Acetylcholine, ATP, and Proteoglycan Are Common to Synaptic Vesicles Isolated from the Electric Organs of Electric Eel and Electric Catfish as Well as from Rat Diaphragm

Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9-12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immunocytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.     

A hybrid DNA sequence containing the replication origin of the multicopy yeast plasmid 2 micron circle and an additional repeated sequence can convert maltose-negative into maltose-positive strains

Summary Yeast DNA pools were prepared by ligating partial Sau3A genomic digests from strains carrying various MAL genes into the BamHI site of the yeast-Escherichia coli shuttle vector YRp7. They were used to transform recipient yeast strains that could not utilize maltose since they lacked a classical MAL gene. Transformants were obtained that could use maltose and also formed normal levels of maltase. They were unstable. They would lose the selective marker TRP1 of YRp7 alone, together with the ability to utilize maltose or only the ability to utilize maltose. The insertion of one of the plasmids was used as a hybridization probe for the others and found to share homologous sequences with all. They were then shown to contain the replication origin of the yeast 2 m circle plasmid and additional genomic digests of total yeast DNA. They hybridized at various degrees of efficiency with several bands, indicating that they were part of a family of repeated sequences. Apparently, it was the combination of the replication origin of the 2 m circles with the additional sequences that promoted maltose utilization.     

Yeast mutants without phosphofructokinase activity can still perform glycolysis and alcoholic fermentation

Summary Mutants of Saccharomyces cerevisiae without detectable phosphofructokinase activity were isolated. They were partly recessive and belonged to two genes called PFK1 and PFK2. Mutants with a defect in only one of the two genes could not grow when they were transferred from a medium with a nonfermentable carbon source to a medium with glucose and antimycin A, an inhibitor of respiration. However, the same mutants could grow when antimycin A was added to such mutants after they had been adapted to the utilization of glucose. Double mutants with defects in both genes could not grow at all on glucose as the sole carbon source. Mutants with a single defect in gene PFK1 or PFK2 could form ethanol on a glucose medium. However, in contrast to wild-type cells, there was a lag period of about 2 h before ethanol could be formed after transfer from a medium with only nonfermentable carbon sources to a glucose medium. Wild-type cells under the same conditions started to produce ethanol immediately. Mutants with defects in both PFK genes could not form ethanol at all. Mutants without phosphoglucose isomerase or triosephosphate isomerase did not form ethanol either. Double mutants without phosphofructokinase and phosphoglucose isomerase accumulated large amounts of glucose-6-phosphate on a glucose medium. This suggested that the direct oxidation of glucose-6-phosphate could not provide a bypass around the phosphofructokinase reaction. On the other hand, the triosephosphate isomerase reaction was required for ethanol production. Experiments with uniformly labeled glucose and glucose labeled in positions 3 and 4 were used to determine the contribution of the different carbon atoms of glucose to the fermentative production of CO₂. With only fermentation operating, only carbon atoms 3 and 4 should contribute to CO₂ production. However, wild-type cells produced significant amounts of radioactivity from other carbon atoms and pfk mutants generated CO₂ almost equally well from all six carbon atoms of glucose. This suggested that phosphofructokinase is a dispensable enzyme in yeast glycolysis catalyzing only part of the glycolytic flux.     

Zur Charakterisierung neuronaler und gliöser Elemente im Epithel des Saccus vasculosus von Knochenfischen

Zusammenfassung Das Epithel des Saccus vasculosus des Flußbarsches Perca fluviatilis besteht aus Krönchenzellen, bipolaren Liquorkontaktneuronen (Zahlenverhältnis etwa 41) und Stützzellen. Im Bereich des Saccus kommen Macrophagen vor. Die Krönchenzellen wurden unter verschiedenen Fixierungsbedingungen untersucht. Die Globuli enthalten schlauchförmige Zisternen, die nicht mit den Zisternen des Zellapex in Verbindung stehen. Im Zytoplasma des Zellapex und der Globuli wird bei langdauernder OsO₄-Imprägnation Osmium gebunden. Die Krönchenzellen werden basal von Ausläufern der Stützzellen unterlagert. Sie werden nicht innerviert und entsenden keine Axone.Die bipolaren Neurone sind durch einen im Liquor endigenden Dendriten und ein Axon gekennzeichnet, das in eines der Faserbündel des Epithels eintritt. Der Dendrit trägt 1 bis 2 Zilien. Die Zelle ist reich an Vesikeln und kann am Perikaryon wie an den Ausläufern Synapsen tragen. Im extrazellulären Raum um die Neurone und in vesikulären Strukturen des Apex wird Acetylcholinesterase nachgewiesen.Der Nervus sacci vasculosi dürfte nur afferente Axone von Liquorkontaktneuronen und efferente Fasern, die diese innervieren, enthalten.

