Relationship between the host cell mitochondria and the parasitophorous vacuole in cells infected with Encephalitozoon microsporidia

Encephalitozoon microsporidia proliferate and differentiate within a parasitophorous vacuole. Using the fluorescent probe, calcein, and the mitochondrial probe, MitoTracker-CMXRos, a vital method was developed that confirmed ultrastructural reports that the host cell mitochondria frequently lie in immediate proximity to the parasitophorous vacuole. Morphometry failed to demonstrate any infection-induced increase in host cell mitochondria as there was no correlation between the mitochondrial volume and the extent of infection as judged by the parasitophorous vacuole volume. The total ATP concentration of infected cells did not differ from that of uninfected cells in spite of the increased metabolic demands of the infection. Treatment with 10(-6) M albendazole, more than ten times the antiparasitic IC50 dose, and demecolcine had no subjective effect on the proximity of mitochondria to the parasitophorous vacuole membrane when studied by either transmission electron microscopy or by confocal microscopy even though these drug concentrations affected microtubule structure. Thus, once the association between mitochondria and the parasitophorous vacuole has been established, host cell microtubule integrity is probably not required for its maintenance. It is unlikely that the antimicrosporidial action of albendazole involves physically uncoupling developing parasite stages from host cell organelle metabolic support.     

VCAM-1-induced inwardly rectifying K(+) current enhances Ca(2+) entry in human THP-1 monocytes

Hyperpolarization in human leukemia THP-1 monocytes adherent tovascular cell adhesion molecule (VCAM)-1 is due to an induction ofinwardly rectifying K⁺ currents(I_ir) (Colden-Stanfield M and Gallin EK,Am J Physiol Cell Physiol 275: C267-C277, 1998).We determined whether the VCAM-1-induced hyperpolarization issufficient to augment the increase in intracellular free calcium([Ca²⁺]_i) produced by Ca²⁺ storedepletion with thapsigargin (TG) and readdition of external CaCl₂ in fura 2-loaded THP-1 monocytes. Whereas there was a2.1-fold increase in [Ca²⁺]_i in monocytesbound to glass for 5 h in response to TG and CaCl₂ addition, adherence to VCAM-1 produced a 5-fold increase in[Ca²⁺]_i. Depolarization of monocytes adherentto VCAM-1 by I_ir blockade or exposure to high[K⁺] abolished the enhancement of the peak[Ca²⁺]_i response. In monocytes bound toglass, hyperpolarization of the membrane potential with valinomycin, aK⁺ ionophore, to the level of hyperpolarization seen incells adherent to VCAM-1 produced similar changes in peak[Ca²⁺]_i. Adherence of monocytes to E-selectinproduced a similar peak [Ca²⁺]_i to cellsbound to glass. Thus monocyte adherence to the physiological substrateVCAM-1 produces a hyperpolarization that is sufficient to enhanceCa²⁺ entry and may impact Ca²⁺-dependentmonocyte function.

     

The narrow sheath duplicate genes: sectors of dual aneuploidy reveal ancestrally conserved gene functions during maize leaf development

The narrow sheath mutant of maize displays a leaf and plant stature phenotype controlled by the duplicate factor mutations narrow sheath1 and narrow sheath2. Mutant leaves fail to develop a lateral domain that includes the leaf margins. Genetic data are presented to show that the narrow sheath mutations map to duplicated chromosomal regions, reflecting an ancestral duplication of the maize genome. Genetic and cytogenetic evidence indicates that the original mutation at narrow sheath2 is associated with a chromosomal inversion on the long arm of chromosome 4. Meristematic sectors of dual aneuploidy were generated, producing plants genetically mosaic for NARROW SHEATH function. These mosaic plants exhibited characteristic half-plant phenotypes, in which leaves from one side of the plant were of nonmutant morphology and leaves from the opposite side were of narrow sheath mutant phenotype. The data suggest that the narrow sheath duplicate genes may perform ancestrally conserved, redundant functions in the development of a lateral domain in the maize leaf.     

