Polar organizers mark division axis prior to preprophase band formation in mitosis of the hepaticReboulia hemisphaerica (Bryophyta)

Summary Changes in the pattern of microtubules during the cell cycle of the hepaticReboulia hemisphaerica (Bryophyta) were studied by indirect immunofluorescence using conventional and confocal laser scanning microscopy (CLSM). The first indication that a cell is preparing for division is fusiform shaping of the nucleus accompanied by the appearance of well-defined polar organizers (POs) at the future spindle poles. Microtubules emanating from the POs ensheath the nucleus and eventually develop into the half-spindles of mitosis. Some of the microtubules from each PO pass tangential to the nucleus and interact in the region of the future mitotic equator. A preprophase band (PPB) forms in this region later in prophase and coexists with the prophase spindle. Thus, the plane of division appears to be determined by interaction of opposing arrays of microtubules emanating from POs. Prometaphase is marked by disappearance of the POs, loss of astral microtubules, and conversion of the fusiform spindle of prophase to a truncated, barrel-shaped spindle more typical of higher plants. Restoration of cortical microtubules in daughter cell occurs on the cell side distal to the new cell plate, but nucleation of microtubules is associated with the nuclear envelope and not with organized POs. At the next division POs appear at opposite poles of preprophase nuclei with no evidence of division and migration that is characteristic of cells with centriolar centrosomes. These data lend additional support for the view that mitosis in hepatics is transitional between green algae and higher plants.Abbreviations AMS axial microtubule system - CLSM confocal laser scanning microscopy - MTOC microtubule organizing center - PO polar organizer - PPB preprophase band of microtubules - QMS quadripolar microtubule system - TEM transmission electron microscopy     

In vitro evaluation of dissolution behavior for a colon-specific drug delivery system (CODES) in multi-pH media using United States Pharmacopeia apparatus II and III

United States Pharmacopeia dissolution apparatus II (paddle) and III (reciprocating cylinder) coupled with automatic sampling devices and software were used to develop a testing procedure for acquiring release profiles of colon-specific drug delivery system (CODES^™) drug formulations in multi-pH media using acetaminophen (APAP) as a model drug. System suitability was examined. Several important instrument parameters and formulation variables were evaluated. Release profiles in artificial gastric fluid (pH 1.2), intestinal fluid (pH 6.8), and pH 5.0 buffer were determined. As expected, the percent release of APAP from coated core tablets was highly pH dependent. A release profile exhibiting a negligible release in pH 1.2 and 6.8 buffers followed by a rapid release in pH 5.0 buffer was established. The drug release in pH 5.0 buffer increased significantly with the increase in the dip or paddle speed but was inversely related to the screen mesh observed at lower dip speeds. It was interesting to note that there was a close similarity (f ₂=80.6) between the release profiles at dip speed 5 dpm and paddle speed 100 rpm. In addition, the release rate was reduced significantly with the increase in acid-soluble Eudragit E coating levels, but lactulose loading showed only a negligible effect. In conclusion, the established reciprocating cylinder method at lower agitation rates can give release profiles equivalent to those for the paddle procedure for CODES^™ drug pH-gradient release testing. Apparatus III was demonstrated to be more convenient and efficient than apparatus II by providing various programmable options in sampling times, agitation rates, and medium changes, which suggested that the apparatus II approach has better potential for in vitro evaluation of colon-specific drug delivery systems.     

Organization of cortical microtubules at the plasma membrane in Arabidopsis

 To understand the role of microtubules in the regulation of cell elongation, we characterized microtubule patterns in fass, a cell shape mutant of Arabidopsis thaliana (L.) Heynh. Examining microtubule patterns via immunocytochemistry, we found that fass cells were able to organize their microtubules into mitotic spindles and phragmoplasts. During interphase or preprophase, fass cells had cortical microtubules, verified by transmission electron microscopy, but these microtubules were not organized into the cortical array or preprophase band. Using chromatin condensation and tubulin localization on the nuclear envelope as preprophase stage markers, we found that although fass cells lacked the preprophase band and cortical array, their cell division cycle appeared normal. To pinpoint the defect in fass cells, we delineated the sequential events leading to cortical array formation in Arabidopsis cells and found that fass cells initiated and recolonized cortical microtubules in the same manner as wild-type cells, but failed to order them into the cortical array. Taken together, these results suggest fass cells are impaired in a component of the microtubule organizing center(s) required for the proper ordering of cortical microtubules at the plasma membrane. Received: 23 August 1996 / Accepted: 25 September 1996     

