Effect of ACTH on the nucleolar size of adrenal cortical cells and of liver cells. An electron-microscopic study using a quantitative biometrical method.
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Adrenal Glands↗
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Functional and molecular characterization of a volume-activated chloride channel in rabbit corneal epithelial cells.
We characterized the functional and molecular properties of a volume-regulated anion channel (VRAC) in SV40-immortalized rabbit corneal epithelial cells (tRCE), since they mediate a robust regulatory volume decrease (RVD) response during exposure to a hypotonic challenge. Whole-cell patch clamp-monitored chloride currents and light-scattering measurements evaluated temporal cell-volume responsiveness to hypoosmotic challenges. Exposure to 200 mOsm medium elicited an outwardly-rectifying current (VACC), which was reversible upon reperfusion with isotonic (300 mOsm) medium. VACC and RVD were chloride-dependent because either chloride removal or application of NPPB (100 microM) suppressed these responses. VACC behavior exhibited voltage-dependent inhibition in the presence of DIDS (500 microM), whereas inhibition by both NPPB (100 microM) and niflumic acid (500 microM) was voltage-independent. VACC was insensitive to glibenclamide (250 microM), verapamil (500 microM) or removal of extracellular calcium. Phorbol dibutyrate, PDBu, (100 nM) had no effect on activated VACC. However, preincubation with PDBu prior to hypotonic challenge prevented VACC and RVD responses as well as prolonged characteristic time. An inactive phorbol ester analogue had no effect on RVD behavior. Moreover, Northern blot analysis verified expression of ClC-3 gene transcripts. The presence of ClC-3 transcripts along with the correspondence between the effects of known ClC-3 inhibitors on VACC and RVD suggest that ClC-3 activation underlies these responses to hypotonic-induced cell swelling.
Animals↗
Association of ClC-3 channel with Cl- transport by human nonpigmented ciliary epithelial cells.
Electrophysiologic and volumetric evidence link the swelling-activated Cl- channels [gCl(Vol)] of nonpigmented ciliary epithelial (NPE) cells with the Cl(-)-channel/Cl(-)-channel regulator protein pICln. However, inhibitors (verapamil and dideoxyforskolin) of another Cl- channel/regulator (MDR1) have been found to inhibit the volume-activated transport response [the regulatory volume decrease (RVD)] of bovine NPE cells. We have addressed the possible molecular basis for the NPE Cl- channels by volumetric measurements of ODM human NPE cells in hypotonic and isotonic test solutions, and by polymerase chain reaction (PCR) cloning and Northern analyses of the same cells. Verapamil and dideoxyforskolin did inhibit the RVD. However, at a concentration (100 microM) which blocks > 90% of the MDR1-associated Cl- currents, forskolin had no effect on the volume-activated Cl- channels or on the inhibition of those channels by protein kinase C. High concentrations of ATP (3.5 and 10 mM) and niflumic acid (IC50 approximately 200 microM) also block [gCl(Vol)]. The RVD is inhibited by 9-phenylanthranilic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), unaffected by anthracene-9-carboxylic acid (9-AC), and stimulated by ionomycin. The Cl(-)-channel blockers NPPB, niflumic acid, DPC and 9-AC, and the Ca2(+)-ionophore ionomycin had qualitatively similar effects on the rate of staurosporine-activated isotonic cell shrink-age. These results support the concept that the volume-sensitive protein pICln regulates the Cl- channels, and that the same conduits subserve volume- and staurosporine-activated Cl- release. Of the cloned and sequenced Cl- channels, ClC-3 uniquely conforms to the stationary currents and PKC sensitivity of the NPE Cl- channels. PCR amplifications of human cDNA libraries from ciliary body, NPE cells and retina with primers based on human ClC-3 and ClC-4 cDNA, and Northern analyses using the products generated indicated that ciliary epithelial cells express transcripts for ClC-3 (but not ClC-4). We suggest that ClC-3 provides the same conduit for both volume-activated and isotonically staurosporine-activated Cl- channels of human nonpigmented ciliary epithelial cells.
Alkaloids↗
Na+/H+ and CI-/HCO3-antiporters of bovine pigmented ciliary epithelial cells.
