Calcium dynamics associated with a single action potential in a CNS presynaptic terminal.

Calcium dynamics associated with a single action potential were studied quantitatively in the calyx of Held, a large presynaptic terminal in the rat brainstem. Terminals were loaded with different concentrations of high- or low-affinity Ca2+ indicators via patch pipettes. Spatially averaged Ca2+ signals were measured fluorometrically and analyzed on the basis of a single compartment model. A single action potential led to a total Ca2+ influx of 0.8-1 pC. The accessible volume of the terminal was about 0.4 pl; thus the total calcium concentration increased by 10-13 microM. The Ca(2+)-binding ratio of the endogenous buffer was about 40, as estimated from the competition with Fura-2, indicating that 2.5% of the total calcium remained free. This is consistent with the peak increase in free calcium concentration of about 400 nM, which was measured directly with MagFura-2. The decay of the [Ca2+]i transients was fast, with time constants of 100 ms at 23 degrees C and 45 ms at 35 degrees C, indicating Ca2+ extrusion rates of 400 and 900 s-1, respectively. The combination of the relatively low endogenous Ca(2+)-binding ratio and the high rate of Ca2+ extrusion provides an efficient mechanism for rapidly removing the large Ca2+ load of the terminal evoked by an action potential.     

Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA. II. The organization of sequences in petite mitochondrial DNAs carrying genetic markers from the 21S region.

We have investigated the organization of sequences in ten rho- petite mtDNAs by restriction enzyme analysis and electron microscopy. From the comparison of the physical maps of the petite mtDNAs with the physical map of the mtDNA of the parental rho+ strain we conclude that there are at least three different classes of petite mtDNAs: I. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA. II. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA with a terminal inverted duplication. III. Mixed repeats of an (almost) continuous rho+ mtDNA segment. In out petite mtDNAs of the second type, the inverted duplications do not cover the entire conserved rho+ mtDNA segment. We have found that the petite mtDNAs of the third type contain a local inverted duplication at the site where repeating units can insert in two orientations. At least in one case this local inverted duplication must have arisen by mutation. The rearrangements that we have found in the petite mtDNAs do not cluster at specific sites on the rho+ mtDNA map. Large rearrangements or deletions within the conserved rho+ mtDNA segment seem to contribute to the suppressiveness of a petite strain. There is also a positive correlation between the retention of certain segments of the rho+ mtDNA and the suppressiveness of a petite strain. We found no correlation between the suppressiveness of a petite strain and its genetic complexity. The relevance of these findings for the mechanism of petite induction and the usefulness of petite strains for the physical mapping of mitochondrial genetic markers and for DNA sequence analysis are discussed.     

The kinetoplast DNA of Trypanosoma equiperdum

We have analyzed the kinetoplast DNA for Trypanosoma equiperdum (American Type Culture Collection 30019) and two dyskinetoplastic strains derived from it. The DNA networks from the kinetoplastic strain are made up of catenated mini-circles and maxi-circles, like the networks from the closely-related Trypanosoma brucei. The mini-circles of T. equiperdum lack the pronounced sequence heterogeneity of T. brucei mini-circles, as shown by the fragment distribution of restriction digests and by the predominance of well-matched duplexes in electron micrographs of renatured DNA. The electrophoretic analysis of kinetoplast DNA digested with various restriction endonucleases shows the maxi-circle of T. equiperdum to consist of circular DNA molecules of 8.4 x 10(6) daltons, without size or sequence heterogeneity or repetitious segments. A comparison of the sequence by restriction endonuclease fragmentation and hybridization shows extensive sequence homology. The size difference between both maxi-circles is due to the deletion of one continuous segment of 5.10(6) daltons. In the two dyskinetoplastic strains, we cannot detect DNA sequences that hybridize with kinetoplast DNA from T. brucei or from the kinetoplastic strain of T. equiperdum. In one of these strains, a 'low-density' DNA fraction contained a simple sequence DNA, cleaved by restriction endonuclease HindIII into fragments of 180 base-pairs and multimers of this. The relation of this DNA to kinetoplast DNA, if any, is unknown.     

Ortstreue, Altersaufbau und Mortalit?t einer Population des Baumpiepers(Anthus t. trivialis)

Zusammenfassung 1. Von den in einer intensiv untersuchten Population des Baumpiepers in Nordbelgien beringten Nestlingen kehrten 24 junge und 3 junge zurück. 12 von 17 farbberingten Jungvögeln stammten dabei aus Erstbruten.2. 6 und die beiden waren Heimatansiedler, 1 Fremdansiedler, 6 geburtsortstreu und 2 Geburtsorts-Rücksiedler.3. Die mittlere Entfernung des Revieres vom Geburtsnest betrug bei den geburtsortstreuen 184±118 m, bei den anderen 818±368 m.4. Alte zeigen größere Umsiedlungsentfernungen als alte .5. Einjährige haben in der Population einen sehr hohen Anteil.6. 79 % der und 50 % der kehrten wieder in die Population zurück.7. Die Population umfaßte durchschnittlich 42 Brutpaare. Der Anteil an ledigen Altvögeln betrug 7 %. Die Siedlungsdichte erreichte einen Wert von 3,6 Brutpaaren pro 10 ha (ohne Gewässer).8. Angaben zu Paartreue, Paarauflösung, Bigynie und Paarbildung werden mitgeteilt.9. Aus den Rückkehrzahlen errechnet sich eine durchschnittliche Mortalität der jungen von etwa 65 %, eine Lebenserwartung von einem Jahr und ein Durchschnittsalter von 1,5 Jahren.10. 2,5 % der gelegten Eier bzw. 4,6 % der ausgeflogenen Jungvögel erbrachten brutreife Rückkehrer.11. Für alte errechnet sich eine Mortalität von 47,5 %, für alte von 66,7 % und eine Lebenserwartung von 1,6 bzw. 1,0 Jahren.12. Zum Erhalt der Population müssen jährlich etwa 46 % der ausgeflogenen Jungvögel bis zum nächsten Jahr überleben.13. Die untersuchte Population ist durchschnittlich aus 4,6 % geburtsortstreuen Jungvögeln, 41,3 % fremden Jungvögeln, 37,4 % ortstreuen Altvögeln und 16,7 % unbekannter Altvögel zusammengesetzt.

