The cytology of phytophthora infestans

Summary Phytophthora infestans has three kinds of somatic nuclei: an oval shaped nucleus (approx. 3.1×2.7 ) which stains diffusely except for a crescent shaped Feulgen positive cap which stains intensely; a granular nucleus whose contents are organized into a fairly constant number of stained bodies, and, a deeply staining condensed nucleus. The capped nucleus is thought to be metabolic or resting and the granular nucleus is thought to be dividing as it is most commonly found in hyphal tips. Attenuated forms of all three kinds of nuclei are found.Nuclear division is mitotic and intranuclear. Eight—ten chromosomes are seen at metaphase.Sporangia have a mean of 6.3 nuclei which is constant for age and strain of culture. Sporangia become multinucleate as a result of nuclear migration and not by division in the developing sporangium. Zoospores are usually uninucleate.The nuclear cap is persistent throughout nuclear division when it also divides. It is associated with flagella production and nuclear migration and has some of the properties of a blepharoplast.     

Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

Ultrastructure and behaviour of the spindle pole body of the aphid-pathogenic fungusErynia neoaphidis

Summary A detailed account of the ultrastructure and behaviour of the spindle pole body (SPB) of the entomophthoraceous fungusErynia neoaphidis is presented for the first time.The SPB consists of extranuclear (ENC) and intranuclear (INC) components. The ENC is a saucepan-shaped structure which lies in a pocket of the nuclear envelope. It is composed of a forked, fibrillar handle and a shallow, cylindrical pan. The pan has a wall of two layers, both of which are thickened with a regular periodicity so that they appear to be beaded. It is postulated that the pan is formed from rough endoplasmic reticulum and that it synthesizes the amorphous, electron-dense material coating the ENC.The INC is a saucer-shaped, electron-dense plaque in which the ends of the spindle microtubules terminate. During metaphase, a clear zone separates the INC from the nuclear envelope and persists until telophase. The roles of the amorphous, electron-dense material and the clear zone as well as the method of SPB replication are discussed.     

Lateral hydrodynamic effects of rotating filaments

On the basis of the filament rotation model that was elaborated for interpretations in cell motility, the lateral hydrodynamic effects of rotating filaments have been investigated by large-scale model experiments. Helices were rotated by small electric motors in a medium of high viscosity (honey or polyethyleneglycol). The observed effects, hitherto not investigated in detail by hydrodynamics, show some features that were attributed to the indefinable formative power or vital force of the past. The main effects generated by the rotating filaments are (1) flows and flow patterns with impact zones where flows collide, (2) regions of excessive pressure and negative pressure (corner effect) along a wall, (3) grooves and smoothly shaped ridges on a free fluid surface, and (4) rolling motions of freely hanging filaments. All effects and flow patterns depend on the appropriate distribution of rotating and counterrotating filaments. Each change of the rotational direction means a dramatic alteration. The application of the observed effects explains largely the function of the microtubule/microfilament hoops or helices during the cytokinesis of a plant cell. Interpretations or simulations are described for events as the formation of secondary wall thickenings, the orientation of their microfibrils, the motion of the preprophase band microtubules, the formation of the phragmosome, the migration, stationary position and shape of the preprophase nucleus, the girdle-, septum- and H-piece formation of cell walls in algae and some events of morphogenesis inMicrasterias. Further interpretations are related to the lateral flows and to invaginations of free cell membranes, to lateral filament motions, to the right-left problem, to the selfintertwining of filaments, to the rotation of a cell body by its flagellum, to the repulsion of chromatids during meiosis and to the tetragonal and hexagonal arrangement of filaments.Dedicated to Prof. DrLothar Geitler on the occasion of the 90th anniversary of his birthday.     

The distribution and form of the endoplasmic reticulum during spermatogenesis in the crayfish,Cambaroides japonicus

Summary The form and differentiation of the endoplasmic reticulum has been studied in the developing sperm of the crayfish, Cambaroides japonicus. Throughout development a relationship between the nuclear envelope and cytoplasmic portion of the endoplasmic reticulum has been shown to exist. Furthermore, large contributions of material from the nuclear envelope to extranuclear cytoplasmic systems has been noted in the development of early spermatids and nearly mature sperm.A sequential predominance of several types of endoplasmic reticulum has been described in the differentiating sperm. An agranular vesicular reticulum is the most common in the early stages although annulate lamellar stacks and rough surfaced stacks are scattered randomly throughout the cytoplasm. Blebs of the nuclear envelope appear to contribute rough surfaced reticulum to the cytoplasmic system in the early spermatid. A fusion of vesicular elements results in the formation of the dense filamentous reticulum which is typical of the nearly mature sperm. Densely packed lamellae develop on the nuclear envelope in the maturing sperm and are connected to both the nuclear envelope and filamentous endoplasmic reticulum. The possible relationships of these lamellar groups to mitochondria or Golgi is discussed.Supported in part by Grant No B-2314 of the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service.Predoctoral Research Fellow of the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service.     