Medical therapy of glaucoma commonly aims at slowing aqueous humor formation by the ocular ciliary epithelial bilayer, but underlying mechanisms are poorly understood. The first step in secretion is NaCl uptake from the stroma into the pigmented ciliary epithelial (PE) cell layer by electroneutral transporters. After crossing gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, solute is released into the aqueous humor. Published data have indicated that both paired Na+/H+ and Cl-/HCO3- antiporters and the Na+-K+-2Cl- symporter are involved in net uptake. The molecular identities of the paired antiporters have not been elucidated. We have studied continuously cultured bovine PE cells. Acid-activated 22Na+ uptake was inhibited by cariporide, EIPA (ethyl-isopropyl-amiloride) and amiloride, at concentrations characteristic of the NHE-1 isoform. Videomicroscopy of BCECF-loaded PE cells verified the presence of an EIPA-inhibitable Na+/H+ antiporter. Removing external Cl- also triggered an alkalinization, which was Na+-independent and could be inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Application of hypotonicity followed by return to isotonicity triggered a regulatory volume increase, which was pharmacologically similar to the uptake mechanisms described for intact rabbit ciliary epithelium. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of RNA from the human ciliary body detected expression of the AE2 Cl-/HCO3- exchanger, but not of AE1, cAE3 or bAE3. Immunostaining of bovine PE cells also revealed the presence of AE2 epitope. We conclude that paired NHE-1 Na+/H+ and AE2 Cl-/HCO3- antiporters are important components in the initial step in aqueous humor formation.
Animals↗
Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice.
Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthe grisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.
Animals↗
Analysis of the melon genome in regions encompassing TIR-NBS-LRR resistance genes.
Plant genomes contain numerous genes (R-genes) that play a role in initiating defence measures against their particular pathogens. Defence mechanisms controlled by R-genes have been the focus of extensive research over the past several years. The majority of the R-genes described so far belong to a super-family of genes (150-600 members) that encode proteins with a nucleotide binding site (NBS), some leucine-rich repeats (LRR) and an N-terminal domain that shows similarity to the Toll and Interleukin-1 receptors (TIR) or a N-terminal coiled-coil (CC) domain. Analysis of four regions of the melon (Cucumis melo) genome, including two sequenced BACs, identified 14 TIR-NBS-LRR genes. Known disease resistance genes have been mapped in three of these regions. Transcriptional expression was detected for predicted genes that are possibly involved in defence responses to pathogen attack. TIR-NBS-LRR genes appear to be clustered in the melon genome. They contain all the conserved motifs that have previously been described for their counterparts in other species, although differences were also detected. The results presented here may contribute to a better understanding of the genomic distribution and evolution of this group of resistance gene homologues and their variability.
Amino Acid Sequence↗
Apoptosis is not correlated with the presence of CD57+ NK-cells in brain metastases.
BACKGROUND: Cell apoptosis in solid tumours has been related to immunological attack by NK-cells. The purpose of the present study is to verify in the tissue of brain metastases a possible relationship among the degree of NK-cell infiltration and the number of apoptotic tumour cells. METHODS: Twenty brain metastases whose tumour cells expressed CD95 (Fas/APO1) have been studied. NK-cells were identified by using the monoclonal antibody to CD57, and apoptotic tumour cells by means of the immunostain with the anti-ssDNA monoclonal antibody F7-26. The Spearman rank correlation test was used to study the relationship between the degree of CD57-NK-cell infiltration and the apoptosis labelling-index. FINDINGS: Positivity to F7-26 was present in all tumour samples, but the number of immunostained cells showed a wide variability, with a mean apoptosis labelling-index of 11.48%. All the studied tumours showed CD57 immunostained cells, with a number that ranged between 4 and 20 per microscopical field at 200x (mean+/-standard deviation: 8.4+/-3.7). Statistical studies showed that there was no correlation between the number of CD57 immunostained NK-cells and the apoptosis labelling-index (p>0.05). INTERPRETATION: These findings suggest that in brain metastases, apoptosis related to immune response is mainly mediated by activated tumour-infiltrating mononuclear cells other than CD57(+) NK-cells.
Adenocarcinoma↗
Imbalance between apostain expression and proliferative index can predict survival in primary glioblastoma.
