Regulation of immediate-early gene expression in rat retinal ganglion cells after axotomy and during regeneration through a peripheral nerve graft

To determine mechanisms of structural plasticity in adult CNS neurons, we investigated the expression of immediate early genes (IEGs) in the rat retina. Gene products of different IEG families (JUN and FOS proteins) and cAMP-responsive element binding protein (CREBP) were examined by immunohistochemistry under three different paradigms. Normal rats which were not axotomized were compared with axotomized animals, where retinal ganglion cells (RGCs) were axotomized by intraorbital optic nerve cut and retrogradely labeled with fluorogold (FG). Under these circumstances, RGCs show only transient sprouting, followed by continuous retrograde RGC degeneration. In the third group, after the optic nerve lesion, adult rats additionally received a sciatic nerve graft to the transected optic nerve stump. This allows some RGCs to regenerate an axon into the grafted nerve. In both groups, the time course of RGC survival and JUN, CREB, and FOS protein expression was monitored. In normal animals, JUN-Immunoreactivity (JUN-Ir) was not detectable in the retinal ganglion cell layer. JUN-Ir was induced in about 70% of all FG-positive RGCs 5 days after axotomy. The expression of JUN-Ir started to decline 8 days after axotomy. Only a few JUN-Ir-positive RGCs were found after 2 weeks. In transplanted animals, however, the numbers of JUN-Ir-positive RGCs were significantly higher 2 and 3 weeks after transplantation compared to animals that exclusively received axotomy. Furthermore, in grafted rats about 70% of the regenerating RGCs expressed JUN-Ir 2 weeks after grafting as compared to only 38% JUN-positive RGCs among the surviving but not regenerating RGCs. In normal animals CREBP-Ir was constitutively expressed in nearly all cells of the retinal ganglion cell layer. The decline in number of CREBP-Ir-positive cells paralleled the axotmy-induced RGC death. FOS-Ir-positive cells were not found in the ganglion cell layer at any time. These results demonstrate a selective and transient JUN expression of RGCs after axotomy which is sustained during axonal regeneration. This suggests that sciatic nerve grafts are able to regulate the expression of JUN proteins in axotomized RGCs of adult rats. 1994 John Wiley & Sons, Inc.     

Single-channel analysis of the anion channel-forming protein from the plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense

The anion channel protein from Clavibacter michiganense ssp. nebraskense (Schürholz, Th. et al. 1991, J. Membrane Biol. 123: 1-8) was analyzed at different concentrations of KCl and KF. At 0.8 M KCl the conductance G(V_m) increases exponentially from 21 pS at 50 mV up to 53 pS at V_m = 200 mV, 20°C. The concentration dependence of G(V_m) corresponds to a Michaelis-Menten type saturation function at all membrane voltage values applied (0-200 mV). The anion concentration K_0.5, where G(V_m) has its half-maximum value, increases from 0.12 M at 50 mV to 0.24 M at 175 mV for channels in a soybean phospholipid bilayer. The voltage dependence of the single channel conductance, which is different for charged and neutral lipid bilayers, can be described either by a two-state flicker (2SF) model and the Nernst-Planck continuum theory, or by a two barrier, one-site (2B1S) model with asymmetric barriers. The increase in the number of open channels after a voltage jump from 50 mV to 150 mV has a time constant of 0.8 s. The changes of the single-channel conductance are much faster (<1 ms). The electric part of the gating process is characterized by the (reversible) molar electrical work ΔG^θ_el = ρZ_gFV_m ≈ -1.3 RT, which corresponds to the movement of one charge of the gating charge number |Z_g| = 1 across the fraction ρ = ΔV_m/V_m = 0.15 of the membrane voltage V_m = 200 mV. Unlike with chloride, the single channel conductance of fluoride has a maximum at about 150 mV in the presence of the buffer PIPES (≥5 mM, pH 6.8) with K_0.5 ≈ 1 M. It is shown that the decrease in conductance is due to a blocking of the channel by the PIPES anion. In summary, the results indicate that the anion transport by the Clavibacter anion channel (CAC) does not require a voltage dependent conformation change of the CAC.     

Mechanisms of beta-adrenergic stimulation of cardiac Ca2+ channels revealed by discrete-time Markov analysis of slow gating.

Individual cardiac Ca2+ channels cycle slowly between a mode of gating in which the channel is available to open, and one in which the channel remains silent. The regulation of this multisecond cycling process by isoproterenol was investigated by single-channel recording and the development of a discrete-time Markov model that describes the slow switching among modes in terms of (de) phosphorylation reactions. The results provide evidence that isoproterenol increases Ca2+ channel activity by a reciprocal regulatory mechanism: not only is the phosphorylation rate of the channel increased, but also the dephosphorylation rate decreased. The discrete-time Markov formalism should prove useful as a general tool for understanding the mode switching demonstrated by a number of ionic channels.     

