Connor, C. P. Mandell, L. Lowenstine, R. Munn, and N. C.
Pedersen. 1988. Pathogenesis of experimentally induced feline
immunodeficiency virus infection in cats. Am. J. Vet. Res. 48:
1246-1258.
Zenger, E., E. W. Collisson, R. Barhoumi, R. C. Burghardt, I. R.
Danave, and E. Tiffany-Castiglioni. Laser cytometric analysis of
FIV-induced injury in astroglia. glia, in press.
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ПЕСМЕ ПУТНИКА??? http://www.youtube.com/watch?v=iek8IwFjEec&feature=related
НИСМО МИ НАРОД ОД ЈУЧЕ http://www.youtube.com/watch?v=Pj_oIBoTqPE&feature=related
ДАЛИ СМО СЕ И МИ У ТО ПРЕТВАРАМО??? БЕЗГРЕШНЕ, БОЛЕСНЕ, НЕМОРАЛНЕ УБИЦЕ. ПРЕТВАРАЊЕ ПЕДИГРЕА У БОЛЕСТ http://www.youtube.com/watch?v=NaZpUMgOetY&playnext=1&list=PL1C95E9B7EC090541
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НЕ ЗОВИ МАМА ДОКТОРА, ЊИМА СЕ НЕ МОЖЕ ВИШЕ ВЕРОВАТИ. ОНИ СУ НЕЗНАЛИЦЕ И УБИЦЕ...http://www.youtube.com/watch?v=sOLN5blP4rw&NR=1
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КАКО СЕ ЦЕНИ ЛИЧНОСТ КОЈА ЈЕ НАЈВИШЕ ДОПРИНЕЛА ЧОВЕЧАНСТВУ ОД СВОЈЕГ НАСТАНКА!!! http://www.pula-online.com/article.php?id=2981
ТЕК ПОСЛЕ 150 ГОДИНА
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А ТЕСЛИНО ВРЕМЕ ТЕК ДОЛАЗИ, ЈОШ СЕ ГРЕЈЕМО НА ДРВА И УГАЉ И ТРОШИМО НАФТУ......ЕНЕРГИЈА ЈЕ СВУД ОКО НАС!!!
ДИВНИ ЉУДИ ЖИВЕ СКРОМНО, И НЕЗАПАЖЕНО, А ЗА СОБОМ ОСТАВЉАЈУ ВЕЛИКА ДЕЛА http://www.youtube.com/watch?v=eoY_7mbm5ng
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ДА СУ КАПИТАЛИСТИ ПОСЛУШАЛИ ТЕСЛУ СТРУЈА БИ СВИМА БИЛА БЕСПЛАТНА, А ПЛАНЕТА ЗЕМЉА БИ БИЛА ЧИСТИЈА И ЗДРАВИЈА, АЛИ ОНИ СУ ВОЛЕЛИ ЛОВУ!!!
САДА СЕ ЈАВЉАЈУ КЛИМАТСКЕ ПРОМЕНЕ, ТОПЕ СЕ ПОЛОВИ, И КО ЗНА ШТА ЋЕ СВЕ БИТИ. ЖИВИ БИЛИ ПА ВИДЕЛИ!!!!
А И САДА САМО КУКАЈУ ЗА ПАРЕ!!!!
ШТА ЈОШ РЕЋИ??? http://www.b92.net/tesla/zivot.php?nav_id=202354
Prevremeni izbori loši za evropske integracije, a naročito za koaliciju na vlasti. Kako će živeti bez vlasti kada je vole??? NAROD MOŽE I DA IZUMRE, ALI MI ŽELIMO U EVROPSKU UNIJU!!!