BACKGROUND: Cell proliferation and cell death are opposing processes in tumour growth, with tumour progression reflecting the balance between proliferating and apoptotic cells. The purpose of the present study is to verify the hypothesis that an imbalance between apoptosis and proliferation can predict survival in patients with primary glioblastoma. METHODS: After the immunohistochemical study of Apostain and MIB-1 expression, the index of apoptosis (AI), the index of proliferation (PI), and the ratio AI/PI was recorded for each tumour specimen, in a series of 32 primary glioblastomas. Studies of correlation between AI and PI, between AI and survival, between PI and survival, and between the ratio AI/PI and survival, were performed using the Spearman rank correlation test. Furthermore, a comparative study of survival was performed for subgroups of patients with ratio AI/PI greater or lesser than 1, using the log rank test. FINDINGS: In the present series, values of AI and PI showed a wide distribution, with a mean +/- SD of 8.16 +/- 7.2, and of 12.69 +/- 21.1, respectively. The values for the ratio AI/PI ranged between 0.01 and 6.03 (mean +/- SD: 1.44 +/- 1.60). Statistical study failed to obtain correlation between AI and PI. Survival of patients not correlated with AI neither with PI. The ratio between AI and PI did not correlate with survival either. Nevertheless, when survival for the subgroups of patients showing a ratio AI/PI greater or lesser than 1 was compared, a significant difference was found (p: 0.02). Survival ranged between 50 and 81 weeks (mean of 58.5 +/- 11.05 weeks) for the 12 cases showing a ratio AI/PI greater than 1, and it ranged between 8 and 85 weeks (mean: 38.20 +/- 25.37 weeks) for the 20 cases showing a ratio AI/PI lesser than 1. INTERPRETATION: Our present results suggest that a clear imbalance between cell proliferation and apoptosis can predict outcome in patients operated on for a primary glioblastoma.
Aged↗
Quantification of isomeric equilibria formed by metal ion complexes of 8-[2-(phosphonomethoxy)ethyl]-8-azaadenine (8,8aPMEA) and 9-[2-(phosphonomethoxy)ethyl]-8-azaadenine (9,8aPMEA). Derivatives of the antiviral nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA).
The acidity constants of the two-fold protonated acyclic 9-[2-(phosphonomethoxy)ethyl]-8-azaadenine, H2(9,8aPMEA)(+)(-), and its 8-isomer, 8-[2-(phosphonomethoxy)ethyl]-8-azaadenine, H2(8,8aPMEA)(+)(-), both abbreviated as H2(PA)(+)(-), as well as the stability constants of their M(H;PA)+ and M(PA) complexes with the metal ions M2+=Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+, have been determined by potentiometric pH titrations in aqueous solution at I=0.1 M (NaNO3) and 25 degrees C. Application of previously determined straight-line plots of log K(M)M(R-PO3) versus pK(H)H(R-PO3)for simple phosph(on)ate ligands, R-PO3(2-), where R represents a residue without an affinity for metal ions, proves that for all M(PA) complexes a larger stability is observed than is expected for a sole phosphonate coordination of the metal ion. This increased stability is attributed to the formation of five-membered chelates involving the ether oxygen present in the aliphatic residue (-CH2-O-CH2-PO3(2-)) of the ligands. The formation degrees of these chelates were calculated; they vary between about 13% for Ca(8,8aPMEA) and 71% for Cu(8,8aPMEA). The adenine residue has no influence on complex stability except in the Cu(9,8aPMEA) and Zn(9,8aPMEA) systems, where an additional stability increase attributable to the adenine residue is observed and equilibria between four different isomers exist. This means (1) an open isomer with a sole phosphonate coordination, M(PA)op, where PA(2-)=9,8aPMEA2-, (2) an isomer with a five-membered chelate involving the ether oxygen, M(PA)cl/O, (3) an isomer which contains five- and seven-membered chelates formed by coordination of the phosphonate group, the ether oxygen and the N3 site of the adenine residue, M(PA)cl/O/N3, and finally (4) a macrochelated isomer involving N7, M(PA)cl/N7. For Cu(9,8aPMEA) the formation degrees are 15, 30, 48 and 7% for Cu(PA)op, Cu(PA)cl/O, Cu(PA)cl/O/N3 and Cu(PA)cl/N7, respectively; this proves that the macrochelate involving N7 is a minority species. The situation for the Cu(PMEA) system, where PMEA2- represents the parent compound, i.e. the dianion of 9-[2-(phosphonomethoxy)ethyl]adenine, is quite similar. The relationship between the antiviral activity of acyclic nucleoside phosphonates and the structures of the various complexes is discussed and an explanation is offered why 9,8aPMEA is biologically active but 8,8aPMEA is not.
