Cytosolic and cell-wall-bound acid invertases from leaves of Urtica dioica L.: a comparison

Two different forms of acid invertase (EC 3.2.1.26) were extracted from expanding leaves of the stinging nettle (Urtica dioica L.). One form was soluble and could be localized within the cytosol, whereas the other was ionically bound to the cell-wall and could not be detected in protoplasts. Both forms were purified, the latter to homogeneity. Western blotting with antibodies against the pure enzyme from cell walls was positive with the cell-wall enzyme but negative with the soluble form of acid invertase. Both forms are glycoproteins with identical molecular weights of 58 kDa. The K_m values for sucrose (raffinose) are 5 mM (4.8 mM) for the soluble and 1.2 mM (3.6 mM) for the cell-wall-bound enzyme. The pH optimum of the latter is slightly more acidic (4.5) than that of the soluble invertase (5.5). Both forms could easily be distinguished by their isoelectric points which were determined at pH 4.6 for the soluble and pH 9.3 for the wall-bound enzyme. When extraction and purification were carried out in the absence of protease inhibitors, both acid invertases showed microheterogeneity (multiple forms). However, with benzamidine and phenylmethylsulfonylfluoride as protease inhibitors each invertase produced only one protein band upon isoelectric focusing and gel electrophoresis, respectively.Abbreviations B benzamidine - Con A concanavalin A - FPLC fast protein liquid chromatography - IEF isoelectric focusing - kDa kilodalton - pI isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the Sonderforschungsbereich 137.     

Visualization of RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy

Freeze-etch electron microscopy of pure RecA protein aggregates, as well as of RecA protein complexes on single-stranded and double-stranded DNA formed with various nucleotides, has permitted a clearer discrimination between the two different helical polymers that this protein forms. Both are continuous, single-start, right-handed helices; however, the form observed when ATP or non-hydrolyzable ATP analogs are present has a pitch of 9.5 nm and a diameter of 10 nm, while the other form, observed in the absence of ATP or its analogs, or in the presence of ADP, has a pitch of 6 nm and a diameter of 12 nm. The former "long pitch" helix is found only when RecA protein is bound to DNA. The latter "short pitch" helix is also observed in pure RecA protein polymers (also termed rods) and in the needle-like paracrystals of RecA protein that form in the presence of magnesium or spermidine ions, representing bundles of rods closely packed in register. Addition of ATP or non-hydrolyzable ATP analogs in the absence of DNA dissociates the pure RecA protein crystals, as well as individual helical rods, into short curvilinear chains of attached monomers. These chains typically form closed, circular rings of 7(+/- 1) protein monomers, similar in construction to a single turn of the RecA protein helix, but significantly broader in diameter. The role of ATP in interconverting the various polymeric forms of RecA protein is discussed within the context that ATP functions as a reversible allosteric effector of RecA protein, much as it mediates reversible conformational changes in other vectoral motor proteins such as myosin, dynein, kinesin and the 70,000 Mr "heat shock" ATPases. We discuss how cyclic conversions back and forth between the short- and long-pitch conformations of RecA protein could mediate in reversible single-stranded and double-stranded DNA interactions during the search for homology.     

Ultrastructure and early development of the pore plate sensilla ofGymnomerus laevipes (Shuckard) (Vespoidea,Eumenidae)

Summary The oval pore plates (approx. 17 m long) are separated from the antennal cuticle by a furrow, the inner wall of which is flexible. The thin perforated plates are strengthened by an encircling and a middle ledge, the latter of which branches into about 100 almost parallel rims. Each pore plate is innervated by about 20 sense cells. The dendrites fork into numerous branches occupying the outer receptor lymph cavity below the perforated plate. Each pore plate is associated with one thecogen cell, two trichogen cells, one tormogen cell and one envelope cell 4. A so-called additional cell surrounds the sensillum in the imaginal stage. The envelope cells in the later of the two pupal stages examined, have reached an arrangement which immediately precedes the secretion of the cuticulin layer. The surface of the duplicate trichogen cells is almost equal in area to the completed perforated plate. A dendritic sheath, entirely reduced in the imago, protrudes into the exuvial space, where it encloses a single dendrite.In the younger pupal stage the Sensillenanlage forms a crater, whereby envelope cell 4 overtops the other envelope cells. The distal ends of the trichogen cells are divided into several appendages that form the bottom of the crater.     

