Altering electrical connections in the nervous system of the pteropod mollusc Clione limacina by neuronal injections of gap junction mRNA

Neurons can communicate with each other either via exchange of specific molecules at synapses or by direct electrical connections between the cytoplasm of either cell [for review see Bruzzone et al. (1996) Eur. J. Biochem., 238, 1-27]. Although electrical connections are abundant in many nervous systems, little is known about the mechanisms which govern the specificity of their formation. Recent cloning of the innexins--gap junction proteins responsible for electrical coupling in invertebrates (Phelan et al. (1998) Trends Genet., 14, 348-349], has made it possible to study the molecular mechanisms of patterning of the electrical connections between individual neurons in model systems. Here we demonstrate that intracellular injection of mRNA encoding the molluscan innexin Panx1 (Panchin et al. 2000 Curr. Biol., 10, R473-R474) drastically alters the specificity of electrical coupling between identified neurons of the pteropod mollusc Clione limacina.     

Highly efficient,large volume flow electroporation

Electroporation is widely used to transfect and load cells with various molecules. Traditional electroporation using a static mode is typically restricted to volumes less than 1 mL, which limits its use in clinical and industrial bioprocessing applications. Here we report efficient, large volume transfection results by using a scalable-volume electroporation system. Suspended (Jurkat) and adherent cells (10T1/2 and Huh-7) were tested. A large macromolecule, FITC-conjugated dextran (MW=500 kD) was used to measure cell uptake, while a plasmid carrying the gene coding for enhanced green fluorescence protein (eGFP) was used to quantitate the flow electrotransfection efficiency as determined by flow cytometry. The flow electroloading efficiency of FITC-dextran was >90%, while the cell viability was highly maintained (>90%). High flow electrotransfection efficiency (up to 75%) and cell viability (up to 90%) were obtained with processing volumes ranging from 1.5 to 50 mL. No significant difference of electrotransfection efficiency was observed between flow and static electrotransfection. When 50 mL of cell volume was processed and samples collected at different time points during electroporation, the transgene expression and cell viability results were identical. We also demonstrated that DNA plasmid containing EBNA1-OriP elements from Epstein-Barr virus were more efficient in transgene expression than standard plasmid without the elements (at least 500 too 1000-fold increase in expression level). Finally, to examine the feasibility of utilizing flow electrotransfected cells as a gene delivery vehicle, 10T1/2 cells were transfected with a DNA plasmid containing the gene coding for mIL12. mIL12 transfected cells were injected subcutaneously into mice, and produced functional mIL12, as demonstrated by anti-angiogenic activity. This is the first demonstration of efficient, large volume, flow electroporation and the in vivo efficacy of flow electrotransfected cells. This technology may be useful for clinical gene therapy and large-scale bioprocesses.     

The influence of conditioned medium from mouse intestinal epithelial cells on the proliferative activity of crypt cells: role of granulocyte-macrophage colony-stimulating factor

Background: The aim of this work was to study the influence of soluble factors produced by native mouse intestinal epithelial cells (IECs) on the proliferative activity of freshly isolated intestinal crypt cells. Methods: The crypt cells were cultured with either conditioned medium and its ultrafiltrates or recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence or absence of neutralizing anti-GM-CSF antibodies. GM-CSF in culture medium was identified by the electrochemiluminescence method. Results: It was demonstrated that the IEC conditioned medium contained GM-CSF. This cytokine led to both the upregulation and downregulation of crypt cell proliferative activity, depending on its concentration in the culture medium. The effect of native GM-CSF was reproduced with recombinant mouse GM-CSF: 25 and 5 ng/ml inhibited the proliferative activity, whereas 1 ng/ml led to its significant stimulation. Conclusions: Freshly isolated murine IECs produce GM-CSF, which plays a critical role in crypt cell proliferative activity in vitro. These results suggest the involvement of this factor in the regulation of the crypt proliferative zone, in an autocrine and/or paracrine manner. Received: February 25, 2002 / Accepted: July 26, 2002     

