Exon Mapping by Fiber-FISH or LR-PCR

In this study we systematically assessed the sensitivity limits of fiber-FISH in model experiments. Exonic fragments and cDNAs with exon sizes of ≥200 bp could be mapped on their cognate cosmid. This positional fiber-FISH mapping was validated by long-range PCR. It is expected that these two independent mapping approaches will help to refine current available gene maps and show their applicability in fine mapping of sequence-tagged sites or expressed sequence tags. Also, they will be useful in resolving gene structures by mapping exon and intron locations.     

Calcium-calmodulin signalling is involved in light-induced acidification by epidermal leaf cells of pea, Pisum sativum L.

Pathways of signal transduction of red and blue light-dependentacidification by leaf epidermal cells were studied using epidermalstrips of the Argenteum mutant of Pisum sativum. In these preparationsthe contribution of guard cells to the acidification is minimal.The hydroxypyridine nifedipine, a Ca²⁺-channel blocker, partlyinhibited the response to both blue and red light, while thephenylalkylamine, verapamil, a Ca²⁺-channel blocker that hasbeen shown in plant cells also to block K⁺-channels, causednearly complete inhibition. The Ca²⁺-channel activator S(–)BayK 8644 induced acidification when added in the dark and diminishedthe light-induced lowering of the extracellular pH. The Ca²⁺-ionophores,ionomycin and A23187 [GenBank] , also reduced the light response. Furthermore,the light-induced acidification was inhibited by the calmodulinantagonists W-7 and trifluoperazine, but not by W-5. These calmodulininhibitors completely inhibited the red light-induced acidification,but inhibited the response to blue light by only 60–70%.In general, inhibition by compounds affecting Ca-calmodulinsignalling was always stronger on the red light response thanthat on the blue light response (with the exception of verapamilthat blocked both the red and blue light responses equally well).This differential effect on red and blue light-induced responsesindicates a role for Ca²⁺-CaM signalling in both the red andblue light responses, while a second process, independent ofCa²⁺ is activated by blue light. Key words: Signal transduction, light-induced acidification, epidermal cells, pea     

Estimation of oxygen evolution by marine phytoplankton from measurement of the efficiency of Photosystem II electron flow

The relation between photosynthetic oxygen evolution and Photosystem II electron transport was investigated for the marine algae t Phaeodactylum tricornutum, Dunaliella tertiolecta, Tetraselmis sp., t Isochrysis sp. and t Rhodomonas sp.. The rate of Photosystem II electron transport was estimated from the incident photon flux density and the quantum efficiency of Photosystem II electron transport as determined by chlorophyll fluorescence. The relation between the estimated rate of Photosystem II electron transport and the rate of oxygen evolution was investigated by varying the ambient light intensity. At limiting light intensities a linear relation was found in all species. At intensities approaching light saturation, the relation was found to deviate from linearity. The slope of the line in the light-limited range is species dependent and related to differences in absorption cross-section of Photosystem II. The observed non-linearity at high irradiances is not caused by photorespiration but probably by a Mehler-type of oxygen reduction. The relationship could be modelled by including a redox-state dependent oxygen uptake. In the diatom t Phaeodactylum tricornutum, the photochemical efficiency of dark adapted open Photosystem II centers was found to be temperature-dependent with an optimum near 10°C.     

Should we expect strange attractors behind plankton dynamics - and if so, should we bother?

Chaotic behaviour can easily be generated from simple deterministicmodels of a number of ecological interactions. It is arguedthat these interactions, as well as the conditions necessaryto produce chaos from them, are common in planktonic communitiesFurthermore, attention is drawn to the fact that sustained algalfluctuations are observed in micro-ecosystems that are keptunder constant external conditions for as long as 10 years.These fluctuations are shown to have the same characteristicsas those arising from a simple chaotically behaving model ofcompeting algae. Recently, signs of chaos have also been shownin a series of plankton data from the field. It is concludedthat there are, in fact, several independent reasons to believethat planktonic systems generally have a strange attractor,rather than a static or cyclic equilibrium. The chaotic naturehas some implications that may warrant a reconsideration ofour framework for thinking about plankton dynamics. For instance'(i) the observed fluctuations can be seen as a special typeof equilibrium, the nature of which can be understood from therelationships within the system; and (ii) although the rangeof possible behaviour of the system is well defined, the actualcourse of events is unpredictable, because small differencesin initial condition expand in time exponentially. The conclusionsfrom chaos theory sound quite spectacular, however, many ofthem do not appear to be essentially new to plankton ecologyIt is argued that the discrepancy between the mathematical findingsand present-day knowledge of the mechanisms behind planktondynamics are to a large extent semantic. Nonetheless, the seeminglysmall step of accepting that these systems are fundamentallychaotic implies the acceptance of the fact that we will neverbe able to predict their long-term behaviour accurately     

Interspecific variation in temperature dependence of egg development of five congeneric stonefly species (Protonemura Kempny, 1898, Nemouridae,Plecoptera)

