The Role of Preassembled Cytoplasmic Complexes in Assembly of Flagellar Dynein Subunits

Previous work has revealed a cytoplasmic pool of flagellar precursor proteins capable of contributing to the assembly of new flagella, but how and where these components assemble is unknown. We tested Chlamydomonas outer-dynein arm subunit stability and assembly in the cytoplasm of wild-type cells and 11 outer dynein arm assembly mutant strains (oda1-oda11) by Western blotting of cytoplasmic extracts, or immunoprecipitates from these extracts, with five outer-row dynein subunit-specific antibodies. Western blots reveal that at least three oda mutants (oda6, oda7, and oda9) alter the level of a subunit that is not the mutant gene product. Immunoprecipitation shows that large preassembled flagellar complexes containing all five tested subunits (three heavy chains and two intermediate chains) exist within wild-type cytoplasm. When the preassembly of these subunits was examined in oda strains, we observed three patterns: complete coassembly (oda 1, 3, 5, 8, and 10), partial coassembly (oda7 and oda11), and no coassembly (oda2, 6, and 9) of the four tested subunits with HCβ. Our data, together with previous studies, suggest that flagellar outer-dynein arms preassemble into a complete Mr ≃ 2 × 106 dynein arm that resides in a cytoplasmic precursor pool before transport into the flagellar compartment.

Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1β are differentially regulated in human blood mononuclear cells and mouse spleen cells

Interleukin (IL)-18, formerly called interferon γ (IFN-γ)-inducing factor, is biologically and structurally related to IL-1β. A comparison of gene expression, synthesis, and processing of IL-18 with that of IL-1β was made in human peripheral blood mononuclear cells (PBMCs) and in human whole blood. Similar to IL-1β, the precursor for IL-18 requires processing by caspase 1. In PBMCs, mature but not precursor IL-18 induces IFN-γ; in whole human blood stimulated with endotoxin, inhibition of caspase 1 reduces IFN-γ production by an IL-1β-independent mechanism. Unlike the precursor for IL-1β, precursor for IL-18 was expressed constitutively in PBMCs and in fresh whole blood from healthy human donors. Western blotting of endotoxin-stimulated PBMCs revealed processed IL-1β in the supernatants via an caspase 1-dependent pathway. However, in the same supernatants, only unprocessed precursor IL-18 was found. Unexpectedly, precursor IL-18 was found in freshly obtained PBMCs and constitutive IL-18 gene expression was present in whole blood of healthy donors, whereas constitutive IL-1β gene expression is absent. Similar to human PBMCs, mouse spleen cells also constitutively contained the preformed precursor for IL-18 and expressed steady-state IL-18 mRNA, but there was no IL-1β protein and no spontaneous gene expression for IL-1β in these same preparations. We conclude that although IL-18 and IL-1β are likely members of the same family, constitutive gene expression, synthesis, and processing are different for the two cytokines.

Altered stability of pulmonary surfactant in SP-C-deficient mice

The surfactant protein C (SP-C) gene encodes an extremely hydrophobic, 4-kDa peptide produced by alveolar epithelial cells in the lung. To discern the role of SP-C in lung function, SP-C-deficient (−/−) mice were produced. The SP-C (−/−) mice were viable at birth and grew normally to adulthood without apparent pulmonary abnormalities. SP-C mRNA was not detected in the lungs of SP-C (−/−) mice, nor was mature SP-C protein detected by Western blot of alveolar lavage from SP-C (−/−) mice. The levels of the other surfactant proteins (A, B, D) in alveolar lavage were comparable to those in wild-type mice. Surfactant pool sizes, surfactant synthesis, and lung morphology were similar in SP-C (−/−) and SP-C (+/+) mice. Lamellar bodies were present in SP-C (−/−) type II cells, and tubular myelin was present in the alveolar lumen. Lung mechanics studies demonstrated abnormalities in lung hysteresivity (a term used to reflect the mechanical coupling between energy dissipative forces and tissue-elastic properties) at low, positive-end, expiratory pressures. The stability of captive bubbles with surfactant from the SP-C (−/−) mice was decreased significantly, indicating that SP-C plays a role in the stabilization of surfactant at low lung volumes, a condition that may accompany respiratory distress syndrome in infants and adults.

