Haemochromatosis in a Brazilian tapir (Tapirus terrestris) in an Australian zoo.

A 23-year-old Brazilian, or lowland, tapir with a 6-month history of loss of body condition developed clinical signs and laboratory findings consistent with liver failure. The animal was euthanased and a diagnosis of hepatic haemochromatosis was made based on histopathology. Two other healthy tapirs in the same collection had chronically elevated serum and tissue iron concentrations. The excessive accumulation of iron in tissues with resultant tissue damage (i.e. haemochromatosis) has been reported in a range of captive species. This and other reported cases of haemochromatosis in the Brazilian tapir would suggest that this condition is an important consideration in the management of this species in zoos. Further research into the endogenous regulation of iron metabolism, especially the role of hepcidin, in tapirs and other species at risk of iron storage disorders may be helpful in the prevention of this condition.

Cyclic AMP and LDL trigger a rapid enhancement in gap junction assembly through a stimulation of connexin trafficking.

Given the rapid turnover of connexin proteins, gap junction (GJ) assembly represents an important means of regulating the extent of GJ communication between cells. This report describes an increase in the level of GJ assembly within one hour following treatment with cAMP-elevating reagents or low density lipoprotein (LDL). Dye transfer methods and freeze-fracture with electron microscopy were used to assay junctional permeability and structure, respectively, subsequent to the dissociation, recovery and reaggregation of Novikoff hepatoma cells. Reaggregating cells in the presence of agents that increase cAMP levels (8-Br-cAMP, forskolin and IBMX) enhanced both dye transfer rates between cells and the extent of GJ formation 2- to 3-fold. These data and studies with the protein kinase A inhibitor, H-89, indicate that cAMP signaling plays a key role in enhanced assembly. The response to LDL parallels that to cAMP and relies on the activity of both adenylyl cyclase and protein kinase A. Immunoblot analysis revealed no change in the level of connexin43 (Cx43) or its phosphorylation states over a period of 2.5 hours. However, three agents (brefeldin A, monensin and nocodazole), that inhibit intracellular membrane trafficking by different mechanisms, all blocked the enhanced assembly of GJs when triggered by either elevated cAMP or exposure to LDL. Related studies, which employed trafficking inhibitors at different stages in GJ assembly, suggested that Cx43 trafficking during enhanced assembly is regulated, in part, by cell contact. Intracellular sources of Cx43 were characterized by colabeling for several markers of cytoplasmic membrane systems. We conclude that an increase in GJ assembly: (i) occurs rapidly in the presence of elevated cAMP or LDL, (ii) does not require an increase in Cx43 levels or major changes in Cx43 phosphorylation and (iii) is dependent upon the trafficking of Cx43 from intracellular storage sites.

Inhibitory effects of 12-O-tetradecanoylphorbol-13-acetate on dye leakage from single Novikoff cells and on dye transfer between reaggregated cell pairs.

The inhibitory effects of 12-O-tetradeconylphorbol-13-acetate (TPA) on the Lucifer Yellow leakage in single (nondissociated and dissociated) Novikoff cells in the presence of ethyleneglycotetraacetic acid (EGTA) and transfer between reaggregated cell pairs were investigated. Under treatment with TPA, single Novikoff cells showed inhibition of the dye leakage in the presence of EGTA, especially after 60-min treatment. There were only slight differences between nondissociated and dissociated cells in these experiments. The dye transfer in reaggregated cell pairs were significantly inhibited after 30-min treatment with TPA. In pretreatment with TPA during the recovery period of reaggregation the dye transfer in cell pairs was completely blocked. It was suggested that TPA blocks the assembly of gap junction, while it had no remarkable effect on gating mechanism of the hemichannels.

Comparison of lucifer yellow leakage and cell-to-cell transfer following intracellular injection in normal and antisense Novikoff cells under treatment with low extracellular Ca2+.

