Functional specialization of presynaptic Cav2.3 Ca2+ channels

Ca2+ influx into presynaptic terminals via voltage-dependent Ca2+ channels triggers fast neurotransmitter release as well as different forms of synaptic plasticity. Using electrophysiological and genetic techniques we demonstrate that presynaptic Ca2+ entry through Cav2.3 subunits contributes to the induction of mossy fiber LTP and posttetanic potentiation by brief trains of presynaptic action potentials while they do not play a role in fast synaptic transmission, paired-pulse facilitation, or frequency facilitation. This functional specialization is most likely achieved by a localization remote from the release machinery and by a Cav2.3 channel-dependent facilitation of presynaptic Ca2+ influx. Thus, the presence of Cav2.3 channels boosts the accumulation of presynaptic Ca2+ triggering presynaptic LTP and posttetanic potentiation without affecting the low release probability that is a prerequisite for the enormous plasticity displayed by mossy fiber synapses.     

The role of Suppressor of Hairless in Notch mediated signalling during zebrafish somitogenesis

Suppressor of Hairless (Su(H)) codes for a protein that interacts with the intracellular domain of Notch to activate the target genes of the Delta-Notch signalling pathway. We have cloned the zebrafish homologue of Su(H) and have analysed its function by morpholino mediated knockdown. While there are at least four notch and four delta homologues in zebrafish, there appears to be only one complete Su(H) homologue. We have analysed the function of Su(H) in the somitogenesis process and its influence on the expression of notch pathway genes, in particular her1, her7, deltaC and deltaD. The cyclic expression of her1, her7 and deltaC in the presomitic mesoderm is disrupted by the Su(H) knockdown mimicking the expression of these genes in the notch1a mutant deadly seven. deltaD expression is similarly affected by Su(H) knockdown like deltaC but shows in addition an ectopic expression in the developing neural tube. The inactivation of Su(H) in a fss/tbx24 mutant background leads furthermore to a clear breakdown of cyclic her1 and her7 expression, indicating that the Delta-Notch pathway is required for the creation of oscillation and not only for the synchronisation between neighbouring cells. The strongest phenotypes in the Su(H) knockdown embryos show a loss of all somites posterior to the first five to seven ones. This phenotype is stronger than the known amorphic phenotypes for notch1 (des) or deltaD (aei) in zebrafish, but mimicks the knockout phenotype of RBP-Jkappa gene in the mouse, which is the homologue of Su(H). This suggests that there is some functional redundancy among the Notch and Delta genes. This fact that the first five to seven somites are only weakly affected by Su(H) knockdown indicates that additional genetic pathways may be active in the specification of the most anterior somites.     

The circling behavior of the deafblind LEW-<Emphasis Type="Italic">ci2</Emphasis> rat is linked to a segment of RNO10 containing <Emphasis Type="Italic">Myo15</Emphasis> and <Emphasis Type="Italic">Kcnj12</Emphasis>

The LEW/Ztm-ci2 rat is an autosomal recessive mutant that displays circling behavior, deafness, progressive retinopathy, locomotor hyperactivity, ataxia, and opisthotonus. We performed a genome-wide scan of a (LEW/Ztm-ci2 × BN/Ztm) F₁ × LEW/Ztm-ci2 backcross population with anonymous microsatellite markers to analyze the genetics of this mutant rat. This linkage analysis demonstrated a very strong association of RNO10 SSLP markers to the phenotype with a core region in the central part of the chromosome. The knowledge of genes mapping to this part of the rat genome and their linkage to SSLP markers is still poor. We developed SSLP markers closely linked to genes, which might be responsible for the mutant phenotype by using the growing amount of rat-specific DNA sequences available at World Wide Web databases. Application of this method facilitated the search for candidate genes for the phenotype of the LEW-ci2 rat. We were able to map Myo15 and its neighboring genes, Znf179 and Aldh3a1, to the region of interest and Myo1c to a more distal location on RNO10. Further rat BAC clones were used to create a physical map of the region of interest. This map revealed the position of further genes. Among those is Kcnj12. Owing to their localization on RNO10 and their involvement in a similar pathology in human and mouse, Myo15 and Kcnj12 can be regarded as candidate genes for the deafblind phenotype of the LEW-ci2 rat.     

Phenylbutyrates as potent,orally bioavailable vitronectin receptor (integrin alphavbeta3) antagonists

In our continuing efforts to identify small molecule vitronectin receptor antagonists, we have discovered a series of phenylbutyrate derivatives, exemplified by 16, which have good potency and excellent oral bioavailability (approximately 100% in rats). This new series is derived conceptually from opening of the seven-membered ring of SB-265123.     

