Changes in Metabotropic Glutamate Receptor mRNA Levels Following Global Ischemia: Increase of a Putative Presynaptic Subtype (mGluR4) in Highly Vulnerable Rat Brain Areas

Abstract: Metabotropic glutamate receptors mediate their intracellular response by coupling to G proteins and may be divided into three subfamilies: mGluR1 and mGluR5, which stimulate phosphatidylinositol hydrolysis; mGluR2 and mGluR3, which are negatively coupled to cyclic AMP formation; and mGluR4 and mGluR6, which also inhibit forskolin-stimulated cyclic AMP formation. The mGluR4 subtypes may represent l -2-amino-4-phosphonobutyrate-sensitive presynaptic autoreceptors, and two alternatively spliced variants of the mGluR4 coding for two receptors with different C termini have been identified. Using in situ hybridization, we measured the levels of mGluR1–mGluR5 mRNA in regions of the rat brain 24 h after transient global ischemia, a time point when no neuronal damage can yet be observed morphologically. In the hippocampus, the mRNA levels for mGluR1, mGluR2, and mGluR5 were decreased, mGluR3 mRNA levels were unchanged, and the mGluR4 mRNA levels were strongly increased. The strongest increase appeared to be in the mRNA encoding mGluR4b. The mGluR4 mRNA was also increased in the parietal cortex, whereas the ventral posteromedial thalamic nucleus showed a small decrease in its mRNA content. These results suggest that vulnerable neurons react to an increased extracellular glutamate concentration by differential regulation of the mRNA for pre- and postsynaptically located metabotropic glutamate receptors.     

Fast Cytoplasmic pH Regulation in Acid-Stressed Leaves

Induction of photosynthesis in leaves was prolonged, and steadystate photosynthesis was inhibited by very high CO₂ concentrationswhich cause cytoplasmic acidification. Prolonged exposure tohigh CO₂ relieved initially observed inhibition of photosynthesisat least partially. The sensitivity of carbon assimilation tohigh CO₂ was different in different plant species. Acidificationby CO₂ (or subsequent alkalization) was detected by measuringrapid CO₂-release from the tissue and by monitoring fluorescenceof pH-indicating dyes which had been fed to the leaves throughthe petiole. The results indicate that two different mechanismsoperate in leaves to achieve and maintain pH homeostasis. Rapidand efficient pH-adjustment is provided by proton/cation exchangeacross the tonoplast. Slower and less efficient regulation occursby formation or consumption of base. In the presence of highCO₂ concentrations, protons are pumped from the cytosol intoalready acidic vacuoles. In turn, vacuolar cations replace exportedprotons in the cytosol permitting bicarbonate accumulation andincreasing the pH of the acidified cytosol. Similarly effectiveand fast proton/cation exchange relieves acid-stress in thechloroplast stroma and permits photosynthesis to proceed withhigh quantum efficiency or high light-saturated rates in thepresence of CO₂ concentrations which would, in the absence offast cytoplasmic pH regulation, inhibit photosynthesis. By inference,proton/cation exchange must also occur across the mitochondrialboundary. After cytoplasmic pH adjustment in the presence ofhigh CO₂, removal of CO₂ results in transient cytoplasmic alkalizationand, subsequently, in the return of cytoplasmic pH values tolevels observed prior to acid-stress. In addition to fast pHregulation by rapid proton/cation exchange across biomembranes,slow base production (e.g. NH₃-formation) also contributes torelieving acid stress. Base produced in the presence of highCO₂ is rapidly consumed after removal of CO₂. Implications of the findings in regard to forest damage by potentiallyacidic air pollutants such as SO₂ are briefly discussed. (Received November 8, 1993; Accepted February 3, 1994)     

Microbial transformation of ethylpyridines

Pyridine and its derivatives have been found as pollutants in the environment. Although alkylpyridines constitute the largest class of pyridines contaminating the environment, little information is available concerning the fate and transformation of these compounds. In this investigation ethylpyridines have been used as model compounds for investigating the biodegradability of alkylpyridines. A mixed culture of ethylpyridine-degrading microorganisms was obtained from a soil that had been exposed to a variety of pyridine derivatives for several decades. The enrichment culture was able to degrade 2-, 3-, and 4-ethylpyridine (100 mg/L) at 28° C and pH 7 within two weeks under aerobic conditions. The degradation rate was greatest for 2-ethylpyridine and least for 3-ethylpyridine. Transformation of ethylpyridines was dependent on substrate concentration, pH, and incubation temperature. Studies on the metabolic pathway of 4-ethylpyridine revealed two products; these chemicals were identified by MS and NMR analyses as 4-ethyl-2(1H)-pyridone and 4-ethyl-2-piperidone. 6-Ethyl-2(1H)-pyridone was determined to be a product of 2-ethylpyridine degradation. These results indicate that the transformation mechanism of ethylpyridines involves hydroxylation and reduction of the aromatic ring before ring cleavage.     

Proteomic fingerprinting enables quantitative biodiversity assessments of species and ontogenetic stages in Calanus congeners (Copepoda,Crustacea) from the Arctic Ocean

Species identification is pivotal in biodiversity assessments and proteomic fingerprinting by MALDI-TOF mass spectrometry has already been shown to reliably identify calanoid copepods to species level. However, MALDI-TOF data may contain more information beyond mere species identification. In this study, we investigated different ontogenetic stages (copepodids C1–C6 females) of three co-occurring Calanus species from the Arctic Fram Strait, which cannot be identified to species level based on morphological characters alone. Differentiation of the three species based on mass spectrometry data was without any error. In addition, a clear stage-specific signal was detected in all species, supported by clustering approaches as well as machine learning using Random Forest. More complex mass spectra in later ontogenetic stages as well as relative intensities of certain mass peaks were found as the main drivers of stage distinction in these species. Through a dilution series, we were able to show that this did not result from the higher amount of biomass that was used in tissue processing of the larger stages. Finally, the data were tested in a simulation for application in a real biodiversity assessment by using Random Forest for stage classification of specimens absent from the training data. This resulted in a successful stage-identification rate of almost 90%, making proteomic fingerprinting a promising tool to investigate polewards shifts of Atlantic Calanus species and, in general, to assess stage compositions in biodiversity assessments of Calanoida, which can be notoriously difficult using conventional identification methods.     

COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Sec16p.

Formation of COPII-coated vesicles at the endoplasmic reticulum (ER) requires assembly onto the membrane of five cytosolic coat proteins, Sec23p, Sec24p, Sec13p, Sec31p, and Sar1p. A sixth vesicle coat component, Sec16p, is tightly associated with the ER membrane and has been proposed to act as a scaffold for membrane association of the soluble coat proteins. We previously showed that Sec23p binds to the C-terminal region of Sec16p. Here we use two-hybrid and coprecipitation assays to demonstrate that the essential COPII protein Sec24p binds to the central region of Sec16p. In vitro reconstitution of binding with purified recombinant proteins demonstrates that the interaction of Sec24p with the central domain of Sec16p does not depend on the presence of Sec23p. However, Sec23p facilitates binding of Sec24p to Sec16p, and the three proteins can form a ternary complex in vitro. Truncations of Sec24p demonstrate that the N-terminal and C-terminal regions of Sec24p display different binding specificities. The C terminus binds to the central domain of Sec16p, whereas the N terminus of Sec24p binds to both the central domain of Sec16p and to Sec23p. These findings define binding to Sec16p as a new function for Sec24p and support the idea that Sec16p organizes assembly of the COPII coat.     

Bioenergetics of Carbon Assimilation in Intact Chloroplasts: Coupling of Proton to Electron Transport at the Ratio H+/e=3 Is Incompatible with H+/ATP=3 in ATP Synthesis

During a transition from aerobic to largely anaerobic conditionslight-saturated carbon assimilation of intact chloroplasts wasnot decreased although both the transthylakoid proton gradientand ATP levels declined. After a dark period under anaerobiosis,illumination failed to initiate carbon assimilation. ATP increasedonly transiently in the light and then returned to the darklevel. Under such conditions, the addition of electron acceptorssuch as oxygen, oxalacetate or nitrite resulted in the increaseof ATP levels and carbon assimilation was initiated. Assimilationcontinued under anaerobiosis in the presence of reduced protongradients and reduced ATP levels after electron acceptors addedin addition to bicarbonate were reduced. Cyclic electron transport was inhibited when anaerobiosis didnot permit linear electron transport. It was induced in thissituation by micromolar concentrations of oxygen or when, underanaerobiosis, DCMU decreased PSII activity. Oxygen inhibitedcyclic electron transport by draining electrons from the cyclicpathway only when electron donation from PSII was weak. Theobservations give evidence of the delicate redox balance requiredfor cyclic electron transport. Since H⁺/e=3 in linear electron transport, the observationsof effective carbon reduction under a decreased transthylakoidproton gradient and decreased levels of ATP are incompatiblewith H⁺/ATP=2 or 3. They are compatible with H⁺/ATP=4. (Received May 1, 1995; Accepted October 3, 1995)     

Correlation between apparent activation state of nitrate reductase (NR), NR hysteresis and degradation of NR protein

Nitrate reductase (NR) activity was measured in extracts fromspinach leaves exposed to light or prolonged darkness, and tovarious treatments provoking an artificial activation of theenzyme in the dark. NR activity was determined immediately eitherin the presence of Mg²⁺, which gives an estimation of the putative(actual) activity in situ (NR_act), or in EDTA without preincubation,which gives an intermediate activity (NR_int), or after a 30min preincubation with EDTA plus AMP plus Pi, which gives themaximum NR activity (NR_max). NR_max is thought to reflect totalNR protein contents. In the dark, NR_act was usually very low. Dark inactivation wasprevented or reversed by feeding AICAR (5-aminoimidazole-4-carboxiamideribonucleoside), or by anaerobiosis, acid treatment or additionof uncoupler. During prolonged darkness, NR_max decreased, indicatingnet protein degradation with a half-time of 21 h. Conditionswhich caused an activation (dephosphorylation) of NR in thedark, slowed down NR protein degradation. This was also confirmedby Western blotting. Blockage of cytosolic protein synthesis with cycloheximide (CHX)did not accelerate NR protein degradation. In contrast, after5 h in the dark, NR_act increased in CHX-treated leaves. As thisincrease was sensitive to PP2A-inhibitors, it was probably dueto NR dephosphorylation. However, extractable NR kinase andNR phosphatase activities were not changed by CHX treatment.Apparently, CHX interacted with the NR regulatory system indirectlyby affecting turnover of another protein. The increase from NR_int to NR_max which occurred during preincubationof the leaf extract with EDTA plus AMP plus Pi was insensitiveto PP2A inhibitors and was interpreted as a hysteretic conversionof NR from an inactive into an active form. Hysteretic activationwas positively correlated to the NR phosphorylation state. Amodel is presented to explain the hysteretic behaviour of NRin relation to NA phosphorylation/ dephosphorylation. Overall, the data indicate that NR protein phosphorylation notonly controls the catalytic activity of NR, but also acts asa signal for NR protein degradation, with phospho-NR probablybeing a better substrate for protein degradation than the dephospho-form. Key words: Enzyme hysteresis, nitrate reductase, posttranslational modification, protein phosphorylation, protein turnover