Single and paired control Novikoff and connexin 43 antisense-transfected Novikoff cells (antisense cells) were used to investigate the differences of intercellular dye transfer and leakage between these two types of cells. Lucifer Yellow was injected into cells by iontophoresis. There was no dye leakage in single cells of both types in Swims solution. The dye transfer from injected cell to recipient cell in the normal solution was different in the two types of cell pairs. The transfer was significantly suppressed in antisense cells; 46/84 cases were blocked vs. 1/84 in Novikoff cell pairs. Under EGTA treatment, the dye leakage in single antisense cells was suppressed showing an increase in the very slow rate of leakage (16/40 vs. 6/40), and the mean rate of leakage was also significantly low. This suggests that dye transfer and leakage are suppressed in antisense cells and demonstrates that connexin 43 plays an important part both in dye transfer in paired cells and dye leakage in single cells.

Syringolide 1 Triggers Ca2+ Influx, K+ Efflux, and Extracellular Alkalization in Soybean Cells Carrying the Disease-Resistance Gene Rpg4.

Alleles of avirulence gene D (avrD) specify the production by bacteria of syringolides that elicit the hypersensitive response in soybean (Glycine max) plants carrying the disease-resistance gene Rpg4, but not rpg4 plants. Syringolide 1 caused extracellular alkalization, K+ efflux, and Ca2+ influx about 30 min after addition to suspension-cultured cells of two Rpg4 cultivars, Harosoy and Flambeau, but not in two rpg4 cultivars, Acme and Merit. All responses were sustained for at least 1.5 h and were inhibited by La3+, which blocks certain Ca2+ channels. These results suggest that syringolide 1 activates a Ca2+ influx-dependent signaling pathway only in Rpg4 soybean cells.

Psychiatric clinical nurse specialists as intensive case managers for the seriously mentally ill.

The government estimates the number of seriously mentally ill as high as three million, with annual cost for the care of this population approaching billions of dollars. This article describes a program for the seriously mentally ill in the community using psychiatric clinical nurse specialists as intensive case managers. Interventions regarding disease relapse symptomology and early recognition of medication side effects assist clients to seek earlier intervention. Desired outcomes include earlier hospitalizations at less serious stages, reduced length of stay, higher functioning, improved self-care skills, and improvement in obtaining and receiving medical service. With clinical nurse specialists in case management roles, clients receive treatment at more appropriate and less costly service levels.

Mechanical stimulation initiates cell-to-cell calcium signaling in ovine lens epithelial cells.

Although abnormalities in calcium regulation have been implicated in the development of most forms of cataract, the mechanisms by which Ca2+ is regulated in the cells of the ocular lens remain poorly defined. Cell-to-cell Ca2+ signaling was investigated in primary cultures of ovine epithelial cells using the Ca(2+)-reporter dye fura-2 and fluorescence microscopy. Mechanical stimulation of a single cell with a micropipette initiated a propagated increase in cytosolic free Ca2+ that spread from the stimulated cell through 2-8 tiers of surrounding cells. During this intercellular Ca2+ wave, cytosolic Ca2+ increased 2- to 12-fold from resting levels of approximately 100 nM. Nanomolar extracellular Ca2+ did not affect the cell-to-cell propagation of the Ca2+ wave, but reduced the magnitude of the cytosolic Ca2+ increases, which was most evident in the mechanically-stimulated cell. Depletion of intracellular Ca2+ stores with thapsigargin eliminated the propagated intercellular Ca2+ wave, but did not prevent the cytosolic Ca2+ increase in the mechanically-stimulated cell, which required extracellular Ca2+ and was attenuated by the addition of the Ca2+ channel blockers Ni2+, Gd3+ and La3+ to the medium. These results are most easily explained by a mechanically-activated channel in the plasma membrane of the stimulated cell. The propagated increase in cytosolic Ca2+ appeared to be communicated to adjacent cells by the passage of an intracellular messenger other than Ca2+ through gap junction channels. However, if the plasma membrane of the mechanically-stimulated cell was ruptured such that there was loss of cytosolic contents, the increase in cytosolic Ca2+ in the surrounding cells was elicited by both a messenger passing through gap junction channels and by a cytosolic factor(s) diffusing through the extracellular medium. These results demonstrate the existence of intercellular Ca2+ signaling in lens cells, which may play a role in regulating cytosolic Ca2+ in the intact lens.

Cyclic AMP modifies the cellular distribution of connexin43 and induces a persistent increase in the junctional permeability of mouse mammary tumor cells.

