A direct-colouring, metal precipitation method for the demonstration of arylsulphatases A and B

Summary A new, direct-colouring, metal precipitation method for the light microscopical demonstration of arylsulphatases A and B is described. It is based on the reducing capacity of nitrocatechol liberated by arylsulphatases fromp-nitrocatechol sulphate. The reaction is carried out in Karnovsky-Roots' copper ferricyanide incubation mixture at pH 5.0 or 5.5; the nitrocatechol liberated reduces ferricyanide to ferrocyanide, which in turn forms a brown precipitate with copper that indicates enzyme activity. Enzyme activity was localized in cytoplasmic particles compatible with a lysosomal localization of the enzyme. The histochemical reaction was inhibited by phosphate, which has been shown to inhibit arylsulphatases A and B in biochemical determinations. The method described is technically simple, and sections can be mounted in synthetic resin after dehydration.     

Electron flow through metalloproteins

Electron transfers in photosynthesis and respiration commonly occur between metal-containing cofactors that are separated by large molecular distances. Understanding the underlying physics and chemistry of these biological electron transfer processes is the goal of much of the work in our laboratories. Employing laser flash-quench triggering methods, we have shown that 20 Å, coupling-limited Fe(II) to Ru(III) and Cu(I) to Ru(III) electron tunneling in Ru-modified cytochromes and blue copper proteins can occur on the microsecond timescale both in solutions and crystals; and, further, that analysis of these rates suggests that distant donor-acceptor electronic couplings are mediated by a combination of sigma and hydrogen bonds in folded polypeptide structures. Redox equivalents can be transferred even longer distances by multistep tunneling, often called hopping, through intervening amino acid side chains. In recent work, we have found that 20 Å hole hopping through an intervening tryptophan is several hundred-fold faster than single-step electron tunneling in a Re-modified blue copper protein.     

Model membranes: Spherical shells bounded by one bimolecular layer of phospholipids

Summary The conditions are described which lead to the formation of small spherical vesicles bounded by singlelipid membranes. These membranes appear as triple-layered structures in electron micrographs and thus resemble theunit membrane configuration of cellular boundaries. The theory of light scattering by spherical shells is found applicable to the vesicles described here; size determinations by electron microscopy (r=195 Å) and by light scattering (r=225 Å) give reasonably close values. The refractive index of the membranes as determined by differential refractometry is found to be 1.46 for green incident light of wavelength 5461 Å.The results of this work were presented in part at the Symposium on Biophysical Aspects of Permeability, Jerusalem, Israel, July 2–9, 1968.I gratefully acknowledge the help provided me during this work by Dr Walter Stoeckenius, and I also thank Dr Dominic Dziewiatkowski for the permission to use his light scattering photometer.This work was supported by grant No. GB-4871 from the National Science Foundation.     

Spermatozoen und Spermiohistogenese vonGraphidostreptus spec. (Myriapoda,Diplopoda)

Zusammenfassung Die reifen Spermatozoen des afrikanischen TausendfüßlersGraphidostreptus spec. (Diplopoda) wurden licht- und elektronenmikroskopisch untersucht.Die hutförmigen Spermatozoen sind paarweise in Spermatophoren vereinigt. Sie bestehen aus einer den scheibenförmigen Kern enthaltenden Platte und einem Zylinder, in dem sich der Akrosomkomplex befindet. — Der freie Rand des Hutes und eine basale Schicht unter dem Karyoplasma werden von sehr elektronendichtem Zytoplasma gebildet, das basal halbkugelige Vorwölbungen besitzt. Das basale Zytoplasma enthält helle Röhrchen von ca. 100 Å Durchmesser. — Dem Karyoplasma fehlt eine Kernmembran. Es besteht aus einer elektronendichten Substanz und ist vollständig von langen, 400 Å dicken hellen Röhrchen durchsetzt. Die Röhrchen enthalten einen elektronendichten Tubulus von 200 Å Durchmesser, in dessen Achse ein ca. 40 Å dickes Filament liegt. — In den Rand des Kernes ist dorsal ein Ring von Mitochondrien eingelassen. — Der den Zylinder bildende Akrosomkomplex besteht aus einem Mantel streifigen Materials unter der Zellmembran, aus der nageiförmigen Akrosomvakuole, einem im Schnitt M-förmigen Körper, einer halbkugeligen Kalotte und einem zentral gelegenen Kristalloid.