Subcellular localization of a high affinity binding site for D-myo-inositol 1,4,5-trisphosphate from Chenopodium rubrum

It is now generally accepted that a phosphoinositide cycle is involved in the transduction of a variety of signals in plant cells. In animal cells, the binding of D-myo-inositol 1,4,5-trisphosphate (InsP(3)) to a receptor located on the endoplasmic reticulum (ER) triggers an efflux of calcium release from the ER. Sites that bind InsP(3) with high affinity and specificity have also been described in plant cells, but their precise intracellular locations have not been conclusively identified. In contrast to animal cells, it has been suggested that in plants the vacuole is the major intracellular store of calcium involved in signal induced calcium release. The aim of this work was to determine the intracellular localization of InsP(3)-binding sites obtained from 3-week-old Chenopodium rubrum leaves. Microsomal membranes were fractionated by sucrose density gradient centrifugation in the presence and absence of Mg(2+) and alternatively by free-flow electrophoresis. An ER-enriched fraction was also prepared. The following enzymes were employed as specific membrane markers: antimycin A-insensitive NADH-cytochrome c reductase for ER, cytochrome c oxidase for mitochondrial membrane, pyrophosphatase for tonoplast, and 1,3-beta-D-glucansynthase for plasma membrane. In all membrane separations, InsP(3)-binding sites were concentrated in the fractions that were enriched with ER membranes. These data clearly demonstrate that the previously characterized InsP(3)-binding site from C. rubrum is localized on the ER. This finding supports previous suggestions of an alternative non-vacuolar InsP(3)-sensitive calcium store in plant cells.     

A primary culture of guinea pig gallbladder epithelial cells that is responsive to secretagogues

We have developed a cell culture of guinea pig gallbladder epithelial cells with which to study ion transport. When grown on permeable supports, the cultured epithelia developed a transepithelial resistance (R(t)) of approximately 500 Omega. cm(2). The epithelial cell origin of the cell culture was further confirmed by immunocytochemical localization of cytokeratin. Ionomycin and forskolin increased transepithelial voltage and short-circuit current (I(sc)) and decreased R(t). The response to ionomycin was transient, whereas that to forskolin was sustained. Both were attenuated by replacement of Cl(-) and/or HCO(3)(-). Mucosal addition of the anion transport inhibitors DIDS or diphenylamine-2-carboxylic acid (DPC) blocked the response to ionomycin. The response to forskolin was blocked by DPC but not by DIDS. Ionomycin, but not forskolin, increased intracellular Ca(2+) concentration in fura 2-loaded cells. PGE(2), histamine, vasoactive intestinal polypeptide, and secretin elicited a sustained increase in I(sc). Responses to ATP and CCK were transient. Thus cultured guinea pig gallbladder epithelia display the range of responses observed in the native tissue and are an appropriate model for studies of ion transport in gallbladder and intestinal epithelia.     

P. carinii induces selective alterations in component expression and biophysical activity of lung surfactant

Studies of Pneumocystis carinii pneumonia (PCP) suggest an important role for the surfactant system in the pathogenesis of the hypoxemic respiratory insufficiency associated with this infection. We hypothesized that PCP induces selective alterations in alveolar surfactant component expression and resultant biophysical properties. PCP was induced by intratracheal inoculation of 2 x 10(5) P. carinii organisms into C.B-17 scid/scid mice. Six weeks after inoculation, large (LA)- and small (SA)-aggregate surfactant fractions were prepared from bronchoalveolar lavage fluids and analyzed for expression of surfactant components and for biophysical activity. Total phospholipid content was significantly reduced in LA surfactant fractions from mice infected with PCP (53 +/- 15% of uninfected mice; P < 0.05). Quantitation of hydrophobic surfactant protein (SP) content demonstrated significant reductions of alveolar SP-B and SP-C protein levels in mice with PCP compared with those in uninfected mice (46 +/- 7 and 19 +/- 6%, respectively; P < 0.05 for both). The reductions in phospholipid, SP-B, and SP-C in LA fractions measured during PCP were associated with an increase in the minimum surface tension of LAs as measured by pulsating bubble surfactometer (13.1 +/- 1.1 vs. 5.4 +/- 1.8 mN/m; P < 0.05). In contrast to decreases in the hydrophobic SPs, SP-D content in the SA fraction was markedly increased (343 +/- 30% of control value; P < 0. 05) and SP-A levels in LA surfactant were maintained (93 +/- 26% of control value) during P. carinii infection. In all cases, the changes in SP content were reflected by commensurate changes in the levels of mRNA. We conclude that PCP induces selective alterations in surfactant component expression, including profound decreases in hydrophobic protein contents and resultant increases in surface tension. These changes, demonstrated in an immunologically relevant animal model, suggest that alterations in surfactant could contribute to the hypoxemic respiratory insufficiency observed in PCP.     

