Participation of Ca2+ in cessation of cytoplasmic streaming induced by membrane excitation inCharaceae internodal cells

Summary The mechanism of the cessation of cytoplasmic streaming upon membrane excitation inCharaceae internodal cells was investigated.Cell fragments containing only cytoplasm were prepared by collecting the endoplasm at one cell end by centrifugation. In such cell fragments lacking the tonoplast, an action potential induced streaming cessation, indicating that an action potential at the plasmalemma alone is enough to stop the streaming.The active rotation of chloroplasts passively flowing together with the endoplasm also stopped simultaneously with the streaming cessation upon excitation. The time lag or interval between the rotation cessation and the electrical stimulation for inducing the action potential increased with the distance of the chloroplasts from the cortex. The time lag was about 1 second/15 m, suggesting that an agent causing the rotation cessation is diffused throughout the endoplasm.Using internodes whose tonoplast was removed by replacing the cell sap with EGTA-containing solution (tonoplast-free cells,Tazawa et al. 1976), we investigated the streaming rate with respect to the internal Ca²⁺ concentration. The rate was roughly identical to that of normal cells at a Ca²⁺ concentration of less than 10^–7 M. It decreased with an increase in the internal Ca²⁺ concentration and was zero at 1 mM Ca²⁺.The above results, together with the two facts that Ca²⁺ reversibly inhibits chloroplast rotation (Hayama andTazawa, unpublished) and the streaming in tonoplast-free cells does not stop upon excitation (Tazawa et al. 1976), lead us to conclude that a transient increase in the Ca²⁺ concentration in the cytoplasm directly stops the cytoplasmic streaming. Both Ca influxes across the resting and active membranes were roughly proportional to the external Ca²⁺ concentration, which did not affect the rate of streaming recovery. Based on these results, several possibilities for the increase in Ca²⁺ concentration in the cytoplasm causing streaming cessation were discussed.     

A polarized light and electron microscopic study of the birefringent fibrils inPhysarum plasmodia

Summary The birefringent fibrils in thin-spread plasmodium ofPhysarum polycephalum have been investigated with both polarizing and electron microscopes. The birefringent fibrils were classified into three groups by polarized light microscopy. The first type of fibril is observed in the advancing frontal region as a mutual orthogonal array. The birefringence changes rhythmically in accordance with the shuttle streaming. The second type of birefringent fibril is located in the strand region and runs parallel or somewhat oblique to the strand axis. The third type is observed in the strand region always perpendicular to the streaming axis. Electron microscopy confirmed that all these fibrils are composed of microfilaments, which range in densities in the cross view of the fibril from 1.2 to 1.7 × 10³/m² (1.5 × 10³/(xm² on the average).     

Ion fluxes inAcetabularia: Vesicular shuttle

Summary Ion flux relations in the unicellular marine algaAcetabularia have been investigated by uptake and washout kinetics of radioactive tracers (²²Na⁺,⁴²K⁺,³⁶Cl^– and⁸⁶Rb⁺) in normal cells and in cell segments with altered compartmentation (depleted of vacuole or of cytoplasm). Some flux experiments were supplemented by simultaneous electrophysiological recordings. The main results and conclusions about the steady-state relations are: the plasmalemma is the dominating barrier for translocation of K⁺ with influx and efflux of about 100 nmol·m^–2·sec^–1×K⁺ passes three- to sevenfold more easily than Rb⁺ does. Under normal conditions, Cl^– (the substrate of the electrogenic pump, which dominates the electrical properties of the plasmalemma in the resting state) shows two efflux components of about 17 and 2 mol·m^–2·sec^–1, and a cytoplasmic as well as vacuolar [Cl^–] of about 420mm ([Cl^–] _o =529mm). At 4°C, when the pump is inhibited, both influx and efflux, as well as the cellular [Cl^–], are significantly reduced. Na⁺ ([Na⁺] _i : about 70mm, [Na⁺] _o : 461mm), which is of minor electrophysiological relevance compared to K⁺, exhibits rapid and virtually temperature-insensitive (electroneutral) exchange (two components with about 2 and 0.2 mol·m^–2·sec^–1 for influx and efflux). Some results with Na⁺ and Cl^– are inconsistent with conventional (noncyclic) compartmentation models: (i) equilibration of the vacuole (with the external medium) can be faster than equilibration of the cytoplasm, (ii) absurd concentration values result when calculated by conventional compartmental analysis, and (iii) large amounts of ions can be released from the cell without changes in the electrical potential of the cytoplasm. These observations can be explained by the particular compartmentation of normalAcetabularia cells (as known by electron micrographs) with about 1 part cytoplasm, 5 parts central vacuole, and 5 parts vacuolar vesicles. These vesicles communicate directly with the central vacuole, with the cytoplasm and with the external medium.     

