Cell shaping and microtubules in developing mesophyll of wheat (Triticum aestivum L.)

Summary Differentiated mesophyll cells ofTriticum aestivum (cv. Star) exhibit a lobed outline resembling tube-shaped balloons with almost regularly spaced constrictions. It was shown that these constrictions are probably the result of hoops of wall reinforcements laid down during early stages of cell expansion. It appears that these hoops prevent expansion in the corresponding regions and thus give rise to the peculiar cell shape. The comparatively thin cell walls of the bulges are uniformly reinforced after the lobed shape is established.By using immunofluorescence techniques a change in the pattern of cortical microtubule arrangement was observed which corresponded to the pattern of cell wall deposition. Discrete bands of microtubules were found beneath the sites of hoop reinforcement. These bands disintegrated during late stages of cell expansion with microtubules fanning out into the almost empty regions of the bulges.Abbreviations DMSO dimethyl sulfoxid - EGTA ethylene glycol bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanat - MSB microtubule stabilizing buffer - PBS phosphate buffered saline - PIPES 1,4-piperazine diethanesulfonic acid - PMSF phenylmethyl sulfonylfluoride     

Microtubules and cell shaping in the mesophyll ofNigella damascena L.

Summary Cell shaping in the mesophyll ofNigella damascena was investigated with the aim of determining the origin of the arm-like protrusions, which are characteristic of, e.g., arm-palisade cells. It was found that hoops of cell wall were deposited during the early stages of cell expansion. The hoops were interconnected, thus embracing the cells with a wide-meshed net of local wall reinforcement. The pattern of wall deposition in the extra-cellular matrix correlated with a pattern of bands of microtubules in the cortical cytoplasm of the cells. During lateral expansion bulges were forced through the comparatively thin walls of spaces between the meshes, giving rise to the arm-like protrusions. After establishing the cell shape the bands of microtubules disintegrated and cell wall was uniformly deposited. The results are discussed in the context of the mode of cell shaping observed in the mesophyll of other systems and of a previous, classical hypothesis on the origin of arms in mesophyll cells.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - PME phosphate-magnesium-EGTA-buffer     

The<Emphasis Type="Italic"> Trox-2</Emphasis> Hox/ParaHox gene of<Emphasis Type="Italic"> Trichoplax</Emphasis> (Placozoa) marks an epithelial boundary

Hox and ParaHox genes are implicated in axial patterning of cnidarians and bilaterians, and are thought to have originated by tandem duplication of a single ProtoHox gene followed by duplication of the resultant gene cluster. It is unclear what the ancestral role of Hox/ParaHox genes was before the divergence of Cnidaria and Bilateria, or what roles the postulated ProtoHox gene(s) played. Here we describe the full coding region, spatial expression and function of Trox-2, the single Hox/ParaHox-type gene identified in Trichoplax adhaerens (phylum Placozoa) and either a candidate ProtoHox or a ParaHox gene. Trox-2 is expressed in a ring around the periphery of Trichoplax, in small cells located between the outer margins of the upper and lower epithelial cell layers. Inhibition of Trox-2 function, either by uptake of morpholino antisense oligonucleotides or by RNA interference, causes complete cessation of growth and binary fission. We speculate that Trox-2 functions within a hitherto unrecognized population of possibly multipotential peripheral stem cells that contribute to differentiated cells at the epithelial boundary of Trichoplax.Edited by D. Tautz     