Influence of alterations in culture condition and changes in perfusion parameters on the retention performance of a 20 μm spinfilter during a perfusion cultivation of a recombinant CHO cell line in pilot scale

Since 1969 much attention has been devoted to the useof spinfilter systems for retention of mammalian cellsin continuous perfusion cultivations. Previousinvestigations dealt with hydrodynamic conditions,fouling processes and upscaling. But hydrodynamicconditions and fouling processes seem to have asecondary importance in spinfilter performance duringauthentic perfusion cultivations. Obviously,alterations in culture condition are more relevantespecially during long-term processes. Therefore, ourpratical approach focussed on the performance qualityof a commercially available 20 m spinfilterduring a perfusion cultivation of a recombinant CHOcell line in pilot scale regarding the followingissues: 1) retention of viable cells in thebioreactor; 2) removal of dead cells and cell debrisfrom the bioreactor; 3) alterations in culturecondition; and 4) changes in perfusion mode.Furthermore, we tested the performance of 20 mspinfilters in 2 and 100 l pilot scale using solidmodel particles instead of cells. Our investigationsshowed that retention of viable cells in pilot scalewas independent of spinfilter rotation velocity andperfusion rate; the retention increased from 75 to 95%corresponding to operation time, enlarging celldiameter and enhanced formation of aggregates in theculture during the perfusion cultivation. By means ofthe Cell Counter and Analyzer System (CASY) anoperation cut off of 13 m was determined forthis spinfilter. Using solid model particles in 2 lscale, optimal retention was achieved at a tip speedof 0.43 m s^-1 (141 rpm) – furtherenhancement of spinfilter rotation velocity up to0.56 m s^-1 (185 rpm) decreased the retentionrapidly. In pilot scale best retention performance wasobtained with tip speeds of 0.37 m s^-1(35 rpm) and 1.26 m s^-1 (120 rpm). Hence,significant retention in pilot scale could already beachieved with low agitation. Therefore, the additionof shear force protectives could be avoided so thatthe purification of the target protein from thesupernatant would be facilitated.     

PDGF receptor kinase blocker AG1295 attenuates interstitial fibrosis in rat kidney after unilateral obstruction

The current study was designed to investigate possible effects of the platelet-derived growth factor (PDGF) receptor kinase blocker AG1295 on the development of interstitial fibrosis in rats with unilateral ureteral obstruction (UUO), monitored by ED-A⁺ fibronectin expression, the number of macrophages, and the presence of myofibroblasts as visualized by immunohistochemistry with monoclonal antibodies (mAb) IST9, mAb ED1, and mAb 1A4, respectively; interstitial fibrosis was quantified by Sirius-Red staining and computer-aided image analysis. Without AG1295 treatment, the Sirius-Red stained area of the control kidneys comprised 6.8ǃ.3% of the totally inspected area and increased to 19.0ǃ.9% in animals by 14 days and to 23.4ǃ.7% by 21 days after UUO. The number of macrophages increased from 4.3ǃ.1 in controls to 16.6DŽ.6 in animals at 14 days and to 23.2dž.4 at 21 days after UUO. This was accompanied by an increase in both ED-A⁺ fibronectin deposition and !-smooth muscle actin expression. Treatment with AG1295 (12 mg/kg body weight, daily i.p.) significantly reduced interstitial fibrosis as verified by a smaller Sirius-Red stained area (15.7ǃ.9% in animals at 14 days and 17.0ǂ.7% at 21 days after UUO) and also by a reduced number of macrophages (12.8ǃ.4 in animals at 14 days and 15.5Dž.8 at 21 days after UUO), and by the ED-A⁺ fibronectin deposition and the number of cells positive for !-smooth muscle actin. The study indicates that the PDGF receptor kinase blocker AG1295 is able to decrease interstitial fibrosis in the rat UUO model significantly. The diminution of early fibrosis mediators, i.e., macrophages, ED-A⁺ fibronectin, and myofibroblast phenotype, points to a modulated fibrosis process via a blockade of PDGF actions.     

