G proteins are involved in cellular signalling and regulate a variety of biological processes including differentiation and development. We have generated mice deficient for the G protein subunit alpha i2 (G alpha i2) by homologous recombination in embryonic stem cells. G alpha i2-deficient mice display growth retardation and develop a lethal diffuse colitis with clinical and histopathological features closely resembling ulcerative colitis in humans, including the development of adenocarcinoma of the colon. Prior to clinical symptoms, the mice show profound alterations in thymocyte maturation and function. The study of these animals should provide important insights into the pathogenesis of ulcerative colitis as well as carcinogenesis.
The segregation of voltage-dependent sodium channels to specialized regions of the neuron is crucial for propagation of an action potential. Studies of their lateral mobility indicate that sodium channels are freely mobile on the neuronal cell body but are immobile at the axon hillock, presynaptic terminal and at focal points along the axon. To elucidate the mechanisms that regulate sodium channel topography and mobility, we searched for specific proteins from the brain that associate with sodium channels. Here we show that sodium channels labelled with 3H-saxitoxin (STX) are precipitated in the presence of exogenous brain ankyrin by anti-ankyrin antibodies and that 125I-labelled ankyrin binds with high affinity to sodium channels reconstituted into lipid vesicles. The cytoplasmic domain of the erythrocyte anion transporter competes for the latter interaction. Neither the neuronal GABA (gamma-aminobutyric acid) receptor channel complex nor the dihydropyridine (DHP) receptor bind brain ankyrin. The results indicate that brain ankyrin links the voltage-dependent sodium channel to the underlying cytoskeleton and may help to maintain axolemmal membrane heterogeneity and control sodium channel mobility.
Transgenic mice were generated by microinjection of a construct containing a self-activating human TGF-beta1 cDNA driven by the lens-specific alphaA-crystallin promoter. Seven transgenic founder mice were generated, and four transgenic lines expressing TGF-beta1 were characterized. By postnatal day 21, mice from the four families exhibited anterior subcapsular cataracts. The lenses in these mice developed focal plaques of spindle-shaped cells that expressed alpha-smooth muscle actin, and that resembled the plaques seen in human anterior subcapsular cataracts. Transgenic mice showed additional ocular defects, including corneal opacification and structural changes in the iris and ciliary body. The corneal opacities were associated with increased exfoliation of the squamous layer of the corneal epithelium and increased DNA replication in the basal epithelium.
Mice deficient for the G protein subunit Gi2 alpha were obtained by gene targeting. They displayed a growth retardation that was apparent at 6 weeks of age. They subsequently developed diffuse colitis with clinical and histopathological features closely resembling those of ulcerative colitis in humans. Seven of 20 Gi2 alpha-deficient mice with colitis also developed adenocarcinomas of the colon. Gi2 alpha-deficient thymocytes displayed two- to fourfold increases in mature CD4+8- and CD4-8+ phenotypes, an approximately threefold increase in high-intensity CD3 staining and enhanced proliferative responses to T-cell receptor stimuli. Stimulation of Gi 2 alpha-deficient peripheral T cells induced a hyperresponsive profile of interleukin-2, tumour necrosis factor, and interferon-gamma production, which may reflect a heightened response of primed cells or a defective negative regulation. We suggest that Gi 2 alpha-deficient mice may represent a useful animal model for dissecting the pathomechanisms of inflammatory bowel disease and also for the development of novel therapeutic strategies.
Strychnine is one of the most potent antagonists of glycine-mediated inhibitory conductances in the mammalian spinal cord. In order to examine the distribution of glycine receptors (GlyRs) on neuronal cells, 2 novel fluorescent strychnine derivatives were synthesized and characterized chemically, spectroscopically, and biologically. Both compounds retain their biological activity after derivatization and are potent inhibitors of 3H-strychnine binding to bovine spinal cord membranes and membranes from rat spinal cord cultures. Using these fluorescent strychnine analogs, the cellular distribution and lateral mobility of GlyRs on cultured rat spinal cord neurons were studied by digital fluorescence imaging and photobleach recovery microscopy. On these neurons, even in the absence of observable synaptic contact and early in development GlyRs are predominantly localized to cell bodies with sparse labeling of neuritic processes. Although GlyRs are confined to the neuronal cell body, approximately 50% of the receptors are very mobile, with lateral diffusion coefficients of 1.15 +/- 0.05 x 10(-9) cm2/sec, a value which is characteristic of unrestricted protein lateral diffusion. However, the remaining fraction of these receptors are immobile on the neuronal cell body. More than 70% of the GlyRs distributed on neuronal processes are immobile, while 30% are laterally mobile, with diffusion rates of 5.50 +/- 0.1 x 10(-10) cm2/sec. The results indicate that even early in development GlyRs are expressed and segregated to the cell body, where they are confined within a domain that restricts their redistribution.
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