Plant growth regulatory metabolites from novel actinomycetes

Metabolites from 796 isolates of aerobic actinomycetes were screened for plant growth regulatory properties using an algal bioassay. These included 266 isolates ofStreptomyces, 28 unidentified actinomycetes, and 502 isolates of novel actinomycetes represented by 18 genera. Algal growth inhibition of 30% was observed with 60 isolates, 37 of which belonged to the genusStreptomyces. Among other inhibitors were 8 isolates ofActinomadura, 6 ofActinoplanes, 2 each of the generaThermomonospora, Streptoverticillium, andPromicromonospora, and 3 unidentified. Metabolites from 70 isolates promoted algal growth by 20%. These included 13 isolates ofMicromonospora, 11 ofStreptomyces, 6 ofNocardia, 5 ofActinomadura, and 4 each ofRhodococcus andThermomonospora. Sixteen unidentified isolates; 3 isolates ofPromicromonospora; 2 isolates each ofActinoplanes, Streptosporangium, andOerskovia; and 1 of Thermoactinomyces peptonophilus-like organism andSaccharomonospora viridis also promoted the algal growth by 20%. The plant growth inhibitory properties of 9 actinomycetes and the growth promoting properties of 6 were demonstrable during the secondary screening on higher plants using chemicals extracted from the culture broth. The metabolites fromMicromonospora, Nocardia, Rhodococcus, Streptosporangium, andOerskovia isolates were associated with plant growth promotion only; those fromStreptomyces were most frequently involved with the growth inhibition.This is Michigan Agriculture Experiment Station Journal Article No. 12191.     

The binding of epidermolytic toxin from Staphylococcus aureus to mouse epidermal tissue

Summary Fluorescein-labelled epidermolytic toxin (FTC-toxin) ofStaphylococcus aureus and ferritin—toxin conjugate have been prepared and purified. FTC-toxin bound selectively to cryostat and resin-impregnated sections of neonatal mouse skin. Binding was localized at the keratohyalin granules and in the stratum corneum. In an epidermal cell (granular, spinous and basal) preparation, only keratohyalin granules of the granular cells bound FTC-toxin. Ferritin—toxin conjugate bound to skin sections at the same two sites as FTC-toxin and was competitive with the binding of free toxin. Keratohyalin granules in unstained sections had a novel patched appearance under the electron microscope, and the ferritin—toxin conjugate bound preferentially to the electron-lucent areas. In the stratum corneum it was shown by quantitative estimation that the target density decreased as the surface of the tissue was approached.     

Growth and maturation of melanosomes in the melanophores of a teleost,Oryzias latipes

Summary Formation of melanosomes in melanophores of a teleost, Oryzias latipes, was studied by means of electron microscopy. Two distinct types of premelanosomes are observed in the same cell: (i) multivesicular premelanosomes, which later develop into melanosomes with electron-lucent hollows in the center, appear at early embryonic stages; (ii) premelanosomes with highly organized, fibrous internal structure are formed at later stages of development and give rise to melanosomes with a filamentous center. Melanosomes are generally ellipsoid in shape, and the difference in the dimensions of fibrillar premelanosomes, melanosomes in the cells at younger developmental stages and those developed fully in melanophores of adults indicates that these organelles grow during development. The growth is achieved by fusion of small unmelanized vesicles or fibrillar premelanosomes to preformed melanosome and by fusion of two or more premelanosomes to form a larger organelle. The addition of the matrix of fibrillar premelanosomes around preformed melanosomes, which are derived from either multivesicular or fibrillar premelanosomes, forms a concentric outer deposit, and the fusion of small vesicles produces electron-lucent pits which are scattered irregularly in mature melanosomes.     

The<Emphasis Type="Italic"> ssgB</Emphasis> gene,encoding a member of the regulon of stress-response sigma factor σ<Superscript>H</Superscript>, is essential for aerial mycelium septation in<Emphasis Type="Italic"> Streptomyces coelicolor</Emphasis> A3(2)

The Streptomyces coelicolor A3(2) gene ssgB belongs to the regulon of stress-response sigma factor ^H. By integrative transformation via double cross-over, a stable null mutant of ssgB was obtained. This mutation had no obvious effect on vegetative growth, but critically affected aerial mycelium septation. The S. coelicolor ssgB mutant produced aerial hyphae without any signs of septation into spore compartments. The mutation was complemented in trans by wild-type ssgB including the ^H-dependent ssgBp promoter. The results proved that ssgB belongs a developmental branch of the ^H regulon.     