Germination and parasitation of the resting sporangia ofSynchytrium endobioticum

Summary The cytoplasmic organization of the long-lived, thick walled resting stage of the sporangium ofSynchytrium endobioticum (Schilb.) Perc. is described. The cytoplasm of the resting sporangium contains a large number of closely packed lipid bodies and irregular electron dense bodies, which are interspaced with fine channels of cytoplasm. These ultrastructural observations are discussed in relation to the hypothesis ofBally (1912) andCurtis (1921) that zoospore primordia are already present during the resting stage. It is shown that the zoospore primordium is actually a lipid body and an osmiophilic body and the strands postulated to connect the individual zoospore primordia are actually the fine channels of cytoplasm.A new inner wall layer is laid down prior to the start of the germination. It is this wall layer which will protrude to form the vesicle in which sporogenesis takes place. The germination process observed, protrusion of a vesicle through a crack in the sporangial wall, the migration of the sporangial content into the vesicle, and the formation of a single, membrane-bound sporangium within this vesicle, is in full agreement with the recent light microscopic studies ofSharma andCammack (1976). These observations support the transfer ofS. endobioticum from the subgenusMesochytrium to the subgenusMicrosynchytrium (bothsensu Karling 1964).A major objective of the study, to obtain ultrastructural evidence for the location of the meiotic divisions in the life cycle, was not fulfilled.Three different fungi were observed to parasitize the resting sporangium ofS. endobioticum. These infections are discussed in relation to other mycoparasites of plant pathogenic fungi. The possibility of using a mycoparasite for the biological control of potato wart disease is considered to be without practical relevance.     

Kernkontrollierte Lactatdehydrogenase inAcetabularia

Zusammenfassung In kernhaltigen, aber auch in kernlosen Zellen vonAcetabularia wird wÄhrend des Wachstums die LDH-AktivitÄt bis zum Beginn der Hutbildung vermehrt. Verschiedene Arten vonAcetabularia weisen bezüglich der LDH entweder durch Zahl der Banden oder Position der Banden Unterschiede voneinander auf. Heterologe Transplantate bzw. Implantate lassen einige Wochen nach der Operation erkennen, da\ die artspezifischen Isozymbanden des Cytoplasmas verschwinden und die vom Kern determinierten erscheinen. Zumindest ein Teil der LDH-AktivitÄt ist an die Chloroplasten gebunden. Dieses wurde mit Hilfe der Saccharose-Dichtegradienten-Zentrifugation und auf histochemischem Wege nachgewiesen.

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Nucleus dependent lactic dehydrogenase inAcetabularia

Summary In nucleate as well as in anucleate cells ofAcetabularia LDH activity is increased until the cap is formed. Different species ofAcetabularia can be distinguished by the electrophoretic behavior of the LDH. Heterologous grafts of nucleus containing rhizoids or of isolated nuclei to anucleate cytoplasm result in the disappearance of the LDH pattern of the cytoplasm and in the appearance of a new one which is specific for the species of the nucleus. At least part of the enzyme activity is linked to the chloroplasts as was shown by sucrose density gradient centrifugation and by histochemical techniques.

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Wir danken Herrn Dr. G.Werz für Anregungen beim histochemischen Nachweis und Herren H.Kretschmer für seine Hilfe bei der PrÄparation der Transplantate und Implantate.     