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase1 : IV. Fusicoccin Induces the Association between the Plasma Membrane H+-ATPase and the Fusicoccin Receptor

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H+-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H+-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H+-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H+-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H+-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H+-ATPase antiserum and with an anti-14–3-3 antiserum indicated an FC-induced association of FCBP with the PM H+-ATPase. Analysis of the activation state of PM H+-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.

Molecular identity and cellular distribution of advanced glycation endproduct receptors: relationship of p60 to OST-48 and p90 to 80K-H membrane proteins.

Advanced glycation endproducts (AGEs) are derivatives of nonenzymatic reactions between sugars and protein or lipids, and together with AGE-specific receptors are involved in numerous pathogenic processes associated with aging and hyperglycemia. Two of the known AGE-binding proteins isolated from rat liver membranes, p60 and p90, have been partially sequenced. We now report that the N-terminal sequence of p60 exhibits 95% identity to OST-48, a 48-kDa member of the oligosaccharyltransferase complex found in microsomal membranes, while sequence analysis of p90 revealed 73% and 85% identity to the N-terminal and internal sequences, respectively, of human 80K-H, a 80- to 87-kDa protein substrate for protein kinase C. AGE-ligand and Western analyses of purified oligosaccharyltransferase complex, enriched rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membranes from rat liver or RAW 264.7 macrophages yielded a single protein of approximately 50 kDa recognized by both anti-p60 and anti-OST-48 antibodies, and also exhibited AGE-specific binding. Immunoprecipitated OST-48 from rat rough endoplasmic reticulum fractions exhibited both AGE binding and immunoreactivity to an anti-p60 antibody. Immune IgG raised to recombinant OST-48 and 80K-H inhibited binding of AGE-bovine serum albumin to cell membranes in a dose-dependent manner. Immunostaining and flow cytometry demonstrated the surface expression of OST-48 and 80K-H on numerous cell types and tissues, including mononuclear, endothelial, renal, and brain neuronal and glial cells. We conclude that the AGE receptor components p60 and p90 are identical to OST-48, and 80K-H, respectively, and that they together contribute to the processing of AGEs from extra- and intracellular compartments and in the cellular responses associated with these pathogenic substances.

Identification and characterization of a RING zinc finger gene (C-RZF) expressed in chicken embryo cells.

To identify changes in gene expression that occur in chicken embryo brain (CEB) cells as a consequence of their binding to the extracellular matrix molecule cytotactin/tenascin (CT/TN), a subtractive hybridization cloning strategy was employed. One of the cDNA clones identified was predicted to encode 381 amino acids and although it did not resemble any known sequences in the nucleic acid or protein data bases, it did contain the sequence motif for the cysteine-rich C3HC4 type of zinc finger, also known as a RING-finger. This sequence was therefore designated the chicken-RING zinc finger (C-RZF). In addition to the RING-finger, the C-RZF sequence also contained motifs for a leucine zipper, a nuclear localization signal, and a stretch of acidic amino acids similar to the activation domains of some transcription factors. Southern analysis suggested that C-RZF is encoded by a single gene. Northern and in situ hybridization analyses of E8 chicken embryo tissues indicated that expression of the C-RZF gene was restricted primarily to brain and heart. Western analysis of the nuclear and cytoplasmic fractions of chicken embryo heart cells and immunofluorescent staining of chicken embryo cardiocytes with anti-C-RZF antibodies demonstrated that the C-RZF protein was present in the nucleus. The data suggest that we have identified another member of the RING-finger family of proteins whose expression in CEB cells may be affected by CT/TN and whose nuclear localization and sequence motifs predict a DNA-binding function in the nucleus.

Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells.

Proteins of the 14-3-3 family can associate with, and/or modulate the activity of, several protooncogene and oncogene products and, thus, are implicated in regulation of signaling pathways. We report that 14-3-3 is associated with another important transducing enzyme, phosphatidylinositol 3-kinase (PI3-K). A recombinant 14-3-3 fusion protein bound several tyrosine-phosphorylated proteins from antigen receptor-stimulated T lymphocytes. PI3-K was identified by immunoblotting and enzymatic assays as one of the 14-3-3-binding proteins in resting or activated cells. Moreover, endogenous 14-3-3 and PI3-K were coimmunoprecipitated from intact T cells. Far-Western blots of gel-purified, immunoprecipitated PI3-K with a recombinant 14-3-3 fusion protein revealed direct binding of 14-3-3 to the catalytic subunit (p110) of PI3-K. Finally, anti-phosphotyrosine immunoprecipitates from activated, 14-3-3-overexpressing cells contained lower PI3-K enzymatic activity than similar immunoprecipitates from control cells. These findings suggest that association of 14-3-3 with PI3-K in hematopoietic (and possibly other) cells regulates the enzymatic activity of PI3-K during receptor-initiated signal transduction.

