Dense-core vesicles and non-synaptic exocytosis in the central body of the crayfish brain

Summary The central body in the median protocerebrum of the brain of the crayfish Cherax destructor is a distinctive area of dense neuropile, the nerve fibres of which contain three main types of vesicles: electronlucent vesicles (diameter 35 nm), dense-core vesicles (diameter 64 nm), and large structured dense-core vesicles (diameter 98 nm, maximum 170 nm). Different vesicle types were found together in the same neurons. Electronlucent vesicles were seen at presynaptic sites and rarely observed in the state of exocytosis. Exocytosis of densecore and structured dense-core vesicles was a regular feature on non-synaptic release sites either close to, or at some distance from pre- and subsynaptic sites. Non-synaptic exocytotic sites are more often observed than chemical synapses. Different forms of exocytosis seen at non-synaptic sites included the release of single densecore vesicles, packets of dense-core vesicles, and rows of dense-core vesicles lined up along cell membranes and around fibre invaginations. Swelling and the enhanced electron density of extracellular non-synaptic spaces may mark the positions of prior exocytotic events. In vitro treatment of the brain with tannic acid buffer solution followed by conventional double fixation resulted in the augmentation of non-synaptic exocytosis. Electron microscopy of proctolin- and serotonin-immunoreactive nerve fibres shows them to contain dense-core and electron-lucent vesicles and to be surrounded by many unlabelled profiles similarly laden with dense-core vesicles and electron-lucent vesicles, indicating the presence of other, not yet identified, neuroactive compounds.     

Mitochondrial mutations restricting spontaneous translational frameshift suppression in the yeast Saccharomyces cerevisiae.

Summary The +1 frameshift mutation, M5631, which is located in the gene (oxi1) for cytochrome c oxidase II (COXII) of the yeast mitochondrial genome, is suppressed spontaneously to a remarkably high extent (20%–30%). The full-length wild-type COXII produced as a result of suppression allows the mutant strain to grow with a leaky phenotype on non-fermentable medium. In order to elucidate the factors and interactions involved in this translational suppression, the strain with the frameshift mutation was mutated by MnCl₂ treatment and a large number of mutants showing restriction of the suppression were isolated. Of 20 mutants exhibiting a strong, restricted, respiration-deficient (RD) phenotype, 6 were identified as having mutations in the mitochondrial genome. Furthermore, genetic analyses mapped one mutation to the vicinity of the gene for tRNA^Pro and two others to a region of the tRNA cluster where two-thirds of all mitochondrial tRNA genes are encoded. The degree of restriction of the spontaneous frameshift suppression was characterized at the translational level by in vivo ³⁵S-labeling of the mitochondrial translational products and immunoblotting. These results showed that in some of these mutant strains the frameshift suppression product is synthesized to the same extent as in the leaky parent strain. It is suggested that more than one +1 frame-shifted product is made as a result of suppression in these strains: one is as functional as the wild-type COXII, the other(s) is (are) non-functional and prevent leaky growth on non-fermentable medium. A possible mechanism for this heterogenous frameshift suppression is discussed.     

An unusual cell in the central nervous system of Hirudo medicinalis L.: a neuron with ribbons and flags

Summary A neuron (cell 151) with the ability to silence efferent activity in the roots of a leech segmental ganglion was filled with horseradish peroxidase and studied by light-and electron microscopy. The neurites of cell 151 penetrate all areas of the neuropile except for a thin ventral layer. The branching pattern of the secondary neurites is highly variable. Post-and presynaptic structures of chemical synapses with clear vesicles of 25 nm diameter were identified. Neurites are mostly wrapped in glia and run in bundles among other axons. They frequently form ribbons which are 20–40 nm thick, extend several microns away from the dendrite and are followed up to 3 m in depth. They also form flags which are 0.5 m thick, spread out 10–20 m horizontally and run up to 80 m laterally. Both structures lie adjacent to or wrap around axons of other neurons, forming a gap of 8–10 nm. Flags and ribbons are typical for glia but have not been described previously as structures of neurons. Contralateral cells 151 appose each other in the commissures with a gap of 5–10 nm. The possible functional significance of these findings is discussed with respect to electrical coupling and to reception of strain.     

Non-destructive assay systems for detection of β-glucuronidase activity in higher plants

β-glucuronidase (GUS) can be qualitatively assayed in seedlings and fully grown plants without injury or irreversible damage by short term incubations in X-gluc or by spraying 4-MUG.     

Trypsin activity in the small intestine of rats after application of copper and zinc

In vivo experiments with Sprague-Dawley rats were conducted in order to explore the influence of Cu²⁺, Zn²⁺ as well as of the combinations of both on the activity of trypsin. The solutions of the trace elements were given per os, the animals were killed 30 min after the applications, and the activity of trypsin was determined in the juice of the small intestine by usingN ^α-benzoyl-L-arginine-p-nitroanilide (L-BAPA) as the substrate. The activity of trypsin depends on the concentration of the trace elements. When Cu²⁺ ions are applied, there is a minimum activity at 10⁻⁵ mol Cu²⁺/L and a maximum at 10⁻⁴ mol Cu²⁺/L. When giving Zn²⁺ ions, a minimum of trypsin activity is found at 10⁻⁵ mol Zn²⁺/L and a maximum at 5×10⁻⁶ mol Zn²⁺/L. On the whole, the trypsin activity is lower when the Cu²⁺/Zn²⁺ combinations are applied compared to the addition of the single trace elements. On principle, a good conformity of the in vivo results was found with in vitro results.     