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Spermatozoa and spermiohistogenesis in Graphidostreptus spec. (Myriapoda, Diplopoda)I. The mature spermatozoa

Summary The mature spermatozoa of the African millipedeGraphidostreptus spec. (Diplopoda) were studied by light and electron microscopy. The hat-like spermatozoa are combined two by two in spermatophores. They consist of a plate containing a disk-like nucleus, and a cylinder containing the acrosomal complex. — The free border of the hat and a basal layer below the caryoplasma are formed by a very electron-dense cytoplasma with hemispheric protrusions on the basal side. The basal cytoplasma contains light canaliculi with a diameter of about 100 Å. — There is no membrane around the caryoplasma which is composed of an electrondense substance. It is completely filled up with long and light canaliculi with a diameter of 400 Å. These canaliculi contain an electron-dense tubulus of about 200 Å diameter, in the axis of which a filament of about 40 Å diameter is to be found. — In the border of the nucleus on its dorsal side there is a circle of mitochondria. — The acrosomal complex building up the cylinder is composed of an envelope of striated material below the cell membrane, of a nail-like acrosomal vacuole, of a body appearing in section like a M, of a hemispheric calotte, and of a cristalloid lying in the center.

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Wir danken der Deutschen Forschungsgemeinschaft und der Joachim Jungius-Gesellschaft der Wissenschaften in Hamburg für Sachbeihilfen.     

The ultrastructure of the human sex vesicle

A correlated light and electron microscopical study has been made on the sex vesicle of human spermatocytes. The human sex vesicle contains no RNA and no ultrastructural granular element. The sex vesicle is formed at zygotene by the two heteropycnotic sex chromosomes that come into end-to-end contact at this stage. Neither the main nor secondary nucleoli are formed in connection with the human sex vesicle. The ultrastructure of the sex vesicle shows two main components: chromatin threads 250 of Å in diameter, separated by interthread spaces, and the filament or core, 700 Å wide and smoothly curved. No synaptinemal complexes have been observed in the human sex vesicle, although the filaments can become parallel for short segments. The absence of synaptinemal complexes is discussed in relation to the observations on other mammals.     

Ultrahistochemical localization of adenylate cyclase activity in the electric organ ofTorpedo marmorata

Summary The lead pyrophosphate precipitation technique was used to visualize adenylate cyclase activity with the electron microscope in unfixed electric organ and synapto-somes ofTorpedo marmorata, with special attention to presynaptic membranes. Specificity of the deposition of reaction product was ensured by using 5′-adenylyl imidodiphosphate as substrate and 5′-guanylyl imidodiphosphate and sodium fluoride as activators. Under suitable conditions a reaction product was deposited on the Schwann cell, on presynaptic vesicles, on the inner side of membranes of cisternae and on glycogen granules of the presynaptic region of the endplate. In some cases, a precipitate was also found on postsynaptic membranes of the synaptic cleft and on mitochondria. In isolated synaptosomes localization of the reaction product was identical with that of minced tissue. However, most strikingly, on presynaptic membranes no precipitate was ever found, neither in pieces of electric organ nor in isolated synaptosomes. Furthermore, the extended membrane system of the postsynaptic region of the electroplax remained always free of leed pyrophosphate precipitate.     

The chemistry of the CuB site in cytochrome c oxidase and the importance of its unique His-Tyr bond

The Cu_B metal center is at the core of the active site of the heme-copper oxidases, comprising a copper atom ligating three histidine residues one of which is covalently bonded to a tyrosine residue. Using quantum chemical methodology, we have studied the Cu_B site in several redox and ligand states proposed to be intermediates of the catalytic cycle. The importance of the His-Tyr crosslink was investigated by comparing energetics, charge, and spin distributions between systems with and without the crosslink. The His-Tyr bond was shown to decrease the proton affinity and increase the electron affinity of both Tyr-244 and the copper. A previously unnoticed internal electronic equilibrium between the copper atom and the tyrosine was observed, which seems to be coupled to the unique structure of the system. In certain states the copper and Tyr-244 compete for the unpaired electron, the localization of which is determined by the oxygenous ligand of the copper. This electronic equilibrium was found to be sensitive to the presence of a positive charge 10 Å away from the center, simulating the effect of Lys-319 in the K-pathway of proton transfer. The combined results provide an explanation for why the heme-copper oxidases need two pathways of proton uptake, and why the K-pathway is active only in the second half of the reaction cycle.     

Electron-microscopic histochemical study of myosin ATP-ase activity

Summary The sites of calcium-activated myosin ATP-ase reaction have been determined by a method modified from that of Padykula and Hermann. On coherent muscle preparations the enzyme was found to be mainly active in the segment A, although reaction was sometimes observed in the strip Z also. In several cases the precipitate appeared in a spherical shape in the segment A. Since no such phenomenon was observed in isolated bundles of myofibrils it is supposed to be an artefact. The reaction appeared in fibre bundles at 300 to 400 Å intervals, and a certain spatial periodicity was observed also on isolated thick filaments.     

