Complementation of null CF mice with a human CFTR YAC transgene.

We have made transgenic mice carrying a 320 kb YAC with the intact human cystic fibrosis transmembrane regulator (CFTR) gene. Mice that only express the human transgene were obtained by breeding with Cambridge null CF mice. One line has approximately two copies of the intact YAC. Mice carrying this transgene and expressing no mouse cftr appear normal and breed well, in marked contrast to the null mice, where 50% die by approximately 5 days after birth. The chloride secretory responses in these mice are as large or larger than in wild-type tissues. Expression of the transgene is highly cell type specific and matches that of the endogenous mouse gene in the crypt epithelia throughout the gut and in salivary gland tissue. However, there is no transgene expression in some tissues, such as the Brunner's glands, where it would be expected. Where there are differences between the mouse and human pattern of expression, the transgene follows the mouse pattern. We have thus defined a cloned fragment of DNA which directs physiological levels of expression in many of the specific cells where CFTR is normally expressed.     

Proof-of-concept human laboratory study for protracted abstinence in alcohol dependence: effects of gabapentin

There is a need for safe medications that can effectively support recovery by treating symptoms of protracted abstinence that may precipitate relapse in alcoholics, e.g. craving and disturbances in sleep and mood. This proof-of-concept study reports on the effectiveness of gabapentin 1200 mg for attenuating these symptoms in a non-treatment-seeking sample of cue-reactive, alcohol-dependent individuals. Subjects were 33 paid volunteers with current Diagnostic and Statistical Manual of Mental Disorders-IV alcohol dependence and a strength of craving rating 1 SD or greater for alcohol than water cues. Subjects were randomly assigned to gabapentin or placebo for 1 week and then participated in a within-subjects trial where each was exposed to standardized sets of pleasant, neutral and unpleasant visual stimuli followed by alcohol or water cues. Gabapentin was associated with significantly greater reductions than placebo on several measures of subjective craving for alcohol as well as for affectively evoked craving. Gabapentin was also associated with significant improvement on several measures of sleep quality. Side effects were minimal, and gabapentin effects were not found to resemble any major classes of abused drugs. Results suggest that gabapentin may be effective for treating the protracted abstinence phase in alcohol dependence and that a randomized clinical trial would be an appropriate next step. The study also suggests the value of cue-reactivity studies as proof-of-concept screens for potential antirelapse drugs.     

The effect of heat and smoke on the soil seed banks of heathlands on permanent freshwater swamps

In South Australia, Swamps of the Fleurieu Peninsula are critically endangered due to past vegetation clearance and changes in hydrology, but still contain a high diversity of threatened plant species. This vegetation community provides habitat for 82 threatened ground-stratum plant species, including the nationally critically endangered subshrub Hibbertia tenuis which is endemic to these swamps. With infrequent burning, native ferns and taller shrubs outcompete these species. We conducted glasshouse trials to determine the potential of fire to regenerate threatened and other swamp plant species. Soil samples from eight swamps were used in germination trials with half of each sample treated with heat plus smoke, and half left untreated. Samples were spread onto trays (n = 188), and seedling emergence was recorded for twelve months. Emergence was dominated by native species (2649 seedlings m⁻²) compared to exotics (675 seedlings m⁻²). In total, 48 native and 25 introduced species germinated, with 21% of germinated native species absent from the above-ground vegetation. The dominant native fern Gleichenia microphylla did not emerge from any soil samples, indicating that recovery likely lags behind that of ground-strata species, giving them time to establish and set seed before being outcompeted. Thirty-four rare or threatened species germinated, including five species absent from above-ground vegetation. Of all native species that emerged, only five were confined to heat plus smoke treatments, suggesting most species will regenerate without fire if overstorey competition is reduced. However, seedling emergence of native shrubs/subshrubs more than doubled with the fire treatment. Of particular importance, H. tenuis showed an 18-fold increase in germination when treated with heat plus smoke. This study supports the utility of ecological burns as a management tool to regenerate threatened plant species in long-undisturbed peaty heathlands on permanent swamps.     

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.     

The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression.

The cystic fibrosis gene product, CFTR, and the multidrug resistance P-glycoprotein (encoded by the MDR1 gene) are structurally related proteins and both are associated with epithelial chloride channel activities. We have compared their cell-specific expression in the rat by in situ hybridization. In all tissues examined the two genes were found to have complementary patterns of expression, demonstrating exquisite regulation in both cell-specific and temporal fashions. Additionally, a switch in expression from one gene to the other was observed in certain tissues. For example, expression in the intestine switches from CFTR to MDR1 as the cells migrate across the crypt-villus boundary. A switch from CFTR to MDR1 expression was also observed in the uterine epithelium upon pregnancy. These data suggest that CFTR and P-glycoprotein serve analogous roles in epithelial cells and provide additional evidence that P-glycoprotein has a physiological role in regulating epithelial cell volume. The patterns of expression suggest that the regulation of these two genes is coordinately controlled.     

Continuous-time modeling of cell fate determination in Arabidopsis flowers

Background  

The genetic control of floral organ specification is currently being investigated by various approaches, both experimentally and through modeling. Models and simulations have mostly involved boolean or related methods, and so far a quantitative, continuous-time approach has not been explored.