Comparative studies on the teliospore morphology and ontogeny ofSpumula serispora,spec. nova,andRavenelia texensis (Uredinales,Raveneliaceae)

A new rust fungus,Spumula serispora, is described. The morphology of the teliospores and the telia was studied. In the teliospores ofS. serispora andS. quadrifida, the type species of the genus, sterile cells could be found, which are comparable to apical cells of otherRaveneliaceae. Similar sterile cells were also present in the telial heads ofRavenelia texensis. The ontogeny of the teliospores ofS. serispora and the teliospore heads ofRavenelia texensis was studied and compared. In both species the ontogeny of the spores heads was similar. InR. texensis hygroscopic cysts sustaining the spore heads were produced by division and not by conversion of basal sterile cells of the teliospore heads.Part 114 of the series Studies inHeterobasidiomycetes.     

An attachment tip and pili-like structures in insect- and plant-pathogenic spiroplasmas of the class Mollicutes

Ultrastructural studies using scanning electron microscopy (SEM), negative-staining transmission electron microscopy (TEM), and thin-sectioning TEM on four species of Spiroplasma, in vitro and/or in vivo, indicated that their helices commonly possess one tapered end (tip structure) and one blunt or round end. These tip structures appeared morphologically different from the rest of the helix, exhibiting an electron-dense conical or rod-shaped core. In thin sections of the midgut of the leafhopper Dalbulus elimatus, the tip structures of Spiroplasma kunkelii in the midgut lumen were mostly aligned between microvilli, perpendicular to the apical plasma membrane of epithelial cells. These tip structures appeared frequently attached or closely apposed to the plasma membrane, in which cup-shaped invaginations close to the tips were observed. Pleomorphic forms of spiroplasma, enclosed in membranous vesicles, were found in the cytoplasm of the midgut epithelial cells. These findings suggest that the tip structure may be involved in the orientation and attachment of spiroplasma helices in relation to their host cells, and thus may be functionally comparable to the attachment organelle of mycoplasmas. Additionally, pili-like structures were observed by negative-staining TEM on the surface of Spiroplasma melliferum, and in thin sections of S. kunkelii infecting the leafhopper vector Dalbulus gelbus. Abbreviations CSS Corn stunt spiroplasma - SEM Scanning electron microscopy - TBS Tris-buffered saline - TEM Transmission electron microscopy     

Characterization ofGandalf, a new inverted-repeat transposable element ofDrosophila koepferae

The cloning and characterization ofGandalf, a new DNA-transposing mobile element obtained from theDrosophila koepferae (repleta group) genome is described. A fragment ofGandalf was found in a middle repetitive clone that shows variable chromosomal localization. Restriction, Southern blot, PCR and sequencing analyses have shown that mostGandalf copies are about 1 kb long, are flanked by 12 by inverted terminal repeats and contain subterminal repetitive regions on both sides of the element. As with other elements of the DNA-transposing type (known as the Ac family), theGandalf element generates 8 by direct duplications at the insertion point. Coding region analysis has shown that the longer open reading frame found inGandalf copies could encode part of a protein. However, whether or not the 1 kb copies of the element are actually the active transposons remains to be elucidated.Gandalf shows a very low copy number inD. buzzatii, a sibling species ofD. koepferae. An attempt to induce interspecific hybrid dysgenesis in hybrids of these two species has been unsuccessful.     

Constant copy numbers of plasmids inSaccharomyces cerevisiae hosts with different ploidies

Summary The cellular -galactosidase activities produced by thelac'Z gene ofEscherichia coli, cloned on YEp, YRp, or YCp-type plasmids in host cells ofSaccharomyces cerevisiae with different ploidies, and which was expressed by a modified jeastHIS5 promoter, showed characteristic differences depending on the plasmid. But for any given plasmid, the isogenic diploid and tetraploid transformants showed slightly lower enzyme activities than their respective haploid transformants. This was due to the similar copy numbers of the plasmids in host cells. Since the cell number per unit volume of the culture decreased with increasing cell ploidy, the enzyme activity per unit volume of the culture decreased significantly. The holding stability of plasmids increased with increasing ploidy of the host cell, especially that of the YRp plasmid. On the YRp plasmid, thelac'Z gene showed higher productivity withTRP1 thanLEU2 as the selection marker for the plasmid.     

