Chemotropism and branching as alternative responses of Achlya bisexualis to amino acids

Hyphae of Achlya bisexualis growing on lean media orient their extension towards a source of amino acids, and also put forth branches. Micropipettes were used to generate gradients of amino acids in the vicinity of individual hyphae. Phenylalanine and methionine were the most powerful attractants: 0.04 mM amino acid in the pipette produced reorientation, and higher concentrations made the hyphae curl around the pipette and grow into its tip. Hyphae detected gradients as low as 5% across their width. Methionine and phenylalanine appeared to bind to different receptors. Local application of these amino acids also elicited the emergence of single branches, next to the pipette and on the high side of the gradient; comparison of diverse amino acids and their analogues suggested that branching and chemotropism share common receptors. By contrast, cytochalasin A and various ionophores induced branches at random sites, without receptor involvement. We propose that binding of amino acids to their receptors determines the site of precursor vesicle exocytosis, and consider possible mechanisms.     

Membrane H+ Conductance of Clostridium thermoaceticum and Clostridium acetobutylicum: Evidence for Electrogenic Na+/H+ Antiport in Clostridium thermoaceticum

H⁺ conductance in de-energized cells of Clostridium thermoaceticum and Clostridium acetobutylicum was determined from the rate of realkalinization of the medium after an acid pulse. In both organisms, cell membrane proton permeability was increased by fermentation end products and ionophores. In C. thermoaceticum, H⁺ conductance was increased by Na⁺ ions compared with K⁺ as counterions. In these cells, addition of Na⁺, but not K⁺, elicited efflux of H⁺; H⁺ efflux was stimulated by SCN⁻ and decreased by various ionophores. We concluded that C. thermoaceticum possesses an electrogenic Na⁺/H⁺ antiporter. In contrast, C. acetobutylicum cells did not have an electrogenic Na⁺/H⁺ antiporter.     

Polyphosphoinositol lipids inChlamydomonas eugametos gametes

InChlamydomonas eugametos gametes, phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) comprised 0.4 and 0.3% of the whole-cell phospholipids. They were concentrated in the plasma membrane around the cell body and were present in low concentrations in the flagellar membrane. When gametes were fed³²PO ₄ ^- , the label was rapidly incorporated into PtdInsP and PtdInsP₂ and only slowly incorporated into structural lipids such as phosphatidylethanolamine and phosphatidylglycerol. Similarly, when a pulse of³²PO ₄ ^- was chased with PO ₄ ^- , the label was rapidly lost from the polyphosphoinositol lipids but not from the structural lipids. The major fatty acids in the polyphosphoinositides were C-22 carbon polyenoic acids (70%). The significance of these results in relationship to intracellular signalling via inositol phosphates and Ca²⁺ is discussed.Abbreviations InsP₃ inositol 1,4,5-trisphosphate - mt^–/mt⁺ mating-type plus or minus - PtdA phosphatidic acid - PtdEtn phosphatidylethanolamine - PtdGro phosphatidylglycerol - PtdIns phosphatidylinositol - PtdInsP phosphatidylinositol 4-phosphate; - PtdInsP₂ phosphatidylinositol 4,5-bisphosphate - TCA trichloroacetic acid We thank Frank Schuring for Fig. 5A and Susan Kenter, Hans Kruisselbrink, Saskia Bijvank and Nelleke Corbett for their enthousiastic assistance.     

In vitro growth and differentiation of human kidney tubular cells on a basement membrane substrate

