Bending the MDCK Cell Primary Cilium Increases Intracellular Calcium

We tested the hypothesis that the primary cilium of renal epithelia is mechanically sensitive and serves as a flow sensor in MDCK cells using differential interference contrast and fluorescence microscopy. Bending the cilium, either by suction with a micropipette or by increasing the flow rate of perfusate, causes intracellular calcium to substantially increase as indicated by the fluorescent indicator, Fluo-4. This calcium signal is initiated by Ca²⁺-influx through mechanically sensitive channels that probably reside in the cilium or its base. The influx is followed by calcium release from IP₃-sensitive stores. The calcium signal then spreads as a wave from the perturbed cell to its neighbors by diffusion of a second messenger through gap junctions. This spreading of the calcium wave points to flow sensing as a coordinated event within the tissue, rather than an isolated phenomenon in a single cell. Measurement of the membrane potential difference by microelectrode during perfusate flow reveals a profound hyperpolarization during the period of elevated intracellular calcium. We conclude that the primary cilium in MDCK cells is mechanically sensitive and responds to flow by greatly increasing intracellular calcium. Received: 4 April 2001/Revised: 28 June 2001     

Stand structure and regeneration in a Kamchatka mixed boreal forest

Abstract. A 1‐ha plot was established in a Betula platyphylla‐Picea ajanensis mixed boreal forest in the central Kamchatka peninsula in Russia to investigate stand structure and regeneration. This forest was relatively sparse; total density and stand basal area were 1071/ha and 25.8 m²/ha, respectively, for trees > 2.0 cm in trunk diameter at breast height (DBH). 25% of Betula regenerated by sprouting, and its frequency distribution of DBH had a reverse J‐shaped pattern. In contrast, Picea had a bimodal distribution. The growth rates of both species were high, reaching 20 m in ca. 120 yr. The two species had clumped distributions, especially for saplings. Betula saplings were not distributed in canopy gaps. Small Picea saplings were distributed irrespective of the presence/absence of gaps, while larger saplings aggregated in gaps. At the examined spatial scales (6.25–400 m²) the spatial distribution of Betula saplings was positively correlated with living Betula canopy trees and negatively with dead Picea canopy trees. This suggests that Betula saplings regenerated under the crowns of Betula canopy trees and did not invade the gaps created by Picea canopy trees. The spatial distribution of Picea saplings was negatively correlated with living and dead Betula canopy trees and positively with dead Picea canopy trees. Most small Picea seedlings were distributed under the crowns of Picea trees but not under the crowns of Betula trees or in gaps. This suggests that Picea seedlings establish under the crowns of Picea canopy trees and can grow to large sizes after the death of overhead Picea canopy trees. Evidence of competitive exclusion between the two species was not found. At a 20 m × 20 m scale both skewness and the coefficient of variation of DBH frequency distribution of Picea decreased with an increase in total basal area of Picea while those of Betula were unchanged irrespective of the increase in total basal area of Betula. This indicates that the size structure of Picea is more variable with stand development than that of Betula on a small scale. This study suggests that Betula regenerates continuously by sprouting and Picea regenerates discontinuously after gap formation and that the species do not exclude each other.     

Implication of connexins 40 and 43 in functional coupling between mouse cardiac fibroblasts in primary culture

Cardiac fibroblasts contribute to the structure and function of the myocardium. However their involvement in electrophysiological processes remains unclear; particularly in pathological situations when they proliferate and develop fibrosis. We have identified the connexins involved in gap junction channels between fibroblasts from adult mouse heart and characterized their functional coupling. RT-PCR and Western blotting results show that mRNA and proteins of connexin40 and connexin43 are expressed in cultured cardiac fibroblasts, while Cx45 is not detected. Analysis of gap junctional communications established by these connexins with the gap-FRAP technique demonstrates that fibroblasts are functionally coupled. The time constant of permeability, k, calculated from the fluorescence recovery curves between cell pairs is 0.066 ± 0.005 min^− 1 (n = 65). Diffusion analysis of Lucifer Yellow through gap junction channels with the scrape-loading method demonstrates that when they are completely confluent, a majority of fibroblasts are coupled forming an interconnecting network over a distance of several hundred micrometers. These data show that cardiac fibroblasts express connexin40 and connexin43 which are able to establish functional communications through homo and/or heterotypic junctions to form an extensive coupled cell network. It should then be interesting to study the conditions to improve efficiency of this coupling in pathological conditions.     