Studies on Baker's yeasts of East Pakistan II

Summary 88 strains of yeast and 4 strains of fungi imperfecti have been isolated from dough from three more districts of East Pakistan. The yeasts comprise 53 strains ofSaccharomyces carlsbergensis, 15 strains ofCandida krusei, 8 strains ofCandida guilliermondii var.membranaefaciens, 6 strains ofTorulopsis colliculosa, 2 strains ofTorulopsis globosa, and 4 strains ofHansenula anomala. Of the four strains of fungi imperfecti, 3 belonged toCladosporium butyri and one toSporophora sp. TheSporophora sp. is considered to be a contaminant.The 92 isolates have been tested for their capacity to ferment -methyl glucoside. The possibility of the utilisation of the fermentation of -methyl glucoside as an additional character in yeast taxonomy has been discussed.Tests for the syntheses of various members of vitamin B-complex have shown that all the 92 isolates are more or less autotrophic.     

Distribution of cell wall components in<Emphasis Type="Italic"> Sphagnum</Emphasis> hyaline cells and in liverwort and hornwort elaters

Spiral secondary walls are found in hyaline cells of Sphagnum, in the elaters of most liverworts, and in elaters of the hornwort Megaceros. Recent studies on these cells suggest that cytoskeletal and ultrastructural processes involved in cell differentiation and secondary wall formation are similar in bryophytes and vascular plant tracheary elements. To examine differences in wall structure, primary and secondary wall constituents of the hyaline cells of Sphagnum novo-zelandicum and elaters of the liverwort Radula buccinifera and the hornwort Megaceros gracilis were analyzed by immunohistochemical and chemical methods. Anti-arabinogalactan–protein antibodies, JIM8 and JIM13, labeled the central fibrillar secondary wall layer of Megaceros elaters and the walls of Sphagnum leaf cells, but did not label the walls of Radula elaters. The CCRC-M7 antibody, which detects an arabinosylated (16)-linked -galactan epitope, exclusively labeled hyaline cells in Sphagnum leaves and the secondary walls of Radula elaters. Anti-pectin antibodies, LM5 and JIM5, labeled the primary wall in Megaceros elaters. LM5 also labeled the central layer of the secondary wall but only during formation. In Radula elaters, JIM5 and another anti-pectin antibody, JIM7, labeled the primary wall. The distribution of arabinogalactan–proteins and pectic polysaccharides restricted to specific wall types and stages of development provides evidence for the developmental and functional regulation of cell wall composition in bryophytes. Monosaccharide-linkage analysis of Sphagnum leaf cell walls suggests they contain polysaccharides similar to those of higher plants. The most abundant linkage was 4-Glc, typical of cellulose, but there was also evidence for xyloglucans, 4-linked mannans, 4-linked xylans and rhamnogalacturonan-type polysaccharides.Abbreviations AGP Arabinogalactan–protein - Araf Arabinofuranose - Fucp Fucopyranose - GalAp Galacturonopyranose - Galp Galactopyranose - GlcAp Glucuronopyranose - HGA Homogalacturonan - Manp Mannopyranose - RG Rhamnogalacturonan - Rhap Rhamnopyranose - XG Xyloglucan - Xylp Xylopyranose     

Resistance ofHER2/neu-overexpressing tumor targets to lymphokine-activated-killer-cell-mediated lysis: evidence for deficiency of binding and post-binding events