Pan-Arctic soil moisture control on tundra carbon sequestration and plant productivity

Long-term atmospheric CO₂ concentration records have suggested a reduction in the positive effect of warming on high-latitude carbon uptake since the 1990s. A variety of mechanisms have been proposed to explain the reduced net carbon sink of northern ecosystems with increased air temperature, including water stress on vegetation and increased respiration over recent decades. However, the lack of consistent long-term carbon flux and in situ soil moisture data has severely limited our ability to identify the mechanisms responsible for the recent reduced carbon sink strength. In this study, we used a record of nearly 100 site-years of eddy covariance data from 11 continuous permafrost tundra sites distributed across the circumpolar Arctic to test the temperature (expressed as growing degree days, GDD) responses of gross primary production (GPP), net ecosystem exchange (NEE), and ecosystem respiration (ER) at different periods of the summer (early, peak, and late summer) including dominant tundra vegetation classes (graminoids and mosses, and shrubs). We further tested GPP, NEE, and ER relationships with soil moisture and vapor pressure deficit to identify potential moisture limitations on plant productivity and net carbon exchange. Our results show a decrease in GPP with rising GDD during the peak summer (July) for both vegetation classes, and a significant relationship between the peak summer GPP and soil moisture after statistically controlling for GDD in a partial correlation analysis. These results suggest that tundra ecosystems might not benefit from increased temperature as much as suggested by several terrestrial biosphere models, if decreased soil moisture limits the peak summer plant productivity, reducing the ability of these ecosystems to sequester carbon during the summer.     

The unexpected long period of elevated CH4 emissions from an inundated fen meadow ended only with the occurrence of cattail (Typha latifolia)

Drainage and agricultural use transform natural peatlands from a net carbon (C) sink to a net C source. Rewetting of peatlands, despite of high methane (CH₄) emissions, holds the potential to mitigate climate change by greatly reducing CO₂ emissions. However, the time span for this transition is unknown because most studies are limited to a few years. Especially, nonpermanent open water areas often created after rewetting, are highly productive. Here, we present 14 consecutive years of CH₄ flux measurements following rewetting of a formerly long-term drained peatland in the Peene valley. Measurements were made at two rewetted sites (non-inundated vs. inundated) using manual chambers. During the study period, significant differences in measured CH₄ emissions occurred. In general, these differences overlapped with stages of ecosystem transition from a cultivated grassland to a polytrophic lake dominated by emergent helophytes, but could also be additionally explained by other variables. This transition started with a rapid vegetation shift from dying cultivated grasses to open water floating and submerged hydrophytes and significantly increased CH₄ emissions. Since 2008, helophytes have gradually spread from the shoreline into the open water area, especially in drier years. This process was periodically delayed by exceptional inundation and eventually resulted in the inundated site being covered by emergent helophytes. While the period between 2009 and 2015 showed exceptionally high CH₄ emissions, these decreased significantly after cattail and other emergent helophytes became dominant at the inundated site. Therefore, CH₄ emissions declined only after 10 years of transition following rewetting, potentially reaching a new steady state. Overall, this study highlights the importance of an integrative approach to understand the shallow lakes CH₄ biogeochemistry, encompassing the entire area with its mosaic of different vegetation forms. This should be ideally done through a study design including proper measurement site allocation as well as long-term measurements.     

Environmental vibrio phage–bacteria interaction networks reflect the genetic structure of host populations

Phages depend on their bacterial hosts to replicate. The habitat, density and genetic diversity of host populations are therefore key factors in phage ecology, but our ability to explore their biology depends on the isolation of a diverse and representative collection of phages from different sources. Here, we compared two populations of marine bacterial hosts and their phages collected during a time series sampling program in an oyster farm. The population of Vibrio crassostreae, a species associated specifically to oysters, was genetically structured into clades of near clonal strains, leading to the isolation of closely related phages forming large modules in phage–bacterial infection networks. For Vibrio chagasii, which blooms in the water column, a lower number of closely related hosts and a higher diversity of isolated phages resulted in small modules in the phage–bacterial infection network. Over time, phage load was correlated with V. chagasii abundance, indicating a role of host blooms in driving phage abundance. Genetic experiments further demonstrated that these phage blooms can generate epigenetic and genetic variability that can counteract host defence systems. These results highlight the importance of considering both the environmental dynamics and the genetic structure of the host when interpreting phage–bacteria networks.     

Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation inEscherichia coli

To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 inEscherichia coli, theinfC, rpmI andrpIT genes encoding IF3, L35 and L20, respectively, were placed under the control oflac promoter/operator sequences. Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombinant lambda phages that express eitherrpmI andrplT orinfC andrpmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations. At low IPTG concentrations in the IF3-limited strain, the cellular concentration of IF3, but not L20, decreases and the growth rate slows. Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo. During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis. As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation ofinfC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein. In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41–43S is seen. Previous studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepresses the expression of anrplT-lacZ gene fusion, thus confirming autogenous regulation by L20.