Coagulation,fibrinolysis and angiogenesis: new insights from knockout mice

Angiogenesis plays a key role in a broad array of physiologic and pathologic processes. Two major systems--coagulation and fibrinolysis--maintaining hemostasis, have recently been implicated in angiogenesis. Generation of mice deficient in components of coagulation and plasminogen systems has provided an extraordinary opportunity to define the role of each of these systems in vivo and to elucidate molecular mechanisms involved in angiogenesis. It appears that several factors of the coagulation system, such as the tissue factor, the factor V and the thrombin receptor, play an important role in embryonic vessel formation, most probably in the formation of the primitive vascular wall. In addition, the plasminogen system appears to play a significant role in angiogenesis in adulthood, regulating the migration of endothelial and smooth muscle cells, the degradation of the extracellular matrix and activity of the metalloproteinase system. These new revelations open a possibility for future therapeutic strategies to specifically control angiogenesis in different pathological processes where abnormalities of tissue vascularization are pathogenetically prominent.     

Concomitant focal fibrocartilaginous dysplasia of the tibia and eosinophilic granuloma of the jaw in a child

This 2-year-old child presented with concomitant eosinophilic granuloma of the lower jaw and focal fibrocartilaginous dysplasia of the right tibia. Her eosinophilic granuloma was diagnosed on the basis of the clinical picture, imaging studies and the characteristic histologic appearance. Focal fibrocartilaginous dysplasia was revealed incidentally during the eosinophilic granuloma staging process. After chemotherapy, all signs of eosinophilic granuloma subsided, but focal fibrocartilaginous dysplasia remained without signs of clinical or radiographic progression. The importance of differentiating these two conditions is stressed in order to avoid ineffective and inappropriate treatment of focal fibrocartilaginous dysplasia.     

The difference between partitioning and distribution from a thermodynamic point of view: NSAIDs as an example.

Solubility and solvation of some NSAIDs were studied in their non-ionic (aqueous buffers of pH 2.0) and ionic molecular form (pH 7.4) over a wide temperature interval. Absolute scale values for the thermodynamic terms (Gibbs energy, enthalpy and entropy) were obtained. Thermodynamic parameters of the transfer of the molecules from one buffer to the other (representing protonation/deprotonation) were derived. It has been found that the thermodynamic characteristics of solvation (hydration) of (+)- and (+/-)-IBP in the buffers show a difference, which is larger than the experimental error. This may be explained by differences in the association states of the molecules in solution. For the other NSAIDs studied, a correlation between the Gibbs energy of transfer, deltaG(tr) (pH 7.4-->pH 2.0) and the pK(a)-value, and a compensation effect between the enthalpic and entropic terms have been revealed. Thermodynamic aspects of the transfer process from the buffers to n-octanol were analysed. The two types of the transfer processes (non-dissociated molecule to octanol (partitioning), and dissociated form to octanol (distribution)) have essentially different driving forces: partitioning is enthalpy driven, whereas the transfer of the ionic form is entropy driven. The following points are discussed: (a) significance of using water-octanol systems (logP as a measure of drug lipophilicity) to describe biological membranes (lipid systems); (b) differences in thermodynamic aspects of the partitioning/distribution processes of these systems; (c) advantages of the present transfer method approach in comparison with temperature dependencies of logP to analyse the driving forces of partitioning/distribution.     

Neurospecific S-100 protein content in brains of different mouse strains

Total whole brain concentrations of S-100 protein and of its water-soluble fraction were determined in 11 inbred mouse straine: DBA/2J, AKR/J, CBA/Lac, C57BL/6J, C57BL/6J-Ay, C3H/He, C3H/f, DD, A/He, BALB/cLac, CC57BR/Mv, and in cerebral cortex, cerebellum and hippocampus in DBA/2J, AKR/J and CBA/Lac strains. Highly significant differences in the concentrations of the water-soluble S-100 protein were found between some strains. Slight differences were found in total S-100 protein content in whole brains between the strains (0.01 less that P less than 0.05). The DBA/2J mice had the highest brain S-100 protein content, and were characterized by a higher learning rate in shuttle-box as compared to CBA/Lac and AKR/J mice, who had a low content of this neurospecific protein.