Politika : Cvetković: Prevremeni izbori loši za evropske integracije : POLITIKA
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У Србији се све изгледа своди на чекање. ТАКО САМ ЈА ЧЕКАО СРПСКЕ "СТРУЧНЕ" ЛЕКАРЕ ДА ИЗЛЕЧЕ МОЈУ ГОГУ А ВРАТИЛИ СУ ЈЕ У САНДУКУ http://bs.wikiquote.org/wiki/%C4%8Cekanje_%28Borges%29
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КАДА СВИ ПРОКОЦКАЈУ НАШЕ ПОВЕРЕЊЕ ТРЕБА СЕ ОСЛОНИТИ НА СЕБЕ. ТАДА СИ БАР САМ КРИВ ЗА СВОЈЕ ГРЕШКЕ. И ОНИ СВИ ГРЕШЕ, А ПРАВЕ СЕ ДА НИСУ КРИВИ ИЛИ ДА ЈЕ ПО ЗАКОНУ. http://www.b92.net/biz/komentari.php?nav_id=476487
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ПОБЕДИЋУ ЗВЕРИ 100% http://sr.wikipedia.org/sr-el/%D0%A1%D0%B0%D0%BC%D0%BE%D0%BF%D0%BE%D1%83%D0%B7%D0%B4%D0%B0%D1%9A%D0%B5
YouTube - How to Build Your Self Confidence
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ДЕБИЛНИ МЕДИЦИНСКИ "СТРУЧЊАЦИ" УБИЈАЈУ НАШЕ НАЈМИЛИЈЕ НЕКАЖЊЕНО, ЗАР НЕ??? http://goran.forumotion.net/t5258-eutanazija-ubistvo-iz-milosra
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KONTROLA BOLA I KOMUNIKACIJA http://www.kewego.fr/video/iLyROoafYv0i.html
SISTEM U HIBERNACIJI!!! Kada će u našoj jadnoj Srbiji, raditi odgovorne poslove ljudi koji su profesionalci, a ne bolesne neznalice koje ubijaju neznanjem. Uvek sam se trudijo da u svom poslu budem vrhunski, nisam lagao, ni varao ljude. Uvek sam im govorio istinu. Srpski lekari nažalost to nisu. Lažu, varaju, pljačkaju, i onda tvrde da su u pravu. Imaju licence, imaju diplome, imaju komoru, koja se brine sama o sebi, ali nemaju znanje i moral. Izmišljamo razne gluopsti insajdere, novinare, proizvodimo tračeve, ali pravog znanja i intelekta nema. Sve se svodi na entuzijazam pojedinaca. Pravde nema, zakona nema, države nema. Sve je slabo, jadno i virtualno. Prosimo po svetu da bi preživeli, a imamo divan, pametan i sposoban narod, koji nema šta da radi. Policija štrajkuje, školstvo štrajkuje, kažu da će i zdravstvo, radnička klasa gladuje, a ne hvataju se ubice, ne razlikuju prirodnu i nasilnu smrt, deca neznaju da misle, lekari ubijaju bezgrešno. Kažnjavaju za pogresno parkiranje i pušenje, teraju na robiju za maltretiranje jadnih, pasa, a naše najmilije ubijaju doktori nakaze i ne može im se ništa.Čemu sluzi takva, glupa, nemoralna, nakazna država. Štaće nam Evropa kad umiru deca i ljudi, i tako hiljade pitanja, a ni jedan odgovor. Postaćemo nebeski narod,ići ćemo kod Manitua u večna lovišta, pa nek vladaju sami sa sobom... Bez svrhe je nauka, tehnologija, znanje, svrha je samo pljačka, pljačka, pljačka...... Tablice, dozvole, porezi, takse, zemljarine, PDV, forma, energija, internet, tv pretplata, .....+beskonačno...... Gde je nestala ljudska svest i moral???
ДАЛИ СРПСКИ МЕДИЦИНСКИ РАДНИЦИ, ПРАВНИЦИ, СУДИЈЕ, ПОЛИТИЧАРИ ИМАЈУ РАЗУМ??? http://sr.wikipedia.org/sr-el/%D0%A0%D0%B0%D0%B7%D1%83%D0%BC
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ПРОФЕСОРИ УНИВЕРЗИТЕТА НЕЗНАЛИЦЕ И УБИЦЕ НАШИХ НАЈМИЛИЈИХ!!!
ОВЕ ЗВЕРИ ВАСПИТАВАЈУ БУДУЋЕ "МОРАЛНЕ" ЛЕКАРЕ!!!
ОНДА НАША НАЈБОЉА ПРИЈАТЕЉИЦА КАЖЕ:"НИШТА ЈОЈ НИЈЕ ИШЛО НА РУКУ" И ШТА ОНДА РЕЋИ???
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ДА НЕЗНАЛИЦЕ НАУЧЕ ЦЕО УНИВЕРЗУМ ЈЕ КАО ВЕЛИКИ ИНФОРМАЦИОНИ СИСТЕМ, ПА И ВИРУСИ И БАКТЕРИЈЕ КОМУНИЦИРАЈУ СА ОРГАНИЗМОМ УЗ ПОМОЋ ЕЛЕТРОМАГНЕТНИХ ТАЛАСА!!!
JOURNAL OF VIROLOGY, Oct. 1994,p. 6745-6750 Vol. 68, No. 10
0022-538X/94/$04.00+0
Copyright C 1994, American Society for Microbiology NOTES
Feline Immunodeficiency Virus Decreases Cell-Cell Communication
and Mitochondrial Membrane Potential INDRANI R. DANAVE,1 EVELYN TIFFANY-CASTIGLIONI,2 ELISABETH ZENGER,2 ROLA BARHOUMI,2
ROBERT C. BURGHARDT,2 AND ELLEN W. COLLISSON3*
Departments of Biology,1 Veterinary Anatomy and Public Health, 2 and Veterinary Pathobiology,3
Texas A&M University, College Station, Texas 77843
Received 25 April 1994/Accepted 6 July 1994
The in vitro effects of viral replication on mitochondrial membrane potential (MMP) and gap junctional intercellular communication (GJIC) were evaluated as two parameters of potential cellular injury. Two distinct cell types were infected with the Petaluma strain of feline immunodeficiency virus (FIV). Primary astroglia supported acute FIV infection, resulting in syncytia within 3 days of infection, whereas immortalized Crandell feline kidney (CRFK) cells of epithelial origin supported persistent FIV infection in the absence of an obvious
cytopathic elect. An examination of cells under conditions that included an infection rate of more than 90% for either population revealed that the astroglia produced about four times more virus than the CRFK cells.