Direct communication between cells via gap junctions is thought to be an important component of homeostasis and coordinated cellular responses to external signals. We investigated how the second messenger cAMP exerts its effects on junctional communication in a mouse mammary tumor cell line, MMT22. Junctional permeance was quantitatively assessed using dye microinjection and video microscopy. An increase of permeance was found after exposure to 8-bromo-cAMP, being detectable after 30 minutes of treatment and attaining a fourfold higher level of permeance by 24 hours. This elevated level was maintained with continuous exposure to 8-bromo-cAMP for seven days. The permeability change was accompanied by an increase in gap junctions as shown by freeze-fracture electron microscopy and by confocal microscopy using antibodies directed against the gap junction protein, connexin43. The amount of detergent-insoluble connexin43 also increased with 8-bromo-cAMP treatment, and most of the increase could be attributed to an increase of slower migrating (i.e. phosphorylated) species of connexin43. However, connexin43 mRNA and the total cellular content of connexin43 did not change over this period of exposure to 8-bromo-cAMP, as shown by densitometric analyses of northern and western blots. We conclude that 8-bromo-cAMP affects the distribution of connexin43 such that a greater proportion of the protein is utilized for channel formation. Since these changes were relatively slow to develop and persisted with prolonged exposure to 8-bromo-cAMP, it is possible that the junctional permeability of these mammary tumor cells is linked to the 'basal' level of cAMP, i.e. levels maintained by the cells in accordance with a particular cell state.

Intracellular lucifer yellow leakage from Novikoff cells in the presence of ATP or low extracellular Ca: evidence for hemi-gap junction channels.

Lucifer Yellow was microinjected into Novikoff hepatoma cells and leakage was investigated under treatment with ATP (5 mM) and EGTA (5 mM) in the culture medium. In control conditions, there was no leakage in single or paired cells, except a few cases which showed very slow leakage (defined as slope < -0.0007/sec). Slow leakage rate (slope > -0.0008 but < -0.009/sec) and quick leakage rate (slope > -0.01) of intracellular dye were not seen. Dye transfer between cell pairs after Lucifer Yellow was injected into one cell was divided into two groups: quick transfer rates (4 cases, slope = -0.151 +0.0032) and slow transfer rates (15 cases, slope = -0.041 +0.0018). Under ATP treatment the intracellular dye leakage was observed in single cells (16 of 31 cases) and in cell pairs (20 of 57 cases). Extracellular low Ca2+ (EGTA treatment) enhanced the dye leakage much more: 30 of 40 cases in single cells and 21 of 36 cases in cell pairs. The leakage rates of intracellular dye under these treatments were similar to the transfer rates of the dye between cell pairs with quick and slow rates. It is suggested that the dye leakage from Novikoff cells under treatment with ATP or low [Ca2+]o shares the same mechanism as dye transfer through gap junctions, suggesting that the hemichannels in the plasma membrane can be opened under certain conditions.

Measurement of gap junctional communication by fluorescence activated cell sorting.

Cell-to-cell communication via gap junctions has played a fundamental role in the orderly development of multicellular organisms. Current methods for measuring this function apply mostly to homotypic cell populations. The newly introduced Fluorescence Activated Cell Sorting (FACS) method, albeit with some limitations, is simple, reliable, and quantitative in measuring the dye transfer via gap junctions in both homotypic and heterotypic cell populations. In the homotypic setting, the result in dye transfer from the FACS method is comparable to the scrape-loading and microinjection methods. Using this FACS method, we observed a decline of cell-to-cell communication in transformed and cancer cells. We also observed a differential degree of communication between two heterotypic cell populations depending on the direction of dye transfer.

Intercellular communication is reduced by TPA and Ki-ras p21 in quiescent, but not proliferating, NRK cells.