The estimation of the motive force for protoplasmic streaming inNitella

Summary By use of a theoretical model for a section of a cell ofNitella and assumptions regarding the form of the stress/rate of strain relation for the streaming protoplasm, it has been possible to determine possible velocity profiles for the streaming in normal and disturbedNitella cells. A match of velocities from these theoretical studies to those measured in real systems has led to a re-estimation of the motive force and of the viscosity coefficient as well as to a first estimate of the thickness of the layer over which the force must be distributed.These new results show the motive force field to be restricted to a layer of about 0.1m thickness alongside the sol/gel interface (the outside boundary of the streaming layer), the force per unit area of this interface to be about 0.36 Nm^–2 (3.6 dyne cm^–2) and a possible stress/rate of strain relation to be of the form (stress)=(viscosity coefficient) × (rate of strain)1/3.Although this latter relation is similar to that obtained by Kamiya and Kuroda (1965) for isolated protoplasm, their viscosity coefficient is about twelve times the present estimate (0.027 Nm^–2s 1/3) suggesting that the fluidin situ is much less viscous than their isolated material. The estimate for the motive force is about double that of previous workers. (Kamiya andKuroda 1958,Tazawa 1968).     

Morphologische Veränderungen in Chloroplasten und Mitochondrien von verdunkelten Acetabularia-Zellen

Zusammenfassung Zelldifferenzierungen werden in verdunkelten Acetabutaria-Zellen nicht realisiert, obgleich die Potenzen dafür vorhanden sind. Im Gegensatz zur Zelle differenzieren junge Zellorganellen — Chloroplasten und Mitochondrien — von kernhaltigen und kernlosen Zellen unter Dunkelbedingungen charakteristische Strukturkomponenten. Dieses Verhalten weist darauf hin, daß diese Zellorganellen eigene Differenzierungsmechanismen besitzen. In Analogie zu Prozessen der Zelldifferenzierung, die auf spezifischen Funktionen von Kern-DNS basieren, scheinen die Differenzierungsvorgänge in den Zellorganellen auf Wirkungen ihrer eigenen DNA zu beruhen.

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Morphological changes in chloroplasts and mitochondria of darkened Acetabularia cells

Summary Cell differentiation is not realized in darkened Acetabularia cells, although the potentials concerned are present. In contrast to cell differentiation, young chloroplasts and mitochondria of nucleated as well as of enucleated cells are able for the differentiation of characteristic structural components under dark conditions. This behaviour indicates that these cell organelles possess own differentiation mechanisms. In analogy to cell differentiation which is based on actions of nuclear DNA, the differentiation processes within the organelles are proposed to be dependent on actions of their own DNA.

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Herrn Professor Dr. J. Hämmerling zu seinem 65. Geburtstage gewidmet.     

Ribonuclease-Aktivität in Acetabularia

Zusammenfassung In Acetabularia mediterranea, Acetabularia crenulata und Polyphysa cliftonii wurde das Vorhandensein von Ribonuclease-Aktivität nachgewiesen. Es wurde gezeigt, daß das Enzym hitzestabil ist und bei pH 7,2 und im Bereich von pH 5 Maxima aufweist. Selbst durch eine Zentrifugation nit 105000 x g über 120 min wurde die Enzym-Aktivität nicht sedimentiert.In kernlosen Zellen von Acetabularia nimmt die Ribonuclease-Aktivität zu, allerdings in geringerem Maße als in kernhaltigen Zellen.Die Stabilität der genetischen Information im Cytoplasma von kernloser Acetabularia kann nicht durch das Fehlen von Ribonuclease-Aktivität erklärt werden.Fräulein A. Grohs sei für die geschickte und sorgfältige Hilfe bei der Durchführung der Experimente gedankt.

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Activity of ribonuclease in Acetabularia

Summary In Acetabularia mediterranea, Acetabularia crenulata and Polyphysa cliftonii ribonuclease activity was demonstrated. The enzyme is heat stabile and exhibits maxima at pH 7.2 and at the region of pH 5. Even centrifugation with 100000 x g for 120 minutes does not result in sedimentation of enzyme activity.Anucleate cells of Acetabularia are capable of increasing their ribonuclease activity, but to a smaller extent than nucleate cells do.The stability of the genetic information in the cytoplasm of anucleate Acetabularia cannot be explained by a lack in ribonuclease activity.