The mitochondrial uptake of the cationic fluorescent dye rhodamine 123 in infected astroglia was less than 45% of that of normal control cells, whereas the MMP of the CRFK cells, which produced about one-fourth as much virus, was 80.8% of that of the normal cells. Cell-cell communication between adjacent cells was determined by the recovery of fluorescence following photobleaching of a single cell. In spite of the lower level of innate cell-cell communication among cultured CRFK cells than among astroglia, viral replication resulted in a 30%Yo
decrease in the GJIC of both astroglia and CRFK cells. These studies indicate that cell injury, as defined by an inhibition of MMP and GJIC, can occur as a result of persistent and acute infection with the Petaluma strain of FIV.
Feline immunodeficiency virus (FIV) is a common pathogen of domestic cats throughout the world (24, 25, 37). Clinical signs of FIV infection, such as wasting, diarrhea, lymphoid atrophy, and opportunistic infections, resemble those attribut-able to human immunodeficiency virus (HIV) infection in humans (24, 37, 38, 42, 46). FIV, however, apparently has a broader range of tropism than HIV, with certain strains readily infecting cells of epithelial origin (such as Crandell feline kidney [CRFK] cells) as well as CD8+ and CD4+ T lymphocytes, macrophages, and astroglia (2, 3, 15, 37). FIV and HIV-1 have also been associated with similar neurologic signs and
central nervous system lesions (6, 11, 13-15, 43). FIV has been isolated from both cerebrospinal fluid and brain tissues of naturally and experimentally infected cats (45, 46).
Like HIV, FIV replicates in vitro in astroglia and microglia of the central nervous system but apparently not in oligodendrocytes or neurons (12, 13, 15). Neurologic disease could be the result of FIV- or HIV-induced alterations in astroglial or microglial function. Astroglia, the most abundant cells of the central nervous system, are macroglia that maintain ion concentrations, take up and metabolize neurotransmitters, regulate the differentiation and function of myelinated axons, and aid in the formation of tight junctions between endothelial cells in the blood-brain barrier (21, 27-29, 41). They are also a source for cytokines that are involved in cellular signaling,
inflammatory responses, and antigen presentation (19, 20, 22, 34).
The cellular mechanisms for lentivirus-induced lesions are not well studied, although these viruses often induce cell-cell
fusion, resulting in multinucleated cells and limited viability in susceptible target cells. In addition, there is evidence suggest-ing that HIV infection alters membrane integrity and that membrane-HIV interactions play a critical role in the pathogenesis of AIDS (7, 17, 23, 33). Membrane interactions of the host cell with HIV or viral proteins are postulated to cause permeability changes in the membrane, interference with the normal homeostasis of the cell, alteration of ion fluxes, elevation of intracellular Ca2+ concentrations, and an increase in membrane unsaturated oleic acid (7, 8, 17, 23, 30, 33, 36).
Virally induced effects of gap junctional intercellular communication (GJIC) and the dissipation of mitochondrial membrane potential (MMP) are two critical end points of cellular injury which are very sensitive to changes in intracellular Ca2+ levels. MMP is essential in the generation of energy for cellular homeostasis. Whereas the outer mitochondrial membrane is permeable to most ions, the inner membrane houses the electron transport chain-driven proton pump. The resulting electrochemical gradient facilitates a continuous cycling of H+,
allowing the transport of certain ions, such as Ca2' and K+, and high-energy phosphates between the cytosol and the mitochondria (31).
Direct intercellular communication occurs through gap junctions within the cytoplasmic membrane. These junctions are aggregates of cell-cell channels which join adjacent cells and mediate bidirectional diffusion of inorganic ions and small molecules (<1 kDa) between cells but do not mediate the passage of macromolecules (32). Individual channels between adjacent cells are formed by the alignment of two hexameric
*
Corresponding author. Phone: (409) 845-4122. Fax: (409) 845-
9972. Electronic mail address: EWC0087@Venus.TAMU.EDU.
6745
6746
NOTES
aggregates of proteins called connexons which form a hydro-
pore about 1 nm wide. GJIC is involved in the mainte-
philic
nance of cellular homeostasis, growth control, cell repair, and
neoplastic transformation (18).
In order to evaluate potential molecular targets of FIV-
induced cellular injury, fluorescent probes for specific cellular
functions were used to examine the in situ effects of viral
replication on MMP and GJIC. The Petaluma strain of FIV
acutely infects primary astroglia, producing obvious syncytia
and cell death, whereas this strain chronically infects the
immortalized CRFK cells in the absence of a cytopathic effect
(9, 10, 14, 15, 44).
FIV infection of CRFK cells and astroglia. MMP and GJIC
in astroglia acutely infected and CRFK cells persistently
infected with the Petaluma strain of FIV were measured.