Ultrastructure and localization of wall polymers during regeneration and reversion of protoplasts ofSchizophyllum commune

Summary Cell-wall regeneration and reversion of protoplasts ofSchizophyllum commune were investigated using electron microscopic methods and X-ray diffraction.After 3 hours of regeneration protoplasts have formed a loosely organized wall which does not react with Thiéry's stain for periodic acid sensitive carbohydrates. This wall largely consists of chitin microfibrils which are adpressed to the plasmalemma and which are covered by loose aggregates of alkali-soluble S-glucan (-1,3-glucan). Both components are microcrystalline, at least partly. Walls formed in the presence of polyoxin D only consist of thick loose fibers of S-glucan.From 3 hours onward the inner chitin microfibrils of the wall of the primary cells become embedded in alkali-insoluble material that stains heavily with the Thiéry reagent and probably is similar to the R-glucan of the mature wall (i.e., -1,3--1,6-glucan). The outer chitin microfibrils remain free of this matrix and are covered by S-glucan only.Bud-like structures that arise have the same wall architecture as the primary cells,i.e., only the inner chitin microfibrils are embedded in R-glucan and the S-glucan forms a fluffy coat. The walls of hyphal tubes that arise are distinct, however, in that all chitin microfibrils are embedded in R-glucan and the S-glucan forms a compact coat.Cytoplasmic vesicles are sparse in primary cells except at the sites of emergence of budlike structures and hyphae. They continue to be present in the apex of growing hyphae.     

Labor and the materiality of the sign: Beyond dualist theories of culture [1]

Conclusion What I hope to have shown is that the labor theory of culture developed here has much to contribute to an understanding of culture as meaning, and that there need not be a contradiction between pragmatic and symbolic approaches to culture as a result. It may appear that this reconciliation depends upon a break with the economic theory of labor as instrumental action, which is implicit in the very operation of capitalist society, and therefore impossible to transcend at will. No doubt the lived weight of the mental/manual division of labor in our society will continue to exercise an inordinate influence on popular opinion and common sense for some time to come. But I have nevertheless argued that what is truly impossible are the theories of meaning which result from this division of labor, whose attempts to definitively separate meaning from practice could only be realized by abolishing both. We have already seen that the notion of culture as pure reason has never been anything more than an ideology. By the same token, no specimen of completely instrumentalized labor has ever been discovered, and would not even be worth exploiting if it did exist: in any physical work, even the most degraded and mechanical, there exists a minimum of technical qualification, that is, a minimum of creative intellectual activity [126].The integrity of labor is therefore at least as demonstrable as its division, and is finally a more primordial and insuperable fact. Despite the way it has been stigmatized by dualist thought, labor continues to maintain our world, not just in a material sense, but also in a meaningful sense, as it has always done. This is something that we can point to in the here-and-now as the basis for an alternative cultural theory, one which is both more realistic and more optimistic than dualism. For in revindicating this neglected reality of labor, our concept of culture is also affirmed in new and important ways.Peter Gose is Professor of Anthropology, Department of Anthropology, University of Lethbridge, Canada.     