Summary Kidney cortical tubular cells, mainly proximal tubular cells, isolated from human kidney and grown either on a basement membrane substrate in chemically defined medium or on plastic in serum-supplemented medium, had substantial proliferative potential and could be propagated for more than 10 generations or 8 passages before senescence. Basement membrane produced on a plastic substrate by the HR-9 endodermal cell line could replace serum supplementation in promoting tubular cell growth. Tubular cells grown on an HR-9 basement membrane substrate exhibited stable epithelial morphology over an extended period of time; in the presence of 5% serum they differentiated into organized structures such as hemicysts and cell cords. Cells grown on plastic failed to differentiate and gradually degenerated. Tubular cells on HR-9 basement membrane were characterized by densely packed microvilli, abundant rough endoplasmic reticulum and free polysomes, basal cell membrane interdigitations, a well-developed endocytotic apparatus, and conspicuous junctional complexes—all features of the proximal tubular cell. Compared with cells on plastic substrate, there were higher levels of the brush border enzymes γ-glutamyl transpeptidase,l-leucine aminopeptidase, and alkaline phosphatase in cells maintained on an HR-9 basement membrane substrate, further supporting the conclusion that a basement membrane substrate promoted differentiation of tubular cells. These data and morphological observations indicate that a basement membrane substrate can promote growth and both functional and morphologic differentiation of human kidney tubular cells. This work was supported by the Veterans Administration.     

An antigen-specific DTH-initiating cell clone. Functional, phenotypical, and partial molecular characterization

The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is due to the sequential action of two different Ag-specific Thy-1+ cells. An early-acting DTH-initiating cell in the lymphoid organs produces a circulating, Ag-specific factor that is functionally analogous to IgE antibody and initiates DTH by sensitizing the local tissue for release of the vasoactive amine serotonin. In picryl chloride (PC1) or oxazolone (OX) contact sensitivity, this DTH-initiating factor is called PC1-F and OX-F respectively, and is Ag-specific, but MHC-unrestricted. The phenotype of polyclonal DTH-initiating cells was recently shown to be unusual for an Ag-specific cell. The phenotype was: Thy-1+, Lyt-1+ (CD5), triple negative (CD4-, CD8-, and CD3-), B220+ (Ly-5, CD45RA), positive for IL-3 receptors, but not IL-2 receptors, and positive for antibodies that react with a putative constant or framework portion of DTH-initiating factors such as anti-PC1-F antibodies and 14-30 mAb. We report here the generation of an Ag-specific DTH-initiating cell clone from nude mice that were immunized and boosted by contact sensitization with OX. By flow microfluorometry analysis, this clone has a similar unique surface phenotype, and by in vivo assay has the same functional abilities, as polyclonal DTH-initiating cells. The clone produces Ag-specific OX-F that acts in an Ag-specific manner to initiate DTH. Moreover, specific cDNA probes and Northern blot analysis of the clone demonstrated that the Ag-specific DTH-initiating cells are Thy-1+, CD3-, and IL-3R+. Thus, DTH initiation is due to an Ag-specific lymphoid cell, that produces an Ag-specific factor, and that has a unique surface phenotype for Ag-specific cells; namely, Thy-1+, CD5+, sIg-, CD4-, CD8-, CD3-, CD45RA+, IL-2R-, and IL-3R+.     

Evolution of collagen IV genes from a 54-base pair exon: A role for introns in gene evolution

The exon structure of the collagen IV gene provides a striking example for collagen evolution and the role of introns in gene evolution. Collagen IV, a major component of basement membranes, differs from the fibrillar collagens in that it contains numerous interruptions in the triple helical Gly-X-Y repeat domain. We have characterized all 47 exons in the mouse alpha 2(IV) collagen gene and find two 36-, two 45-, and one 54-bp exons as well as one 99- and three 108-bp exons encoding the Gly-X-Y repeat sequence. All these exons sizes are also found in the fibrillar collagen genes. Strikingly, of the 24 interruption sequences present in the alpha 2-chain of mouse collagen IV, 11 are encoded at the exon/intron borders of the gene, part of one interruption sequence is encoded by an exon of its own, and the remaining interruptions are encoded within the body of exons. In such "fusion exons" the Gly-X-Y encoding domain is also derived from 36-, 45-, or 54-bp sequence elements. These data support the idea that collagen IV genes evolved from a primordial 54-bp coding unit. We furthermore interpret these data to suggest that the interruption sequences in collagen IV may have evolved from introns, presumably by inactivation of splice site signals, following which intronic sequences could have been recruited into exons. We speculated that this mechanism could provide a role for introns in gene evolution in general.     