Cultured CRFK cells maintained in Dulbecco's modified Ea-
gle's medium with 10% calf serum, lx minimal essential
medium (Gibco, Gaithersburg, Md.), and 1 mM sodium pyru-
vate with penicillin and streptomycin (Sigma, St. Louis, Mo.)
had been persistently infected for more than 6 months with the
Petaluma strain of FIV (NIH AIDS Repository). Astroglia
from fetal domestic cats at 50 to 60 days of gestation were
prepared as described by Zenger et al. (47) and maintained in
Dulbecco's modified Eagle's medium F12 (1:1, vol/vol) with
15% fetal bovine serum. A glial fibrillary acidic protein assay
with rabbit anti-bovine glial fibrillary acidic protein antibody
(Dako, Carpinteria, Calif.) was used to confirm the astroglial
nature of the cultured central nervous system cells (47).
Astroglia were inoculated by incubation for 1 h at 37°C with
viral stock from supernatant fluid of Petaluma strain-infected
CRFK cells having a reverse transcriptase activity of 7.8 x 106
cpm/ml. The astroglia in a 75-cm flask were infected at
approximately 50% confluency with 1 ml of the stock virus or
with medium. After incubating at 37°C for 1 h, the cells were
given fresh medium and observed daily for the presence of
syncytia. Uninfected cells were added two or three times per
week to maintain viability. Infected astroglia were analyzed at
15 to 20 days postinfection. Both cell types were subcultured at
a density of 2 x 105 viable cells in 35-mm-diameter plastic
tissue culture dishes 48 h prior to evaluation of MMP or GJIC.
An ACAS 570 interactive laser cytometer (Meridian Instru-
ments, Okemos, Mich.) permitted evaluations of these param-
eters in individual cells as well as in populations of cells.
Therefore, conditions were selected so that a high percentage
of cells in the sample population were supporting viral repli-
cation. Unlike infected CRFK cells and normal control cells,
astroglia infected with the Petaluma strain of FIV began
forming syncytia 3 days after subculturing with added viable,
uninfected cells. The cultured CRFK cells and astroglia were
examined 48 h after subculturing in order to avoid measuring
changes that might reflect membrane fusion in the latter cells.
At this time, evaluation of the infected cells by indirect
immunofluorescence assay indicated that 95% of the CRFK
cells and 91% of the astrocytes were infected with FIV (40)
and microscopic examination indicated that the cells were well
attached and were not showing signs of a cytopathic effect.
Although similar percentages of astroglia and CRFK cells were
infected, the amount of virus in the supernatants as measured
by the reverse transcriptase assay (2) was more than four times
greater in the infected astroglia (15.1 x 105 cpm/106 cells) than
in the CRFK cells (3.49 x 105 cpm/106 cells). The intensity of
FIV-specific immunofluorescence was also consistently greater
in the infected astroglia (data not shown). Therefore, the
amount of virus produced by the astroglia was considerably
greater than the amount produced by the persistently infected
CRFK cells. For each experiment, the cell cultures were
J. VIROL.
divided into one control and at least two treated groups, with
three to six culture dishes per group. Means were compared by
Duncan's multiple range test for variables and the General
Linear Models procedure. The P values of <0.05 were consid-
ered significant.
Effects of viral replication on MMP. The mitochondrial
incorporation of the cationic fluorescent dye rhodamine 123 is
dependent on the presence of an intact electrochemical gradi-
ent across the mitochondrial inner membrane. Therefore, a
decrease in fluorescence values indicates a dissipation of MMP
(4, 16). The cationic fluorescent dye rhodamine 123, prepared
as a 2-mg/ml stock in ethanol, was used as a probe for
examining the dissipation of MMP (4, 16). After overnight
incubation at 37°C, the infected and uninfected control cul-
tured cells were washed four times with Dulbecco's phosphate-
buffered saline (PBS) and incubated for 30 min at 37°C with
rhodamine 123 diluted from the stock solution to 5 ,ug/ml in
serum-free and phenol red-free medium. The cells were sub-
sequently washed four times in serum-free medium without
phenol red, and the fluorescence intensity was immediately
evaluated with the ACAS 570 laser cytometer at an excitation
wavelength of 488 nm. Eight areas per dish in three dishes per
experimental group were examined in experiments from two
different days. The mean fluorescence intensity was deter-
mined for 50 to 60 cells per treatment group to provide a
relative measure of MMP based on the Nermstian distribution
of the dye (16).
A significant difference (P < 0.05) in the incorporation of
rhodamine 123 in the infected CRFK cells and astroglia
compared with the uninfected cells was observed (Fig. 1). The
mean fluorescence intensity in the infected astroglia was 44.9%
of that of the uninfected controls, and the intensity in the
infected CRFK cells was 80.8% of that of the uninfected
controls (Fig. 2). While the MMPs of normal astroglia and
CRFK cells were comparable, the incorporation of rhodamine
123 in infected astroglia was consistently less than that in the
infected CRFK cells. In addition, although equivalent numbers
of cells were infected, the amount of virus produced as
measured by reverse transcriptase activity was about four times
greater in the acutely infected astroglia. MMP may be quan-
titatively affected by the amount of replication because infec-
tion of astroglia which produced more virus than the CRFK
cells resulted in nearly twice the decrease in MMP compared
with that in corresponding uninfected control cells. Alterna-
tively, the greater impact of infection on the astroglia could be
the result of fundamental differences in the two cell types. The
inhibition of MMP may not, however, be a requirement for a
productive FIV infection because under conditions in which
the MMP in infected cells could be maintained at a normal
level (with camptothecin treatment), the amount of virus
produced was equivalent to that in infected CRFK cells with
the dissipation of MMP (unpublished data).