On progressive evolution and competitive extinction

Synopsis Extinction may be caused by abiotic and biotic factors in the environment. In the present essay the focus is on extinction caused by biotic factors, and the problem is discussed with reference to two theories of evolution, micromutation and macromutation. According to the former, evolution is adaptive, implying that the only kind of extinction is pseudoextinction or gradual extinction, reflecting the mechanism through which evolution advances in small steps. The macromutation theory asserts that two kinds of evolution exist, divergent (adaptive) and progressive. The latter steadily gives rise to more dominant forms in the phylogenetic hierarchy, and whenever these have a chance to compete with inferior organisms, they will cause the extermination of the latter. However, primitive forms may survive in isolation. The predictions of the macromutation theory are shown to be in better agreement with the fossil record and other observations than are those of the micromutation theory.Invited Editorial     

Adhesion by Paired Pectoral and Pelvic Fins in a Mountain-Stream Catfish, Pseudocheneis sulcatus (Sisoridae)

We describe the adhesive nature of the pectoral and pelvic fins in the catfish Pseudocheneis sulcatus, as examined by scanning electron microscopy. The outer rays of these fins are modified into structures that bear prominent transverse ridges and grooves. The outer epidermal cells of the ridges are thrown into elongated spines. Mucous pores (openings of mucous glands) are frequently present (100m apart) in the epidermis of the ridges and show entangled mucus droplets. In the pectoral fins, they are present towards the contour areas of the outer rays, but they are absent in the pelvic fins. In the latter, mucous pores are present near the base of the ridges (distal to the inner rays). Spines as well as mucous pores are absent in the cells that line the groove between two adjacent ridges. We suggest that in this species adhesion is effected by suction pressure generated by the musculature attached to the grooves and ridges, and that mucus and the spines aid in this process.     

Microanatomy of the subumbrellar motor innervation inAglantha digitale (Hydromedusae: Trachylina)

Summary The hydrozoan medusaAglantha digitale (Müller 1776) has eight syncytical giant motor axons, up to 40 m in diameter, running from the margin, up the inside of the bell towards the apex. Giant motor axons injected with Lucifer Yellow CH are connected with lateral neurons running circumferentially across the subumbrellar muscle. These processes fill with the dye. Bundles of 20 to 50 small dye-coupled neurons extend circumferentially along the margin for up to 0.85mm. Giant motor axons injected with horseradish peroxidase divide into a few short branches on entering the inner nerve ring. Here the giant motor axon forms both chemical synapses and gap junctions with neurons that also send their axons into the inner nerve ring. In this region the inner and outer nerve ringe are connected by axons passing through openings in the intervening mesoglea.     

A conceptual model of ecosystem restoration triage based on experiences from three remote oceanic islands

A conceptual model, that illustrates restoration, ecological landscaping, rehabilitation and regreening, is developed. It considers biocentric, historical, aesthetic and engineering aspects. The term ecosystem restoration triage is used because the first step is to decide whether to do nothing (because, on the one hand, the system is too degraded to warrant restoration, or, on the other, because biological integrity is relatively intact and therefore either none, or minimal, restoration is required) or to do something (because restoration is worthwhile, urgent and feasible). This approach hinges on the definition that restoration in the strictist sense is a biocentric activity that returns the original compositional, structural and functional diversity, along with its dynamics and natural evolutionary potential. Original is a difficult qualifier as it depends on just how far back in time we go. Where human values are involved, this is not restoration in the pure sense of restoring ecological integrity, but is ecological landscaping, rehabilitation or regreening. Experience from three remote oceanic islands [Easter Island, Cousine Island (Seychelles), Marion Island (Sub-Antarctic)] and which represent near extremes of this model are used to illustrate it.     

蚤类感觉板的细微结构

本文利用扫描电镜观察了4科18属26种蚤的感觉板.通过对其内、外表面及横断面细微结构的详细观察,发现前人称之为杯陷的结构,实为一中空的圆丘,分内、外两层,各具若干小室,作者称其为感觉室.该室在感觉板上具有一定的分布型.感觉毛着生于内室底部,通过中央孔及外室顶孔向外伸出.蚤类成虫具有坚韧的外骨骼,因此,在制备扫描电镜标本时可不经特殊处理直接进行观察.此外,对感觉室的功能及在分类上的意义进行了讨论.     