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Herrn Prof. J. Hämmerling zum 65. Geburtstag gewidmet.     

Differenzierung und Zellwandbildung in isoliertem Cytoplasma ausAcetabularia

Zusammenfassung Die vorliegende Arbeit beschreibt die Isolierung kernhaltigen und kernfreien Cytoplasmas aus den Hutkammern vonAcetabularia und dessen morphogenetisches Verhalten unterin vitro-Bedingungen.Kernhaltiges Cytoplasma (Protoplasten) bildet stets Cysten und cystenspezifische Zellwand.Die Fähigkeit kernlosen Cytoplasmas (Cytoplasten) zur Differenzierung und zur Synthese von Cystenwand hängt von der Einwirkungszeit des Kernes vor der Isolierung ab. Daher zeigen Cytoplasten aus Hutkammern, die keine Kerne enthielten, weder Differenzierung noch Bildung von Cystenwand. Differenzierung und Bildung von Cystenwand findet in gewissem Ausmaß dann statt, wenn Cytoplasten aus Hutkammern isoliert werden, in denen das Cytoplasma unter Kerneinfluß einen höheren Grad der Differenzierung erreicht hatte.

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Differentiation and cell wall formation in isolated cytoplasm ofAcetabularia

Summary The present paper reports on the isolation of nucleate and anucleate cytoplasm of cap rays ofAcetabularia and with its morphogenetic behaviour underin vitro conditions.The nucleate cytoplasm (protoplasts) always differentiates cysts and cyst specific cell wall.The ability of anucleate cytoplasm (cytoplasts) to differentiate and to synthesize cyst wall depends on the time of nuclear actions prior to its isolation. Therefore, cytoplasts of cap rays which did not contain nuclei, show neither differentiation nor cyst wall formation. A certain degree of differentiation and of cyst wall formation takes place if cytoplasts are isolated from cap rays in which the cytoplasm had obtained a higher degree of differentiation eunder nuclear actions.

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Fräulein R.Sandfuchs danke ich für die Aufzucht der Versuchspflanzen, Herrnstud. rer. nat. K.Ertl für seine Hilfe bei der Durchführung der Experimente.     

Development of the resting sporangia ofSynchytrium endobioticum,the causal agent of potato wart disease

Summary An ultrastructural study of the development of the resting sporangium ofSynchytrium endobioticum (Schilb.) Perc. infecting potato cells is presented. The resting sporangium is found to have a single large, centrally placed nucleus with a prominent nucleolus through its entirein situ development. The cytoplasmic organization of the resting sporangium is further characterized by numerous membrane-bound lipid bodies and osmiophilic bodies. The latter have a characteristic sieve-like appearance, probably because certain storage components have been extracted during preparation for electron microscopy. Because of the similar location and appearance of these osmiophilic bodies it is suggested that they are identical to what has earlier (based on light microscopy) been described as chromatin granules; and the ultrastructural studies presented here show that nucleolar discharge which was described from light microscopic observations as leading to chromatin granules in the cytoplasm, and finally forming the nuclei of the zoospores (bally 1912,curtis 1921,percival 1910) simply does not occur.The appearance of dense fibrillar-like structures on the sporangial surface at an early stage of resting sporangium development ultrastructurally distinguishes the resting sporangium from the zoosporangium. The development of the layered portion of the thick sporangial wall is shown to be due to the fusion of vacuoles containing pre-made wall fibrils with the cell membrane. It is suggested that the inner compact wall layer which is essentially substructureless is formed by the membrane itself.The characteristic wings of the matureS. endobioticum resting sporangium originate from the potato host cell wall. Remnants of host cell organelles in the outermost layer of the resting sporangium wall show that degradation of the host cell cytoplasm contributes to wall formation of the parasite.     