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Site-tenacity, age structure and mortality in a population of the tree pipit(Anthus t. trivialis) in northern Belgium

Summary 1. 24 first year and 3 first year , ringed as nestlings, returned in next years. 12 of 17 colour-ringed birds came from first broods.2. 6 and the 2 were Heimatansiedler, 1 Fremdansiedler, 6 geburtsortstreu and 2 Geburtsorts-Rücksiedler.3. The mean distances between the first territory and the birth nest of the geburtsortstreue and of the other were 818±368 m and 184±118 m respectively.4. Adult showed greater settling distances than adult .5. The percentage of first year was very high.6. 79 % and 50 % returned to their breeding population in next years.7. The mean density of the population was 42 pairs or 3,6 pro 10 ha. Unpaired adults amounted 7 %.8. Data on pair formation, mate-faithfulness and bigyny are treated.9. Calculation of mortality from the data of returned birds yielded a mortality of young of 65 %, a life expectancy of one year and a mean life time of 1,5 years.10. Sexual mature individuals derived from 2,5 % of all eggs laid and from 4,6 % of all youngs fledged.11. The computed mortality of adult is 47,5 % and of adult 66,7 %. The life expectancy is 1,6 and 1,0 years for adult and respectively.12. Allowing for the annual losses of adults a survival rate of first year birds of about 46 % is necessary.13. The mean annual composition of the population should be: 4,6 % geburtsortstreue juveniles, 41,3 % non-autochthonous juveniles, 37,4 % ortstreue adults and 16,7 % unknown adults.

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Mit Unterstützung des Nat. Fonds v. Wetenschappelijk Onderzcek, Brüssel.     

Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein

Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation.     

Multidrug resistance-associated protein 9 (ABCC12) is present in mouse and boar sperm

In order to search for novel components of lipid membrane microdomains involved in neural signalling pathways, mAbs (monoclonal antibodies) were raised against the detergent-insoluble membrane fraction of PC12 (pheochromocytoma) cells. Among the 22 hybrid clones, mAb PR#1 specifically detected a fucoganglioside Fuc(Gal)-GM1 [a-fucosyl(a-galactosyl)-GM1], a ganglioside homologous with GM1a (II3NeuAc,GgOse4Cer), as a novel member of microdomain components with biological functions. In the presence of mAb PR#1 in the culture medium, the outgrowth of neurites was induced in PC12 cells in a dose-dependent manner, with no effects on cell proliferation, suggesting that Fuc(Gal)-GM1 is preferentially involved in PC12 cell neuritogenesis. Effects through Fuc(Gal)-GM1 were different from those through GM1a during differentiation, e.g. under PR#1 treatment on Fuc(Gal)-GM1, round cell bodies with thinner cell processes were induced, whereas treatment with CTB (cholera toxin B subunit), a specific probe for GM1a, produced flattened cell bodies with thicker pro-cesses. Molecular analysis demonstrated that the PR#1-Fuc(Gal)-GM1 pathway was associated with Fyn and Yes of the Src family of kinases, although Src itself was not involved. No association was found with TrkA (tropomyosin receptor kinase A) and ERKs (extracellular-signal-regulated kinases), which are responsible for GM1a-induced differentiation. From these findings, it is suggested that a fucoganglioside Fuc(Gal)-GM1 provides a functional platform distinct from that of GM1a for signal transduction in PC12 cell differentiation.     

Lung response to <Emphasis Type="Italic">Bordetella pertussis</Emphasis> infection in mice identified by gene-expression profiling

Host genetics determines the course of Bordetella pertussis infection in mice. Previously, we found four loci, Tlr4 and three novel loci, designated Bps 1–3, that are involved in the control of B. pertussis infection. The purpose of the present study was to identify candidate genes that could explain genetic differences in the course of B. pertussis infection, assuming that such genes are differentially regulated upon infection. We, therefore, studied the course of mRNA expression in the lungs after B. pertussis infection. Of the 22,000 genes investigated, 1,841 were significantly differentially expressed with 1,182 genes upregulated and 659 genes downregulated. Upregulated genes were involved in immune-related processes, such as the acute-phase response, antigen presentation, cytokine production, inflammation, and apoptosis, while downregulated genes were mainly involved in nonimmune processes, such as development and muscle contraction. Pathway analysis revealed the involvement of granulocyte function, toll-like receptor signaling pathway, and apoptosis. Nine of the differentially expressed genes were located in Bps-1, 13 were located in Bps-2, and 62 were located in Bps-3. We conclude that B. pertussis infection induces a wide and complex response, which appears to be partly specific for B. pertussis and partly nonspecific. We envisage that these data will be helpful in identifying polymorphic genes that affect the susceptibility and course of B. pertussis infection in humans. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Raw and normalized data of the experiment can be accessed at the online database ArrayExpress http://www.ebi.ac.uk/arrayexpress/