Ribozyme as an approach for growth suppression of human pancreatic cancer

Ribozymes (catalytic RNAs, RNA enzymes) are effective modulators of gene expression because of their simple structure, site-specific cleavage activity, and catalytic potential, and have potentially important implications for cancer gene therapy. Point mutations in the K-ras oncogene are found in approx 90% of human pancreatic carcinomas, and can be used as potential targets for specific ribozyme-mediated reversal of the malignant phenotype. In this study, we focused on in vitro manipulation of ribozyme targeting of the mutated K-ras oncogene in a human pancreatic carcinoma cell line. We evaluated the efficacy of an anti-K-ras hammerhead ribozyme targeted against GUU-mutated codon 12 of the K-ras gene in cultured pancreatic carcinoma cell lines. The anti-K-ras ribozyme significantly reduced cellular K-ras mRNA level (GUU-mutated codon 12) when the ribozyme was transfected into the Capan-1 pancreatic carcinoma cells. The ribozyme inhibited proliferation of the transfected Capan-1 cells. These results suggested that this ribozyme is capable of reversing the malignant phenotype in human pancreatic carcinoma cells.     

Radial leaves of the maize mutant ragged seedling2 retain dorsiventral anatomy

ragged seedling2 (rgd2) is a novel, recessive mutation affecting lateral organ development in maize. The mutant phenotype of homozygous rgd2-R leaves is variable. Mild leaf phenotypes have a reduced midrib and may be moderately narrow and furcated; severe Rgd2-R(-) leaves are filamentous or even radial. Despite their radial morphology, severe Rgd2-R(-) mutant leaves develop distinct adaxial and abaxial anatomical features. Although Rgd2-R(-) mutants exhibit no reduction in adaxial or abaxial cell types, areas of epidermal cell swapping may occur that are associated with misaligned vascular bundles and outgrowths of ectopic margins. Scanning electron microscopy of young primordia and analyses of leaf developmental-marker gene expression in mutant apices reveal that RGD2 functions during recruitment of leaf founder cells and during expansive growth of leaf primordia. Overall, these phenotypes suggest that development is uncoordinated in Rgd2-R(-) mutant leaves, so that leaf components and tissues may develop quasi-independently. Models whereby RGD2 is required for developmental signaling during the initiation, anatomical patterning, and lateral expansion of maize leaves are discussed.     

The interaction of lipophilic drugs with intestinal fatty acid-binding protein

Intestinal fatty acid-binding protein (I-FABP) is a small protein that binds long-chain dietary fatty acids in the cytosol of the columnar absorptive epithelial cells (enterocytes) of the intestine. The binding cavity of I-FABP is much larger than is necessary to bind a fatty acid molecule, which suggests that the protein may be able to bind other hydrophobic and amphipathic ligands such as lipophilic drugs. Herein we describe the binding of three structurally diverse lipophilic drugs, bezafibrate, ibuprofen (both R- and S-isomers) and nitrazepam to I-FABP. The rank order of affinity for I-FABP determined for these compounds was found to be R-ibuprofen approximately bezafibrate > S-ibuprofen > nitrazepam. The binding affinities were not directly related to aqueous solubility or partition coefficient of the compounds; however, the freely water-soluble drug diltiazem showed no affinity for I-FABP. Drug-I-FABP interaction interfaces were defined by analysis of chemical shift perturbations in NMR spectra, which revealed that the drugs bound within the central fatty acid binding cavity. Each drug participated in a different set of interactions within the cavity; however, a number of common contacts were observed with residues also involved in fatty acid binding. These data suggest that the binding of non-fatty acid lipophilic drugs to I-FABP may increase the cytosolic solubility of these compounds and thereby facilitate drug transport from the intestinal lumen across the enterocyte to sites of distribution and metabolism.