Changes in density of chromatin in the meristematic cells ofSinapis alba during transition to flowering

Summary Changes in the density of nuclear chromatin in the shoot apical meristem ofSinapis alba L. during floral transition (floral evocation) are described using Feulgen-stained 2 m thick semi-thin sections and scanning cytophotometric techniques. In both G1 and G2 nuclei the chromatin becomes less heterogeneous and less dense in evoked meristems compared to vegetative meristems. When chromatin is resolved into two fractions the dispersed fraction increases relative to the condensed fraction at evocation. This decondensation process occurs earlier in G1 than in G 2 nuclei. These chromatin changes are presumably closely related to the dramatic stimulation of biosynthetic activity and cell division during floral transition.     

Conformation of the circular dumbbell d〈pCGC-TT-GCG-TT〉: Structure determination and molecular dynamics

Summary The circular DNA decamer 5-dpCGC-TT-GCG-TT-3 was studied in solution by means of NMR spectroscopy and molecular dynamics in H₂O. At a temperature of 269 K, a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4) is present. The L2L2 form contains three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature from 269 K to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted G(anti)-C(syn) closing base pair in the 5-GTTC-3 loop with only one remaining (solvent-accessible) hydrogen bond between NH of the cytosine dC(1) and O6 of the guanine dG(8). The opposite 5-CTTG-3 loop remains stable. The two conformers occur in slow equilibrium (rate constant 2–20 s^–1). Structure determination of the L2L2 and L2L4 forms was performed with the aid of a full relaxation matrix approach (IRMA) in combination with restrained MD. Torsional information was obtained from coupling constants. Coupling constant analysis (³J_HH, ³J_HP, ³J_CP) gave detailed information about the local geometry around backbone torsion angles , , and , revealing a relatively high flexibility of the 5-GTTC-3 loop. The values of the coupling constants are virtually temperature-independent. Weakly constrained molecular dynamics in solvent was used to sample the conformational space of the dumbbell. The relaxation matrices from the MD simulation were averaged over r^–3 to predict dynamic NOE volumes. In order to account for the 1:1 conformational mixture of L2L2 and L2L4 present at 271 K, we also included S² factors and r^–6 averaging of the r^–3-averaged relaxation matrices. On matrix averaging, the agreement of NOE volumes with experiment improved significantly for protons located in the thermodynamically less stable 5-GTTC-3 loop. The difference in stability of the 5-CTTG-3 and 5-GTTC-3 loops is mainly caused by differences in the number of potential hydrogen bonds in the minor groove and differences in stacking overlap of the base pairs closing the minihairpin loops. The syn conformation for dC(1), favored at high temperature, is stabilized by solvation in the major groove. However, the conformational properties of the dC(1) base, as deduced from R-factor analysis and MD simulations, include a large flexibility about torsion angle .     

Ultrastructural analysis of the sperm cells of mature pollen of maize,Zea mays

Summary Ultrastructural analysis of the mature viable unhydrated pollen of maize,Zea mays from dehiscent anthers shows that the sperm cells are physically distant, each bounded by an envelope comprising their own plasma membrane and the inner plasma membrane of the vegetative cell. The chondriome is unusual in containing one or more filamentous complexes, up to 12m in length appressed to the side of the sperm nucleus. The extensions at each end of the elongate sperm cells contain longitudinally-oriented arrays of endoplasmic lamellae. In a three-dimensional reconstruction of serial thin sections, there is a long J-shaped sperm, c. 35 × 5m and up to 1m in thickness, sited within pointed evaginations of the vegetative nucleus and a second shorter sperm c. 20 × 5m and up to 3.5m in thickness.Abbreviations PA-TCH-SP periodic acid-thiocarbohydrazide-silver proteinate - DAPI 4,6-diamino-2-phenylindole - SC sperm cell - Sn sperm nucleus - Ua-Pb Uranyl acetate-lead citrate staining - ER endoplasmic reticulum     

Affinity Profiles of Novel δ-Receptor Selective Benzofuran Derivatives of Non-Peptide Opioids

Highly selective heterocyclic opioid ligands with potent -antagonist activity have been developed on the basis of the message-address concept. Using this strategy, benzofuran derivatives corresponding to the non-selective opioid antagonist, naloxone, and to the -opioid receptor selective agonists, oxymorphone and oxycodone, were synthesized. In vitro opioid receptor binding profiles and agonist/antagonist character of these compounds were determined in rat brain membrane preparations with highly selective radioligands. All three benzofuran derivatives displayed high affinities for the -opioid receptor, much less potency toward the -binding site, and were the least effective at the -site. The results indicated that the addition of the bezofuran moiety to these fused ring opioids confers -receptor selectivity. The Na⁺ indices suggested a partial agonist character for oxymorphone- and oxycodone-benzofuran, and an antagonist character for naloxone-benzofuran. These compounds were capable of irreversible inhibition of opioid binding sites in a dosedependent.     