Association of a transglutaminase-related antigen with intermediate filaments.

A mouse monoclonal antibody, G92.1.2, raised against guinea pig liver transglutaminase (TGase) recognizes an antigen present in primary mouse dermal fibroblasts. A filamentous pattern, bearing remarkable similarity to the vimentin intermediate filament (IF) network, is seen when these cells are fixed and processed for indirect immunofluorescence with the antibody. Double-label immunofluorescence reveals that the antigen reacting with the antibody colocalizes precisely with vimentin IF and that this colocalization is retained after the treatment of fibroblasts with colchicine, which induces a redistribution of the majority of IFs into perinuclear aggregates. These morphological observations are further supported by the finding that the protein reacting with G92.1.2 is retained in IF-enriched cytoskeletal preparations made by using nonionic detergent-containing high ionic strength solutions. Western blots of the IF fraction show that G92.1.2 recognizes a major band of approximately 280 kDa and does not cross react with vimentin. Furthermore, when the antibody is microinjected into live dermal fibroblasts, it causes a collapse of the vimentin IF network in the majority of injected cells. The results suggest that a form of TGase, or a TGase-related antigen, is closely associated with the vimentin IF network of primary cultures of mouse dermal fibroblasts.

Bronchiolar nonciliated secretory (Clara) cells: source of guanylin in the mammalian lung.

The peptide guanylin, which has recently been isolated from the intestine, is involved in the regulation of fluid secretion in the intestinal epithelium by activation of guanylate cyclase C, the putative guanylin receptor. Since the latter protein is also expressed in airway epithelia, we investigated the lung of three mammalian species for the presence and cellular localization of guanylin by immunoblot (Western blot) analyses and light and electron microscopical immunocytochemistry. In Western blots of bovine, guinea pig, and rat lung extracts, three different guanylin antisera directed against the midportion and against the C terminus of the precursor molecule identified a peptide band corresponding to the apparent molecular mass of guanylin. Localization studies in the lung revealed that guanylin is exclusively confined to nonciliated secretory (Clara) cells in the lining of distal conducting airways. The presence of guanylin in the lung and particularly its specific localization to Clara cells indicate that these cells may play a pivotal role in the local (paracrine) regulation of electrolyte/water transport in airway epithelia.

The kappa-opioid receptor is primarily postsynaptic: combined immunohistochemical localization of the receptor and endogenous opioids.

Antisera were raised against a synthetic peptide corresponding to the carboxyl terminus of the kappa-opioid receptor (KOR1). Specificity of the antisera was verified by staining of COS-7 cells transfected with KOR1 and epitope-tagged KOR1 cDNAs, by recognition by the antisera of proteins on Western blots of both transfected cells and brain tissue, by the absence of staining of both brain tissue and transfected cells after preabsorption of the antisera with the cognate peptide, and on the strong correlation between the distribution of KOR1 immunoreactivity and that of earlier ligand binding and in situ hybridization studies. Results indicate that KOR1 in neurons is targeted into both the axonal and somatodendritic compartments, but the majority of immunostaining was seen in the somatodendritic compartment. In sections from rat and guinea pig brain, prominent KOR1 staining was seen in the ventral forebrain, hypothalamus, thalamus, posterior pituitary, and midbrain. While the staining pattern was similar in both species, distinct differences were also observed. The distribution of preprodynorphin and KOR1 immunoreactivity was complementary in many brain regions, suggesting that KOR1 is poised to mediate the physiological actions of dynorphin. However, the distribution of KOR1 and enkephalin immunoreactivity was complementary in some regions as well. These results suggest that the KOR1 protein is primarily, but not exclusively, deployed to postsynaptic membranes where it mediates the effects of products of preprodynorphin and possibly preproenkephalin.

