Odours stimulate neuronal activity in the dorsolateral area of the hippocampal formation during path integration

The dorsolateral area of the hippocampal formation of birds is commonly assumed to play a central role in processing information needed for geographical positioning and homing. Previous work has interpreted odour-induced activity in this region as evidence for an ‘olfactory map’. Here, we show, using c-Fos expression as a marker, that neuronal activation in the dorsolateral area of the hippocampal formation of pigeons is primarily a response to odour novelty, not to the spatial distribution of odour sources that would be necessary for an olfactory map. Pigeons exposed to odours had significantly more neurons activated in this area of the brain than pigeons exposed to filtered air with odours removed. This increased activity was observed only in response to unfamiliar odours. No change in activity was observed when pigeons were exposed to home odours. These findings are consistent with non-home odours activating non-olfactory components of the pigeon''s navigation system. The pattern of neuronal activation in the triangular and dorsomedial areas of the hippocampal formation was, by contrast, consistent with the possibility that odours play a role in providing spatial information.     

Locus-specific integration of extrachromosomal transgenes in C. elegans with the CRISPR/Cas9 system

We established a method to generate integration from extrachromosomal arrays with the CRISPR/Cas9 system. Multi-copy transgenes were integrated into the defined loci of chromosomes by this method, while a multi-copy transgene is integrated into random loci by previous methods, such as UV- and gamma-irradiation. The effects of a combination of sgRNAs, which define the cleavage sites in extrachromosomes and chromosomes, and the copy number of potential cleavable sequences were examined. The relative copy number of cleavable sequences in extrachromosomes affects the frequency of fertile F1 transgenic animals. The expression levels of the reporter gene were almost proportional to the copy numbers of the integrated sequences at the same integration site. The technique is applicable to the transgenic strains abundantly stored and shared among the C. elegans community, particularly when researchers use sgRNAs against common plasmid sequences such as β-lactamase.     

Multigene expression in stable CHO cell pools generated with the piggyBac transposon system

Heterogenous populations of recombinant cells (cell pools) stably expressing 1–4 transgenes were generated from Chinese hamster overy (CHO) cells with the piggyBac (PB) transposon system. The cell pools produced different combinations of three model proteins—enhanced green fluorescent protein (EGFP), secreted alkaline phosphatase (SEAP), and a monoclonal IgG1 antibody. Each transgene was present on a separate PB donor plasmid with either the same or a different selection gene. In both cases, we obtained PB‐derived cell pools with higher recombinant protein yields than from cell pools generated by conventional gene delivery. In PB‐derived cell pools generated using a single selection agent, both protein production and the number of integrated copies of each transgene declined as the number of transfected transgenes increased. However, the total number of integrated transgenes was similar regardless of the number of different transgenes transfected. For PB‐derived cell pools generated by selection of each transgene with a different selection agent, the total number of integrated transgenes increased with the number of transfected transgenes. The results suggest that the generation of cell pools producing multiple recombinant proteins is feasible and that the method is more efficient when each individual transgene is selected with a different marker. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1308–1317, 2016     

Recombinant protein expression in aDrosophila cell line: comparison with the baculovirus system

In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinantDrosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by theDrosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [³H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed.     

The EGF-like homeotic protein dlk affects cell growth and interacts with growth-modulating molecules in the yeast two-hybrid system

Levels of dlk, an EGF-like homeotic protein, are critical for several differentiation processes. Because growth and differentiation are, in general, exclusive of each other, and increasing evidence indicates that Dlk1 expression changes in tumorigenic processes, we studied whether dlk could also affect cell growth. We found that, in response to glucocorticoids, Balb/c 3T3 cells with diminished levels of dlk expression develop foci-like cells that have lost contact inhibition, display altered morphology, and grow faster than control cell lines. Balb/c 3T3 cells spontaneously growing more rapidly are also dlk-negative cells. Moreover, screening by the yeast two-hybrid system, using Dlk1 constructs as baits, resulted in the isolation of GAS1 and acrogranin cDNAs. Interestingly, these proteins are cysteine-rich molecules involved in the control of cell growth. Taken together, these observations suggest that dlk may participate in a network of interactions controlling how the cells respond to growth or differentiation signals.     

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.     

