荧光能量转移和偏振研究小牛胸腺DNA与组蛋白在高低离子盐溶液中的离合

本文研究小牛胸腺DNA和组蛋白在体外低、高离子强度盐溶液中的动态缔合与解离。 实验结果是低离子溶液中荧光给体DANsyl-Cl-组蛋白在发射峰位上荧光强度降低,荧光受体吖啶橙-DNA在发射峰位上的荧光强度增高。此两峰位上荧光受体的荧光增量比值是2.7/1(大于1),有能量转移发生,DNA和组蛋白缔合。高离子溶液中两峰位上受体的荧光增量比值降到1.6/1,能量转移减少,DNA和组蛋白解离。 低离子溶液中所得的吖啶橙-DNA的荧光偏振度P值小而高离子溶液中的P值大。说明解离的DNA硷基排列比缔合的DNA硷基排列有序程度强,进一步证明低离子溶液中DNA和组蛋白是缔合的,而高离子溶液中它们是解离的。     

AN ANALYSIS OF DESMOSOME SHAPE, SIZE, AND ORIENTATION BY THE USE OF HISTOMETRIC AND DENSITOMETRIC METHODS WITH ELECTRON MICROSCOPY

The probable shape, size, and orientation of desmosomes of the cells comprising the secretory tubules in rat submaxillary gland was determined by statistical and algebraic methods applied to electron micrographs. It was concluded that these desmosomes are discrete ellipsoidal discs whose principal axes are in the order of 4100 and 2500 angstrom units, and that they are preferentially oriented with their long axis more or less parallel to the base-apex axis of the cell. Densitometric interpretation agrees with the statistically based reconstruction of desmosomal shape. By densitometric analysis it was also determined that the peak to peak distances between layers within these desmosomes are in essential agreement with other reported findings. The approach described may have general applications to problems in the analysis of submicroscopic morphology.     

Genome Mining in Sorangium cellulosum So ce56: IDENTIFICATION AND CHARACTERIZATION OF THE HOMOLOGOUS ELECTRON TRANSFER PROTEINS OF A MYXOBACTERIAL CYTOCHROME P450*