Cell-wall development in freeze-fixed pollen: Intine formation of Ledebouria socialis (Hyacinthaceae)

The structure and development of the inner pectocellulosic pollen wall, the intine, was re-examined using high-pressure freezing with subsequent freeze substitution in Ledebouria socialis Roth, a monocotyledonous angiosperm. The bilayered intine is formed immediately after differentiation of the endexine. Similar to somatic cell walls, intine matrix substances originate from the Golgi apparatus and leave the cytoplasm via exocytosis. Exintine development starts with the apposition of intine matrix substances to the inner polysaccharide layer of the endexine (termed inner endexine), leading to irregular cell-wall ingrowths. Subsequently the inner endexine becomes intensely infiltrated with intine matrix substances; this process is interpreted as transformation of the inner endexine into intine. Along the aperture region, cell-wall matrix substances are unevenly deposited to such an extent that more or less radially oriented tubules filled with cytoplasm remain within the growing exintine. These tubules subsequently become cut off from the microspore cytoplasm by selective membrane fusions, leading to the incorporation of ground cytoplasm and ribosomes into the exintine. Exintine formation is completed prior to the first mitotic division of the pollen grain whereas the endintine is formed as a homogeneous thin layer after mitosis. Both transformation of the inner endexine by infiltration and passive incorporation of cytoplasm and ribosomes into the exintine by membrane fusions are novel features and are only observed in optimally freeze-fixed, freeze-substituted samples; general aspects of ultrastructure preservation in high-pressure-frozen, freeze-substituted plant cells are discussed as well. Modifications of the Golgi apparatus and post-Golgi-apparatus structures during pollen wall development are correlated with increasing and decreasing polysaccharide exocytosis, respectively. These evenls strictly coincide with the formation of morphologically and chemically different pollen wall layers and therefore seem to reflect the different deposition patterns of the predominant cell-wall polysaccharides.Abbreviations ER endoplasmic reticulum - FS freeze substitution - HPF high-pressure freezing - MS microspore(s) - PATAg periodic acid-thiocarbohydrazine-silver proteinate - PGS post-Golgi-apparatus structures - UA-Pb uranyl acetatelead I am grateful to Dr. Martin Müller (Institut für Zellbiologie, ETH-Zürich) for the kind permission to use the high-pressure freezer and the freeze-substitution unit at his laboratory. I wish to thank Prof. M. Hesse, Mag. M.G. Schlag (Institut für Botanik, Universität Wien) and Dr. I. Lichtscheidl (Institut für Pflanzenphysiologie, Universität Wien) for helpfull discussions. Thanks are also due to A. Glaser and W. Urbancik for excellent technical assistence and to the Stadtgärtnerei Zürich for providing the plant material. This work was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung.     

A cytoskeletal basis for wood formation in angiosperm trees: the involvement of cortical microtubules

Rearrangements of cortical microtubules (CMTs) during the differentiation of axial secondary xylem elements within taproots and shoots of Aesculus hippocastanum L. (horse-chestnut) are described. A correlative approach was employed using indirect immunofluorescence microscopy of α-tubulin in 6- to 10-μm sections and transmission electron microscopy of ultrathin sections. All cell types – fibres, vessel elements and axial parenchyma – derive from fusiform cambial cells which contain randomly oriented CMTs. At the early stages of development, fibres and axial parenchyma cells possess helically arranged CMTs, which increase in number as secondary wall thickening proceeds and simple pits develop. In contrast, incipient vessel elements are distinguished by the marking out of sites of bordered pits; these sites first appear as microtubule-free regions within the reticulum of randomly oriented CMTs that characterises their precursor fusiform cambial cells. Subsequently, the ring of CMTs which develops at the periphery of the microtubule-free region decreases in diameter as the over-arching pit border is formed. Like bordered pits, large-diameter, non-bordered pits (contact pits) which develop between vessel elements and adjacent contact ray cells originate as microtubule-free regions and are also associated with development of a ring of CMTs at the periphery. In the case of contact pits, however, there is no reduction in the diameter of the CMT ring during pit development. Tertiary cell wall thickenings are also a feature of vessel elements and appear to form at sites where bands of laterally associated, transversely oriented CMTs, separated from each other by microtubule-free zones, are found. Later, these bands of CMTs become narrower, and separate into pairs of microtubule bundles located on each side of the developing wall thickening. Development of perforations between vessel elements is also associated with the presence of a ring of CMTs at their periphery. Received: 13 July 1998 / Accepted: 30 November 1998     

