Peptide analyses of a protein component, 50-8, of 50s ribosomal subunit from erythromycin resistant mutants of Escherichia coli and Escherichia freudii

Summary Chromatographic analyses on a Dowex 50x8 column of tryptic digests of the mutationally altered 50-8 protein component from several erythromycin resistant (ery ^r) mutants of Escherichia coli and Escherichia freundii have been performed. It was found that (1) the difference in the elution profile of the altered components detected with carboxymethyl cellulose column chromatography reflects the difference in their amino acid sequence, (2) the structural change(s) of the 50-8 protein from three E. coli ery ^r mutants examined seems to exist only in the same single peptide fragment and (3) the primary structure of the 50-8(R) protein of E. freundii (ery ^s: wild type) differs from that of E. coli Q13 (ery ^s) and the structural change in 50-8(R) component of E. freundii caused by the ery ^r mutation was found to take place in different peptide fragments from that in which the mutational change of the E. coli 50-8 component occurred.     

Beziehungen zwischen Struktur und chemischer Zusammensetzung der Spermien vom Bergmolch,Triturus alpestris Laur.

Zusammenfassung Aus isolierten Achsenfäden der Spermien von Triturus alpestris Laur. wurden drei Proteinfraktionen extrahiert und nach ihrer Zusammensetzung analysiert. In der leichtlöslichen Fraktion f3 überwog Arginin, daneben fand sich u.a. Cystin. f1+f2b enthielt weniger Arginin, dafür aber mehr Lysin. Den größten Anteil hatte hier Alanin. In jeder der beiden Fraktionen waren nur neun Aminosäuren vorhanden. Die schwerlösliche Fraktion c3 enthielt außer Cystin, Methionin und Tyrosin alle nachweisbaren Aminosäuren. Außerdem wies die Fraktion f1+f2b nicht weniger als fünf unbekannte Komponenten auf, von denen eine ein hellgrüner Farbstoff war. In geringeren Mengen kamen drei dieser Substanzen in f3 und noch eine in c3 vor. Nach den Ergebnissen des enzymatischen Abbaus an Ultradünnschnitten von glutaraldehydfixiertem Material dürfte die argininreiche Fraktion im Inneren des Achsenfadens, und zwar in seinem distalen Anteil zu lokalisieren sein. Die lysinreiche Fraktion wäre demnach im Inneren des proximalen Anteils zu finden, während die schwerlösliche Komponente c3 mit der außerordentlich widerstandsfähigen Rinde des Achsenfadens identisch wäre. Im Halsstück, das aus einem kristallinen Mantel und einem homogenen elektronendichten zentralen Teil besteht, fanden sich mindestens drei Komponenten. Der Mantel enthielt ein leicht extrahierbares Protein und ein stabiles Gerüst, in dem aromatische Aminosäuren vermehrt vorzukommen scheinen. Der zentrale Teil bestand aus argininreichen Verbindungen. Die Befunde werden im Zusammenhang mit der Bildung von Achsenfaden und Halsstück während der Spermiogenese diskutiert.

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Relationships between structure and chemical composition of spermatozoa of the newt, Triturus alpestris Laur.

Summary Axial rods from spermatozoa of Triturus alpestris Laur. have been isolated. Three protein fractions therefrom could be extracted and analysed for their composition. Arginine prevailed in the soluble fraction f3, among others also cystine was found. f1+f2b contained less arginine, but more lysine. The highest percentage was found here for alinine. In each of the two fractions only nine amino acids were present. The sparely soluble fraction c3 contained all identifiable amino acids besides cystine, methionine and tyrosine. Moreover, the fraction f1+f2b showed as many as five unknown components, from which one was a light-green colour. To a lesser degree three of these compounds were also found in fraction f3 and still one in fraction c3. According to the results of the enzymatic digestion carried out on ultrathin section of material fixed with glutaraldehyde, the arginine-rich fraction should be localized in the center of the axial rod in its distal part. The lysine-rich fraction would then be found in the center of the proximal part, while the sparely soluble fraction c3 would be identical with the extremely resistant cortex of the axial rod. Within the neck-piece, which consists of a crystalline mantle and a homogenously dark central part, at least three components were present. The mantle contained an easily extractable protein and a stable frame, in which aromatic amino acids seemed to be increased. In the central part arginine prevailed. The results are discussed with regard to the formation of the axial rod and the neck-piece during spermiogenesis.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Serological Typing of 31 Achromogenic and 40 Melanogenic Pseudomonas aeruginosa Strains

Thirty-one achromogenic and 40 melanogenic Pseudomonas aeruginosa strains were studied with 10 monovalent typing sera (3). Twenty-one of the achromogenic (67.7%) and seven of the melanogenic (17.5%) strains were agglutinated by one of the 10 typing sera. Ten achromogenic and 33 melanogenic strains were not agglutinated by any of the 10 typing sera. As far as this set of antisera is concerned, the typability of achromogenic and melanogenic P. aeruginosa strains appears to be much lower than that of the chromogenic, nonmelanogenic strains of the species reported previously.     