The macronuclear envelope ofTetrahymena pyriformis GL in different physiological states

Summary A simple formula is derived to calculate the nucleocytoplasmic RNA-Efflux per nuclear Pore complex per min (REP-rate) which is generally applicable both for growing and stationary eukaryotic cells. In actively growing cells this REP-rate is mainly dependent on the cytoplasmic RNA-pool, the number of RNA-transporting pores, and the growth constant of RNA. These parameters are determined in logarithmicTetrahymena pyriformis GL. In this organism, 45 molecules both of the larger ribosomal RNA (25s rRNA) and of the smaller (17s rRNA) are transported per pore per min from nucleus to cytoplasm. Pulse-label experiments with³H-uridine indicate that the 25s rRNA is obviously transferred more slowly to the cytoplasm than the 17s rRNA. We postulate a gating hypothesis on the regulation of the nucleocytoplasmic RNA-efflux by nuclear pore complexes. This gating hypothesis suggests that nucleopores are controlling points of secondary importance in the sequence of gene expression, and do not directly control the cytoplasmic protein synthesis in eukaryotic cells.     

Diel Movements of Bat Rays, Myliobatis californica, in Tomales Bay, California: Evidence for Behavioral Thermoregulation?

We used ultrasonic telemetry to examine movement patterns of 11 bat rays, Myliobatis californica, in Tomales Bay, California. Tomales Bay is long (20km) and narrow (1.4km), and is hydrographically separated into outer and inner bay regions. The outer bay (the outermost 8km) is characterized by oceanic conditions while the shallow inner bay (the innermost 12km) features wide seasonal temperature shifts. Five rays were tracked monthly from October 1990 to November 1991 and six rays (four of which carried temperature-sensing transmitters) were tracked daily from 30 June to 16 July 1992. Mean bat ray movement rate was 8.84mmin^–1 (range 4.49 to 13.40mmin^–1) and was not significantly affected by size (p=0.592), tidal stage (p=0.610), or time of day (p=0.327). Movement direction was unrelated to tidal stage (p=0.472) but showed a highly significant diel pattern (p<0.001). From 2:50–14:50h, rays moved toward the warmer and shallower inner bay, while from 14:50–2:50h they moved toward the cooler and deeper outer bay. These telemetry data, along with known bat ray foraging patterns and respiratory temperature-sensitivity, argue for behavioral thermoregulation as the primary influence on this movement pattern.     

Hexagonally ordered “rosettes” of particles in the plasma membrane ofMicrasterias denticulata Bréb. and their significance for microfibril formation and orientation

Summary Cells ofMicrasterias denticulata Bréb. at the stage of secondary wall formation have been studied by freeze-etching. It was found that the plasma membrane exhibits oval areas in which arrays of membrane particles occur. These particles form rosettes which are arranged in a hexagonally ordered lattice with a center to center spacing of 25 nm. Nearly the same periodicities can be found between microfibrils. It is concluded that the rosettes probably together with the thickened area of the plasma membrane below them represent the apparatus for the production and orientation of microfibrils. The hypothesis suggesting the incorporation of membrane templates functional in microfibril formation, originally advanced byKiermayer andDobberstein (1973) has received further support.     

Über die Feinstruktur des Zellkerns von Acetabularia nach Gefrierätzung

Summary The ultrastructure of the primary nucleus of the marine green algae Acetabularia (Polyphysa) cliftonii as revealed by the freeze-etching technique is described.The nuclear envelope is perforated by about 3×10⁶ pores which appear arranged nearly in rows. The nucleus is covered with a layer of cytoplasm which protects it in the isolated state and resists various manipulations. This cytoplasmic layer is protected by an elementary membrane. The latter appears rough on the cytoplasmic side, and smooth on the side facing the cell sap. The cytoplasmic layer and the surrounding perinuclear cytoplasm are connected by cytoplasmic ducts. The nucleus appears irregularly shaped owing to many invaginations in the nuclear envelope. Sometimes mitochondria are observed in these cavities.     

