Proofreading systems of multiple stages for improved accuracy of biological discrimination

The phenomenal accuracy of biological discrimination is due in many cases to specific proofreading mechanisms. We have previously developed a macroscopic theory of such mechanisms and applied it to the case of single-stage proofreading. In this article we apply the theory to systems with multiple stages of proofreading. A specific relationship between improved accuracy due to proofreading and the associated energy cost is given. This is a macroscopic relationship that must be satisfied regardless of the details of the underlying mechanisms. Five factors in the design of such systems are shown to influence their overall accuracy: (1) initial discrimination, (2) number of proofreading stages, (3) proofreading discrimination of each stage, (4) distribution of proofreading effort among the stages, and (5) total energy expended for proofreading. We show that there is an optimal distribution of proofreading effort that, for a given degree of accuracy, minimizes the energy cost of proofreading. We also provide a simple physical interpretation of this minimum condition. These results are used to examine proofreading in two experimental systems for which there is appropriate data available in the literature: the valyl-tRNA synthetase catalyzed misacylation of tRNA^Val with threonine and the isoleucyl-tRNA synthetase catalyzed misacylation of tRNA^Ile with valine. The correlation between the magnitude of a discrimination factor and the size of the corresponding enzymatic cavity is discussed.     

Neue sesquiterpene aus Liabum-arten

The reinvestigation of the roots of Liabum eggersii afforded, in addition to compounds reported before, several sesquiterpene hydrocarbons with unusual carbon skeletons and the first derivative of the propellane modhephene which itself is also present. The roots of L. floribundum contain a tricyclic sesquiterpene with three five-membered rings. This ketone most probably is a derivative of the sesquiterpene silphiperfolene. The structures of the new compounds were elucidated by extensive NMR studies and by some chemical transformations. Two further species only afforded known compounds. The chemotaxonomic situation is discussed briefly.     

The development of the compound eyes ofPenaeus duorarum (Crustacea: Decapoda) with remarks on the nervous system

Summary The development of the compound eyes and nervous system of the penaeid shrimp,Penaeus duorarum, from the first nauplius to the first postlarva, has been studied. The first anlage of the compound eyes is a pair of optic discs on the front of the animal. These increase in size through cell-division until the second protozoea stage, where the eye-stalks appear with ommatidia and optic neuropiles developed. The original neuroectoderm of the optic discs is retained in the shape of a proliferation zone throughout the life of the animal. From the optic discs, develop the ommatidia, the lamina ganglionaris, and the medulla externa. The medullae interna and terminalis develop from cells coming from the brain anlage. From the second protozoea and onwards, the development is less rapid. The final shape of the adult eye is reached during the postlarval stages and includes the appearance of a few more pigments and a perfecting of several features. A scheme for the development of crustacean compound eyes is laid down. Further, the medulla externa of the Malacostraca and the single medulla of non-malacostracan crustaceans are homologized.The continuous growth of the nervous system is traced in the development of the neuropile. The appearance of glomeruli structures is reported, as are also, to some extent, neurosecretory organs. The development of the SPX-organ conforms to that of other decapods.For the sake of simplicity, the findings reported below in Results are grouped under two headings, namely the eye-stalk and the nervous system. Under the eye-stalk will be described both the structures coming from the optic discs comprising the ommatidia, the lamina ganglionaris, and the medulla externa, and the contributions from the nervous system comprising the medulla terminalis and medulla interna. Under the nervous system will be described the rest of the nervous system. The term anlage of the compound eyes is the same as the optic discs and denotes all contributions from this area in the early larva.     

The ultrastructure of the nauplius eye ofSapphirina (Crustacea: Copepoda)

Summary The ultrastructure of the specialized nauplius eye of three species of the copepod genusSapphirina was investigated. The gross morphology described earlier (Elofsson, 1966a) was confirmed. The ventral cup is covered by a red pigment and the lateral cups by a red and a black pigment. The ultrastructural configuration of the pigment granules was found to differ in the two kinds of pigment cells. The black pigment cell, moreover, contains a large number of transversely banded fibrils and is able to produce reflecting crystals. The pigment granules of the black pigment cell show a variation in electron density. An intimate connexion exists between the black pigment cell and large retinula cells in the lateral cups, indicating an exchange of material. The tapetal cells present in all three cups form crystal platelets contained in two sets of membranes. It is suggested that the ventral cup and part of the lateral cups function as thePecten-eye (Land, 1965). The rhabdomeres of the retinula cells are composed of microvilli measuring 400 Å. The orientation of these seems to exclude polarotactic behaviour. The ventral cup and the four small cells of the lateral cups contain some retinula cells with microvilli arranged parallel to the incoming light. The retinula cells further develop an intricate system of membrane-invaginations penetrating deep into the cell and associated with numerous mitochondria. Retinula cells of the ventral cup and part of the lateral cups contain clear portions filled with granular material only. Retinula and other cells contain attenuated mitochondria with parallel tubuli. The proximal lens in front of each lateral cup consists of one cell. A development from the conjunctival cells is suggested. The results are evaluated in terms of function and evolution.This work has been supported by a grant from the Swedish Natural Science Research Council (2760-2).     