HER2/neu-overexpressing tumor cell lines are relatively resistant to lymphokine-activated killer (LAK) cell cytotoxicity when compared toHER2/neu-nonexpressing lines.HER2/neu ⁺ targets were also resistant to binding by LAK large granular lymphocytes (LGL) as shown by visualization at the single-cell level, a target monolayer binding assay and in cold target inhibition experiments.HER2/neu ⁺ LAK-resistant ovarian cell lines demonstrated an absence of ICAM-1 expression while expression of LFA-3, N-CAM, laminin and ₁ integrins was comparable to that ofHER2/neu ^– targets. In contrast, theHER2/neu ⁺ breast cell line, SKBR-3, which was also resistant to lysis and binding by LAK LGL, demonstrated normal expression of ICAM-1. Anti-ICAM-1 antibodies blocked binding and lysis ofHER2/neu ^– carcinoma targets by LAK cells, further supporting the notion that lack of ICAM-1 expression onHER2/neu ⁺ cells contributes to their resistance. The modest binding and lysis ofHER2/neu ⁺ targets by LAK cells was significantly inhibited by anti-LFA-1 antibodies, suggesting the existence of another counter-receptor for LFA-1 onHER2/neu ⁺ targets. The following also supported deficiencies in post-binding events whenHER2/neu ⁺ cells resisted the lytic activity of LAK cells: (a) when the relative resistance to effector cell binding was overcome by exogenous lectin,HER2/neu ⁺ cell lines were still resistant to LAK cytolysis, and (b)HER2/neu ⁺ targets were resistant to perforin-containing granule extracts obtained from the CTLL-R8 cytotoxic lymphocyte cell line. These results indicate that deficiency in effector binding as well as post-binding events contributes to the resistance ofHER2/neu-overexpressing tumor targets to LAK-cell-mediated lysis.Supported by research funds of the Veteran's Administration, the California Institute for Cancer Research and Jonsson Cancer Center core grant CA 16042 funded by NIH     

Temporal and spatial changes of cellulose synthesis inClosterium acerosum (Schrank) Ehrenberg during cell growth

The amount and distribution of wall microfibril synthesis were investigated in the cell-division cycle ofClosterium acerosum. Electron-microscopic examination and a methylation analysis of alkali-extracted wall fragments showed that alkali-extracted wall was mainly composed of microfibrils and that the microfibrils ofC. acerosum were 4-linked glucans, i.e., cellulose. Cellulose synthesis was measured as incorporation of¹⁴C, fed to cells as NaHCO₃, into extracted wall fragments. Extensive cellulose synthesis was coincident with septum formation, continued for more than 6 h and then ceased. It was found by microautoradiography that cellulose synthesis after cell division was essentially restricted to the expanding new semicells. Such a restricted distribution of cellulose synthesis was maintained for more than 6 h after septum formation, i.e., for more than 2 h after the cessation of expansion; afterwards, cellulose synthesis in some, but not all, cells became extended to the old semicells, and then ceased. Considerable cellulose synthesis also took place in the band-like expanding part of non-divided cells, indicating that cell division was not necessarily required for the induction of cellulose synthesis and the latter was coupled with cell expansion. Extension of cellulose synthesis to old semicells was brought about in divided cells by treatment with 3 mM colchicine, 28 M vinblastine, 50 M isopropyl-N-phenylcarbamate or 1 M isopropyl-N(3-chlorophenyl)carbamate, indicating that microtubules are involved in the limitation of cellulose synthesis to the new semicells.Abbreviations CIPC isopropyl-N(3-chlorophenyl)carbamate - DPO 2,5-diphenyloxazole - IPC isopropyl-N-phenylcarbamate     

