Biomarker studies on microbial carbonates: Extractable lipids of a Calcifying Cyanobacterial mat (Everglades, USA)

Summary Biomarker investigations are applied to the free lipid fractions of a naturally grown freshwater microbial mat, constructed by calcifying cyanobacteria (Scytonema sp. andSchizothrix sp.). The absolute and relative concentrations of hydrocarbons, free alcohols and carboxylic acids are studied and their probable biological precursors are discussed. A significant signal of cyanobacterial lipids is recognized by the strong predominance ofn-heptadecane (C₁₇),n-heptadecene, two monomethyl-heptadecanes, and the pentacyclic triterpenoid diploptene. Their occurrences parallel the lipid distributions found in pure cultured cyanobacteria and in recent cyanobacterial mats grown in particular environments (hypersaline, lagoonal, hot spring). The observed compound signature appears to be a suitable reference for environments, where cyanobacteria are directly associated with theloci of carbonate precipitation and thus, rock formation. In the studied material, a significant contribution of organic matter from other sources, especially higher plants is characterized by the occurrence of several specific marker compounds, namely lup-20(29)-ene-3-ol, high molecular weightn-alkanes and carboxylic acids. Although these components comprise a notably high portion of the sample’s lipid inventory, they are shown to be distinguished easily from the signal left by the predominant mat building organisms.     

Voraussetzungen und derzeitige Grenzen der quantitativen elektronenmikroskopischen Autoradiographie bei Kinetikstudien an Drüsenzellen

Zusammenfassung Einer weiblichen Maus wurde 3 Tage post partum 750 C ³H-Leucin i. p. injiziert. Zu verschiedenen Zeiten nach der Leucinapplikation wurden dem leicht narkotisierten Tier Gewebeteile der Milchdrüse entnommen und zu elektronenmikroskopischen Autoradiogrammen verarbeitet. An Hand der dabei gewonnenen Ergebnisse wurde versucht, den zeitlichen Ablauf der Milcheiweißbildung rechnerisch zu erfassen. 5 und 15 min nach der ³H-Leucinapplikation kann die Aktivität über dem rauhen endoplasmatischen Retikulum, nach 30 min über dem Golgi-Feld, und nach 240 min zur Hauptsache über den Lumina der Ausführungsgänge beobachtet werden. Die Halbwertszeit von markierten Proteinen im Ergastoplasma errechnete sich zu etwa 22 min, diejenige im Golgi-Feld zu etwa 3 Std.Die Voraussetzungen und derzeitigen Grenzen einer quantitativen elektronenmikroskopischen Autoradiographie werden diskutiert. Wegen der vielen möglichen Fehlerquellen wird die Berechnung der Kinetik der Milcheiweißbildung lediglich als Modell gewertet.

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Summary A female mouse, 3 days post partum, was injected with ³H-leucine. After various intervals parts of the mammary gland were processed for electronmicroscopic autoradiograms, the results of which were mathematically evaluated in order to understand the temporal course of milk protein formation. After 5 and 15 minutes the leucine-activity is located mainly in the rough endoplasmic reticulum, after 30 minutes in the Golgi field and after 240 minutes in the lumina of the mammary ducts. The half-live time of labelled proteins in the rough endoplasmic reticulum is about 22 minutes, in the Golgi field about 3 hours.The preconditions and limitations of quantitative electronmicroscopic autoradiography are discussed. Because of the many possible sources of error, the calculations of the kinetics of protein synthesis and secretion in the mammary gland are merely regarded as a model.

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Ausgeführt mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Wesentliche Teile der Arbeit werden Von Ute Seitter der Medizinischen Fakultät der Universität Freiburg i. Br. als Inaugural-Dissertation vorgelegt.     

High-performance liquid chromatographic assays for free and phosphorylated derivatives of the creatine analogues beta-guanidopropionic acid and 1-carboxy-methyl-2-iminoimidazolidine (cyclocreatine).