Crystallographic studies of mutant reaction centres from Rhodobacter sphaeroides

X-ray structures have been determined for five mutant reaction centres from Rhodobacter sphaeroides, at resolutions varying between 3.4 Å and 2.3 Å. The aim was to examine the effects of mutagenesis of polar residues in the binding pocket of the reaction centre carotenoid. The number of water molecules identified in each structure depended on the resolution and completeness of the data. In a 2.3 Å structure for a WM115F/FM197R mutant reaction centre, two water molecules partially filled the cavity that was created when the tryptophan residue was replaced by a less bulky phenylalanine. Structures obtained for four reaction centres with mutations of polar residues in the carotenoid binding pocket failed to show any significant change in the structure of the reaction centre carotenoid. Low resolution data for a YM210W mutant reaction centre showed that the overall structure of this complex is well conserved. This finding is discussed in light of the intriguing spectroscopic properties of the YM210W mutant reaction centre, and an alternative pathway for transmembrane electron transfer identified in this mutant.     

Wheat cultivar reactions to deleterious rhizosphere bacteria under gnotobiotic conditions

With the aim of elucidating mechanisms behind bacteria-induced deleterious effects and differential cultivar responses to bacterial inoculations, wheat seedlings were subjected to various tests under gnotobiotic conditions. Inoculation with two deleterious Pseudomonas isolates, Å 112 (fluorescent) and Å 313 (nonfluorescent), induced leaf symptoms and shoot and root growth inhibition, while inoculation with growthneutral bacteria (Serratia liquefaciens andEscherichia coli) had no such effects. Deleterious effects were induced at low inoculum densities (<10³ cells per plant), but required addition of nutrient broth in small amounts for consistency. Effects similar to those obtained with living inoculum could be induced by treating plants with sterile culture filtrates from isolate Å 313 or volatile bacterial metabolites from isolate Å 112. Wheat cultivars previously found to differ in their reaction to inoculation under non-sterile conditions, responded differentially to Å 112 and Å 313 also in the gnotobiotic assay. The results agree with the hypothesis that neither cultivar reaction nor the bacterial effects as such are mediated by interactions with an indigenous rhizosphere microflora.     

Inhibition of Photosystem II in the Green Alga Ankistrodesmus falcatus by Copper

Copper strongly inhibited 2,6-dichloroindophenol (DCIP) photoreduction in the broken cells of the green alga Ankistrodesmus falcatus (C303), and the activity lost could not be restored by adding 1,5-diphenylearbazide (DPC). Inactivation of the DCIP Hill reaction reached 45% after incubation with 10 μM cupric sulfate for 20 min. In the same time, copper (13 μg/mg chlorophyll) was bound to the broken cells. Addition of 10 mM KCl reduced copper binding by about 53%. Fluorescence intensity at room temperature decreased upon addition of cupric sulfate and was partially restored by adding 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), These results suggest that copper inactivates electron transport between the oxidizing side of the reaction center of photosystem II and the electron-donating site of DPC. Further, the effect of light intensity shows that copper mostly affected the reaction rate of the dark step and had less inhibitory effect on the quantum efficiency of the primary reaction of electron transport in photosystem II.     

Reconstruction of the Neurospora crassa pachytene karyotype from serial sections of synaptonemal complexes

Serial sections from isolated asci were used to reconstruct the seven pachytene bivalents of Neurospora crassa. The synaptonemal complex could be traced for its whole length in each bivalent, being attached to the nuclear envelope at both ends in six. The satellite end of the nucleolar chromosome did not appear to be attached to the nuclear envelope. The estimated lengths of the bivalents ranged from 10.7 to 5.1 microns in one nucleus, from 11.5 to 4.2 microns in another, and from 8.5 to 4.4 microns in a third, with total haploid complement lengths of 45.5 microns, 47.3 microns, and 43.9 microns respectively. These values are considerably smaller than published light microscopical measurements.—The synaptonemal complex in N. crassa, as in other ascomycetes, has two banded ca. 400 Å wide lateral components held about 1200 Å apart by a central region containing the ca. 200 Å wide central component. With normal glutaraldehyde/OsO₄-phosphate buffered fixation the chromatin of the pachytene bivalents is poorly contrasted. Occasional local thickenings of the central component into electron dense nodes ca. 1000 × 500 Å in longitudinal section are characteristic of the complex.     