Untersuchungen zur histochemischen Lokalisation der Leucin- und Cystinaminopeptidase (Oxytocinase) in der Placenta

Zusammenfassung Mit einer von Semm und Waidl (1962) angegebenen histochemischen Methode wurde versucht, die Serumoxytocinase in der menschlichen Placenta unter Verwendung von l-Cystin-di--Naphthylamid (CBNA) als Substrat nachzuweisen. Diese Methode war chemisch nicht realisierbar, da das vom Substrat abgespaltene -Naphthylamin mit NaNO₂ bei pH 4,9 diazotiert und anschließend mit N-1(Naphthyl)-äthylendiamin zu einem blauen Farbstoff gekoppelt werden soll. Eine Diazotierung kann in diesem pH-Bereich nicht erfolgen. Erst nach Senkung des pH unter 2,0 konnten die Versuche von Semm und Waidl nachvollzogen werden. Hinsichtlich ihrer Aussagekraft über die Histotopie des gesuchten Ferments waren sie jedoch unbefriedigend und von fehlerhaften Schlüssen abhängig.Mit der Methode nach Nachlas, Crawford und Seligman unter Verwendung des zwar unspezifischeren, dafür aber von der Serumoxytocinase zwanzigmal schneller als CBNA gespaltenen l-Leucin--Naphthylamid (LBNA) als Substrat gelang ein histochemischer Nachweis der Schwangerenserumoxytocinase. Durch Hemmung der Leucinaminopeptidase mit DL-Methionin und der Ocytocinase mit EDTA konnte nachgewiesen werden, daß es sich bei diesen Fermentaktivitäten zum größten Teil um die Serumoxytocinase handelt. Die Verwendung des weitaus spezifischeren Substrates CBNA führte nach allerdings längeren Inkubationszeiten zum selben Ergebnis: Es fand sich ein körniger Farbniederschlag im Trophoblasten und in den X-Zellen der Placentasäulen. Diese Strukturen können daher als Bildungsoder Speicherungsstätte der Oxytocinase angesehen werden.

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Investigations on the histochemical localization of leucine- and cystineaminopeptidase

Summary The histochemical demonstration of oxytocinase in human placenta described by Semm and Waidl (1962) is based on the use of L-cystine-di--naphthylamide (CBNA) as a substrate for enzymatic cleavage, on diazotising the enzymically hydrolysed -naphthalamine, and of the development of a blue color by coupling the diazocompound with N-1 (naphthyl)-ethylenediamine. Yet the method was not realisable chemically, because of the high pH during diazotising -naphthylamine. After lowering the pH under 2,0 we succeeded in obtaining the results described by Semm and Waidl. They have to be regarded however as an artefact for tissue staining was of secondary nature. Diffusion of oxytocinase during incubation period lead to an enzymatic reaction within the solvent and not within the tissue as should be supposed.Following the method of Nachlas, Crawford and Seligman (1957) with L-leucine--naphthylamide (LBNA) as substrate we succeded in the real histochemical demonstration of oxytocinase. The cleavage of LBNA by oxytocinase proceeds twenty times faster than the cleavage of the more specific CBNA. Inhibition of Oxytocinase with EDTA was followed by a nearly complete loss of enzyme specific staining, inhibition of leucineaminopeptidase with D,L-methionine displayed no significant loss in enzyme activities. We can conclude therefore that these aminopeptidase activities being located in the trophoblast and in the X-cells of the human placenta are depending chiefly on oxytocinase.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Elektronenmikroskopische Untersuchungen des ventralen Diaphragmas von Locusta migratoria und der langsamen Kontraktionswelle nach Fixation durch Gefriersubstitution

Zusammenfassung Die Muskulatur des ventralen Diaphragmas von Locusta besteht aus parallel verlaufenden Muskelfasern, die über glanzscheibenartige Strukturen in den Querverbindungen miteinander verbunden sind. Jede Muskelfaser ist von einer dicken Bindegewebshülle umgeben. Die Fasern sind über Hypodermiszellen mittels Tonofibrillen in der Cuticula verankert.Unkontrahierte Sarkomere haben eine Länge von 5 m und mehr. Eine H-Zone ist angedeutet, eine M-Linie nicht vorhanden. Aktin- und Myosinfilamente (Durchmesser 72 bzw. 160 AE) liegen nicht im Register. Daneben existiert ein dritter, sehr dünner Filamenttyp. Die Z-Zone hat einen gewellten Verlauf und faßt die Aktinfilamente in Bündeln zusammen. Mitochondrien liegen beiderseits der Z-Zone. Das T-System faltet sich in Form von Sarkolemmkerben in das Faserinnere ein und setzt sich in Tubuli nach innen fort. Z-Zonen und Sarkolemmkerben sind miteinander verbunden. T-System und sarkoplasmatisches Retikulum treten durch unregelmäßig verteilte Diaden miteinander in Kontakt.Begrenzter Ca⁺⁺-Entzug läßt Kontraktionswellen von der Länge mehrerer Sarkomere entstehen.Die Fixation durch Gefriersubstitution erzeugt gegenüber Standardverfahren Veränderungen wie Schrumpfung des Sarkoplasmas, Verdickung der Myosinfilamente, Vakuolisierung von Mitochondrien und vesikulärem System. In der Kontraktionswelle verkürzt sich die A-Zone mit zunehmender Sarkomerenkontraktion. Der Durchmesser der Myosinfilamente beträgt 172 AE, die Periodik der Cross-bridges von 305 AE bleibt im mittleren Bereich der Filamente konstant.