Ultrastructure and development during meiosis and the tetrad period of sporogenesis in the leptosporangiate fern Alsophila setosa (Cyatheaceae) compared with corresponding stages in Psilotum nudum (Psilotaceae)

The pre-meiotic, meiotic and tetrad stages of development in microsporangia of Alsophila setosa were studied with particular emphasis on the early establishment of patterning in the microspore wall and the subsequent development of the sporoderm. The data obtained were compared with corresponding ontogenetic stages of Psilotum nudum. Tapetal behaviour was also examined. During the tetrad period, only one layer, a thin undulating sheet, appeared alongside the plasma membrane of the tetraspores, and this was evidently formed on a pre-patterned structure – a fibrillar layer, corresponding to a kind of primexine matrix. The early free microspores had a wavy plasma membrane with a parallel, sinusoidal, thin initial sporoderm layer. The proximal apertural fold was observed to be an extended outgrowth of this initial spore envelope. Sporoderm ontogeny during the tetrad period in Alsophila and Psilotum show some common points, but also fundamental differences, mainly in the relative timing of events: in Alsophila the end of the tetrad period is the starting point for exospore development, whereas in Psilotum the exospore is already complete at this stage. Considerable differences were also observed in the tapetum of the two species.     

Functional states and fine structure of the contractile apparatus of the penis retractor muscle (PRM) of Helix pomatia L.

Summary The ultrastructure of the isolated glycerinated penis retractor muscle (PRM) of Helix pomatia was investigated. The diameter distributions of thick myofilaments from fibre cross sections in the relaxed, phasic contracted, tonic contracted, and in the catch states show that a characteristic filament spectrum is formed in the catch state and its preceding active state. The significant structural differences are discussed in relation to earlier hypotheses related to the catch state.I wish to thank Prof. Dr. Fr.-W. Schlote for helpful discussion. I also thank M. Jenninger and E. Grafahrend for their technical assistance.     

The effects of spermidine biosynthetic inhibitor methyl bis-(guanylhydrazone) on spore germination,growth and ethylene production in Alternaria consortiale

The spermidine synthesis inhibitor methyl bis-(guanylhydrazone) was found to reduce spore germination, hyphal and mycelial growth in Alternaria consortiale. The addition of spermidine to the culture medium resulted in a promotion of growth. Methyl bis-guanylhydrazone and spermidine did not change ethylene production.The data suggest that spermidine plays a role in the development of Alternaria consortiale independent of ethylene.Abbreviations MGBG methyl bis-(guanylhydrazone) - SPD spermidine - ACC 1-aminocyclopropane-1-carboxylic acid - PDA potato dextrose agar     

Ins(1,4,5)P3 receptor type 1 associates with AKAP9 (AKAP450 variant) and protein kinase A type IIβ in the Golgi apparatus in cerebellar granule cells

Background information. Interconnections between the Ca²⁺ and cAMP signalling pathways can determine the specificity and diversity of the cellular effects mediated by these second messengers. Most cAMP effects are mediated by PKA (protein kinase A), which is anchored close to its membranous substrates by AKAPs (A kinase‐anchoring proteins). In many cell types, the activation of InsP₃R (inositol 1,4,5‐trisphosphate receptor), an endoplasmic reticulum Ca²⁺ channel, is a key event of Ca²⁺ signalling. The phosphorylation of InsP₃R1 by PKA stimulates Ca²⁺ mobilization. This control is thought to be tight, involving the association of PKA with InsP₃R1. The InsP₃R1 isoform predominates in central nervous tissue and its concentration is highest in the cerebellar microsomes. We investigated the complex formed by InsP₃R1 and PKA in this fraction, vith a view to identifying its components and determining its distribution in the cerebellar cortex. Results. Immunoprecipitation experiments showed that InsP₃R1 associated with PKA type IIβ and AKAP450, the longer variant of AKAP9, in sheep cerebellar microsomes. The co‐purification of AKAP450 with InsP₃R1 on heparin‐agarose provided further evidence of the association of these proteins. Immunohistofluorescence experiments on slices of cerebellar cortex showed that AKAP450 was colocalized with InsP₃R1 and RIIβ (regulatory subunit of PKA IIβ) in granule cells, but not in Purkinje cells. AKAP450 was localized in the Golgi apparatus of these two cell types whereas InsP₃R1 was detected in this organelle only in granule cells. Conclusions. Taken together these results suggest that InsP₃R1 forms a complex with AKAP450 and PKAIIβ, localized in the Golgi apparatus of cerebellar granule cells. In contrast, the association of InsP₃R1 with PKA in Purkinje cells would require a different macromolecular complex.     