Ciliated neurons and different types of synapses in anterior hypothalamic nuclei of reptiles

Summary The magnocellular paraventricular and supraoptic nuclei and the parvocellular preoptic and periventricular nuclei have been studied by light and electron microscopy in Emys orbicularis, Lacerta agilis and Elaphe longissima. The ultrastructure of cerebrospinal fluid (CSF)-contacting neurons was described in the preoptic and periventricular nuclei of Emys and Lacerta species. Single 9×2+0 cilia similar to those of the CSF-contacting dendritic terminals were found on perikarya of non CSF-contacting nerve cells, in all four investigated nuclei. The cilia project from funnel-like invaginations of the perikarya into the intercellular space. In the neurons of the nuclei studied, granular vesicles were found, their size being mainly 1,600 Å in the paraventricular nucleus, about 1,800 Å in the supraoptic nucleus, 1,100 Å in the periventricular nucleus and 800 Å, or up to 1,250 Å in the preoptic nucleus. In general, the neurons possess synapses of the axo-somatic, axo-somatic spine, axo-dendritic and axo-dendritic spine types. In the supraoptic nucleus, multiple interdigitated synapses were observed. Presynaptically, either synaptic vesicles only, or synaptic vesicles and dense core vesicles of different sizes (600 to 800 Å, about 1,100 Å, 1250 Å, and up to 2,000 Å) were found. It is discussed whether the above described 9×2+0 cilia may represent some kind of hypothalamic sensory structure that earlier physiological studies postulated to exist. The ciliated hypothalamic perikarya are considered by the authors to be a more differentiated form of the CSF-contacting neurons. The different types of synapses indicate multilateral connections of the nerve cells of the nuclei studied.Dedicated to Prof. Dr. Berta Scharrer on the occasion of her 70th birthday     

Photochemical Electron Transport in Photosynthetic Reaction Centers from Rhodopseudomonas spheroides: I. Kinetics of the Oxidation and Reduction of P-870 As Affected by External Factors

Photosynthetic reaction centers from Rhodopseudomonas spheroides were prepared with the detergent lauryl dimethylamine oxide (LDAO). In contrast to reaction centers made with Triton X-100, these contained no cytochromes and little or no ubiquinone (UQ). The reduction of P-870, after its photochemical oxidation, was studied in these materials with the following results. In reaction centers made with Triton X-100, slow kinetic components (seconds to minutes) could be attributed to secondary electron acceptors or traps. In reaction centers made with LDAO the kinetics were predominantly fast (half-times, 100 msec or less); slower components could be introduced by adding UQ. Added UQ appeared to become bound to reaction centers made with LDAO, but the binding might have meant only that both components were trapped within detergent micelles. Ferricyanide could retard the reduction of oxidized P-870, apparently by capturing electrons from the reducing side of the photochemical system. Under conditions in which the participation of secondary electron acceptors seemed to have been eliminated, the recovery of P-870 was mainly by a first-order process with a half-time of about 60 msec at room temperature and 20-30 msec at about -80°C and below. The transition with decreasing temperature suggested the presence of a mixed population, exhibiting both the 60 and 20 msec components, but variations in the absorption spectra with temperature did not suggest the presence of a mixed population. Absorption difference spectra in the ultraviolet were compatible with the idea that UQ added to reaction centers became reduced in the light.     

Crystal Structure of Sulfide:Quinone Oxidoreductase from Acidithiobacillus ferrooxidans: Insights into Sulfidotrophic Respiration and Detoxification

Sulfide:quinone oxidoreductase from the acidophilic and chemolithotrophic bacterium Acidithiobacillus ferrooxidans was expressed in Escherichia coli and crystallized, and its X-ray molecular structure was determined to 2.3 Å resolution for native unbound protein in space group P4₂2₁2 . The decylubiquinone-bound structure and the Cys160Ala variant structure were subsequently determined to 2.3 Å and 2.05 Å resolutions, respectively, in space group P6₂22  . The enzymatic reaction catalyzed by sulfide:quinone oxidoreductase includes the oxidation of sulfide compounds H₂S, HS⁻, and S²⁻ to soluble polysulfide chains or to elemental sulfur in the form of octasulfur rings; these oxidations are coupled to the reduction of ubiquinone or menaquinone. The enzyme comprises two tandem Rossmann fold domains and a flexible C-terminal domain encompassing two amphipathic helices that are thought to provide for membrane anchoring. The second amphipathic helix unwinds and changes its orientation in the hexagonal crystal form. The protein forms a dimer that could be inserted into the membrane to a depth of approximately 20 Å. It has an endogenous flavin adenine dinucleotide (FAD) cofactor that is noncovalently bound in the N-terminal domain. Several wide channels connect the FAD cofactor to the exterior of the protein molecule; some of the channels would provide access to the membrane. The ubiquinone molecule is bound in one of these channels; its benzoquinone ring is stacked between the aromatic rings of two conserved Phe residues, and it closely approaches the isoalloxazine moiety of the FAD cofactor. Two active-site cysteine residues situated on the re side of the FAD cofactor form a branched polysulfide bridge. Cys356 disulfide acts as a nucleophile that attacks the C4A atom of the FAD cofactor in electron transfer reaction. The third essential cysteine Cys128 is not modified in these structures; its role is likely confined to the release of the polysulfur product.     

