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.     

Growth and reproduction ofPorphyra columbina Mont. (Bangiales,Rhodophyceae) from southern New Zealand

Changes in biomass and chemical composition, and the reproductive phenology ofPorphyra columbina Mont. were monitored at three sites in southern New Zealand over two growing seasons. Both temporal and spatial variations were found. Seasonal changes in biomass and chemical components were correlated with seawater nitrate concentrations and temperature. The summer decline in biomass was a result of the onset of unsuitable environmental conditions and the release of reproductive tissue. Under more suitable conditions, the decline in biomass was delayed. There was an inverse relationship between vegetative growth and reproduction. Reproductive plants first appeared in August at a time of increasing temperature, irradiance and daylength. Only larger plants which were mainly found in subsites low on the shore became reproductive. Plants sampled from high subsites had a shorter growth season, were generally smaller, had lower nitrogen and pigment content and were non-reproductive.Presented at the XIIIth International Seaweed Symposium, University of British Columbia, Vancouver, Canada, August 1989.     

Tooth wear in captive wild ruminant species differs from that of free-ranging conspecifics

The mesowear method evaluates the wear patterns of herbivore cheek teeth by visually evaluating the facet development of the occlusal surfaces. It thus allows classification of most herbivorous ungulates into browsers, grazers or intermediate feeders, due to the fact that in grazers, tooth wear is characterized by a comparatively high degree of abrasion, most probably due to the presence of silicacious phytoliths in grasses, a higher amount of dust and grit adhering to their forage, or both. It has been suggested that excessive tooth wear could be a particularly limiting factor in the husbandry of captive large browsing species, and major tooth wear was demonstrated in captive as compared to free-ranging giraffe. If this increased tooth wear in captivity was an effect of feeding type and diets fed, then it would be expected that other browsing species are affected in a similar manner. In order to test this hypothesis, we investigated the dental mesowear pattern in captive individuals of 19 ruminant species and compared the results to data on free-ranging animals. Compared to free-ranging populations, captive browsers show a significantly more abrasion-dominated tooth wear signal. The reverse applies to captive grazers, which tend to show a less abrasion-dominated wear in captivity. Captive ruminants were generally more homogenous in their wear signature than free-ranging ruminants. If grit contamination in the natural habitat is a major cause of dental wear in grazers, then diets in captivity, although similar in botanical composition, most likely contain less abrasives due to feeding hygiene. If dental wear is one of the major factors limiting longevity, then captive grazers should achieve longer lifespans than both captive browsers and free-ranging grazers. In particular with respect to browsers, the results suggest that captive feeding regimes could be improved.     

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.     

Ultrastructure of the placentae of the natricine snake,Virginia striatula (Reptilia: Squamata)

Virginia striatula is a viviparous snake with a complex pattern of embryonic nutrition. Nutrients for embryonic development are provided by large, yolked eggs, supplemented by placental transfer. Placentation in this species is surprisingly elaborate for a predominantly lecithotrophic squamate reptile. The embryonic-maternal interface consists of three structurally distinct areas, an omphalallantoic placenta and a regionally diversified chorioallantoic placenta. The chorioallantoic placenta over the embryonic hemisphere (paramesometrial region) of the egg, features close apposition of embryonic and uterine blood vessels because of the attenuate form of the interceding epithelial cells. The periphery of the chorioallantoic placenta, which is adjacent to the omphalallantoic placenta, is characterized by a simple cuboidal uterine epithelium apposed to a stratified cuboidal chorionic epithelium. There are no sites with attenuate epithelial cells and close vascular apposition. The morphology of the omphalallantoic placenta is similar to that of the peripheral chorioallantoic placenta, except that the height of uterine epithelial cells is greater and allantoic blood vessels are not associated with the embryonic epithelium. The functional capabilities of the three placental regions are not known, but structural characteristics suggest that the omphalallantoic placenta and peripheral zone of the chorioallantoic placenta are sites of nutritional provision via histotrophy. The paramesometrial region of the chorioallantoic placenta is also nutritive, in addition to functioning as the primary embryonic respiratory system. The structure of the chorioallantoic placenta of V. striatula is a new placental morphotype for squamate reptiles that is not represented by a classic model for the evolution of reptilian placentation.     