Binding of the Bovine and Porcine Pancreatic Secretory Trypsin Inhibitor (Kazal) To Human Leukocyte Elastase: A Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTT – K_a = 6.3 × 10⁴M^?1, δ5G° = -26.9kJ/mol, δH° = +11.7kJ/mol, and δS° = +1.3 × 10² entropy units; porcine PSTI –K_a = 7.0 × 10³M^?1,δG° = -21.5kJ/mol, δH° = +13.0kJ/mol, and δS° = +1.2 × 10² entropy units (values of K_a δG° and δS° were obtained at 21.0°C; values of δH° were temperature independent over the range (between 5.0°C and 45.0°C) explored). On increasing the pH from 4.5 to 9.5, values of K_a for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from ?7.0, in the free enzyme, to ?5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

柑橘全爪螨种群空间格局的地学统计学分析

应用地学统计学方法分析了柑橘园主要害螨柑橘全爪螨Panonychus citri（McGregor）种群的空间格局及其动态。结果表明,柑橘全爪螨种群具有空间相关性,变程介于1．10～21．0m,其半变异函数主要符合高斯模型,表现为聚集分布,其中3月、8月和9月的聚集强度较大;种群空间格局动态显示,4月、10月为该种群的两个发生高峰期,柑橘全爪螨种群数量快速上升扩散。地学统计学方法能够应用于柑橘全爪螨种群的空间格局分析,并有助于对该害螨进行发生预测与控制处理。     

黑脊倒刺鲃卵子发生中生殖质的产生

采用光学和电子显微镜对黑脊倒刺的卵原细胞和各期卵母细胞中生殖质的形成过程进行观察。仅在卵原细胞和Ⅱ时相卵中观察到了生殖质的产生过程 ,在Ⅲ、Ⅳ时相卵中未观察到生殖质的产生过程。生殖质来源于核仁。它从细胞核中释放出来 ,进入细胞质。卵原细胞和卵母细胞产生生殖质的方式不同。在卵原细胞的生殖质形成过程中 ,生殖质的前体物质先移到核膜内侧 ,核膜解体 ,形成囊泡 ,随后 ,在生殖质前体物质所在处的内侧重新形成核膜 ,这样 ,细胞核表面就形成一个凹陷 ,生殖质前体物质位于核表面的凹陷之中 ,就这样被隔离于细胞核之外而进入细胞质中 ,成为生殖质。生殖质形成后 ,在细胞质中与线粒体相结合。生殖质的这种形成方式与精原细胞中拟染色体的形成方式相似。而在卵母细胞的生殖质形成过程中 ,生殖质的前体物质移到核膜内侧之后 ,核膜并无囊泡化 ,核孔明显 ,生殖质的前体物质通过核孔离开细胞核 ,位于细胞核表面 ,形成生殖质。生殖质产生之后也与线粒体结合。以后 ,生殖质连同线粒体离开细胞核。生殖质最终与线粒体分离 ,分散于细胞质中。在Ⅲ时相卵中 ,生殖质细小 ;在Ⅳ时相卵中 ,几乎见不到生殖质     

Potential of antimicrobial treatment of linear low-density polyethylene with poly((tert-butyl-amino)-methyl-styrene) to reduce biofilm formation in the food industry

Antimicrobial surfaces are one approach to prevent biofilms in the food industry. The aim of this study was to investigate the effect of poly((tert-butyl-amino)-methyl-styrene) (poly(TBAMS)) incorporated into linear low-density polyethylene (LLDPE) on the formation of mono- and mixed-species biofilms. The biofilm on untreated and treated LLDPE was determined after 48 and 168 h. The comparison of the results indicated that the ability of Listeria monocytogenes to form biofilms was completely suppressed by poly(TBAMS) (Δ_168 h 3.2 log₁₀ cfu cm^?2) and colonization of Staphylococcus aureus and Escherichia coli was significantly delayed, but no effect on Pseudomonas fluorescens was observed. The results of dual-species biofilms showed complex interactions between the microorganisms, but comparable effects on the individual bacteria by poly(TBAMS) were identified. Antimicrobial treatment with poly(TBAMS) shows great potential to prevent biofilms on polymeric surfaces. However, a further development of the material is necessary to reduce the colonization of strong biofilm formers.     