Copper sulfide precipitation by yeasts from Acid mine-waters

Two strains of Rhodotorula and one of Trichosporon precipitated dissolved copper with H₂S formed by reducing elemental sulfur with glucose. Iron stimulated this activity under certain conditions. In the case of Rhodotorula strain L, iron stimulated copper precipitation aerobically at a copper concentration of 18 but not 180 μg/ml. Anaerobically, the L strain required iron for precipitation of copper from a medium with 180 μg of copper per ml. Rhodotorula strain L was able to precipitate about five times as much copper anaerobically as aerobically. The precipitated copper was identified as copper sulfide, but its exact composition could not be ascertained. Iron was not precipitated by the H₂S formed by any of the yeasts. Added as ferric iron, it was able to redissolve copper sulfide formed aerobically by Rhodotorula strain L from 18 but not 180 μg of copper per ml of medium. Since the yeasts were derived from acid mine-waters, their ability to precipitate copper may be of geomicrobial importance.     

The ultrastructure of plasmodesmata in the filamentous green alga,Bulbochaete hiloensis (Nordst.) tiffany

Summary The ultrastructure of the plasmodesmata found in the green alga Bulbochaete hiloensis has been examined by electron microscopy of ultra-thin sections. Unlike most other plasmodesmata that have been described recently, there are no internal components such as a desmotubule or a derivative of the endoplasmic reticulum. Each plasmodesma consists of a cylindrical connection between the plasma membranes of adjacent cells. The cylinder is constricted at each end to orifices which may be less than 100 Å in diameter. Within the cylinder the cytoplasmic face of the plasma membrane is lined with material probably consisting of helically arranged particles. The lumen here is 400–450 Å in diameter.The observations are discussed in relation to possible functions in intercellular transport.     

Transcription of chromatin from mouse fibroblasts

Chromatin purified from mouse fibroblasts can be fractionated after shearing by sedimentation in a sucrose gradient into an extended «light and a compact «heavy component. Further purification of these classically described components can be achieved by a second cycle of centrifugation of the light and heavy components through an equilibrium density gradient of metrizamide. The light component purified from sucrose gradient sediments faster (peak I) on metrizamide than its heavy counterpart (peak II).Template activity for DNA directed RNA synthesis in the presence of E. coli RNA polymerase is negligible in peak II but very pronounced in the peak I fraction. The difference in template activity appears to be connected with differences in propagation rather than initiation rates. Comparison of gel electrophoresis patterns of proteins indicate that the active subcomponent includes high molecular weight components not present in the inactive one, but that its histone content is somewhat lower. Using a very highly sensitive automatic recording apparatus for the measurement of melting profiles, no clear cut difference has been observed in the behaviour of active and inactive chromatin subcomponents nor in that of their total DNA.     

A C-Terminal Phosphatase Module Conserved in Vertebrate CMP-Sialic Acid Synthetases Provides a Tetramerization Interface for the Physiologically Active Enzyme

The biosynthesis of sialic acid-containing glycoconjugates is crucial for the development of vertebrate life. Cytidine monophosphate-sialic acid synthetase (CSS) catalyzes the metabolic activation of sialic acids. In vertebrates, the enzyme is chimeric, with the N-terminal domain harboring the synthetase activity. The function of the highly conserved C-terminal domain (CSS-CT) is unknown. To shed light on its biological function, we solved the X-ray structure of murine CSS-CT to 1.9 Å resolution. CSS-CT is a stable shamrock-like tetramer that superimposes well with phosphatases of the haloacid dehalogenase superfamily. However, a region found exclusively in vertebrate CSS-CT appears to block the active-site entrance. Accordingly, no phosphatase activity was observed in vitro, which points toward a nonenzymatic function of CSS-CT. A computational three-dimensional model of full-length CSS, in combination with in vitro oligomerization studies, provides evidence that CSS-CT serves as a platform for the quaternary organization governing the kinetic properties of the physiologically active enzyme as demonstrated in kinetic studies.