Elects of viral replication on GJIC. Fluorescence recovery
in photobleached cells is dependent on the ability of non-
membrane-permeating carboxyfluorescein to flow to bleached
cells from the neighboring cells through gap junctions. GJIC
was evaluated with the ACAS 570 by an assay of fluorescence
recovery after photobleaching (1, 44). The cells were washed
four times with PBS and stained for 15 min at 37°C with
5-carboxyfluorescein diacetate diluted to 10 ,ug/ml (from a
2-mg/ml stock solution in dimethyl sulfoxide in serumless
Dulbecco's modified Eagle's medium without phenol red. The
5-carboxyfluorescein diacetate is taken up by cells and con-
verted to carboxyfluorescein, which is able to diffuse through
gap junctions but not through nonjunctional membrane. The
cultures were rinsed four times and maintained in serumless,
NOTES
VOL. 68, 1994
6747
FIG. 1. Decreased rhodamine 123 uptake in FIV-infected astroglia and CRFK cells indicates a decrease in MMP. The relative fluorescence
intensity scale is shown on the left. Fluorescence from rhodamine 123 is shown in a field of uninfected (a) and infected (b) astroglia and uninfected
(c) and infected (d) CRFK cells. Bar, 50 ,um.
phenol red-free medium before microscopic fields containing
aggregates of cells were selected. Single cells, when present,
served as photobleached negative controls. Single cells or small
isolated groups of nonphotobleached cells were used to mon-
itor potential background photobleaching resulting from image
scans and/or the presence of uncleaved dye. Two to three
abutting cells were selected from each field to monitor fluo-
rescence transfer at an excitation wavelength of 488 nm. One
or two cells from each field were photobleached while a single
cell or small group of cells were left unbleached and demar-
cated as positive controls. Photobleaching was done by deliv-
ering a high-intensity laser beam, which reduces the carboxy-
fluorescein due photochemically in the selected cells and
results in a reduction of fluorescence (20 to 40% of the original
value, depending on the cell type). Sufficient bleaching of
2-
0
0
Astroglia CRFK Cells
FIG. 2. The effects of FIV infection on the mean uptake of
rhodamine 123. The fluorescence intensity means and standard errors
for 68 uninfected astroglia and 232 uninfected CRFK cells are
represented by the solid bars, and those for 57 infected astroglia and
222 infected CRFK cells are represented by the hatched bars.
fluorescence to measure recovery without causing visible cell
damage at the light-microscopic level was the criterion used to
determine laser strength, number of bleaches per cell, and scan
and bleach intensities. A series of five postbleach image scans
were generated. One scan was taken immediately postbleach,
and the subsequent four scans were taken at 1-min intervals
to measure the redistribution of intracellular fluorescence
through gap junctions. At least three analyses of cells from
each of six dishes (15 to 20 cells) per treatment group were
conducted in two separate experiments. Fluorescence levels
were quantified by computer-assisted evaluation of data.
Estimation of a rate constant (k) for fluorescence recovery
was performed by fitting the percent fluorescence intensity at a
given time, F(t), to the following equation: F(t) = Feq(l-e-kt)
+ F(O), where Feq represents the percent fluorescence recov-
ery of the bleached cell at equilibrium, and e-k' is the estimate
of the rate constant at a given time, and F(O) is the percent
fluorescence intensity immediately following photobleaching
(1). The value of Feq depends on the number of contacting
cells and the initial level of bleaching. Data from at least 30
cells from each treatment group were pooled to obtain the
mean k values by curve fitting regression analysis, which
permits extrapolation of fluorescence recovery versus time.
The GJIC was monitored in both uninfected and infected
CRFK cells and astroglia. The cells were examined 24 h before
any syncytia could be detected in the cultured astroglia. Figure
3a and b represents a field of uninfected and of infected
astroglia, respectively, before and immediately after photo-
bleaching and at 4 min after photobleaching, showing the
recovery of fluorescence. The cell labelled 3 in each field was
left unbleached as a positive control, and those labelled 1 and
2 were bleached. The GJIC was lower in the infected astroglia
(cells 1 and 2 in Fig. 3b) than in the uninfected astroglia (cells
1 and 2 in Fig. 3a). Cell 2 of the infected population nearly
ceased to communicate with adjacent cells.
At any time up to 4 min after photobleaching of a population
of either cell type, the mean fluorescent recovery was less in
the infected cells than in the corresponding uninfected cells
6748
J. VIROL.
NOTES
b
FIG. 3. Effects of FIV infection on GJIC in astroglia as measured by the recovery of fluorescence after photobleaching of individual uninfected
(a) and infected (b) astroglia. A single field is shown before (prebleach), immediately after (O min), and at 4 min following bleaching of cells
labelled 1 and 2. Cell 3, which was not bleached, was used as a reference control. The graphs represent the recovery of fluorescence from each
cell. Green line, cell 1; red line, cell 2; yellow line, cell 3.