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Electron microscope studies of the ventral diaphragm of Locusta migratoria and of the slow wave of contraction after fixation by freeze substitution

Summary The muscular system of the ventral diaphragm of Locusta consists of parallel muscle fibers, which are connected by structures like intercalated discs within transverse bridges. Each muscle fiber is enveloped by a thick sheath of connective tissue. The fibers are attached to the cuticule by means of hypodermic cells with tonofibrils.Uncontracted sarcomeres have a length of 5 m and more. The H-band is slightly indicated, a M-line is not visible. Actin and myosin filaments (diameter 72 respectively 160 AE) are out of register. Moreover there is a third and very thin type of filaments. The Z-band has an undulating shape and collects the actin filaments into bundles. Mitochondria lie on either side of the Z-band. The T-system invaginates as sarcolemmal clefts and continues its course inwards as tubuli. The sarcolemmal clefts are connected with the Z-band. The T-system and the sarcoplasmic reticulum are joined by diads of irregular distribution.Limitated deprivation of Ca⁺⁺ causes waves of contraction with the length of several sarcomeres.Contrary to standard methods the freeze-substitution causes some modifications such as shrinking of the sarcoplasm, thickening of the myosin filaments, vacuolization of mitochondria and vesicular system. Within the waves of contraction the A-band shortenes with increasing sarcomere contraction. The diameter of the myosin filaments measures 172 AE, the 305 AE-period of the cross-bridges remains constant within the middle of the filaments.

Auszug aus der Dissertationsarbeit Elektronenmikroskopische Untersuchungen der Muskulatur des ventralen Diaphragmas von Locusta migratoria und der langsamen Kontraktionswelle unter Anwendung der Gefriersubstitution (Mathematisch-Naturwissenschaftliche Fakultät der Universität Göttingen). Auf Anregung von Herrn Prof. Dr. Schlote und mit Unterstützung durch die Deutsche Forschungsgemeinschaft und die Göttinger Akademie der Wissenschaften.     

GC-MS of perdeuteriomethylated flavonoid aglycones

GC-MS of perdeuteriomethylated flavonoid aglycones, singly and in mixtures, yields information about both the aglycone types and their substitution patterns. Fragmentation patterns of flavonoid aglycones are discussed. Acid hydrolysis of perdeuteriomethylated flavonoid glycosides, singly and in mixtures, followed by ethylation with diazoethane provides derivatives suitable for GC-MS; the introduced ethyl groups permit identification of the position of attachment of sugars in flavonoid O-glycosides.     

Transfection of Escherichia coli Spheroplasts III. Facilitation of Transfection and Stabilization of Spheroplasts by Different Basic Polymers

The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia coli spheroplasts by phage DNAs was spermine; poly-l-lysine, poly-l-arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight compounds were effective at lower concentrations, and each compound had a sharp concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamine- or protamine-like, natural competence factor from Streptococci. By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast preparations gave efficiencies of transfection between 10(-3) and 3 x 10(-4) for lambda DNA; between 10(-6) and 3 x 10(-8) for T7 DNA; and between 3 x 10(-6) and 10(-7) for T5 phage DNA. The stability of the spheroplasts was extended from 10 h to between 2 and 5 days, depending on the DNA used for transfection.     

On the Question of Crown-Gall Tumor Initiation by DNA of Bacteriophage PS8

DNA of Agrobacterium tumefaciens bacteriophage PS8 was isolated by using several procedures. Whole phage and phage DNA were tested for tumor-inducing ability on 10 species of plants with various additions to assist such activity. The reported tumorigenicity of phage PS8 DNA could not be confirmed, and no evidence to implicate phage PS8 involvement in tumor initiation was obtained.     

The capillaries of the middle ear mucosa in the guinea pig

Summary The middle ear capillaries of the guinea pig have fenestrated endothelium, and the intercellular clefts are closed by tight junctions. Intracardially injected horseradish peroxidase penetrates the fenestrae of the endothelium and gains access to the extra-cellular space beneath the epithelium, and the intercellular clefts of the epithelium.