Adenine↗
Cyclo-oxygenase-2-specific inhibitors and cardiovascular morbidity.
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Anti-Inflammatory Agents, Non-Steroidal↗
Studies on regulation of the ascorbic acid transporter in a cell line derived from rabbit non-pigmented ciliary epithelium.
A cell line was derived from rabbit non-pigmented ciliary epithelium. The non-pigmented ciliary epithelium is one of the two cell layers which secrete aqueous humor into the eye and concentrate ascorbic acid in the newly-formed fluid. The cultured non-pigmented epithelial cells accumulated ascorbic acid at a rate of 3-5 pmol/micrograms protein per h. As in freshly-isolated native tissue, the ascorbate uptake mechanism was sodium-dependent and could be inhibited by phloretin (apparent Ki = 2-10(-5) M). Phorbol 12,13-dibutyrate (PDBu), a protein kinase C activator, reduced the ascorbate uptake rate. The PDBu effect was concentration-dependent; at a concentration of 10(-6) M, PDBu reduced the ascorbate uptake rate to 65% of the control value. PDBu reduced the maximal rate of ascorbate uptake (determined at 200-500 microM external ascorbate) but caused no detectable change in the Km for ascorbic acid (approx. 80 microM). The PDBu-induced inhibition of ascorbate uptake persisted in the presence of ouabain and in low sodium (25 mM Na) medium, suggesting that the effect is not secondary to a change in the sodium gradient. Furthermore, no detectable elevation of cell sodium content was seen in cells equilibrated with 22Na prior to PDBu treatment. The PDBu-induced inhibition of ascorbate uptake was apparently mediated by protein kinase C because the effect was not observed in the presence of staurosporine (10(-6) M), a protein kinase C inhibitor, or in cells in which protein kinase C was downregulated. These observations suggest that activation of protein kinase C causes inhibition of the ascorbate transporter in this cultured cell line.
Alkaloids↗
Nucleotide sequence of a cDNA for the beta 2 subunit isoform of Na+,K(+)-ATPase from human retina.
Using as probe the entire human liver cDNA clone coding for the beta 2 subunit isoform of the Na+,K(+)-ATPase, which lacks the initiation codon ATG, and the entire 5'-untranslated region (Martin-Vasallo, P., Dackowski, W., Emanuel, J.R. and Levenson, R. (1989) J. Biol. Chem. 264, 4613-4618), we isolated a larger clone from a directional human adult retina cDNA library (Swaroop, A. and Xu, J. (1993) Cytogenet. Cell Genet. 64, 292-294). This clone, pNH beta 2, shows 100% homology with the nucleotide sequence of the human liver cDNA clone and also contains additional 407 nucleotides in the 5'-untranslated region, the initiation codon and a poly(A) tail. Northern blot hybridization analysis reveals that the human mRNA (3.6 kb) is approx. 300 nucleotides larger than the major transcript size expressed in rat (3.3 kb). The larger human size mRNA for the human beta 2 Na+,K(+)-ATPase indicates species differences in gene processing.
Amino Acid Sequence↗
Evidence that the alpha and alpha (+)isoforms of the catalytic subunit of (Na+, K+)-ATPase reside in distinct ciliary epithelial cells of the mammalian eye.
Polyclonal antibodies against the canine kidney (Na+,K+)-ATPase were used to examine the localization and distribution of this protein in intact ciliary processes (CP) from bovine eyes by indirect immunofluorescence. The basolateral surface of non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells was found to be stained specifically for the (Na+,K+)-ATPase. Immunoblot analysis of intact CP, separated PE and NPE cells by density gradients and cultured ciliary epithelial cells, revealed two forms of the catalytic subunit of the (Na+,K+)-ATPase: the alpha and alpha (+). The alpha (+) form was enriched in NPE cells while alpha was in PE cells.
Animals↗
Erythema nodosum and pulmonary solitary nodule as the first manifestations of a non-Hodgkin's lymphoma.
Both erythema nodosum and the pulmonary solitary nodule are unusual manifestations of non-Hodgkin's lymphoma (NHL). In this article, we report the first case in which they were simultaneously associated as the initial manifestations of NHL.