We investigated whether the growth state of NRK cells (proliferating or quiescent by serum deprivation) affected the ability of oncogenic Ki-ras p21 and the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), to alter gap junctional communication. We evaluated gap junctional permeance by rate analysis of the transfer of a fluorescent dye, Lucifer Yellow, between cell pairs. We found that while the gap junctions of proliferating NRK cells were unresponsive to both TPA and to Ki-ras p21, junctional communication in quiescent cells was significantly inhibited by brief exposures to 100 ng/ml TPA. Furthermore, activity of Ki-ras p21 2 h prior to TPA exposure enhanced the inhibitory effect of TPA in quiescent cells. Junctional sensitivity to TPA was transient, with inhibition of junctional communication detected at 10 min and refractory after 60 min of continuous exposure. The suppression of junctional communication by TPA was completely prevented if the oncogenic p21 had been active for a longer period of time (48 h). The application of a phorbol ester derivative (4 alpha-PDD), which does not activate protein kinase C, did not affect the ability of quiescent cells to communicate. From these results we conclude that there is a cell-state dependence of junctional sensitivity to TPA in NRK cells and that ras p21 activity potentiates the junctional response to TPA. One interesting possibility is that this involved a cell-cycle effect.

Micromolar levels of intracellular calcium reduce gap junctional permeability in lens cultures.

PURPOSE: To investigate in bovine and embryonic chicken lens cultures the effects of elevated intracellular calcium on the permeability of gap junctions. To determine the changes in intracellular calcium using fura-2. To detect any changes in the phosphorylation of connexin43 after ionophore treatment. METHODS: Lucifer yellow was micro-injected into individual cells, and dye spread to neighboring cells was evaluated. Intracellular calcium levels were measured using the calcium indicator, fura-2. Cultures were also labeled with 32P-orthophosphate followed by immunoprecipitation with antibodies specific for the gap junction protein, connexin43. RESULTS: Bovine lens cultures incubated in the presence of either A23187 or ionomycin showed a reduction in intercellular dye transfer. The intracellular calcium concentrations in bovine cells were increased from a mean value of 0.11 +/- .009 microM in the controls to a mean of 0.40 +/- .073 microM with ionomycin treatment. Subsequent addition of EGTA to the medium decreased the intracellular calcium concentrations to a mean of 0.26 +/- .113 microM and reversed the inhibition of dye spread found with ionomycin. With ionomycin in the medium, the phosphorylated form of connexin43 was not as prominent as in the controls. In contrast, these same treatments had no detectable effect on junctional permeability in the embryonic chicken lens cultures. Dye spread was equally extensive and rapid under control and ionophore conditions, even though fura studies showed an elevation in intracellular calcium levels. CONCLUSIONS: In the bovine cultures, physiologically relevant changes in the levels of cytoplasmic calcium markedly reduced dye transfer. The increase in cytoplasmic calcium was correlated with a change in the phosphorylation level of connexin43. The regulation of junctional communication in the chick lens cultures appears to differ significantly from that in the bovine system.

Involvement of plasma membrane calcium influx in bacterial induction of the k/h and hypersensitive responses in tobacco.

An early event in the hypersensitive response of tobacco to Pseudomonas syringae pv syringae is the initiation of a K(+)/H(+) response characterized by specific plasma membrane K(+) efflux, extracellular alkalinization, and intracellular acidification. We investigated the role of calcium in induction of these host responses. Suspension-cultured tobacco cells exhibited a baseline Ca(2+) influx of 0.02 to 0.06 micromole per gram per hour as determined from (45)Ca(2+) uptake. Following bacterial inoculation, uptake rates began to increase coincidently with onset of the K(+)/H(+) response. Rates increased steadily for 2 to 3 hours, reaching 0.5 to 1 micromole per gram per hour. This increased Ca(2+) influx was prevented by EGTA and calcium channel blockers such as La(3+), Co(2+), and Cd(2+) but not by verapamil and nifedipine. Lanthanum, cobalt, cadmium, and EGTA inhibited the K(+)/H(+) response in both suspension-cultured cells and leaf discs and prevented hypersensitive cell death in leaf discs. We conclude that increased plasmalemma Ca(2+) influx is required for the K(+)/H(+) and hypersensitive responses in tobacco.

Role of the Plasmalemma H-ATPase in Pseudomonas syringae-Induced K/H Exchange in Suspension-Cultured Tobacco Cells.