Bridging the gap between traditional cell cultures and bioreactors applied in regenerative medicine: practical experiences with the MINUSHEET perfusion culture system

To meet specific requirements of developing tissues urgently needed in tissue engineering, biomaterial research and drug toxicity testing, a versatile perfusion culture system was developed. First an individual biomaterial is selected and then mounted in a MINUSHEET^® tissue carrier. After sterilization the assembly is transferred by fine forceps to a 24 well culture plate for seeding cells or mounting tissue on it. To support spatial (3D) development a carrier can be placed in various types of perfusion culture containers. In the basic version a constant flow of culture medium provides contained tissue with always fresh nutrition and respiratory gas. For example, epithelia can be transferred to a gradient container, where they are exposed to different fluids at the luminal and basal side. To observe development of tissue under the microscope, in a different type of container a transparent lid and base are integrated. Finally, stem/progenitor cells are incubated in a container filled by an artificial interstitium to support spatial development. In the past years the described system was applied in numerous own and external investigations. To present an actual overview of resulting experimental data, the present paper was written.     

Interaction of the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane with the beta-receptor adenylate cyclase system in Chang liver cell membranes

Interaction of the insecticide 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane (DDT) with beta-receptor binding and adenylate cyclase activity of biological membranes has been studied. Following exposure of cultured Chang liver cells to DDT, maximal binding of the catecholamine antagonist [125I]-iodohydroxybenzylpindolol (HYP) to isolated cell membranes was decreased by 30% whereas the dissociation constant remained unchanged. Both basal activity and maximal isoproterenol-stimulated activity of adenylate cyclase were not altered. The isoproterenol concentration required for half-maximal stimulation of the enzyme was increased about 2-fold as was the agonist concentration required for half-maximal displacement of the antagonist HYP at the beta-receptor binding site. Thus, coupling efficiency of hormone-stimulated adenylate cyclase activity was not influenced by the presence of DDT in these membranes. The data show that interaction of DDT with the beta-receptor adenylate cyclase complex is restricted to the receptor component. Enzyme activity is directly linked to changes of agonist binding at the beta-receptor. Interference of DDT with signal transduction via 'fluidization' of membrane lipids has not been detected.     

Interaction of epidermal growth factor with initiators and complete carcinogens in the C3H10T1/2 cell culture system

Unlike 12-O-tetradecanoylphorbol-13-acetate, epidermal growth factor (EGF) could not promote the appearance of type III foci from initiated C3H10T1/2 cells. At appropriate concentrations, EGF induced the formation of type II colonies in the absence of any initiator. At higher concentrations, EGF suppressed the induction of both type II and type III colonies elicited by methylcholanthrene.     

A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy.

Summary A new procedure is described to recombine plasmid-bornelacZ fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. The procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries DNA sequence homology to the regions flankinglacZ fusions present in multicopy promotor-probe vectors, which can be mobilized via RP4-mediated transfer but are unable to replicate in non-enteric bacteria. Double recombination between the promoter-probe vectors and the chromosomal homology region of the transposon is genetically selected by reconstruction and expression of wild-type sequences from truncatedlacZ andaadA (streptomycin/spectinomycin) resistance genes in the homology fragment and from an amber mutation carryinglacZ andaadA genes present in the plasmid vectors. The structure of desired clones is confirmed by screening for loss of the transposon-encoded kanamycin resistance marker. We have used this procedure to assemble in monocopy inPseudomonas putida the regulatory elements controlling expression of the Xy1S-activatedPm promoter of the TOL catabolic plasmid pWWO. We show here that thePm promoter undergoes a Xy1S-independent, strictly growth-phase-controlled activation by benzoate but not meta-toluate. In the presence of XylS, however, activation by both effectors involves a combination of growth phase-dependent and -independent controls.     

Inhibition of the growth of human hepatoma cell line both in vitro and in vivo by transducing CKI gene p21WAF-1 with GE7 targeting gene delivery system

The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous gene pCEP-p21WAF-1 into human hepatocellular carcinoma cell both in vitro and in vivo. After in vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. The in vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untransfected control. The average tumor weight of the experiment group was (0.083 ± 0.043) g, while that of the control group was (0.281± 0.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous gene pCEP-p21WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growth with high efficacy both in vivo and in vitro.