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Neuronal and glial cell elements within the epithelium of the Saccus vasculosus in teleosts

Summary The epithelium of the Saccus vasculosus of Perca fluviatilis consists of coronet cells and bipolar liquor contact neurones in a 41 ratio, and supporting cells. The organ also contains macrophages.The coronet cells have been studied after different kinds of fixation. The globules of these cells contain tubelike cisternae, which do not connect with cisternae in the cell's apical protrusion. The cytoplasm of the apical protrusion and to a greater extent of the globules, is stained by longlasting OsO₄-impregnation. The coronet cells have no direct contact with the basement membrane of the organ. They are neither innervated nor have axons.The dendrites of the bipolar nerve cells end with a bulbous structure protruding into the cerebrospinal fluid. The dendrites contain vesicles and each bears 1–2 cilia. The axons join the fiber bundles of the epithelium. There are synaptic contacts on the surface of the neurons and their processes. In some vesicular structures within the apices and more conspicuously within the extracellular space around these cells indications of acetylcholinesterase activity are found.It appears that the nervus sacci vasculosi contains only afferent axons of the bipolar liquor-contact neurons and efferent fibres which form synaptic contacts with these neurons.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Mutagen specificity in the induction of karyotic versus cytoplasmic respiratory deficient mutants in yeast by nitrous acid and alkylating nitrosamides

Summary In the haploid eukaryotic organism Saccharomyces cerevisiae the induction of cytoplasmic and genic (karyotic) RD mutants was studied, using nitrous acid, nitrosomethylurethane (NMU) and nitrosoimidazolidone (NIL).The cytoplasmic or genic origin of the induced RD mutants was determined by prescreening in complementation tests with ^– and wild type tester strains. Among the mutants of all three agents we could thus score the incidence of three RD mutant types: genic, suppressive and cytoplasmic (both primary and secondary). The final identification of the cytoplasmic type was only possible through tetrad analysis, performed in the cases of HNO₂ and NMU.A distinct difference in cytoplasmic versus genic mutagen specificity was observed between HNO₂ and NMU. HNO₂ was unable to induce cytoplasmic RD mutants but it proved to be highly efficient in the induction of genic RD mutants. In contrast, NMU induced more cytoplasmic effects was it possible to detect mutagenic specificities which, solely on the basis of karyotic action, were not detectable.     

Modelluntersuchungen zur Struktur des purpurnen Farbstoffkomplexes der Giemsa-Färbung