Myxobacteria, especially members of the genus Sorangium, are known for their biotechnological potential as producers of pharmaceutically valuable secondary metabolites. The biosynthesis of several of those myxobacterial compounds includes cytochrome P450 activity. Although class I cytochrome P450 enzymes occur wide-spread in bacteria and rely on ferredoxins and ferredoxin reductases as essential electron mediators, the study of these proteins is often neglected. Therefore, we decided to search in the Sorangium cellulosum So ce56 genome for putative interaction partners of cytochromes P450. In this work we report the investigation of eight myxobacterial ferredoxins and two ferredoxin reductases with respect to their activity in cytochrome P450 systems. Intriguingly, we found not only one, but two ferredoxins whose ability to sustain an endogenous So ce56 cytochrome P450 was demonstrated by CYP260A1-dependent conversion of nootkatone. Moreover, we could demonstrate that the two ferredoxins were able to receive electrons from both ferredoxin reductases. These findings indicate that S. cellulosum can alternate between different electron transport pathways to sustain cytochrome P450 activity.The cytochrome P450 (CYP)² enzymes constitute a superfamily of external monooxygenases. The catalytic versatility of the family members explains their involvement in such diverse biological processes as biosynthesis of steroid hormones, carbon source assimilation, and metabolism of xenobiotics. In addition, cytochrome P450 enzymes have been reported to be involved in the biosynthesis of many pharmaceutically interesting secondary metabolites from a variety of microorganisms (1–4). Cytochromes P450 are usually dependent on an external electron donor. With respect to their electron transport system they can be divided into several classes, with class I (the mitochondrial/bacterial cytochrome P450 systems) being the predominant form in prokaryotes (5). In this system the electrons required for the enzymatic reaction originate from NAD(P)H and are delivered to the cytochrome P450 via a ferredoxin reductase and a ferredoxin. In a number of examples, the heterologous reconstitution of the electron transfer chain has been shown to be ineffective, if possible at all (5). Thus, it is desirable to identify the natural redox partners, especially if genomic sequence information is available. However, even then the identification of the correct interaction partners remains challenging because the encoding genes are frequently located at genomic loci distant to the cytochrome P450 genes (6, 7). Interestingly, members of both the [2Fe-2S] and the non-[2Fe-2S] ferredoxins have been reported to sustain cytochrome P450 catalyzed reactions. The latter group is further subdivided into mono- and dicluster ferredoxins (i.e. the [3Fe-4S] or [4Fe-4S] and the [3Fe-4S] + [4Fe-4S] or [4Fe-4S] + [4Fe-4S] ferredoxins). Remarkably, cytochrome P450 systems depending on non-[2Fe-2S] ferredoxins have been found exclusively in bacteria to date (8, 9).To fulfill the role as electron mediator, the ferredoxin component of any given cytochrome P450 system has to be reduced. This reduction is achieved by a ferredoxin reductase, which in turn takes up electrons from NAD(P)H. The ferredoxin reductase is often the least characterized constituent of the cytochrome P450 system because these flavoproteins may be unstable (i.e. easily lose their cofactor) and usually show a relatively low level of expression (10).Sorangium cellulosum So ce56 is a genome-sequenced myxobacterial model strain. Because of their biotechnological potential as producers of secondary metabolites, the myxobacteria attract attention from both the academic community and the pharmaceutical industry. To date, more than 100 new basic structures and some 500 derivatives have been reported (11), with almost half of the newly discovered natural products being isolated from the genus Sorangium (11, 12). The potent anti-cancer agent epothilone, for example, was discovered from S. cellulosum So ce90 (13, 14). Epothilone is one of so far seven known myxobacterial compounds, the biosynthesis of which involves cytochromes P450 (15). Besides the epothilones, these are the antifungal leupyrrins (16) and the cytotoxic spirangienes (17) (also from S. cellulosum), the antibiotic myxovirescin from Myxococcus (18), the electron transport inhibitor stigmatellin (19) and the antibiotic aurafuron (20) from Stigmatella aurantiaca, and the antifungal ajudazols from Chondromyces crocatus (21).The recently genome-sequenced myxobacterium S. cellulosum So ce56 (12) shows great potential for biotechnological applications, as judged on the basis of its capacity for the production of secondary metabolites. Three biologically active compounds have been described so far, namely the fungicidal chivosazoles, the macrolide antibiotic etnangien, and the iron chelator myxochelin (12). Moreover, the bioinformatic analysis of the So ce56 genome has revealed numerous biosynthetic gene clusters of yet unknown function (11, 12). With a size of more than 13 Mbp, the genome of S. cellulosum So ce56 is to date the largest sequenced prokaryotic genome (12). It has been shown to harbor 21 cytochrome P450 genes. In light of the significance of S. cellulosum as a viable source of bioactive secondary metabolites (14) and the role of cytochromes P450 in the synthesis of natural products (2), it is of great interest to elucidate the function of these enzymes.Therefore, the investigation of the S. cellulosum So ce56 cytochrome P450 systems opens a fascinating field not only with regard to basic research but also to exploit the biotechnological potential of this model strain. To achieve this goal it is important to provide a functional electron transport chain. Thus, the main objective of this work was to identify a myxobacterial ferredoxin/ferredoxin reductase couple able to support reactions catalyzed by S. cellulosum So ce56 cytochromes P450.     

ZONE BEHAVIOR OF ENZYMES : ILLUSTRATED BY THE EFFECT OF DISSOCIATION CONSTANT AND DILUTION ON THE SYSTEM CHOLINESTERASE-PHYSOSTIGMINE