Regeneration of a Gentiana pneumonanthe population in an oligotrophic wet meadow

Abstract. The aim of this study was to evaluate the critical phases of the life cycle of Gentiana pneumonanthe, the marsh gentian, a threatened species of the Bohemian flora. The effect of various conditions on germination and seedling establishment and the possible effect of competition on the performance of the species were tested. Seeds were sown in plots which were subjected to four treatments in a randomized complete blocks experiment: unmanaged meadow, mown meadow, burned meadow, and meadow with cut sod. The significantly highest recruitment was found in plots with cut sod, the lowest in the unmanaged control plots. Seedling survival also differed among the treatments. In the following year surviving individuals were only found in plots with cut sod. The influence of neighbouring vegetation on target gentian individuals was evaluated by removing the surrounding vegetation and comparing the performance of these individuals with controls. The initial height of each individual was measured and used as a covariable. No significant effect of neighbouring vegetation on performance was detected. Thus, the establishment phase appears to be critical for population persistence and is also more influenced by the management regime than other stages of the life cycle.     

Long-term changes in a Wisconsin Fagus-Acer forest in relation to glaze storm disturbance

Changes in the composition of a Fagus-Acer (Beech-Sugar maple) forest in southeastern Wisconsin over a 16-yr period from 1971 to 1987 are analyzed in relation to a severe glaze (ice) storm disturbance occurring within the census period. Landscape topography created ‘windward’ and ‘leeward’ forest aspects with respect to storm severity, which resulted in greater canopy opening on the windward aspect. In the tree stratum, most species remained stable in density and most of the common species increased in basal area into larger size classes. However, Fagus grandifolia, Ulmus rubra, and the small tree Ostrya virginiana suffered net losses that suggest synergistic effects between glaze storm disturbance and other factors upon tree mortality. In the sapling stratum, canopy opening strongly promoted release of shade-to levant Acer sac-charum. On the windward forest aspect, sapling densities of less shade-tolerant species also increased, in contrast to the absence of such increases on the leeward forest aspect. In the shrub (regeneration) stratum, species responses were heterogeneous. Regeneration of most species increased over the 16-yr period, and some less shade-tolerant species showed increased regeneration differentially on the windward forest aspect. Overall, disturbance appears to have accelerated forest succession toward increased dominance by A. saccharum and persistence of both Fagus and Tilia americana through their capacities for root sprouting. However, forest succession was retarded somewhat on the windward aspect through increased recruitment of less-shade tolerant species. These results parallel those of other studies of glaze storm disturbance, and they illustrate how spatially heterogeneous disturbance intensity may contribute to maintenance of forest diversity.     

A quantitive evaluation of gap junctions and their morphological alteration during differentiation of amphibian notochord cells

Summary The early development of notochord cells may be divided into three phases according to the quantitative evaluation of gap junctions: late gastrula, neurula and from tailbud to tadpole. In late gastrula, the percentage of the area of gap junctions to total membrane is 0.054% and most of the gap junctions are small in size. During the stages of neurulation, the ratios of gap junctions to total membrane area increase and remain high (0.106–0.181 %), and the majority of the gap junctions are of medium and large size. The high ratios of gap junctions to membrane area during neurulation suggests that intercellular communication via gap junctions is important during this period. In the stages from tailbud to tadpole the ratios decrease and drop drastically to 0.001 % and most of the gap junctions found are small in size. It is in the last phase that gap junctions of altered configuration appear.     

The importance of species range attributes and reserve configuration for the conservation of angiosperm diversity in Western Australia

In order to better understand the relationship between reserve design and the species represented by such designs, we examined the effectiveness of the Western Australian reserve system for conserving angiosperm diversity, and examined the characteristics of those species conserved. We overlayed species distribution data for 14 plant lineages with the distribution of the reserve system (8.5% of the State’s area) and identified the species that remained unprotected. We found that, depending upon the method employed, between 174 (5.7%) and 570 (18.7%) of species were not included within the reserve system. Two main unprotected regions were identified, one of which was also a centre of high diversity. Geographical range sizes of unprotected species were six times smaller than those species that were protected, while species richness of small-ranged endemic species coincided with general patterns of species richness. At the level of Western Australia’s bioregions we found that conservation effectiveness was most dependent on characteristics of the reserve system rather than characteristics (size and positioning) of species ranges. At this scale, the most effective way to conserve more species in Western Australia would be to conserve more land, while conservation would be most successful in a uniformly dispersed reserve system. Our results highlight the fact that reserve systems may take on two design approaches based on scale––at continental scales, reserves should be clustered around the hotspots of endemic species, while within regions, an evenly distributed reserve system will most adequately sample species.     

Temporal variation in the woody understory of an old-growth Fagus-Acer forest and implications for overstory recruitment

Abstract. Changes in woody vegetation were examined over eight years, using a 1.05-ha permanent plot in which the location of every shrub and tree > 1m height was mapped. There was little change in the overstory vegetation, as expected for an old-growth forest. Much greater change occurred in the understory, primarily related to a 40 % increase in density. Differences occurred among species in the under-story, as Acer saccharum and Prunus serotina increased and Fraxinus americana and Fagus grandifolia decreased. Canopy gap dynamics are implicated in differences among species in the establishment and growth of individuals in the understory and their recruitment into the overstory. It is concluded that because understory is temporally variable, overstory recruitment from the understory may take different courses at different times in the same forest.