Integrated Solar City: Application of the Ecological Risk Assessment Paradigm to Inform Reclamation

The Integrated Solar City (ISC) is a large solar project that has been proposed for development in the western state of Gujarat, India. The project will be the largest solar project in the world. It will require the use of large land resources to construct. An ecological risk assessment (ERA) is used to assess potential impacts from project construction and operation. Previous research suggests that a solar project of this scale would require the removal of vegetation along with other negative effects on vegetation and soil. The ERA was used to lay out a revegetation plan that would help mitigate the long-term environmental impacts in the Banaskantha and Kachchh regions of Gujarat, India.

Geodynamic implications derived from Numidian-like distal turbidites deposited along the Internal–External Domain Boundary of the Betic Cordillera (S Spain)

New data reveal Early Burdigalian ‘Numidian-like lithofacies’ in successions of the internal (southernmost) part of the South Iberian Margin (SIM) and the south-western margin of the Mesomediterranean Microplate (MM). The well-known Numidian Formation was deposited in the external (Massylian) sub-domain of the Maghrebian Flysch Basin (a south-western branch of the Tethys Ocean). The anomalous occurrence of ‘Numidian-like lithofacies’ is induced by the particular Early Miocene palaeogeographical and geodynamic complexity of the sector. This consisted of a ‘triple point’ with a dextral transform fault between the SIM and the MM-Maghrebian Flysch Basin system. In this framework, the ageing of Iberian reliefs and the MM collapse, coupled with an African Margin upbulging, and a shortening of the Maghrebian Flysch Basin (both related to the subduction), could have resulted in the arrival of the Numidian depositional system from so far away.

Expression and cellular localization of the voltage-gated calcium channel α2δ3 in the rodent retina

High-voltage-activated calcium channels are hetero-oligomeric protein complexes that mediate multiple cellular processes, including the influx of extracellular Ca2+, neurotransmitter release, gene transcription, and synaptic plasticity. These channels consist of a primary α1 pore-forming subunit, which is associated with an extracellular α2δ subunit and an intracellular β auxiliary subunit, which alter the gating properties and trafficking of the calcium channel. The cellular localization of the α2δ3 subunit in the mouse and rat retina is unknown. In this study using RT-PCR, a single band at ∼305 bp corresponding to the predicted size of the α2δ3 subunit fragment was found in mouse and rat retina and brain homogenates. Western blotting of rodent retina and brain homogenates showed a single 123-kDa band. Immunohistochemistry with an affinity-purified antibody to the α2δ3 subunit revealed immunoreactive cell bodies in the ganglion cell layer and inner nuclear layer and immunoreactive processes in the inner plexiform layer and the outer plexiform layer. α2δ3 immunoreactivity was localized to multiple cell types, including ganglion, amacrine, and bipolar cells and photoreceptors, but not horizontal cells. The expression of the α2δ3 calcium channel subunit to multiple cell types suggests that this subunit participates widely in Ca-channel-mediated signaling in the retina.

Compositional signatures of the Cenozoic sedimentary succesions of the Malaguide Complex (Betic Cordillera, Spain)

The studied Cenozoic sedimentary successions consist of deposits from continental/shallow-water to deep-marine environments of the Malaguide Complex (Betic Cordillera) outcropping in the Sierra Espuña area (SE Spain). The aim of this study is to characterize the composition, source area(s) provenance and weathering processes of these sedimentary successions from the pre-orogenic (Paleocene-Early Oligocene) to the syn-orogenic (Late Oligocene-Early Miocene) stage using petrological and geochemical methodologies. The studied sandstones are mainly quartzolithic with abundant metamorphic and sedimentary lithic fragments. In particular, the composition of samples from the pre-orogenic cycle is mainly carbonate with important siliciclastic components that occur within the medium to fine grained arenites. The composition of samples from the syn-orogenic cycle is characterized by a sharp change from carbonate to siliciclastic terms. Thus, the composition of the overall sandstone samples is very heterogeneous and suggests a source area mainly characterized by the Malaguide basement and lower units of the Internal Betic Zone, that partially compose the Mesomediterranean Microplate. The geochemical proxies suggest a provenance mainly from felsic source area with a minor supply from mafic rocks in some samples of the syn-orogenic stage. Furthermore, palaeoweathering indices indicate low to moderate weathering conditions for the sources. The Cenozoic sedimentary successions of the Malaguide Complex played an important role in the geodynamic evolution of the Betic Cordillera that represents the key tectonic element of the western domains of the Mesomediterranean Microplate.

Bulletin : report of Agricultural Experiment Station, Agricultural and Mechanical College, Auburn, Ala.

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