Activation of a host plasma membrane K(+) efflux/net H(+) uptake exchange by pathogenic pseudomonads plays an important role in the development of hypersensitivity in tobacco (Nicotiana tabacum). Involvement of the plasmalemma H(+)-pumping ATPase in this response was investigated. The exchange response of suspension-cultured tobacco cells to Pseudomonas syringae pv syringae was reduced 90% or more by ATPase inhibitors including vanadate, N-ethylmaleimide, and N,N'-dicyclohexylcarbodiimide. The exchange was also strongly inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone and by slightly alkaline external pH. Respiratory inhibitors such as oligomycin and sodium azide reduced the exchange by 50% to 75%, while glycolysis inhibitors such as sodium arsenite and sodium iodoacetate decreased exchange by approximately 90%. These results suggest that plasmalemma H(+)-ATPase activity is required for the exchange response and that this may reflect a requirement for a plasmalemma pH and/or electrical potential gradient.

Altered junctional permeability between cells transformed by v-ras, v-mos, or v-src.

Junctional permeability in normal and transformed NRK cells was quantitatively assessed by microinjecting fluorescent dye into one cell of a pair, digitizing the changes of fluorescence intensity using video analysis techniques, and applying the digital values to a solution of Fick's diffusion equation. We show that this approach reliably estimates the junctional permeance of a cell pair. Cells that are temperature sensitive for transformation were shown to also be temperature sensitive vis-a-vis junctional permeance. Thus permeance values were reduced approximately 80-90% on transformation by either a mutant Rous sarcoma virus or a mutant Moloney murine sarcoma virus. Cells transformed by wild-type Kirsten sarcoma virus were also shown to possess levels of junctional permeance significantly lower than nontransformed controls. The transformed junctional phenotype could be observed as early as 15 min after shifting to transformation-permissive conditions. Our results suggest that the oncogenes src, ras, and mos exert their effects on NRK cell junctions via converging pathways, of which one may be phosphorylation of junctional proteins.

Molecular cloning of a Pseudomonas syringae pv. syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants.

A cosmid clone isolated from a genomic library of Pseudomonas syringae pv. syringae 61 restored to all Tn5 mutants of this strain studied the ability to elicit the hypersensitive response (HR) in tobacco. Cosmid pHIR11 also enabled Escherichia coli TB1 to elicit an HR-like reaction when high levels of inoculum (10(9) cells per ml) were infiltrated into tobacco leaves. The cosmid, which contains a 31-kilobase DNA insert, was mobilized by triparental matings into Pseudomonas fluorescens 55 (a nonpathogen that normally causes no plant reactions), P. syringae pv. syringae 226 (a tomato pathogen that causes the HR in tobacco), and P. syringae pv. tabaci (a tobacco pathogen that causes the HR in tomato). The plant reaction phenotypes of all of the transconjugants were altered. P. fluorescens(pHIR11) caused the HR in tobacco and tomato leaves and stimulated an apparent proton influx in suspension-cultured tobacco cells that was indistinguishable from the proton influx caused by incompatible pathogenic pseudomonads. P. syringae pv. tabaci(pHIR11) and P. syringae pv. syringae 226(pHIR11) elicited the HR rather than disease symptoms on their respective hosts and were no longer pathogenic. pHIR11 was mutagenized with TnphoA (Tn5 IS50L::phoA). One randomly chosen mutant, pHIR11-18, no longer conferred the HR phenotype to P. fluorescens. The mutation was marker-exchanged into the genomes of P. syringae pv. syringae strains 61 and 226. The TnphoA insertions in the two pseudomonads abolished their ability to elicit any plant reactions in all plants tested. The results indicate that a relatively small portion of the P. syringae genome is sufficient for the elicitation of plant reactions.

Permeance of Novikoff hepatoma gap junctions: quantitative video analysis of dye transfer.

Fluorescent dyes are commonly used to study permeable (gap) junctions, but only rarely have quantitative values for junctional dye permeability been determined. In the present study, junctional permeance (PA, i.e., the product of the junctional permeability coefficient, P, times the junctional area, A) to Lucifer Yellow CH (LY) has been obtained for pairs of Novikoff hepatoma cells. Dye was microinjected into one cell and the subsequent transfer monitored by a SIT camera and recorded on video tape. The intensities of fluorescence in the injected and "recipient" cell were measured using a Digisector (Microworks) digitizing board and an Apple II Plus computer to analyze the video records. These changes in intensity, along with an estimate of volume of the spherical cells, were used to calculate the junctional permeance (PA) of cell pairs according to Fick's diffusion equation. Junctional permeances show considerable variation ranging from 0.08 X 10(-11) to 27.0 X 10(-11) cm3/sec. Using the mean PA and a previous estimate of the mean number of junctional channels per interface in the Novikoff cultures, a value for diffusion coefficient of LY through gap junctions is calculated to be about 1.4 X 10(-6) cm2/sec. There is a general proportionality between mean PA and cell volume for hepatoma cell pairs of a certain size range. Such a relationship between cell volume and junctional capacity suggests one source of variation of PA. Other possible sources, e.g., related to position in the cell cycle, are discussed.