Characterization of a chloroplast inner envelope P-ATPase proton pump

P-ATPases such as the plasma membrane proton pump are known to generate a phosphorylated intermediate as a step in their reaction mechanism; phosphoenzyme formation is a basis for classification of an ATPase as a member of this subfamily of ion pumps. The chloroplast inner envelope is known to contain a H+-ATPase which acts to maintain an alkaline stroma and, thus, optimal photosynthesis. Our characterization of this chloroplast envelope proton pump described in this report focused on determining whether purified chloroplast inner envelope membrane protein preparations containing this ATPase form a phosphorylated intermediate. Incubation of envelope membranes with [-³²P]ATP documented the formation of P-type ATPase phosphoenzyme intermediates by these membrane protein preparations. Our work cannot discount the possibility that more than one chloroplast inner envelope ATPase contributes to this phosphoenzyme formation. However, the kinetics of this phosphoenzyme formation, along with the sensitivity of phosphoenzyme formation to inhibitors and other assay conditions suggested that one of the envelope membrane proteins which is covalently radiolabeled by [-³²P]ATP is a P-type H+-ATPase. Autoradiography of chloroplast envelope membrane proteins size fractionated on lithium dodecyl sulfate-PAGE indicated that the phosphoenzyme intermediate corresponds to a 103 kDa polypeptide. P-type proton pumps are known to be comprised of a single type of 100 kDa subunit. Experimental evidence presented in this report is consistent with the classification of a chloroplast inner envelope H+-ATPase as a P-type proton pump.     

Seed development and function inArtabotrys hexapetalus (Annonaceae)

Until now seeds ofAnnonaceae were characterized as mesotestal only. The seed ofArtabotrys hexapetalus, however, is meso- and endotestal. An outer mechanical layer which surrounds the seed as a lignified fibrous tissue is derived from the mesotesta. A complex inner mechanical layer develops from a partially multi-layered endotesta built up by crystal-containing stone cells. The multi-layered endotesta forms a prominent seed plug in the micropylar region which is prolonged along both sides of the perichalaza as inner walls. The endotesta is one-layered on the sides of the seed and participates in rumination. In addition the endotesta may serve for deposition of end-products of metabolism. The complex growing process of the perichalaza and its surrounding tissues is described in detail. The perichalazal pad of tanniferous cells forms an U-shaped septum, in conjunction with the endotesta, dividing the seed into two chambers. During seed development it functions as transmitter of nutrients from the outer chamber filled with starch grains to the nucellus, endosperm and embryo contained in the inner one. During germination this pad probably serves for the uptake of water. — At the initial phases of germination the seed dehisces into two valves along the raphal and antiraphal side. Later on an additional parenchymatous operculum covering the seed apex disintegrates and the endotestal plug fixed by its two prolongations splits along a preformed fracture line into two parts to release the seedling. Rudiments of an aril are recognizable in young seeds only. — The data obtained fromArtabotrys hexapetalus are discussed and compared with published information on seeds in other annonaceous taxa. For systematic considerations the necessity to define the origin of the annonaceous seed plug from one or the other integument is emphasized as it may prove to be an important differential character withinAnnonaceae.     

A possible localization of α-glycerophosphate dehydrogenase to the inner boundary membrane of mitochondria in livers from rats fed with ethanol

Summary The morphology and respiration of liver mitochondria were studied in rats fed with ethanol for eight months. Morphometric analysis shows an increase of the volume fraction of mitochondria, a decrease of the density of crista membranes but the density of surface area remained unaltered in the ethanol treated rats as compared to the controls. The ethanol treatment caused a reduced capacity of the mitochondria to oxidize succinate. The capacity to oxidize -glycerophosphate remained unchanged.As it is known that succinate dehydrogenase as well as -glycerophosphate dehydrogenase are bound to the inner mitochondrial membrane, different localization is proposed of the two enzymes in this membrane. Thus, there is a good correlation between the reduction of inner mitochondrial membrane and succinate oxidation. As the activity of -glycerophosphate dehydrogenase remained unchanged, this enzyme should be localized to that part of the inner mitochondrial membrane, which is called inner boundary membrane, and which is not altered by the ethanol treatment.The work is a part of investigations made possible by financial support from the Swedish Medical Research Council, Project No. B71-12Y-2364-04.     