Summary Nuclei of Giemsa stained cells show a purple coloration, which is generated by a complex of DNA, azure B (AB) and eosin Y (EY). The structure of this complex is unknown. Its absorption spectrum shows a sharp and strong band at 18 100 cm^–1 (552 nm), the so called Romanowsky band (RB). It is possible to produce the complex outside of the cell, but it is cubersome to handle. Easier to handle is a purple complex composed of chondroitin sulfate (CHS), AB and EY, which also shows a sharp and strong RB at 18100 cm^–1 in the absorption spectrum. This CHS-AB-EY complex is a model for the DNA-AB-EY complex of Giemsa stained cell nuclei. We tried to investigate its structure.In the first step of the staining procedure CHS binds AB cations forming a stable CHS-AB complex. In the case of saturation each anionic SO ₄ ^– - and COO^–-binding site of CHS is occupied by one dye cation and the complex has 1:1 composition. It has a strong and broad absorption band with its maximum at ca. 18000 cm^–1 (556 nm). In the second step the CHS-AB complex additionally binds EY dianions forming the purple CHS-AB-EY complex with its RB at 18100 cm^–1. This band can be clearly distinguished from the broad absorption of the bound AB cations. RB is generated by the EY chromophore, whose absorption is shifted to longer wavelength by the interaction with the CHS-AB framework.The CHS chains of the CHS-AB and CHS-AB-EY complexes can be mechanically aligned in a preferred direction k. Fine films of highly orientated complexes were prepared with a special technique and studied with a microspectrophotometer equipped with a polarizer and an analyzer. They are birefringent and dichroic-the more birefringent, the better the mechanical orientation. The sites of best orientation within the film were selected according to the quality of the birefringence. We measured the absorption of these regions with linearly polarized light. By setting the polarizer (e _p parallel () or perpendicular () to k, we found that the transition moment m _AB of the long wave-length absorption of AB in the CHS-AB and the CHS-AB-EY complexes is polarized almost perpendicular to the preferred direction k, m _AB k. But the transition moment m _EY of EY in CHS-AB-EY is polarized parallel to k, m _EY k. The transition moments m _AB and m _EY lay in the molecular plane in the direction of the long axes of the AB and EY chromophores, respectively. Therefore, in both CHS-AB and CHS-AB-EY the long axes of the AB molecules are approximately perpendicular to the CHS chain; but in CHS-AB-EY the long axes of the EY chromophore are parallel to the chain of the biopolymer. This structure is somewhat surprising. In the CHS-AB-EY dye complex the chromophores of AB and EY are not parallel but approximately perpendicular to each other.     

Intra- and interspecies analyses of the carcinoembryonic antigen (CEA) gene family reveal independent evolution in primates and rodents

Summary Various rodent and primate DNAs exhibit a stronger intra- than interspecies cross-hybridization with probes derived from the N-terminal domain exons of human and rat carcinoembryonic antigen (CEA)-like genes. Southern analyses also reveal that the human and rat CEA gene families are of similar complexity. We counted at least 10 different genes per human haploid genome. In the rat, approximately seven to nine different N-terminal domain exons that presumably represent different genes appear to be present. We were able to assign the corresponding genomic restriction endonuclease fragments to already isolated CEA gene family members of both human and rat. Highly similar subgroups, as found within the human CEA gene family, seem to be absent from the rat genome. Hybridization with an intron probe from the human nonspecific cross-reacting antigen (NCA) gene and analysis of DNA sequence data indicate the conservation of noncoding regions among CEA-like genes within primates, implicating that whole gene units may have been duplicated. With the help of a computer program and by calculating the rate of synonymous substitutions, evolutionary trees have been derived. From this, we propose that an independent parallel evolution, leading to different CEA gene families, must have taken place in, at least, the primate and rodent orders.     

A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.     

Isolation and expression in Escherichia coli of hepB and hepC, genes coding for the glycosaminoglycan-degrading enzymes heparinase II and heparinase III, respectively, from Flavobacterium heparinum.

Upon induction with heparin, Flavobacterium heparinum synthesizes and secretes into its periplasmic space heparinase I (EC 4.2.2.7), heparinase II, and heparinase III (heparitinase; EC 4.2.2.8). Heparinase I degrades heparin, and heparinase II degrades both heparin and heparan sulfate, while heparinase III degrades heparan sulfate predominantly. We isolated the genes encoding heparinases II and III (designated hepB and hepC, respectively). These genes are not contiguous with each other or with the heparinase I gene (designated hepA). hepB and hepC were found to contain open reading frames of 2,316 and 1,980 bp, respectively. Enzymatic removal of pyroglutamate groups permitted sequence analysis of the amino termini of both mature proteins. It was determined that the mature forms of heparinases II and III contain 746 and 635 amino acids, respectively, and have calculated molecular weights of 84,545 and 73,135, respectively. The preproteins have signal sequences consisting of 26 and 25 amino acids. Truncated hepB and hepC genes were used to produce active, mature heparinases II and III in the cytoplasm of Escherichia coli. When these enzymes were expressed at 37 degrees C, most of each recombinant enzyme was insoluble, and most of the heparinase III protein was degraded. When the two enzymes were expressed at 25 degrees C, they were both present predominantly in a soluble, active form.