Heavy metal-nucleotide interactions. 15. Reactions of calf thymus DNA with the electrophiles methylmercury(II) nitrate, cis-dichlorodiammineplatinum(II), and trans-dichlorodiammineplatinum(II) studied using Raman difference spectroscopy. Evidence for the formation of c-DNA upon metalation

This study details the reactions of the electrophiles CH3Hg(NO3), cis-[PtCl2(NH3)2] (cis-DDP) and trans-[PtCl2(NH3)2] (trans-DDP) with calf thymus DNA using Raman and Raman difference spectroscopy. The order of CH3Hg(II) binding to calf thymus DNA is G > T > C > A. The electrophilic attack of cis- and trans-DDP on calf thymus DNA produces different orders of binding: cis-DDP-G>C approximately AT, trans-DDP-G approximately C approximately AT. The reaction of CH3Hg(II) with DNA results in a decrease in the percentage of B-form DNA. whereas the reactions of cis- and trans-DDP with DNA decrease the percentage of B-DNA and cause the formation of C-DNA structure.     

Cambial reactivation in locally heated stems of the evergreen conifer Abies sachalinensis (Schmidt) Masters

A study was made of cambial activity, the localization of storage starch around the cambium, and the localization and occurrence of microtubules in cambial cells from dormancy to reactivation in locally heated (22–26 °C) stems of the evergreen conifer Abies sachalinensis. Heating induced localized reactivation of the cambium in the heated portions of the stem. Erect ray cambial cells resumed cell division 1 d prior to the reactivation of fusiform cambial cells and procumbent ray cambial cells. The re-initiation of the division of fusiform cambial cells occurred first on the phloem side. During the heat treatment, the amount of storage starch decreased in procumbent ray cambial cells and in the phloem parenchyma adjacent to the cambium but increased in fusiform cambial cells. Preprophase bands of microtubules, spindle microtubules and phragmoplast microtubules were observed both in erect ray cambial cells and in procumbent ray cambial cells. By contrast, no evidence of the presence of such preprophase bands of microtubules was detected in fusiform cambial cells. The results suggest that the localized heating of stems of evergreen conifers might provide a useful experimental model system for studies of the dynamics of cambial reactivation in intact trees. Received: 25 May 2000 / Accepted: 12 July 2000     

Spinning behaviour and morphology of the spinning glands in male and female Aposthonia ceylonica (Enderlein, 1912) (Embioptera: Oligotomidae)

Embioptera (webspinners) are unique among insects in that juvenile and adults of both sexes spin silk. They possess spinning apparatuses in the basitarsomeres of their prothoracic legs, which they use to build galleries as habitat and protection. Embioptera are primitively social and cooperate in building the galleries. They also show sexual dimorphism that comprises modifications of the mandibles in males, the winglessness of the females and differences in the morphology of the forelegs. In the present investigation we address the correlation of spinning behaviour and sexual dimorphism in the spinning apparatus of Aposthonia ceylonica (Enderlein, 1912). To analyse spinning behaviour we conducted video observations of Ap. ceylonica in artificial habitats. We observed females and males alone as well as female-male pairs to cover possible effects of interactions between sexes. The morphology of the spinning apparatus was analysed and reconstructed using high resolution X-ray computed tomography (SRμCT). The observations show that during trials of 24 h adult males and females produce similar amounts of silk per body weight, despite the fact that adult males do not feed, perhaps due to modifications of their mandibles related to courtship that interfere with feeding. Spinning glands in males are distinctly smaller than in females in absolute values, which reflect the general size difference in females and males. Despite their smaller body size, the volumes of reservoirs of spinning glands are larger in males in relative as well as in absolute values. Together with spinning behaviour and the amount of silk production, this indicates that males produce and store gland secretions in the large reservoirs prior to their final moult for later use.     

Polyribosome formation and poly(A)-containing RNA in embryos of the sand dollar,Dendraster excentricus

Summary The pattern of appearance of ribosomes, newly synthesized mRNA, and poly(A)-containing mRNA in polyribosomes has been examined in sand dollar embryos. From early blastula until shortly before hatching small polyribosomes engaged in histone synthesis predominate. At the time of hatching, when the rate of cell increase is maximal, the proportion of poly(A)-containing RNA in polyribosomes is low. After hatching a new class of large polyribosomes appears and the amount of poly(A)-containing polyribosomal RNA increases. Cordycepin, an inhibitor of RNA adenylylation, prevents the appearance of the large polyribosomes after hatching as well as the increase in poly(A)-containing polyribosomal RNA.