(Fig. 4a). The mean communication rates values (Fig. 4b)
indicated that the GJIC between infected cells was significantly
less than that between the corresponding uninfected cells (P,
<0.03 for CRFK cells and <0.02 for astroglia). The rates of
recovery for the infected CRFK cells and infected astroglia
were 71.5 and 70.1%, respectively, of the rates for the corre-
sponding uninfected cells.
Although the magnitude of virally induced reduction in both
cell types was similar, GJIC in infected and uninfected astro-
glia was greater than that in the CRFK cells examined. The
mean percent recovery of astroglia ranged from about 37% at
zero time to between 67 and 77% at 4 min after bleaching
compared with less than 20% to between 35 and 45% in the
CRFK cells. The difference in the communication rates of the
two types of cells was statistically significant (P < 0.0001).
It has been suggested that the closing of gap junctions is a
mechanism for isolating injured cells (32). Although the GJIC
of infected cells within tissues is not known, a possible expla-
nation for the GJIC decrease in infected cell culture could be
that cells tend to seal themselves off as a response to infection,
in general, rather than as a function of the presence of a virus
or viral replication in the cell.
Digital imaging procedures that have proven valuable in
examining mechanisms of mycotoxin and heavy metal cytotox-
icity have been exploited to examine the effects of viral
replication on critical cellular functions (5, 39). These sensitive
Minutes
4
Astroglia
CRFK Cells
FIG. 4. Effects of FIV infection on the GJIC of a population of cells. (a) Mean percent fluorescence recoveries following photobleaching of
32 uninfected (closed circles) and 29 infected (open circles) astroglia and 26 uninfected (closed squares) and 27 infected (open squares) CRFK
cells. (b) Mean communication rates (per minute) calculated from panel a, with standard errors. Uninfected cells are represented by the solid bars;
infected cells are represented by the hatched bars.
molecular procedures utilize vital fluorescent probes to deter-
mine virally induced alterations in cellular homeostasis. Rep-
lication of the Petaluma strain of FIV was consistently associ-
ated with quantitative decreases in the MMP and GJIC in
cultured cells. It appears that these alterations are general
consequences of infection with this virus because decreases
were observed in two cell types which differ in origin, function,
and susceptibility to FIV replication. The astroglia were pri-
mary cell cultures that supported an acute infection, producing
large multinucleated cells, and the CRFK were immortalized
cultured cells supporting persistent FIV infection in the ab-
sence of syncytia or other signs of a cytopathic effect. Since an
ultrastructural analysis of gap junctions was not done, it is not
possible to determine whether reduced GJIC was due to
disruption of gap junctions or to reduced permeability of
channels due to other causes, such as cytoplasmic acidification
or altered phosphorylation of channel proteins.
The contributions of these alterations to a cytopathic effect
are not known. Membrane-associated changes could be related
to cell-cell fusion and syncytium formation. Viral replication
may directly alter GJIC and MMP or, alternatively, alter the
homeostasis of interacting end points. Zenger et al. (47) have
found other indicators of cell injury in astroglia; e.g., FIV
infection of astroglia decreased cytoplasmic glutathione levels
and altered Ca2' homeostasis (47). Because glutathione plays
a crucial role in protecting cells against free radicals and
electrophiles and in maintaining membrane integrity (26, 35),
its reduced activity could contribute to the observed injury im
mitochondrial membranes. Differences in intracellular Ca2+
stores were detected following ionophore-induced Ca2+ fluxes.
Decreases in MMP could, therefore be due to mitochondrial
uptake of Ca2' and/or changes in plasma membrane function.
These differences in the sequestering of calcium could be
associated with a calcium-induced loss of MMP (47).
These studies examined intracellular molecular alterations
induced by the replication of the Petaluma strain of FIV.
Similar studies with other strains of FIV will determine
whether these cellular changes are a common property of FIV
or a peculiarity of this strain. Such indicators of cell injury
could provide the tools to characterize the mechanisms for
acute and persistent infections and discriminate the vital
functions that determine virus-induced injury and cell death.
This work was funded by National Institute of Allergy and Infectious
Diseases grants AI 32360-01 and AI 90221-OlAl.
REFERENCES
1. Barhoumi, R., J. A. Bowen, L S. Stein, J. Echol, and R. C.
Burghardt. 1993. Concurrent analysis of intracellular glutathione
content and gap junctional intercellular communication. Cytom-
etry 14:747-756.
2. Brown, W. C., L. Bissey, K. S. Logan, N. C. Pedersen, J. H. Elder,
and E. W. Collisson. 1991. Feline immunodeficiency virus infects
both CD4+ and CD8+ T lymphocytes. J. Virol. 65:3359-3364.
3. Brunner, D., and N. C. Pedersen. 1989. Infection of peritoneal
macrophages in vitro and in vivo with feline immunodeficiency
virus. J. Virol. 63:5483-5488.
4. Bunting, J. R., T. V. Phan, E. Kamali, and R. M. Dowben. 1989.
Fluorescent cationic probes of mitochondria: metrics and mecha-
nism of interaction. Biophys. J. 56:979-993.