Transient Activation of Plasmalemma K Efflux and H Influx in Tobacco by a Pectate Lyase Isozyme from Erwinia chrysanthemi.

A purified pectate lyase isozyme derived from Erwinia chrysanthemi induced rapid net K(+) efflux and H(+) influx in suspension-cultured tobacco cells. Comparable fluxes of other ions (Na(+), Cl(-)) were not observed. The K(+) efflux/H(+) influx response began within 15 minutes after addition of enzyme to cell suspensions and continued for approximately 1 hour after which cells resumed the net H(+) efflux exhibited prior to enzyme treatment. The response was not prolonged by a second enzyme dose 1 hour after the first. The K(+)/H(+) response was characterized by saturation at low enzymic activity (2 x 10(-3) units per milliliter), and inhibition by the protonophore, carbonyl cyanide m-chlorophenylhydrazone, and was not associated with membrane leakiness caused by structural cell wall damage. The total K(+) loss and H(+) uptake induced by enzyme was one-fourth to one-third that induced by Pseudomonas syringae pv. pisi and did not reduce cell viability. These results indicate that pectate lyase induces a K(+) efflux/H(+) influx response in tobacco similar to but of shorter duration than that induced by P. syringae pv. pisi during the hypersensitive response. Pectate lyase or other cell wall degrading enzymes may therefore influence the induction of hypersensitivity.

Modification of gap junctions in cells transformed by a temperature-sensitive mutant of Rous sarcoma virus.

Prompted by our observation that a reduction in junctional permeance is one of the earlier events in the process of neoplastic transformation of a cell line by Rous sarcoma virus, we analyzed the gap junctions from these cells to determine if the basis of the reduction is a loss of junctional channels. The cells (normal rat kidney, or NRK) are infected with a temperature-sensitive mutant of Rous sarcoma virus, allowing one easily to manipulate the cells into and out of the transformed state, and hence also to manipulate the junctional permeance. Using freeze-fracture electron microscopy, we found that the number and size of the junctions did not change in parallel with the permeance changes we had previously characterized. There is, however, a significant rearrangement of the junctional particles to a more random configuration when the cells are transformed and a reversal to the more ordered pattern when the cells are shifted back to the normal phenotype. These changes do parallel the changes in junctional permeance. We conclude that the permeance of existing junctional channels is modified and that the change in permeance may involve a change in the interaction of the junctional channels with each other and/or the surrounding lipid domain.

The Hypersensitive Reaction of Tobacco to Pseudomonas syringae pv. pisi: Activation of a Plasmalemma K/H Exchange Mechanism.

Net electrolyte efflux from suspension-cultured tobacco cells undergoing the hypersensitive reaction to Pseudomonas syringae pv. pisi resulted from a specific efflux of K(+) which was accompanied by an equimolar net influx of H(+). These fluxes began 60 to 90 minutes after inoculation of tobacco cells with bacteria, reached maximum rates of 6 to 9 micromoles per gram fresh weight tobacco cells per hour within 2.5 to 3 hours, and dropped below 4 micromoles per gram per hour within 5 hours. Tobacco cells lost approximately 35% of total K(+) during this period, and average cellular pH declined by approximately 0.75 pH unit. These events were accompanied by a 30% decrease in cellular ATP. K(+) and H(+) fluxes were inhibited by the protonophore (p-trifluoromethoxy)carbonyl cyanide phenylhydrazone and by increasing the K(+) concentration of the external solution. Tobacco leaf discs inoculated with the bacterium also exhibited a specific net K(+) efflux and H(+) influx. These results suggest that induction of the hypersensitive reaction in tobacco proceeds through the activation of a passive plasmalemma K(+)/H(+) exchange mechanism. It is hypothesized that activation of this exchange is a major contributing factor in hypersensitive plant cell death.