1. The kinetics of the reversible combination of one enzyme center with one molecule of a substrate or inhibitor is treated as a true bimolecular instead of a pseudomonomolecular reaction. The general equations describing such a reaction are presented and analyzed algebraically and graphically. 2. A new term, "specific concentration," is introduced to denote the concentration of reactants in units equal to the dissociation constant. Its use makes the kinetic equations universally applicable to all reversible systems of the given type. 3. It is shown that such a system exhibits three "zones" of behavior. Each zone is characterized and shown to exhibit significant differences in the function relating the concentrations of the components of the system at equilibrium. The zone boundaries are rigorously defined in terms of the specific enzyme concentration, for the mathematical error tolerable with a given experimental accuracy; and approximate boundaries for practical use are proposed. 4. The classical treatment of enzyme kinetics is shown to be a limiting case valid only for low specific enzyme concentrations (zone A) and to be inapplicable in a number of systems whose dissociation constants are very small or whose molar enzyme concentrations are very great, and in which, therefore, the specific enzyme concentrations are large. See Table I for a summary of zone differences. 5. In an enzyme system containing substrate or inhibitor, dilution before determination of reaction velocities is shown to be a crucial operation, entailing large changes in the fraction of enzyme in the form of a complex. The changes in fractional activity or inhibition with dilution are shown to be a function of specific enzyme concentration, the dilution factor, and the fraction of enzyme initially in the form of complex. Equations are given permitting the calculation of the state of the system at any concentration. The errors introduced into physiological work by failure to take the dilution effect into account are pointed out. 6. Experimental data are presented showing that the system composed of serum cholinesterase and physostigmine behaves as predicted by the dilution effect equations. 7. Two other conclusions of practical pharmacological importance are drawn from the theory of zone behavior: (a) The finding that a biological response is a linear function of the dose of a drug does not necessarily mean that the reaction is irreversible, but only that if reversible, the reactant with which the drug combines has a high specific concentration. (b) If a tissue enzyme has a high specific concentration, all reversible inhibitors will be equally potent in combining with it, regardless of their relative potency in dilute systems; provided only that their dissociation constants are within certain broad limits. 8. It is shown how the type of analysis here applied to bimolecular reactions can be applied in toto to systems of the type E + nX ⇋ EX_n, where n molecules of substrate or inhibitor unite with one enzyme center. The zone boundaries and the magnitude of the dilution effect change with n, but the general characteristics of the zones are the same for all values of n. 9. Since the analysis is based only on mass law assumptions, it is applicable to any system that is formally analogous to the one here treated.     

LIGHT AND ELECTRON MICROSCOPY OF GRAZING BY POTERIOOCHROMONAS MALHAMENSIS (CHRYSOPHYCEAE) ON A RANGE OF PHYTOPLANKTON TAXA1

Grazing of fluorescent latex beads, bacteria, and various species of phytoplankton by Poterioochromonas malhamensis (Pringsheim) Peterfi (about 8.0 μm in diameter) was surveyed. The alga ingested fluorescent beads and various live or killed and nomnotile or motile organisms including bacteria, blue-green algae, green algae, diatoms, and chrysomonads. The size range of grazed prey was from 0.1 to 6.0 μm for latex beads and from 1.0 μm (bacteria) to about 21 μm (Carteria inverse) for organisms. As many as 17 latex beads (2.0 μm) or more than 10 Microcystis cells (5–6 μm) were ingested by a single P. malhamensis cell. Following such grazing, the cell increased in volume by up to about 30-fold. The range of cell volume of ingested prey was from 0.52 μm³ (bacteria) to about 3178 μm³(Carteria inversa). This study demonstrates for the first time that P. malhamensis is capable of grazing algae 2–3 times larger in diameter than its own cell and of grazing intact motile algae. Poterioochromonas malhamensis is an omnivorous grazer. Food vacuole formation and digestion processes were examined. The membrane that was derived from the plasma membrane and surrounded the prey disappeared sometime after ingestion. The food vacuole was then formed by successive fusion of numerous homogeneous vesicles accumulated around the prey. The prey was enclosed in a single membrane-bound food vacuole and then digested.     

Proteome of Acidic Phospholipid-binding Proteins: SPATIAL AND TEMPORAL REGULATION OF CORONIN 1A BY PHOSPHOINOSITIDES*

Reversible interactions between acidic phospholipids in the cellular membrane and proteins in the cytosol play fundamental roles in a wide variety of physiological events. Here, we present a novel approach to the identification of acidic phospholipid-binding proteins using nano-liquid chromatography-tandem mass spectrometry. We found more than 400 proteins, including proteins with previously known acidic phospholipid-binding properties, and confirmed that several candidates, such as Coronin 1A, mDia1 (Diaphanous-related formin-1), PIR121/CYFIP2, EB2 (end plus binding protein-2), KIF21A (kinesin family member 21A), eEF1A1 (translation elongation factor 1α1), and TRIM2, directly bind to acidic phospholipids. Among such novel proteins, we provide evidence that Coronin 1A activity, which disassembles Arp2/3-containing actin filament branches, is spatially and temporally regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Whereas Coronin 1A co-localizes with PI(4,5)P₂ at the plasma membrane in resting cells, it is dissociated from the plasma membrane during lamellipodia formation where the PI(4,5)P₂ signal is significantly reduced. Our in vitro experiments show that Coronin 1A preferentially binds to PI(4,5)P₂-containing liposomes and that PI(4,5)P₂ antagonizes the ability of Coronin 1A to disassemble actin filament branches, indicating a spatiotemporal regulation of Coronin 1A via a direct interaction with the plasma membrane lipid. Collectively, our proteomics data provide a list of potential acidic phospholipid-binding protein candidates ranging from the actin regulatory proteins to translational regulators.     