Immunoaffinity Purification of Epitope-Tagged Human β2-Adrenergic Receptor to Homogeneity

To obtain large quantities of pure human β₂-adrenergic receptor (β₂-AR) needed for structural studies, an efficient method for β₂-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus. This epitope is recognized by KT3-monoclonal antibody. The epitope tagged β₂-AR was expressed in Sf9 cells with a specific activity of 5–20 pmol/mg of membrane protein. The epitope-tagged and wild-type receptors had identical ligand binding properties. The tagged receptor was solubilized using dodecyl-β-maltoside with a quantitative yield. Solubilized epitope-tagged receptors were partially purified by KT3-mAb immunoaffinity in 60–70% yield. Further purification of the receptors on an alprenolol-affinity column resulted in a homogenous preparation with an overall yield of >30%. The purified receptor was concentrated to >1 mg/ml without loss of ligand binding activity.     

Tumor Necrosis Factor-α (TNF-α), Interferon-γ, and Interleukin-6 but Not TNF-β Induce Differentiation of Neuroblastoma Cells: The Role of Nitric Oxide

Abstract: Tumor necrosis factor-a (TNF-α), interferon-γ (IFN-7), and interleukin-6 (IL-6), but not TNF-β, can induce the in vitro differentiation of the neuroblastoma cell line N103 in a dose-dependent manner. Differentiation of N103 was accompanied by the arrest of cell growth and neurite formation. The induction of neuroblastoma cell differentiation by TNF-α and IFN-γ can be specifically inhibited by a nitric oxide (NO) synthase inhibitor, l -N^G-monomethylarginine. In contrast, the differentiation of N103 cells by IL-6 was not affected by l -N^G-monomethylarginine. These results indicate that TNF-α and IFN-γ, but not IL-6, induce the differentiation of neuroblastoma cells via NO. This is confirmed by the finding that the culture super- natants of N103 cells induced by TNF-α and IFN-γ, but not that by IL-6, contained high levels of NO₂⁻, the production of which was inhibited by l - N ^G-monomethylarginine. Furthermore, the differentiation of N103 cells can be induced directly in a dose-dependent manner by the addition of nitroprusside, a generator of NO, into the culture medium. These data therefore indicate that NO may be an important mediator in the induction of neuronal cell differentiation by certain cytokines such as TNF-α and IFN-γ and that neuronal cells, in addition to the macrophagelike brain cells, can be induced by immunological stimuli to produce large quantities of NO.     

PHYLOGENYAND HISTORICAL ECOLOGY OF THE DESMARESTIACEAE (PHAEOPHYCEAE) SUPPORT A SOUTHERN HEMISPHERE ORIGIN1

Phylogenetic relationships in the Desmarestiales (Phaeophyceae) were inferred among the monotypic Arthrocladia (Arthrocladiaceae) and 27 isolates from Desmarestiaceae, representing 17 taxa of Desmarestia and the monotypic Antarctic genera Himantothallus and Phaeurus. Phaeurus and Arthrocladia were used as outgroups. Parsimony analyses of nuclear ribosomal DNA internal transcribed spacer (ITS1 and ITS2) sequences, in which gaps were both included and excluded, yielded well-resolved trees with a consistent general branching pattern. A parallel analysis of nine morphological and life-history characters and three ecological characters yielded a similar tree but provided little resolution in the terminal clades. The position of the monotypic Arthrocladia villosa within the Desmarestiales is consistent with monophyly for the order, but its position as the most primitive desmarestialean is not resolvable from the molecular data set. The basal position of Phaeurus, the Antarctic Desmarestia species, and Himantothallus is consistent with the hypothesis of a Southern Hemisphere origin for the family Desmarestiaceae. The more recent Northern Hemisphere “aculeata” clade evolved from an Antarctic ancestor. A “D. aculeata-like” species was ancestral to a lineage characterized by annual sporophytes with high sulfuric acid content, which radiated into many species, widely distributed in both hemispheres. Mapping of morphological and ecological characters onto the molecular tree confirm the informativeness of sulfuric acid-containing vacuoles and unilocular sporangial types. There is good congruence between phylogenetic tree topology and temperature impints in relation to biogeographic distribution, supporting the theory that temperature tolerance is a conservative trait.