A method for recording the circadian rhythm of the oxygen balance in a single cell ofAcetabularia mediterranea

Summary A polarographic method using a platinum electrode permits continuous recording of the oxygen balance of a singleAcetabularia cell during several days. The amount of oxygen which is consumed by the electrode is immeasurably small. The accuracy of the method is definitely better than 0.05l O₂/ml medium. The resolution in time is less than 5 min. The length of one period in the circadian rhythm of photosynthesis inAcetabularia has been found to be about 27 hours.     

Use of antisera to localize callose,xylan and arabinogalactan in the cell-plate,primary and secondary walls of plant cells

Antibodies to cellobiose, L-arabinopyranose, L-arabinofuranose, D-galactose, oligosaccharides containing 14 xylose, oligosaccharides containing 14 glucose, and oligosaccharides containing 13 glucose have been raised in rabbits. The antisera have been characterized to show the specificity of binding to particular polysaccharides. They have been used for immunocytology using the electron microscope to locate the polymers in dividing and differentiating cells of bean (Phaseolus vulgaris L.) root, bean callus tissue and cells of Zinnia elegans L. in vitro. Arabinogalactans have been shown to be present in the cell-plate and primary walls but not in secondary thickening. Xylan as distinct from xyloglucan was found in the primary walls but not in the cell-plate. It was present in large amounts in the secondary thickening. Callose was found in the cell plate and also in the young growing wall. In the wall it was specifically located at the plasmodesmata. The use of the antibody against L-arabinofuranose enabled a specific organelle to be detected which was membranous and which occurred within the cytoplasm and also within the vacuole of the cells. Membranes carrying polymers containing L-arabinofuranose were also found in layers just under the plasmamembrane.Abbreviations L-Araf L-arabinofuranose - L-Arap L-arabinopyranose - BSA bovine serum albumin - Gal galactose - D-Galp D-galactopyranose - Glc glucose - Xyl xylose     

Nuclear division in the cyst,and white spot nuclei preceding cyst formation,inAcetabularia wettsteinii

Summary Electron microscopy of nuclear division in young cysts ofAcetabularia wettsteinii shows that the dividing nucleus hat two additional cisternae of endoplasmic reticulum immediately outside the nuclear envelope. These additional cisternae are attached to, and apparently formed from a membrane body which develops outside the nucleus in early prophase. The interphase nucleus does not have the additional cisternae. The nucleoli are extruded from the nucleus at anaphase, the nucleolar bodies remaining in the peri-nuclear cytoplasm. The chromosomes have localized centromeres; the stratified ultrastructure characteristic of some chlorophycean and animal kinetochores has not been found inAcetabularia, although the kinetochore appears distinct, projecting from the chromatid, and has attached microtubules. The condensed bodies of the white spot nucleus are discussed.     

A preliminary phylogeny for the NeotropicalRubiaceae

The sequence of subfamilies,Cinchonoideae, Antirheoideae andRubioideae, attemps to show their natural affinities and phylogeny. The subfamilies are those ofVerdcourt, and the order in which they are presented is that ofBremekamp. A list is presented of the subfamilies, tribes and genera of theRubiaceae to be utilized in the Catálogo Ilustrado de las plantas de Cundinamarca, Colombia.     

