78 research outputs found
Transfection of newt blastema mesenchyme using the techniques of lipofection and direct gene transfer
The regenerating amphibian limb provides a useful system for
studying genes involved in the establishment of positional
information. While a number of candidate genes that may playa role
in pattern formation have been identified, their function in vivo is
unknown in this system.
To better ascertain the role of these genes, it would be useful
to be able to alter their normal patterns of expression in vivo and to
assess the effects of this misexpression on limb pattern. In order
to achieve this, a method of introducing a plasmid containing the
eDNA of a gene of interest into a newt blastema (a growth zone of
mesenchymal progenitor cells) is needed. Unfortunately, most
commonly used transfection techniques cannot be used with newt
blastema cells.
In this study, I have used the techniques of lipofection and
direct gene transfer to introduce plasmid DNA containing reporter
genes into the cells of a regenerating newt limb. The technique of
lipofection was most effective when the blastema cells were
transfected in vitro. The optimal ratio for transfection was shown
to be 1:3 DNA:Lipofectin (W/w) , and an increase in the amount of DNA
present in the mixture (1:3 ratio maintained) resulted in a
corresponding increase in gene expression.
The technique of direct gene transfer was used to transfect
newt blastema cells with and without prior complex formation with
Lipofectin. Injection of plasmid DNA alone provided the most
3
promising results. It was possible to introduce plasmid DNA
containing the reporter gene ~-galactosidase and achieve significant
gene expression in cells associated with the injection site. In the
future, it would be interesting to use this technique to inject
plasmid DNA containing a gene which may have a role in pattern
formation into specific areas of the newt blastema and to analyze
the resulting limb pattern that emerges
Neuronal calcium sensor-1 enhancement of InsP3 receptor activity is inhibited by therapeutic levels of lithium
Author Posting. Ā© American Society for Clinical Investigation, 2006. This article is posted here by permission of American Society for Clinical Investigation for personal use, not for redistribution. The definitive version was published in Journal of Clinical Investigation 116 (2006): 1668-1674, doi:10.1172/JCI22466.Regulation and dysregulation of intracellular calcium (Ca2+) signaling via the inositol 1,4,5-trisphosphate receptor (InsP3R) has been linked to many cellular processes and pathological conditions. In the present study, addition of neuronal calcium sensor-1 (NCS-1), a high-affinity, low-capacity, calcium-binding protein, to purified InsP3R type 1 (InsP3R1) increased the channel activity in both a calcium-dependent and -independent manner. In intact cells, enhanced expression of NCS-1 resulted in increased intracellular calcium release upon stimulation of the phosphoinositide signaling pathway. To determine whether InsP3R1/NCS-1 interaction could be functionally relevant in bipolar disorders, conditions in which NCS-1 is highly expressed, we tested the effect of lithium, a salt widely used for treatment of bipolar disorders. Lithium inhibited the enhancing effect of NCS-1 on InsP3R1 function, suggesting that InsP3R1/NCS-1 interaction is an essential component of the pathomechanism of bipolar disorder.This work was supported by a grant from the NIH (GM63496 to B.E. Ehrlich), German National Merit Foundation scholarships (C. Schlecker and W. Boehmerle), and a National Kidney Foundation Fellowship (A. Varshney)
Entamoeba histolytica Infection and Secreted Proteins Proteolytically Damage Enteric Neurons āæ
The enteric protozoan parasite Entamoeba histolytica causes amebic colitis through disruption of the mucus layer, followed by binding to and destruction of epithelial cells. However, it is not known whether ameba infections or ameba components can directly affect the enteric nervous system. Analysis of mucosal innervations in the mouse model of cecal amebiasis showed that axon density was diminished to less than 25% of control. To determine whether amebas directly contributed to axon loss, we tested the effect of either E. histolytica secreted products (Eh-SEC) or soluble components (Eh-SOL) to an established coculture model of myenteric neurons, glia, and smooth muscle cells. Neuronal survival and axonal degeneration were measured after 48 h of exposure to graded doses of Eh-SEC or Eh-SOL (10 to 80 Ī¼g/ml). The addition of 80 Ī¼g of either component/ml decreased the neuron number by 30%, whereas the axon number was decreased by 50%. Cytotoxicity was specific to the neuronal population, since the glial and smooth muscle cell number remained similar to that of the control, and was completely abrogated by prior heat denaturation. Neuronal damage was partially prevented by the cysteine protease inhibitor E-64, showing that a heat-labile protease was involved. E. histolytica lysates derived from amebas deficient in the major secreted protease EhCP5 caused a neurotoxicity similar to that of wild-type amebas. We conclude that E. histolytica infection and ameba protease activity can cause selective damage to enteric neurons
- ā¦