CONTACT TRANSFER OF DIFLUBENZURON (DIMILIN®) BY BOLL WEEVILS1 AND THE RELATION OF SITE OF APPLICATION AND EFFECT ON EGG HATCH

The growth-regulating chemical diflubenzuron (tested as TH=6040, now formulated as Dimilin^®), N(4-chlorophenyl)-N-(2,6-difluorobenzoyl)urea) was (1) applied to tarsi or to genital sternites of male or female boll weevils, Anthonomus grandis Boheman, that were then placed with untreated weevils of the opposite sex; (2) applied to genital sternites of either males or females and then placed with the egglaying females; (3) placed directly on the genital sternites of egg-laying females; or (4) applied directly to the soft integument under the elytra.TH-6040 applied to the soft dorsal integument produced a higher level of inhibition of egg hatch than did tarsal contact treatment. Application to males had a distinct effect but much smaller, on egg hatch of eggs from their mates than did direct application to the females. Application to hard cuticle (genital sternites) was less effective than to soft integument (i.e., there was less inhibition of hatch, and the rate of recovery was faster).

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Zusammenfassung Der Wirkstoff Diflubenzuron (geprüft als TH-6040 und nun formuliert als Dimilin^®, Thomson Hayward Chemical Co., Baton Rouge, La., U.S.A.) N(4-Chlorophenyl)-N-(2,6,difluorobenzoyl)harnstoff) wurde ausgewachsenen Baumwollkapselkäfern (Anthonomus grandis. Boh.) mit zwei Verfahren auf die Epidermis appliziert. Dabei sollte bei eierlegenden Weibchen der Effekt des Stoffes und die Ubertragung des Stoffes von andern unterschiedlich behandelten Insekten geprüft werden. Tarsalkontakt wurde erreicht, indem Männchen und Weibchen in behandelten Glasschalen gehalten wurden. Ferner wurde 2,8 g des Wirkstoffs am Genitalsternit oder am weichen Integument unter den Deckflügeln appliziert. So behandelte Weibchen wurden mit eierlegenden Weibchen zusammengebracht. Behandelte Männchen wurden während eines Tages mit eierlegenden Weibchen gehalten. Eierlegende Weibchen wurden auch direkt behandelt.Die Ergebnisse zeigen, dass die Applikation an den Tarsen und am weichen Integument ähnliche Hemmwirkungen auf das Eischlüpfen ausübt. Die Applikation am Genitalsternit war weniger erfolgreich. Der Stoff wurde von behandelten Männchen auf Weibchen übertragen und reduzierte das Eischlüpfen. Der Effekt war aber schwächer und kürzer dauernd als bei direkter Applikation. Auch vonWeibchen zu Weibchen erfolgte eine Übertragung, allerdings in geringerem Ausmass.Bei Anwendung des Wirkstoffs im Feld kann nicht mit einer grossen Übertragung von Insekt zu Insekt gerechnet werden. Der Stoff muss direkt auf die Weibchen appliziert werden, oder es muss eine ausreichende behandelte Laubmasse für Tarsalkontakt vorhanden sein.

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Coleoptera, Curculionidae     

CELL KINETICS IN MOUSE THYMUS STUDIED BY SIMULTANEOUS USE OF 3H-THYMIDINE AND COLCHICINE

Following an injection of ³H-thymidine to mice there is no initial incorporation in small thymocytes, only in larger ones. In the course of time small thymocytes aquire the label. Whether the delayed uptake in small thymocytes is due to a direct cell to cell transfer of labelled nuclear material from inititally labelled larger cells to small thymocytes, or whether it is due to small thymocytes being formed from larger cells by mitotic division was investigated by the administration of Colcemid® immediately after one injection of ³H-thymidine. In the absence of cell division no labelled small thymocytes appeared with time. This finding does not support the idea of a cell to cell transfer of DNA; it rather lends support to the view that small thymocytes arise by mitotic division of larger cells in the thymus. During the treatment with Colcemid® the migration of cells took place from peripheral to central cortex just like under normal conditions.     