5. Burghardt, R. C., R. Barhoumi, E. H. Lewis, R. H. Bailey, K. A.
Pyle, B. A. Clement, and T. D. Phillips. 1992. Patulin-induced
cellular-cytotoxicity: a vital fluorescence study. Toxicol. Appl.
Pharmacol. 112:235-244.
6. Cheng-Mayer, C., J. T. Rutka, M. L Rosenblum, T. McHugh, T. P.
Stites, and J. A. Levy. 1987. Human immunodeficiency virus can
productively infect cultured human glial cells. Proc. Natl. Acad.
Sci. USA 84:3526-3530.
7. Cloyd, M. W., and W. S. Lynn. 1991. Perturbation of host cell
membrane is a primary mechanism of HIV cytopathology. Virol-
ogy 181:500-511.
8. Cloyd, M. W., W. S. Lynn, K. Ramsey, and S. Baron. 1989.
Inhibition of human immunodeficiency virus (HIV-1) by diphenyl-
hydantion (dilantin) implicates the role of cellular calcium in the
viral life-cycle. Virology 173:581-590.
9. Crandell, R. A., and E. Q. Despeau. 1959. Cytopathology of feline
viral rhinotracheitis virus in tissue cultures of feline renal cells.
Proc. Soc. Exp. Biol. Med. 154:1403-1418.
10. Crandell, R A., C. G. Fabricant, and W. A. Nelson-Rees. 1973.
Developmental, characterization and viral susceptibility of Felis
catus renal cell line (CRFK). In Vitro (Rockville) 9:176.
11. Dewhurst, S., J. Bresser, M. Stevenson, K. Sakai, M. J. Evinger-
Hodges, and D. J. Volsky. 1987. Susceptibility of human glial cells
to human immunodeficiency virus (HIV). FEBS Lett. 213:138-143.
6750
J. VIROL.
NOTES
12. Dickson, D. W., L. A. Mattiace, K. Kure, K. Hutchins, W. D.
Lyman, and C. F. Brosana. 1991. Microglia in human disease, with
emphasis on the human immune deficiency syndrome. Lab. Invest.
64:135-156.
13. Dow, S. W. 1989. Retrovirus infection of the central nervous
system. Proceedings of the 7th American College Veterinary
Internal Medicine Forum, San Diego, Calif.
14. Dow, S. W., M. J. Dreitz, and E. A. Hoover. 1992. Feline immu-
nodeficiency virus neurotropism: evidence that astrocytes and
microglia are the primary target cells. Vet. Immunol. Immuno-
pathol. 35:23-35.
15. Dow, S. W., M. L. Poss, and E. A. Hoover. 1990. Feline immuno-
deficiency virus: a neurotropic lentivirus. J. Acquired Immune
Defic. Syndr. 3:658-668.
16. Ehrenberg, B., V. Montana, M. D. Wei, J. P. Wuskell, and L. M.
Loew. 1988. Membrane potential can be determined from individ-
ual cells from the Nernstian distribution of cationic dyes. Biophys.
J. 53:785-794.
17. Fermin, C. D., and R. F. Garry. 1992. Membrane alterations linked
to early alterations of HIV with the cell surface. Virology 191:941-
946.
18. Fletcher, W. H., C. V. Byus, and D. A. Walsh. 1987. Receptor
mediated action without receptor occupancy: a function for cell-
cell communication in ovarian follicles, p. 299-323. In V. B.
Mahesh, D. S. Dhindsa, E. Anderson, and S. P. Kalra (ed.),
Regulation of ovarian and testicular function. Plenum Publishing
Corp., New York.
19. Fontana, A., F. Kristensen, R Dubs, D. Gemsa, and E. Weber.
1982. Production of prostaglandin E and an interleukin I like
factor by cultured astrocytes and C6 glioma cells. J. Immunol.
129:2413-2419.
20. Frei, K., S. Bodmer, C. Schwerdel, and A. Fontana. 1986. Astro-
cyte derived interieukin 3 as a growth factor for microglia cells and
peritoneal macrophages. J. Immunol. 137:3521-3527.
21. Frohman, E. M., S. Van Der Noort, and S. Gupta. 1989. Astrocytes
and intracerebral immune responses. J. Clin. Immunol. 9:1-9.
22. Giulian, D., J. Woodward, D. G. Young, J. F. Krebs, and L. B.
Lachman. 1988. Interleukin 1 injected into mammalian brain
stimulates astrogliosis and neovascularization. J. Neurosci.
8:2485-2490.
23. Grewe, C., A. Beck, and H. R Gelderblom. 1990. HIV: early
virus-cell interactions. J. Acquired Immune Defic. Syndr. 10:965-
974.
24. Harbour, D. A., P. D. Williams, T. J. Gruffydd-Jones, J. Burbridge,
and G. R Pearson. 1988. Isolation of a T lymphotropic lentivirus
from a persistently leucopenic domestic cat. Vet. Rec. 122:84-
86.
25. Ishida, T., T. Washizu, K. Toriyabe, S. Motoyoshi, L. Tomoda, and
N. C. Pedersen. 1989. Feline immunodeficiency virus in cats of
Japan. JAMA 194:221-225.