Self-Transmissible Mercury Resistance Plasmids with Gene-Mobilizing Capacity in Soil Bacterial Populations: Influence of Wheat Roots and Mercury Addition

A set of mercury resistance plasmids was obtained from wheat rhizosphere soil amended or not amended with mercuric chloride via exogenous plasmid isolation by using Pseudomonas fluorescens R2f, Pseudomonas putida UWC1, and Enterobacter cloacae BE1 as recipient strains. The isolation frequencies were highest from soil amended with high levels of mercury, and the isolation frequencies from unamended soil were low. With P. putida UWC1 as the recipient, the isolation frequency was significantly enhanced in wheat rhizosphere compared to bulk soil. Twenty transconjugants were analyzed per recipient strain. All of the transconjugants contained plasmids which were between 40 and 50 kb long. Eight selected plasmids were distributed among five groups, as shown by restriction digestion coupled with a similarity matrix analysis. However, all of the plasmids formed a tight group, as judged by hybridization with two whole-plasmid probes and comparisons with other plasmids in dot blot hybridization analyses. The results of replicon typing and broad-host-range incompatibility (Inc) group-specific PCR suggested that the plasmid isolates were not related to any previously described Inc group. Although resistance to copper, resistance to streptomycin, and/or resistance to chloramphenicol was found in several plasmids, catabolic sequences were generally not identified. One plasmid, pEC10, transferred into a variety of bacteria belonging to the β and γ subdivisions of the class Proteobacteria and mobilized as well as retromobilized the IncQ plasmid pSUP104. A PCR method for detection of pEC10-like replicons was used, in conjunction with other methods, to monitor pEC10-homologous sequences in mercury-polluted and unpolluted soils. The presence of mercury enhanced the prevalence of pEC10-like replicons in soil and rhizosphere bacterial populations.The potential use of genetically modified bacteria in agriculture has raised questions pertaining to the spread of introduced recombinant DNA through soil bacterial communities. Gene transfer in soil via conjugation has received much attention, and the focus of most studies has been the transfer and fate of introduced plasmids (6, 22, 27–29, 39). Under favorable conditions, in specific soil microhabitats, or under selection conditions, both self-transmissible and mobilizable plasmids present in introduced hosts can be transferred to introduced recipients, as well as to a variety of indigenous bacteria (15, 20, 27, 28, 33). In particular, rhizospheres of crop plants, such as wheat and sugar beet, provide conditions conducive to conjugal plasmid transfer between bacterial inhabitants (15, 36). When genetically modified bacteria are developed as inoculants for the rhizosphere, insertion of heterologous DNA into non-self-transmissible plasmids or the chromosome might restrict conjugal transfer of this DNA to members of the indigenous bacterial community. However, mobilizing or retromobilizing (33) plasmids present in indigenous soil bacteria could potentially still effect the transfer of the less mobile heterologous DNA via chromosome or plasmid mobilization, which may involve cointegration (9, 19, 31). Such plasmids might thus be responsible for the escape of heterologous DNA from genetically modified bacteria introduced into soil.There is a paucity of knowledge concerning the incidence of plasmids with mobilizing capacity in soils and rhizospheres, as well as concerning the effects of soil factors, such as stresses resulting from pollution or from natural causes (e.g., rhizosphere acidity), on plasmid prevalence and transfer (e.g., reference 38). Whereas it has been suggested that chemical stress often does not enhance plasmid incidence in selected soil bacterial populations (40), pollution in river water or mines (in particular mercury pollution) has been found to exert a selective (enhancing) effect (4, 13).Plasmids of environmental bacteria have classically been obtained by endogenous isolation procedures (20). Endogenous isolation implies that putative plasmid hosts with the phenotype of interest are isolated from soil, after which plasmids are extracted from pure cultures of these strains. On the other hand, pioneering studies performed with river stone epilithon (9) and later extended to soil and sediment (32) have shown that plasmids can be obtained directly from indigenous bacterial communities in new hosts by exogenous isolation. In this approach, plasmids are captured in selectable recipient strains by using mating between these strains and the total bacterial community obtained from an environmental sample. Following incubation, the mating mixture is plated with selection for the recipient and an additional marker gene presumedly located on a plasmid present in the indigenous bacteria (6). The advantage of the exogenous isolation procedure is that no culturing step is required in the mating, which thus allows isolation of plasmids from nonculturable hosts. Furthermore, plasmids are directly selected for their transfer capacity, in addition to the presence of a specific selectable marker.In this study, exogenous plasmid isolation was employed to obtain transferable plasmids from soil bacteria by using mercury resistance as the selectable marker. The objective of this work was to gain insight into the potential present in soil bacterial populations to (retro)mobilize genes out of introduced bacteria into members of the soil bacterial community. Since the incidence of plasmids in soil bacteria is likely influenced by soil ecological factors and selection pressure, the presence of wheat roots and selection by mercury (25) were studied as experimental variables.