The recognition of three different epitopes for the H-type 2 human blood group determinant by lections ofUlex europaeus,Galactia tenuiflora andPsophocarpus tetragonolobus (Winged Bean)

The chemical mapping of the regions of H-type 2 human blood group-related trisaccharide (Fuc(1–2)Gal(1–4)GlcNAcMe) that are recognized by three different lectins, the so-called epitopes, are reviewed together with an account of how and why oligosaccharides form specific complexes with proteins as presently viewed in this laboratory. The occasion is used to report the synthesis of the various mono-O-methyl derivatives of the above trisaccharide that were used in these investigations. Also, Fuc(1–2)Gal(1–4)XylMe was synthesized in order to examine whether or not the hydroxymethyl group of the GlcNAc residue participates in the binding reaction.Abbreviations Me methy - Bn benzyl - Ac acetyl - Bz benzoyl - n-Bu n-butyl - NMR nuclear magnetic resonance - the GlcNAc Gal and Fuc residues of the H-type 2 trisaccharide are designated as the a, b and c structural units, respectively. This is paper XV in a series devoted to molecular recognition.     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Histoenzymatic characterization of sub-types of Type I fibres in fast muscles of rats

Summary The histochemical activities of succinic dehydrogenase (SDH), myofibrillar Adenosine triphosphatase (ATPase) and alpha glycerophosphate dehydrogenase were studied in serial sections of rat vastus lateralis (red) (RVL), gastrocnemius and diaphragm muscles. Three main fibre-types were distinguished. The Type I fibres of RVL and gastrocnemius muscles fell into two distinct groups: one category-Type IA showed very low ATPase activity. The second category of Type IB fibres displayed moderate ATPase reaction. The Type IA fibres were divisible into two sub-groups when tested for SDH reaction. Type IA₁ fibres possessed a homogenous distribution of diformazan·granules throughout the fibre: Type IA₂ fibres displayed characteristic moth-eaten pattern of diformazan localization. The diaphragm muscle did not show either Type IB or Type IA₂ varieties. The great majority of Type I fibres were sub-type IA₁ in the three fast muscles studied. It is also demonstrated here that an inherent heterogeneity exists between Type I fibres of diaphragm and leg muscles in regard to -GPD localization. This histochemical data emphasizes the fact that subdivision of Type I striated muscle fibres of mammalian animals into two sub-types is only approximate and that a further subcategorization is possible.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

The development,structure and function of modified synaptonemal complexes in mosquito oocytes

The nucleus of the maturing oocytes expands to a large thin body of 400×140×3 m but the chromosomes remain together in a small sphere, 15 m in diameter. In Aedes aegypti this sphere becomes surrounded by one to several layers of polycomplexes, annulated polycomplexes, and related annulated pseudomembranes. Just prior to egg laying the expanded nucleus disintegrates while the sphere of chromosomes is surrounded by several layers of membranes. In Culex pipiens the elements which normally connect the lateral elements of the synaptonemal complexes become extended so that all bivalents become interconnected by a framework of pseudomembranes. The continuity between the modified synaptonemal complexes and various membranes associated with the karyosphere suggest that a relationship exists, by origin or by specialization, between the synaptic structures and nuclear envelope.     

Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled withp-aminobenzoic acid ethyl ester (ABEE) afterN-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz¹H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6%, Glc1-3Glc1-3Man₉GlcNAc₂; 35.6%, Glc1-3Man_7–9GlcNAc₂). oligomannose type (15.0%, with the structure Man_5–9GlcNAc₂) and biantennary complex type with core structures of-Man1-3(-Man1-6)Man1-4GlcNAc1-4GlcNAc (9.9%),-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4GlcNAc (25.1%) and-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc₁Man_7–9GlcNAc₂) have been located previously in hen IgY.Abbreviations IgG, IgM, IgA, IgY immunoglobulin G, M, A and Y, respectively - ABEE p-aminobenzoic acid ethyl ester     

Microbial transformation of 18β-glycyrrhetinic acid by Sphingomonas paucimobilis strain G5

The effects of the oxygenase inhibitors, 1-aminobenzotriazole (ABT), ketoconazole, metyrapone and proadifen, on the metabolism of 18-glycyrrhetinic acid (18-GRA) in Sphingomonas paucimobilis strain G5 were investigated. Strain G5 transformed 18-GRA into a major new metabolite (M-D) in the presence of 1 mM ABT or metyrapone. M-D was purified and identified as 3-hydroxy-11-oxo-olean-12-en-24,30-dioic acid by NMR and MS. Based on the structure of M-D, we propose the metabolic pathway of 18-GRA in strain G5.