Creatine and phosphocreatine are substrates for creatine kinase which is a key enzyme involved in energy transfer within the cell. Analogues of creatine have been fed to animals to determine the role this enzyme plays in energy metabolism, but progress in interpretation has been hampered by the lack of quantitative techniques to determine tissue content of these compounds. We describe the separation and quantitation of substituted guanidino compounds and their phosphorylated forms by high-performance liquid chromatography. First, a cation-exchange column is used to assay free creatine and its unphosphorylated analogues, and then phosphocreatine and its phosphorylated analogues as well as adenylate content (AMP, ADP, ATP) are assayed on an anion-exchange column. These methods have proven successful in measuring the chemical contents of these compounds in neutralized perchloric acid extracts of mammalian skeletal muscles. The sensitivity of this method ranges from 50 to 200 pmol, which is adequate to provide information from tissue extracts of 5- to 10-mg samples.     

Spadefoot‐tadpole polyphenism: Histological analysis of differential muscle growth in carnivores and omnivores

Understanding the evolution of phenotypic plasticities and the connections among the environment, genotype, and phenotype requires detailed understanding of the proximate mechanisms regulating morphological differences between phenotypes. Spea multiplicata tadpoles can develop into two different phenotypes, i.e. carnivores and omnivores, which differ in many morphological and behavioral traits. One of the major differences is enlargement of the jaw and tail musculature in carnivores relative to those of omnivores. We investigated pattern of muscle enlargement by measuring differences in myofiber number and cross‐sectional area between the phenotypes during early and mid‐development. The data show that both hyperplasia and hypertrophy underlie the carnivores' enlargement of both the orbitohyoideus jaw muscle (OH) and the tail muscle (TL). Carnivores had more OH and TL myofibers than did omnivores at all ages, but the rate of myofiber addition differed, by ～9 and 17 myofibers per day respectively. Carnivores also had larger OH and TL myofibers than did omnivores, at many of the ages studied, and the rate of myofiber cross‐sectional area increase (log‐transformed myofiber cross‐sectional area plotted against age in days) was significantly greater for carnivores than for omnivores in the internal, but not the peripheral, regions for both the OH and TL muscle. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Ubiquitous expression of the rtTA2S-M2 inducible system in transgenic mice driven by the human hnRNPA2B1/CBX3 CpG island

Background  

A sensitive, ubiquitously expressed tetracycline inducible system would be a valuable tool in mouse transgenesis. However, this has been difficult to obtain due to position effects observed at different chromosomal sites of transgene integration, which negatively affect expression in many tissues. The aim of this study was to test the utility of a mammalian methylation-free CpG island to drive ubiquitous expression of the sensitive doxycycline (Dox) inducible rtTA2S-M2 Tet-transactivator in transgenic mice.     

Spatial distribution of the active surveillance of sheep scrapie in Great Britain: an exploratory analysis

Background  

This paper explores the spatial distribution of sampling within the active surveillance of sheep scrapie in Great Britain. We investigated the geographic distribution of the birth holdings of sheep sampled for scrapie during 2002 – 2005, including samples taken in abattoir surveys (c. 83,100) and from sheep that died in the field ("fallen stock", c. 14,600). We mapped the birth holdings by county and calculated the sampling rate, defined as the proportion of the holdings in each county sampled by the surveys. The Moran index was used to estimate the global spatial autocorrelation across Great Britain. The contributions of each county to the global Moran index were analysed by a local indicator of spatial autocorrelation (LISA).     

Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis

The polysaccharide chains of enterobacterial common antigen (ECA) consist of linear trisaccharide repeat units with the structure -->3)- alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1--> 4)-alpha-d-GlcNAc-(1-->, where Fuc4NAc is 4-acetamido-4, 6-dideoxy-d-galactose, ManNAcA is N - acetyl-d- mannosaminuronic acid, and GlcNAc is N -acetyl-d-glucosamine. The major form of ECA (ECAPG) consists of polysaccharide chains that are believed to be covalently linked to diacylglycerol through phosphodiester linkage; the phospholipid moiety functions to anchor molecules in the outer membrane. The ECA trisaccharide repeat unit is assembled as a polyisoprenyl-linked intermediate which has been tentatively identified as Fuc4NAc-ManNAcA-GlcNAc- pyrophosphorylundecaprenol (lipid III). Subsequent chain-elongation presumably occurs by a block-polymerization mechanism. However, the identity of the polyisoprenoid carrier-lipid has not been established. Accordingly, the current studies were conducted in an effort to structurally characterize the polyisoprenyl lipid-carrier involved in ECA synthesis. Isolation and characterization of the lipid carrier was facilitated by the accumulation of a ManNAcA-GlcNAc- pyrophosphorylpolyisoprenyl lipid (lipid II) in mutants of Salmonella typhimurium defective in the synthesis of TDP-Fuc4NAc, the donor of Fuc4NAc residues for ECA synthesis. Analyses of lipid II preparations by fast atom bombardment tandem mass spectroscopy (FAB-MS/MS) resulted in the identification of the lipid-carrier as the 55-carbon polyisoprenyl alcohol, undecaprenol. These analyses also resulted in the identification of a novel glycolipid which copurified with lipid II. FAB-MS/MS analyses of this glycolipid revealed its structure to be 1,2-diacyl- sn -glycero-3-pryophosphoryl-GlcNAc-ManNAcA (DGP- disaccharide). An examination of purified ECAPGby phosphorus-31 nuclear magnetic resonance spectroscopy confirmed that the polysaccharide chains are linked to diacylglycerol through phosphodiester linkage. Thus, DGP-disaccharide does not appear to be an intermediate in ECAPGsynthesis. Nevertheless, although the available evidence clearly indicate that lipid II is a precursor of DGP-disaccharide, the function of this novel glycolipid is not yet known, and it may be an intermediate in the biosynthesis of a molecule other than ECAPG.      

Activity of creatine kinase in a contracting mammalian muscle of uniform fiber type.

We investigated whether the creatine kinase-catalyzed phosphate exchange between PCr and gamma ATP in vivo equilibrated with cellular substrates and products as predicted by in vitro kinetic properties of the enzyme, or was a function of ATPase activity as predicted by obligatory "creatine phosphate shuttle" concepts. A transient NMR spin-transfer method was developed, tested, and applied to resting and stimulated ex vivo muscle, the soleus, which is a cellularly homogeneous slow-twitch mammalian muscle, to measure creatine kinase kinetics. The forward and reverse unidirectional CK fluxes were equal, being 1.6 mM.s-1 in unstimulated muscle at 22 degrees C, and 2.7 mM.s-1 at 30 degrees C. The CK fluxes did not differ during steady-state stimulation conditions giving a 10-fold range of ATPase rates in which the ATP/PCr ratio increased from approximately 0.3 to 1.6. The observed kinetic behavior of CK activity in the muscle was that expected from the enzyme in vitro in a homogeneous solution only if account was taken of inhibition by an anion-stabilized quaternary dead-end enzyme complex: E.Cr.MgADP.anion. The CK fluxes in soleus were not a function of ATPase activity as predicted by obligatory phosphocreatine shuttle models for cellular energetics.     

Further freeze-fracture studies on the uterine epithelium of Salamandra salamandra (L.) (Amphibia,Urodela) using the antibiotic filipin

Summary The apical portion of the uterine lining of the ovoviviparous fire salamander, Salamandra salamandra, was studied by the freeze-fracture technique in conjunction with the polyene antibiotic filipin. Filipin-sterol complexes were found in the luminal plasmalemma and in the membranes limiting the mucous secretory granules typical of this epithelium. In all females, but particularly in non-pregnant females, more or less discrete clusters of filipinsterol complexes were occasionally found overlying heavily affected secretory granules. The findings are discussed with regard to comparable results (Orci et al. 1980) based on the examination of collapsed and stretched urinary bladders of toads.We are indebted to Mrs. K. Ott for excellent technical assistance, to Miss E.S. MacLure for linguistic corrections and to Dr. J.E. Grady of the Upjohn Co., Kalamazoo, Michigan USA, for kindly providing the filipin     

Klinik und Genetik des Joubert-Syndroms

Joubert syndrome generally represents an autosomal recessive and rarely X-linked disorder characterized by hypotonia, an irregular breathing pattern, abnormal eye movements, ataxia, developmental delay and a complex mid-hindbrain malformation causing the molar tooth sign on magnetic resonance imaging (MRI). Many patients have additional features, with nephronophthisis, retinal dystrophy, coloboma and hepatic fibrosis representing the most frequent features. Due to its clinical variability and overlap with other syndromes, the term “Joubert syndrome and related disorders” (JSRD) was proposed. To date 10 genes are known to cause JSRD. The encoded proteins are localized to cilia, linking JSRD to other human ciliopathies.