Erythema Nodosum↗
Ultrastructure of the proximal convoluted tubule in the hibernating garden dormouse (Eliomys quercinus L.).
We have carried out an ultrastructural study of the proximal convoluted kidney tubule in hibernating and nonhibernating garden dormice. Deep hibernation produced the following changes: the nuclei had an irregular shape; saccular cavities appeared near the nucleus and there was an increase in the components of the lysosomal system; hibernation seems to have no influence on the number of mitochondria; hypertrophic mitochondria occur in controls, whereas none were ever seen in hibernating dormice.
Animals↗
Human ciliary epithelia in monolayer culture.
The ciliary epithelia of human (one to 12 months old) ciliary processes were isolated by trypsin and EDTA, cultured in Dulbecco's Modified Eagle Medium (DMEM) with 5% fetal calf serum and examined by phase and electron microscopy. The primary cultures were maintained for three to four months. Only a few non-pigmented epithelia adhered and none of them proliferated. After the first passage the culture seemed to consist of only the pigmented epithelia. Most cells were densely pigmented at first, then became less pigmented during successive proliferations. Half of the cells remained densely pigmented after the first subculture, another half remained less pigmented. The cells started to lose their pigment granules at four to six weeks in culture. After three months of culture, the cell sheets became entirely unpigmented. In thin section, most of the pigment granules in the cells at two weeks in culture were pre-melanosomes, and half of them were at the earliest stage of pre-melanosomes. Monolayer cells possessed basement membranes. At 14 weeks in culture, most cells established an apparent polarity, contained well-developed Golgi apparatus and rough endoplasmic reticulum and intermediate filaments, but no pigment granules. A bundle of intermediate filaments was found in the perinuclear cytoplasm. Multilayer cells presented a typical apex-to-apex and base-to-base configuration , and the extracellular material was detected only in the base-to-base intercellular spaces. Our culture system provided differentiated cells derived from the pigment epithelia of human ciliary processes.
Cells, Cultured↗
Separation of bovine pigmented ciliary epithelial cells by density gradient and further characterization in culture.
Dissociated pigmented epithelial (PE) cells from ciliary processes (CP) of bovine eyes were separated by isopycnic centrifugation in metrizamide gradients and further cultivated. The level of cellular contamination during separation of PE by non-pigmented epithelial (NPE) cells was estimated to be 5-10% and by non-epithelial cells (i.e. endothelial, fibroblasts and blood cells) less than 1%. Bovine PE cells were found to have different serum requirements than NPE to grow in vitro. PE cells were actively stimulated to proliferate in the presence of fetal calf serum for a few generations. Short-term culture of NPE was only obtained in media containing a low concentration of L-tyrosine and no serum. Two morphological markers, namely desmosomes and intermediate-sized filaments (IF) of the vimentin type, were found by immunolabeling to be coexpressed in cultured PE and were used as criteria to define the epithelial identity of these cells. The present study was unable to detect any tyrosinase activity in cultured PE cells.
Animals↗
Volume regulation of cultured, transformed, non-pigmented epithelial cells from human ciliary body.
Electronic cell sizing has been used to measure the volume of cells suspended in isosmotic and in hyposmotic solutions of identical ionic composition. Without inhibitors, the cells displayed a regulatory volume decrease (RVD) following anisosmotic cell swelling with a time constant (tau) of 6.3 +/- 0.9 min (mean +/- S.E.). The RVD was markedly impaired by substituting gluconate for external Cl-, and tau was prolonged by: (i) reducing the chemical gradient favoring K+ loss (by elevating the external [K+] and blocking the Na. K-exchange pump), (ii) blocking the K+ channels with Ba2+, (iii) blocking Cl- channels and Cl-/HCO3 = exchange with DIDS, and (iv) removing external HCO3-. Withdrawing HCO3- may have altered the RVD either directly by inhibiting a Cl-/HCO3- antiport, or indirectly by affecting intracellular pH. The regulatory volume response of ODM Cl-2/SV40 cells is in several respects qualitatively similar to that of non-pigmented epithelial cells of the intact ciliary body. These common characteristics suggest that the cultured cells can serve as a useful model for studying solute and fluid transport across the human ciliary epithelium. The basis for the RVD is likely to be activation of separate K+ and Cl- channels, with or without the parallel operation of coupled K+/H+ and Cl-/HCO3- antiports.
Cell Line, Transformed↗