Embryonal development of the five congeners Protonemura auberti Illies, 1954, P. hrabei Rauser, 1956, P. meyeri (Pictet, 1841), P. nitida (Stephens, 1835), and P. praecox (Morton, 1894) was studied under various laboratory temperatures and different photoperiods.Mean number of eggs in field collected batches was between 470 (P. praecox) and 1211 (P. auberti). Spring species had smaller egg batches than autumn species (Table 1). Mean hatching success in the laboratory was 50–100% at 2–18 °C. In most species hatching success decreased slightly with increasing temperature (Figs. 1a-e). None of the eggs incubated at 24 °C developed. Hatching pattern followed an asymmetric frequency distribution. In general, the hatching periods were the shorter the higher the incubation temperature.Embryonic development of all five species was inversely temperature dependent (Figs. 2a-e), and well described by a power law relationship (Figs. 3a-e). Interspecific differences in incubation periods were notable at nearly all temperatures (Fig. 4). There was a distinct interspecific sequence in length of incubation period (with steps of about 4 days), which was the same as can be seen in the flight periods: The later the species flies the longer the incubation period. Temperature fluctuations and variations in photoperiod had no influence on incubation and hatching periods or hatching success.The thermal demand of the egg stage neither explains the recent geographical distribution of the Protonemura species, nor does it directly correspond to the field temperatures common during their egg development. However, it is optimal in respect to resource partitioning between the five species, with the consequence of temporal displacement of life cycles.Derived from Brittain's (in press) proposal to compare the two constants a and b of the regressions describing the temperature dependence of embryonal development, a new index (Integral Development Time, IDT) indicating the thermal demand was created for easier comparison of numerous species (Table 2). Evaluation of the IDT for various species of Plecoptera (Fig. 5) suggests that species belonging to the family group Systellognatha generally have higher thermal requirements in the egg stage than species belonging to the Euholognatha.     

Glycerol conversion to 1,3-propanediol by newly isolated clostridia

Summary From pasteurized mud and soil samples glycerol-fermenting clostridia that produced 1,3-propanediol, butyrate and acetate were obtained. The isolates were taxonomically characterized and identified as Clostridium butyricum. The most active strain, SH1 = DSM 5431, was able to convert up to 110 g/l of glycerol to 56 g/l of 1,3-propanediol in 29 h. A few Clostridium strains from culture-collections (3 out of 16 of the C. butyricum group) and some isolates of Kutzner from cheese samples were also able to ferment glycerol, but the final concentration and the productivity of 1,3-propanediol was lower than in strain SH1. Strain SH1 grew well in a pH range between 6.0 and 7.5, with a weak optimum at 6.5, and was stimulated by sparging with N₂. Best overall productivity was obtained in fed-batch culture with a starting concentration of 5% glycerol. In all fermentations the yield of 1,3-propanediol in relation to glycerol was higher than expected from NADH production by acid formation. On the other hand the H₂ production was lower than expected, if per mole of acetyl coenzyme A one mole of H₂ is released. The observations point to a substantial transfer of reducing potential from ferredoxin to NAD, which finally results in increased 1,3-propanediol production.     

Identification and modulation of a voltage-dependent anion channel in the plasma membrane of guard cells by high-affinity ligands.

Guard cell anion channels (GCAC1) catalyze the release of anions across the plasma membrane during regulated volume decrease and also seem to be involved in the targeting of the plant growth hormones auxins. We have analyzed the modulation and inhibition of these voltage-dependent anion channels by different anion channel blockers. Ethacrynic acid, a structural correlate of an auxin, caused a shift in activation potential and simultaneously a transient increase in the peak current amplitude, whereas other blockers shifted and blocked the voltage-dependent activity of the channel. Comparison of dose-response curves for shift and block imposed by the inhibitor, indicate two different sites within the channel which interact with the ligand. The capability to inhibit GCAC1 increases in a dose-dependent manner in the sequence: probenecid less than A-9-C less than ethacrynic acid less than niflumic acid less than IAA-94 less than NPPB. All inhibitors reversibly blocked the anion channel from the extracellular side. Channel block on the level of single anion channels is characterized by a reduction of long open transitions into flickering bursts, indicating an interaction with the open mouth of the channel. IAA-23, a structural analog of IAA-94, was used to enrich ligand-binding polypeptides from the plasma membrane of guard cells by IAA-23 affinity chromatography. From this protein fraction a 60 kDa polypeptide crossreacted specifically with polyclonal antibodies raised against anion channels isolated from kidney membranes. In contrast to guard cells, mesophyll plasma membranes were deficient in voltage-dependent anion channels and lacked crossreactivity with the antibody.     

ATPase activity of isolated plasma membrane vesicles of leaves of Elodea as affected by thiol reagents and NADH/NAD+ ratio

The goal of this study was to test the hypothesis that the plasma membrane-bound ATPase activity is influenced by the redox poise of the cytoplasm. Purified plasma membrane vesicles from leaves of Elodea canadensis Michx. and E. nuttallii (Planch.) St. John were isolated using an aqueous polymer two-phase batch procedure. The distribution of marker enzyme activities confirmed the plasma membrane origin of the vesicles. The vesicles exhibited NADH-ferricyanide reductase activity, indicating the presence of a redox chain in the plasma membrane. The K⁺, Mg²⁺-ATPase activity associated with these vesicles was inhibited by the sulfhydryl reagents N-ethylmaleimide and glutathione (GSSG). Furthermore the activity was inhibited by NAD⁺. This inhibition by NAD⁺ was relieved by increasing the NADH/NAD⁺ ratio. The possibility that the ATPase activity is regulated by the cytoplasmic NAD(P)H/ NAD(P)⁺ ratio is discussed, as well as the role of a plasma membrane-bound redox chain.