26. Jain, A., J. Martensson, E. Stole, P. A. M. Auld, and A. Meister.
1991. Glutathione deficiency leads to mitochondrial damage in the
brain. Proc. Natl. Acad. Sci. USA 88:1913-1917.
27. Janzer, R. C., and M. C. Raff. 1987. Astrocytes induce blood brain
properties in endothelial cells. Nature (London) 325:253-257.
28. Kimelberg, H. K. 1986. Occurrence and functional significance of
serotonin and catecholamine uptake by astrocytes. Biochem.
Pharmacol. 35:2273-2281.
29. Kimelberg, M. R., and B. R Ransom. 1986. Physiological and
pathological aspects of astrocyte swelling, p. 129-158. In S. Fed-
eroff and A. Vernadis (ed.), Astrocyte cell biology and pathology
of astrocytes, vol. 3. Academic Press, New York.
30. Kinchington, D., W. Barker, S. Galpin, and K. Apostolov. 1992.
Temperature enhancement of syncytium formation by HIV and
Sendai virus. J. Med. Virol. 36:44-48.
Lehninger, A. L. 1984. Principles of Biochemistry, p. 491-492.
Worth Publishers Inc., New York.
Loewenstein, W. R 1966. Permeability of membrane junctions.
Ann. N. Y. Acad. Sci. 137:441-471.
Lynn, W. S., A. Tweedale, and M. W. Cloyd. 1988. Human
immunodeficiency virus (HIV-1): perturbation of the cell mem-
brane and depression of phospholipid synthesis. Virology 163:43-
51.
Malipiero, U. V., K. Frei, and A. Fontana. 1990. Production of
hemopoietic and colony stimulating factors by astrocytes. J. Im-
munol. 144:3816-3821.
Meister, A., and M. E. Anderson. 1983. Glutathione. Annu. Rev.
Biochem. 52:711-760.
Miller, M. A., R. F. Garry, J. M. Jaynes, and R. C. Montelaro.
1991. A structural correlation between lentivirus transmembrane
proteins and natural cytolytic peptides. AIDS Res. Hum. Retro-
viruses 7:511-519.
Pedersen, N. C., E. W. Ho, M. L. Brown, and J. K. Yamamoto.
1987. Isolation of a T lymphotropic virus from domestic cats with
immunodeficiency-like syndrome. Science 235:790-793.
Pumarola-Sune, T., B. A. Navia, C. Cordon-Cardo, E. S. Cho, and
R W. Price. 1987. HIV antigens in the brain of patients with AIDS
dementia complex. Ann. Neurol. 21:490-496.
Rahn, C. A., D. W. Bombick, and D. J. Doolittle. 1991. Assessment
of mitochondrial membrane potential as an indicator of cytotox-
icity. Fundam. Appl. Toxicol. 16:435-448.
Rey, M. A., B. Spire, D. Dormont, F. Barre-Sinoussi, L. Montag-
nier, and J. C. Chermann. 1984. Characterization of the RNA
dependent DNA polymerase of a new human T lymphotropic
retrovirus (lymphadenopathy associated virus). Biochem. Biophys.
Res. Commun. 121:126-133.
Rodriguez, M., M. L. Pierce, and E. L. Howie. 1987. Immune
response gene products (Ia antigens) on glial and endothelial cells
in virus induced demyelination. J. Immunol. 138:3438-3442.
Sparger, E. E., P. A. Luciw, J. H. Elder, et al. 1989. Feline
immunodeficiency virus is a lentivirus associated with an AIDS-
like disease in cats. J. Acquired Immune Defic. Syndr. 3(1):43-45.
Stavrou, D., P. Mehraein, W. Mellert, K. Bise, K. Schmidtke, E.
Rothemunds, I. Funke, U. Stocher, L. Babaryka, C. Zietz, V. Frydl,
F. Prantl, and V. Erfle. 1989. Evaluation of intracerebral lesions in
patients with acquired immunodeficiency syndrome, neuropatho-
logical findings and experimental data. Neuropathol. Appl. Neu-
robiol. 15:207-222.
Wade, M. H., J. E. Trosko, and M. Schindler. 1986. A fluorescence
photobleaching assay of gap junction mediated communication
between human cells. Science 232:525-528.
Yamamoto, J. K., H. Hansen, E. W. Ho, T. Y. Morishita, T. Okuda,
T. R. Sawa, R. M. Nakamura, W. P. Kau, and N. C. Pedersen.
1989. Epidemiologic and clinical aspects of feline immunodefi-
ciency virus infection of cats from the continental United States
and Canada and possible modes of transmission. J. Am. Vet. Med.
Assoc. 194:213-220.
Yamamoto, J. K., E. Sparger, E. W. Ho, P. R. Anderson, T. P.
O'Connor, C. P. Mandell, L. Lowenstine, R. Munn, and N. C.
Pedersen. 1988. Pathogenesis of experimentally induced feline
immunodeficiency virus infection in cats. Am. J. Vet. Res. 48:
1246-1258.
Zenger, E., E. W. Collisson, R. Barhoumi, R. C. Burghardt, I. R.
Danave, and E. Tiffany-Castiglioni. Laser cytometric analysis of
FIV-induced injury in astroglia. glia, in press.
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