Differentiation of the thermo-/hygrosensitive (no-pore) sensilla on the antenna of Antheraea pernyi (Lepidoptera,Saturniidae): a study of cryofixed material

Summary The morphogenesis of the thermo- and hygro-sensitive sensilla styloconica of Antheraea pernyi was studied, exclusively by cryomethods, during the second half of pupal development. The three major processes taking place during this period are (1) the differentiation of the dendritic outer segments of the sensory cells, especially of the lamellated type-2 receptor, (2) the formation of the receptor-lymph cavities, (3) the formation of tubular structures of unknown function in the inner receptor-lymph cavity, and (4) the elongation of the dendrite sheath. The formation of lamellae in the type-2 dendritic outer segment is achieved by the enfolding of its originally cylindrical cytoplasmic membrane. Autocellular junctions, previously described in the sensilla of adult animals, are found to join the forming lamellae. Close similarities between the junctions and smooth septate junctions are demonstrated. Both the extensive inner and outer receptor-lymph cavities are formed by invagination and folding of the apical cytoplasmic membranes of the three enveloping cells. Formation starts at the most apical projection of the cells and proceeds in a proximal direction. Up to 4-m-long tubular structures appear, exclusively in developmental stages, in the inner receptor-lymph cavity. They are composed of plasma membranes whose inner surface is studded with regularly spaced electron-dense particles. Contacts with the cytoplasmic membrane of the innermost enveloping cell demonstrate that the structures are composed of lipid membranes. During elongation of the dendrite sheath, which in these sensilla is apically attached to the hair wall, an 2-m-long growth-zone is observed at its proximal end. By addition of sheath-forming material to the growth-zone, the latter continuously moves proximally until the sheath is completed.     

Insertion of bitopic membrane proteins into the inner membrane of mitochondria involves an export step from the matrix

The mitochondrial inner membrane contains a large number of polytopic proteins that are derived from prokaryotic ancestors of mitochondria. Little is known about the intramitochondrial sorting of these proteins. We chose two proteins of known topology as examples to study the pathway of insertion into the inner membrane; Mrs2 and Yta10 are bitopic proteins that expose negatively charged loops of different complexity into the intermembrane space. Here we show that both Mrs2 and Yta10 transiently accumulate as sorting intermediates in the matrix before they integrate into the inner membrane. The sorting pathway of both proteins can be separated into two sequential reactions: (i) import into the matrix and (ii) insertion from the matrix into the inner membrane. The latter process was found to depend on the membrane potential and, in this respect, is similar to the insertion of membrane proteins in bacteria. A comparison of the charge distribution of intermembrane space loops in a variety of mitochondrial inner membrane proteins suggests that this mode of "conservative sorting" might be the typical insertion route for polytopic inner membrane proteins that originated from bacterial ancestors.     

Structure and postnatal development of photoreceptors and their synapses in the retina of the tree shrew (Tupaia belangen)

Summary The all cone retina of the tree shrew (Tupaia belangeri) was examined in the adult and early postnatal stages by light and electron microscopy. Rods are not as rare as previously thought, but make up about 4% of the photoreceptors. They are relatively short and narrow cells, which stain (toluidine blue) more intensively and lie more proximal than cones. Among the cones three morphological varieties could be distinguished. Most cones stain lightly but have a light or a dark giant mitochondrion in their inner segment; a third type stains darker but occurs only rarely. All cones possess extensive radial processes (lateral fins) around the basal part of their inner segments. Such fins are well known from reptiles and birds, but have only once been described in a mammal (gray squirrel).The maturation of the retina in Tupaia belangeri proceeds centrifugally, i.e., from the vitreal to the scleral side, as in most mammals. A few synapses are already present at birth in the outer and inner plexiform layers, but seem to be more advanced in the latter. Such early synapses are small and have only few synaptic vesicles; they appear almost mature by day 14. The light-sensitive outer segments develop last. The first disks are seen by day 10, but regular membrane stacks are only present by day 18. Thus, it seems that the retina is functional when the young first open their eyes, which occurs around day 18.     