Ribosomes in thiostrepton-resistant mutants of Bacillus megaterium lacking a single 50 S subunit protein.

A protein required for the binding of thiostrepton to ribosomes of Bacillus megaterium has been purified and further characterized by immunological techniques. This protein, which does not bind the drug off the ribosome, is serologically-homologous to Escherichia coli ribosomal protein L11 and is designated BM-L11. Ribosomes from certain thiostrepton-resistant mutants of B. megaterium appear to be totally devoid of protein BM-L11 as judged by modified immunoelectrophoresis. Such ribosomes are significantly less sensitive than those from wild-type organisms to the action of thiostrepton in vitro but retain substantial protein synthetic activity. Re-addition of protein BM-L11 to ribosomes from the mutants restores them to wild-type levels of activity and thiostrepton sensitivity. Thus ribosomal protein BM-L11 is involved not only in binding thiostrepton but also in determining the thiostrepton phenotype.     

Protein synthesis and transport by the rat choroid plexus and ependyma

Summary Light (LM-ARG) and electron microscope (EM-ARG) autoradiographs were prepared from immature rat choroid plexus and ependyma at 5, 10, 30, and 60 min and 16 h following intraperitoneal administration of [³H]- labeled amino acid mixtures. Intracellular protein synthesis and transport were ascertained in lateral and fourth ventricle choroid plexus epithelium by quantitative EM-ARG at the several post-injection intervals. ARG were also prepared from choroid plexuses cultured for one day, pulse labeled for one hour and reincubated for various periods in nonradioactive media. Significant labeling of both attached and free apical protrusions (blebs) was observed in both choroid plexus and ependyma in vivo and in choroid plexus in vitro. This phenomenon was interpreted as a physiologically significant mechanism for protein transport (apocrine secretion) by epithelia into the cerebrospinal fluid (CSF).This study was supported in part by N.I.H. Research Grant NS 12906     

Licht- und elektronenmikroskopische Untersuchungen am Radulakomplex und zur Radulabildung vonBiomphalaria glabrata Say (=Australorbis gl.) (Gastropoda,Basommatophora)

Zusammenfassung Biomphalaria glabrata besitzt eine Prä- und eine Postradulatasche, sowie eine Sperrkutikula. Das Radulapolster besteht nicht aus Knorpel, sondern aus großen Zellen, die durch zahlreiche Vesikel, Mitochondrien, sowie durch peripher liegende Muskelfasern gekennzeichnet sind, während andere Zellen große Mengen Glykogen speichern. Die Odontoblasten sind charakterisiert durch ungewöhnlich lange Mikrovilli, die bis in die neugebildeten Radulazähne ragen. Die Zahnbildung beginnt über den hinteren Odontoblasten, die zunächst nur kurze Mikrovilli aufweisen. Das Aufrichten eines neugebildeten Zahns dürfte dadurch zustande kommen, daß die Mikrovilli länger werden. Zwischen den Mikrovilli befindet sich elektronendichtes Material, in dem Mikrofibrillen entstehen; diese dürften Chitin enthalten. Die Verflechtung der Mikrofibrillenbündel im ausgebildeten Zahn entspricht offensichtlich der komplizierten Anordnung der langen Mikrovilli während der Zahnbildung. Die Mikrovilli werden schließlich in den neugebildeten Zahn und die Radulamembran integriert. Mehrere Odontoblasten sezernieren gemeinsam einen Radulazahn. Die Radulamembran wird vorwiegend oder ausschließlich vom vordersten Odontoblasten sezerniert. Die Zellen des Deckepithels umschließen die Radulazähne; die sogenannte Sekrethöhle dürfte ein Artefakt sein. Zwischen Deckepithel und Zahn befindet sich elektronendichtes Material, das dem Zahn nicht aufgelagert wird, sondern in die Zähne eingelagert werden dürfte. Die Zellen des Basalepithels zeigen starke sekretorische Aktivität; die Sekrete dürften in Radulamembran und -zähne eingelagert werden.

---

Light and electron microscopic investigations on the radula complex and radula formation ofBiomphalaria glabrata say (=Australorbis gl.) (Gastropoda, Basommatophora)

Summary Biomphalaria (Australorbis) glabrata has a preradular as well as a postradular pocket and a collostyle hood. The odontophore cartilage does not consist of cartilage, but of cells which are characterized by numerous vesicles, mitochondria, and muscle fibres in the periphery; other cells contain large amounts of glycogen. The odontoblasts are characterized by unusually long microvilli which reach into the newly formed radula teeth. The formation of a tooth begins above the posterior odontoblast which has at first only short microvilli. The tooth seems to be raised by the extension of these microvilli. Microfibrils are formed in the electron dense material which is present in the small space between the microvilli; probably these microfibrils contain chitin. Obviously the interlacing of the bundles of microfibrils in a tooth corresponds with the complex arrangement of the long microvilli during formation of the tooth. Finally the microvilli are integrated into the newly formed tooth and radular membrane. Several odontoblasts join to form a single tooth. The radular membrane is secreted mainly or exclusively by the most anterior odontoblast. The cells of the superior epithelium surround the radula teeth. The so-called secretion cavity seems to be an artifact. Electron dense material is present between teeth and superior epithelium which is not apposed to but seems to be integrated into the teeth. The cells of the inferior epithelium show considerable secretory activity; the secretions seem to be incorporated into radular teeth and membrane.