ANALYSIS OF SPATIAL AND TEMPORAL DIVERSITY AND DISTRIBUTION OF PORPHYRA (RHODOPHYTA) IN SOUTHEASTERN NEW ZEALAND SUPPORTED BY THE USE OF MOLECULAR TOOLS1

Molecular studies have shown that New Zealand’s rocky shores are a habitat for >30 species of Porphyra, but little is known of their seasonal and zonal distribution. The spatial and temporal distribution of bladed Porphyra gametophytes at Brighton Beach, southeast New Zealand, were monitored for 32 months. Molecular markers were used for species identification, and a total of nine species was observed as being present during this time. Two species, P. cinnamomea and Porphyra sp. “ROS 54,” were the most common, and both were present for most months, while the remaining seven species were present sporadically, for only a few weeks at a time. P. cinnamomea W. A. Nelson and Porphyra sp. “ROS 54” were most common in the midintertidal, and both showed a similar seasonality with the highest presence during spring. They also showed a similar trend of seasonal dieback resulting in at least 1 month (May) in two consecutive years when they were both absent. This is one of the few studies investigating spatial and temporal distribution within a genus and over a 3‐year period. Our results show no distinct intertidal zonation patterns within the genus, and we conclude that morphologically similar species in a similar habitat rely on physiological mechanisms for survival.     

PERIPHYTON COLONIZATION OF ROCK SURFACES IN A BOREAL FOREST STREAM STUDIED BY SCANNING ELECTRON MICROSCOPY AND TRACK AUTORADIOGRAPHY1

Periphyton colonization of natural rock surfaces and granite tiles was followed experimentally in the Matamek River, an acidic (pH 5.5) sixth order boreal stream in northeast Quebec, Canada. Accumulation of chlorophyll a and freshweight algal biomass was logarithmic over a 25 day colonization period. The major colonizers were Tabellaria flocculosa (Roth) Kütz., T. fenestrata (Lyngb.) Kütz., Mougeotia sp., and Eunotia pectinalis (Kütz) Rabh., and its varieties. The microcolonization sequence on granite tiles, followed over 27 days with scanning electron microscopy, showed an initial accumulation of algal cells on the upstream and downstream margins and in depressions, followed by a gradual filling-in of the flat surfaces. It is hypothesized that the observed slow rate of colonization was due to the high surface tension of the granite substratum and the absence of preconditioning by an organic film. It is further hypothesized that the increase in cellular carbon fixation rates of T. flocculosa measured over a 23 day period using nuclear track autoradiography parallels the development of an algal-detrital microcosm on the granite tile, and is evidence for the establishment of localized tight nutrient spiralling. It is suggested that the periphyton community may be regarded as a nutrient recycling system at a microenvironmental level which may be especially important in oligotrophic systems.     

THE USE OF SPECIFIC ANTIBODY IN ELECTRON MICROSCOPY : I. Preparation of Mercury-Labeled Antibody

The preparation of antimyosin conjugated with mercury and fluorescein is described. The mercury was introduced to permit visualization of the antibody in the electron microscope. An organic mercurial, tetraacetoxymercuriarsanilic acid, was prepared and coupled to the antibody through the diazonium salt. The fluorescein was coupled through the isocyanate by a modification of the procedure described by Coons and Kaplan. The antibody conjugate retained its specificity of reaction with the tissue antigen. This was demonstrated by the staining pattern obtained in fluorescence microscopy.     

CALCIFICATION BY COCCOLITHOPHORIDS: EFFECTS OF pH AND Sr1

Cells of Coccolithus huxleyi which fail to deposit CaCO₃ and form coccoliths often occur as unwanted components in cultures used for studies of calcification. Non-calcified cells generally cannot be made to recalcify, but they can be removed from cultures by treatment at elevated pH or by a method based on faster sinking of calcified cells. Lowering the concentrations of nitrate, phosphate, or trace metals in the medium did not restore calcifying ability of non-calcified cells. However, addition of strontium did promote recalcification of decalcified Cricosphaera carterae grown under calcium limitation. Strontium seemed to promote coccolith attachment to cells rather than to affect calcium uptake or coccolith formation itself.