Morphogenetic processes inAttheya decora (Bacillariophyceae,Biddulphiineae)

The valva of the diatomAttheya decora is formed within a silica deposition vesicle which enlarges centrifugally by the fusion of small vesicles. The silica deposition vesicle can already be seen when the spindle has not yet disappeared completely. Valva formation seems to begin with the shaping of an organic matrix within a silica deposition vesicle. Later, this material silicifies. The complicated shape of the labiate process is preformed by the silica deposition vesicle, the inner membrane of which is associated with electron dense material on both faces. The horns are formed when the expanding silica deposition vesicle has reached the cell corners. They are elaborated without participation of microtubules. Swelling of local depositions of polysaccharides seems to provide the forces that spread the silicified horns during daughter cell separation and to cause the local spontaneous plasmolyses under the valva and along the cell flanks in the region of the intercalary bands. The inner organic wall layers and the organic continuations of the intercalary bands are formed on the surface of the plasmalemma; each of the continuations is produced simultaneously with the intercalary band belonging to it and becomes attached to the latter when the silica deposition vesicle opens.     

Ultrahistochemical study on the ruthenium red surface staining

Summary The cell coat picture effect which is usually obtained with the conventional RR method, that is with the RR/OsO₄ coupled reaction, is investigated. In this first paper, each of conceivable events which might take place between RR, OsO₄ and cell surface membrane is discussed or studied. Various tests are carried out on ascites Ehrlich carcinoma cells and Zajdela ascites hepatoma cells treated with numerous chemical reagents, as also on a few pure proteins. The set of data supports the concept that the staining pattern is due to the combination in surface membranes of RR with a colloidal-like form of OsO₂. The latter might occur during the formation of stable cyclic osmic acid diesters between OsO₄ and membrane unsaturated lipids. A possibility by which the resulting marker is though also to be in a colloidal-like state is put forward. A next report will deal with this problem.     

T.H. Morgan,neither an epistemological empiricist nor a “Methodological” empiricist

T. H. Morgan (1866–1945), the founder of the Drosophila research group in genetics that established the chromosome theory of Mendelian inheritance, has been described as a radical empiricist in the historical literature. His empiricism, furthermore, is supposed to have prejudiced him against certain scientific conclusions. This paper aims to show two things: first, that the sense in which the term empiricism has been used by scholars is too weak to be illuminating. It is necessary to distinguish between empiricism as an epistemological position and the so-called methodological empiricism. I will argue that the way the latter has been presented cannot distinguish an empiricist methodology from a non-empiricist one. Second, I will show that T. H. Morgan was not an epistemological empiricist as this term is usually defined in philosophy. The reason is that he believed in the existence of genes as material entities when they were unobservable entities when they were unobservable entities introduced to account for the phenotypic ratios found in breeding experiments. These two points, of course, are interrelated. If we were to water down the meaning of empiricis, perhaps we could call Morgan an empiricist. But then we would also fail to distinguish empiricism from realism.     

Mitochondrial protein import

Most polypeptides of mitochondria are imported from the cytosol. Precursor proteins contain targeting and sorting information, often in the form of amino-terminal presequences. Precursors first bind to receptors in the outer membrane. Two putative import receptors have been identified: a 19-kilodalton protein (MOM19) inNeurospora mitochondria, and a 70-kilodalton protein (MAS70) in yeast. Some precursors integrate directly into the outer membrane, but the majority are translocated through one or both membranes. This process requires an electrochemical potential across the inner membrane. Import appears to occur through a hydrophilic pore, although the inner and outer membranes may contain functionally separate translocation machineries. In yeast, a 42-kilodalton protein (ISP42) probably forms part of the outer membrane channel. After import, precursors interact with chaperonin ATPases in the matrix. Presequences then are removed by the matrix protease. Finally, some proteins are retranslocated across the inner membrane to the intermembrane space.