Gravity-dependent polarity of cytoplasmic streaming inNitellopsis

Summary The internodal cells of the characean algaNitellopsis obtusa were chosen to investigate the effect of gravity on cytoplasmic streaming. Horizontal cells exhibit streaming with equal velocities in both directions, whereas in vertically oriented cells, the downwardstreaming cytoplasm flows ca. 10% faster than the upward-streaming cytoplasm. These results are independent of the orientation of the morphological top and bottom of the cell. We define the ratio of the velocity of the downward- to the upward-streaming cytoplasm as the polar ratio (PR). The normal polarity of a cell can be reversed (PR<1) by treatment with neutral red (NR). The NR effect may be the result of membrane hyperpolarization, caused by the opening of K⁺ channels. The K⁺ channel blocker TEA Cl^– inhibits the NR effect.External Ca²⁺ is required for normal graviresponsivness. The [Ca²⁺] of the medium determines the polarity of cytoplasmic streaming. Less than 1 M Ca²⁺ resulted in a PR<1 while greater than 1 M Ca²⁺ resulted in the normal gravity response. The voltage-dependent Ca²⁺ -channel blocker, nifedipine, inhibited the gravity response in a reversible manner, while treatment with LaCl₃ resulted in a PR<1, indicating the presence of two types of Ca²⁺ channels. A new model for graviperception is presented in which the whole cell acts as the gravity sensor, and the plasma membrane acts as the gravireceptor. This is supported by ligation and UV irradiation experiments which indicate that the membranes at both ends of the cell are required for graviperception. The density of the external medium also affects the PR ofNitellopsis. Calculations are presented that indicate that the weight of the protoplasm may provide enough potential energy to open ion channels.     

Biosynthesis of the storage polysaccharide from the snail Biomphalaria glabrata,identification and specificity of a branching β1→6 galactosyltransferase

Adult snails synthesize in their albumen glands a storage polysaccharide called galactan which is utilized by the developing embryos. With [6-³H]-uridine 5diphosphogalactose the incorporation of labelled d-galactose into the polysaccharide can be traeed in freshly removed glands maintained in a bathing buffer. After centrifugation of homogenized glands, galactosyltrasferase activity is only found in the insoluble fraction. Chaps extracts of this material retain almost all of their activity and can be used for comparison of the incorporation rates into different native galactans or in various oligosaccharides. A highly efficient -(16) galactosyltransferase was detected when methyl 3-O-(-d-galactopyranosyl)--d-galactopyranoside was offered as acceptor. The substitution at the penultimate residue resulted in a branched trisaccharide as demonstrated by ¹H-NMR-spectroscopy and permethylation analysis of the reaction product. Comparable results were obtained with various oligosaccharides containing an internal galactose unit glycosidically linked 13. Attempts to separate and purify the various enzymes involved resulted in the isolation of a fraction which is able to transfer d-Gal exclusively to native galactan, but not to oligosaccharides. A further fraction was obtained from a different resin with activity for native galactan and 6-O-(-d-galactopyranosyl)-d-galactopyranose. but without any for methyl-3-O-(-d-galactopyranosyl)--d-galactopyranose. It is thus concluded that at least three different enzymes are involved in the biosynthesis of this snail galactan.Abbreviation Gal galactose - glc gas-liquid chromatography - Gro glycerol - tlc thin layer chromatography     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Asialo-α1-acid glycoprotein resialylated with 9-amino-5-N-acetyl-d-neuraminic acid is resistant towards bacterial,viral and mammalian sialidases

We demonstrate that 9-amino-NeuAc transferred to asialo-₁-acid glycoprotein resists cleavage by bacterial, viral and mammalian sialidases. This is the first synthetic sialic acid analogue, which can be activated and transferred to glycoprotein, but is not a sialidase (EC 3.2.1.18) substrate.Abbreviations HPLC high performance liquid chromatography - BSA bovine serum albumin - NeuAc N-acetyl-d-neuraminic acid, 5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid - 9-Amino-NeuAc 9-amino-5-N-acetyl-d-neuraminic acid, 5-acetamido-9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid - CMP-NeuAc cytidine-5-monophospho-N-acetyl-d-neuraminic acid - CMP-9-amino-NeuAc cytidine-5-monophospho-9-amino-5-N-acetyl-d-neuraminic acid - 9-azido-NeuAc 5-acetamido-9-azido-3,5,9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid. Enzymes EC 3.2.1.18 sialidase, acylneuraminylhydrolase - EC 2.4.99.1 Galß1-4GlcNAc a(2-6)-sialytransferase     

Asperula visianii,nova spec., undA. staliana (Rubiaceae), Endemiten der Inseln Mitteldalmatiens

Asperula visianii Korica is described as a new stenoendemic species from the small Central Dalmatian island of Svetac (near Vis). It differs in several morphological features (which remain constant in cultivation) and in its ecology from the closely relatedA. staliana Vis., endemic on the nearby island of Bievo.