The crypticβ-fructofuranosidase ofSaccharomyces rouxii

Summary The synthesis of-fructofuranosidase in synchronously dividing cells ofS. rouxii was continuous (as opposed to periodic) throughout the budding cycle and followed the increase in cell mass. Similar patterns for cell mass and enzyme increases were observed even in phosphate-deprived cells which did not divide. The-fructofuranosidase activity remained physically cryptic throughout the cell cycle as evidenced by analyses on equilibrium density gradient fractions. The-fructofuranosidase activity released from mechanically disrupted cells resisted sedimentation when subjected to 131 000 g for 1 h, thus ruling out membrane association. Ethyl acetate was routinely employed to break the crypticity barrier. Enzyme in cell-free extract or in cells was equally sensitive to inactivation at pH values below 5 in the presence of ethyl acetate, which suggested that this is an inherent property of the enzyme in question and not a reflection of proteolytic inactivation. The status of-fructofuranosidase in selected species of Saccharomyces was compared with that forS. rouxii and a close similarity withS. bisporus var.mellis was noted. The degree of crypticity encountered in genetically defined strains ofS. cerevisiae (e.g. ×2180 a/) was relatively high (42%) compared with that for commercially derived bakers' and brewers' strains (about 6%). Extant data on the cryptic-fructofuranosidase ofS. rouxii are evaluated and the utility of this system for studying enzyme translocation is discussed.     

Highly polymorphic DNA markers to specify strains of the ectomycorrhizal basidiomycete Tricholoma matsutake based on σ marY1 , the long terminal repeat of gypsy-type retroelement marY1

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies—matsutake—in Pinus sp. forest. Here we report that PCR with outward facing primers designed based on sequences comprising _marY1, the long terminal repeat of the gypsy-type retroelement marY1, specifies strains of T. matsutake. PCR with a primer based on the 22-bp sequence conserved at the 5-end of _marY1 conferred 73 reliable bands overall whose profiles depend upon strains of T. matsutake and T. magnivelare, the latter known as American matsutake. This PCR system gave no detectable band in any other species of Tricholoma tested, including T. bakamatsutake and T. fulvocastaneum, symbionts closely related to T. matsutake, as well as a host plant, Pinus densiflora. Similarly, PCR with a set of primers based on 26-bp and 28-bp sequences at bp 48–73 and bp 281–308 of _marY1, internal regions that are mutated in a variant of marY1, conferred 90 reliable bands only in strains of T. matsutake. Theoretically, PCR with the 22-bp primer would allow generation of 2⁷³, or 9.4×10²¹, types of polymorphism, and PCR with a combination of 26- and 28-bp primers, 2⁹⁰, or 1.2×10²⁷ types. The probability of falsely specifying two different isolates as the same strain is <1/10²¹. While polymorphisms conferred by the primer based on the 5 end of _marY1 rather exhibit genetic conservation of a group of T. matsutake, those resulting from primers based on the internal sequences more clearly demonstrate intra-specific diversification. Both systems revealed that T. matsutake is divergent within the species. Ectomycorrhizas formed between P. densiflora and T. matsutake were identified by the PCR systems developed in the present study. This method, using _marY1 as a genetic marker, is useful in analyzing the diversity of T. matsutake, monitoring the behavior of individual mycorrhizas, and specifying the ecological background of fruit bodies traded in markets.     

Fine structure of the Malpighian tubes in the blood-sucking insect,Triatoma infestans Klug

Summary The ultrastructure of the distal cells in the Malpighian tubes ofTriatoma infestans Klug differs from that of the proximal cells in terms of types of striated border, and distribution of mitochondria and laminated concretions. This is in accordance with published data for another blood-sucking insect,Rhodnius prolixus Stahl. Other observations, however, elucidate cytoplasmic structures not yet reported inReduviidae insects. Layered membranous formations ending in spiral configurations are found in both cell types giving rise to layered membranous globules. Bundles of fibers made up of tubuli occur in the apical regions of the proximal cells in fasted animals. Glycogen deposits surround vacuole-like areas which are probably representing a stage in the formation of laminated concretions. Several globule types [lysosome-like structures, and layered membranous globules sometimes containing cell organelles (cytolysomes?)], are present in the distal and proximal cells, whereas laminated concretions are displayed only by the distal cells. The different globules are ascribed to various stages in the excretion of substances and the elimination of organelles. No special ultrastructural findings could be related to the diversified nuclear phenotypes previously described in the Malpighian tubes ofT. infestans.