Glucose-6-phosphate dehydrogenase cytochemistry using a copper ferrocyanide method and its application to rapidly frozen cells

We describe an improved copper ferrocyanide-based method for cytochemical detection of glucose-6-phosphate dehydrogenase (G6PD), which was used to localize the enzyme within the ultrastructure of rat hepatocytes and adrenocortical cells. With this method, glutaraldehyde fixation and the addition of exogenous electron carriers (for example, phenazine methosulfate) to the cytochemical reaction medium were essential. Copper ferrocyanide reaction product showing the distribution of G6PD was readily recognized at the light microscopic level as Hatchett’s brown staining and at the electron microscopic level as electron-dense deposits. Within stained regions, enzyme cytochemical G6PD activity was found to be associated with ribosome-like structures. Because G6PD is a soluble, cytosolic enzyme, its displacement or extraction may occur during conventional fixation. We, therefore, combined a rapid-freezing technique with G6PD enzyme cytochemistry. The resultant rapid-freezing enzyme cytochemistry enabled us to show the subcellular distribution of G6PD in a more life-like state; the localization of G6PD in rapidly frozen cells was in substantial agreement with that in conventionally fixed cells. Accepted: 14 July 1999     

Slp1 and Slp2-a localize to the plasma membrane of CTL and contribute to secretion from the immunological synapse

Rab27a is required for polarized secretion of lysosomes from cytotoxic T lymphocytes (CTLs) at the immunological synapse. A series of Rab27a-interacting proteins have been identified; however, only Munc13-4 has been found to be expressed in CTL. In this study, we screened for expression of the synaptotagmin-like proteins (Slps): Slp1/JFC1, Slp2-a/exophilin4, Slp3-a, Slp4/granuphilin, Slp5 and rabphilin in CTL. We found that both Slp1 and Slp2-a are expressed in CTL. Isoforms of Slp2-a in CTL showed variation of the linker region but conserved the C2A and C2B and Slp homology (SHD) domains. Both Slp1 and Slp2-a interact with Rab27a in CTL, and Slp2-a, but not Slp1, is rapidly degraded when Rab27a is absent. Slp2-a contains PEST-like sequences within its linker region, which render it susceptible to degradation. Both Slp1 and Slp2-a localize predominantly to the plasma membrane of both human and mouse CTLs, and we show that Slp2-a can focus tightly at the immunological synapse formed with a target cell. Individual knockouts of either Slp2-a or Slp1 fail to impair CTL-mediated killing of targets; however, overexpression of a dominant-negative construct consisting of the SHD of Slp2-a, which is 56% identical to that of Slp1, reduces target cell death, suggesting that both Slp1 and Slp2-a contribute to secretory lysosome exocytosis from CTL. These results suggest that both Slp1 and Slp2-a may form part of a docking complex, capturing secretory lysosomes at the immunological synapse.     

Synthesis,cleavage, and antifungal activity of a number of novel,water-soluble ester prodrugs of antifungal triazole CS-758

In this study, the synthesis and evaluation of a number of esters of CS-758 as injectable prodrugs are described. Phosphoryl ester 1a was soluble in water (>30 mg/mL) and was converted to CS-758 in human liver microsome. It was also converted to CS-758 in rats after iv administration, wherein the bioavailability of CS-758 was 53%. Compound 1a (iv) reduced the viable cell counts in kidneys in a murine systemic Candida albicans infection model, wherein the effect was comparable to or slightly superior to that of CS-758 (po). The prodrug 1a proved to be a promising injectable antifungal agent whose further evaluation is warranted.     

Varp Is a Novel Rab32/38-binding Protein That Regulates Tyrp1 Trafficking in Melanocytes

Two small GTPase Rabs, Rab32 and Rab38, have recently been proposed to regulate trafficking of melanogenic enzymes to melanosomes in mammalian epidermal melanocytes; however, the exact molecular mechanism of Rab32/38-mediated transport of melanogenic enzymes has never been clarified, because no Rab32/38-specific effector has ever been identified. In this study, we screened for a Rab32/38-specific effector by a yeast two-hybrid assay using a guanosine triphosphate (GTP)-locked Rab32/38 as bait and found that VPS9-ankyrin-repeat protein (Varp)/Ankrd27, characterized previously as a guanine nucleotide exchange factor (GEF) for Rab21, functions as a specific Rab32/38-binding protein in mouse melanocyte cell line melan-a. Deletion analysis showed that the first ankyrin-repeat (ANKR1) domain functions as a GTP-dependent Rab32/38-binding domain, but that the N-terminal VPS9 domain (i.e., Rab21-GEF domain) does not. Small interfering RNA-mediated knockdown of endogenous Varp in melan-a cells caused a dramatic reduction in Tyrp1 (tyrosinase-related protein 1) signals from melanosomes but did not cause any reduction in Pmel17 signals. Furthermore, expression of the ANKR1 domain in melan-a cells also caused a dramatic reduction of Tyrp1 signals, whereas the VPS9 domain had no effect. Based on these findings, we propose that Varp functions as the Rab32/38 effector that controls trafficking of Tyrp1 in melanocytes.