Involvement of napsin A in the C- and N-terminal processing of surfactant protein B in type-II pneumocytes of the human lung

Surfactant protein B (SP-B) is a critical component of pulmonary surfactant, and a deficiency of active SP-B results in fatal respiratory failure. SP-B is synthesized by type-II pneumocytes as a 42-kDa propeptide (proSP-B), which is posttranslationally processed to an 8-kDa surface-active protein. Napsin A is an aspartic protease expressed in type-II pneumocytes. To characterize the role of napsin A in the processing of proSP-B, we colocalized napsin A and precursors of SP-B as well as SP-B in the Golgi complex, multivesicular, composite, and lamellar bodies of type-II pneumocytes in human lungs using immunogold labeling. Furthermore, we measured aspartic protease activity in isolated lamellar bodies as well as isolated human type-II pneumocytes and studied the cleavage of proSP-B by napsin A and isolated lamellar bodies in vitro. Both, napsin A and isolated lamellar bodies cleaved proSP-B and generated three identical processing products. Processing of proSP-B by isolated lamellar bodies was completely inhibited by an aspartic protease inhibitor. Sequence analysis of proSP-B processing products revealed several cleavage sites in the N- and C-terminal propeptides as well as one in the mature peptide. Two of the four processing products generated in vitro were also detected in type-II pneumocytes. In conclusion, our results show that napsin A is involved in the N- and C-terminal processing of proSP-B in type-II pneumocytes.     

A novel redox mechanism for the glutathione-dependent reversible uptake of a fungal toxin in cells

The fungal metabolite gliotoxin is characterized by an internal disulfide bridge and can exist in either disulfide or dithiol forms. Gliotoxin and other members of the epipolythiodioxopiperazine class of toxins have immunosuppressive properties and have been implicated in human and animal mycotoxicoses. The bridged disulfide moiety is thought to be generally essential for biological activity. Here we show that only the natural (oxidized) form of gliotoxin is actively concentrated in a cell line in a glutathione-dependent manner. Intracellular levels of the toxin can be up to 1500-fold greater than the applied concentration, and toxin in the cells exists almost exclusively in the reduced form. A simple model of toxin entry followed by reduction to the cell-impermeant dithiol explains active uptake, cell density dependence of EC50 values and predicts a value for the maximum concentration of toxin at limiting cell density in agreement with the experiment. Oxidation of the intracellular toxin results in rapid efflux from the cell that also occurs when glutathione levels fall following induction of apoptotic cell death by the toxin. This mechanism allows for minimal production of the toxin while enabling maximal intracellular concentration and thus maximal efficacy of killing in a competitor organism initially present at low cell density. The toxin effluxes from the apoptotic cell exclusively in the oxidized form and can further enter and kill neighboring cells, thus acting in a pseudocatalytic way.     

The location of 5S RNA genes in lampbrush polytene chromosomes from Drosophila

Drosophila polytene chromosomes were transformed into lampbrush-like structures by exposure to solutions of alkali-urea. In this process, the chromosomes shorten and widen, and the bands (chromomeres) extend laterally into loops leaving a central core between the paired homologues. The expanded polytene chromosomes are very similar in appearance to the true lampbrush chromosomes of amphibian oocytes and to ordinary chromosomes in pachytene. The denaturing effects of alkali-urea were partially counteracted by return of the treated chromosomes to Ringer solution. These observations are interpreted in terms of recent findings on protein backbones in chromosomes, and indicate that chromosomes generally may have very similar basic organization, despite differences due to species, polyteny and degree of condensation. To gain more information on the specific location of a structural gene, ¹²⁵I-labelled low molecular weight (containing 5S RNA) was hybridized in situ to normal and lampbrush-like polytene chromosomes. Autoradiography showed silver grain distribution for 5S RNA consistent with hybridization primarily to the loop regions of the lampbrush chromosomes rather than the core. This provides further indirect evidence that structural genes like 5S RNA may be located on the bands (chromomeres) and not the interbands of normal polytene chromosomes.