Disentangling CO2 fluxes: direct measurements of mesocosm‐scale natural abundance 13CO2/12CO2 gas exchange, 13C discrimination,and labelling of CO2 exchange flux components in controlled environments

A ¹³C/¹²C mass spectrometer was interfaced with a open gas exchange system including four growth chambers to investigate CO₂ exchange components of perennial ryegrass (Lolium perenne L.) stands. Chambers were fed with air containing CO₂ with known δ¹³C (δ_CΟ2?2.6 or ?46.8‰). The system did not fractionate C isotopes and no extraneous CO₂ leaked into chambers. The on‐line ¹³C discrimination (Δ) of ryegrass stands in light was independent of δ_CΟ2 when δ_CΟ2 was constant. The δ of CO₂ exchanged by the stands in light (δ_Nd) and darkness (δ_Rn) differed by 0.7‰, suggesting some Δ in dark respiration at the stand‐level. However, Δ decreased by ～ 10‰ when δ_CΟ2 was switched from ?46.8 to ?2.5‰, and increased by ～ 10‰ following a shift from ?2.6 to ?46.7‰ due to isotopic disequilibria between photosynthetic and respiratory fluxes. Isotopic imbalances were used to assess (non‐photorespiratory) respiration in light and the replacement of the respiratory substrate pool(s) by new photosynthate. Respiration was partially inhibited by light, but increased during the light period and decreased in darkness, in association with temperature changes. The labelling kinetics of respiratory CO₂ indicated the existence of two major respiratory substrate pools: a fast pool which was exchanged within hours, and a slow pool accounting for ～ 60% of total respiration and having a mean residence time of 3.6 d.     

Immunolocalization of long-chain acyl-CoAs in plant cells

Long chain acyl-Coenzyme A esters (acyl-CoAs) are key substrates in many enzymic reactions of lipid metabolism. Due to their amphiphilic nature, the membrane localization of these molecules cannot be established by subcellular membrane fractionation and usual biochemical studies. We have developed another approach based on ultrastructural immunogold cytochemistry. To preserve the acyl-CoA membrane content, the plant material was freeze substituted and cryoembedded after short aldehyde fixation followed by quick freezing. Using Arabidopsis thaliana root cells and specific antibodies raised against acyl-CoAs, we show that acyl-CoAs are mainly localized in endoplasmic reticulum membranes.Our results demonstrate the value of cryo-methods for the accurate localization of labile metabolites in plant cells.     

Classical dot–blot format implemented as an amperometric hybridisation genosensor

A new electrochemical hybridisation genosensor has been designed. This genosensor is based on a concept adapted from classical dot–blot DNA analysis, but implemented in an electrochemical biosensor configuration. The use of amperometric transduction and the enzyme label method—that increases the genosensor sensitivity—are the main features of this new approach. The analytical procedure consists of five steps: DNA target immobilisation by adsorption onto a nylon membrane, hybridisation between DNA target and biotin–DNA probe, complexation reaction between biotin-DNA probe and an enzyme (horseradish peroxidase) streptavidin conjugate; integration of the modified membrane onto an electrochemical transducer; and finally, amperometric detection using a suitable substrate for the enzyme labelled duplex. Besides the adapted dot–blot format, a competitive assay in which the target is in solution is reported as well. This procedure, based on amperometric transduction, represents certain advantages with respect to dot–blot analysis: labelled hybrid detection is far simpler, quicker and requires more ordinary or simple reactives; the response obtained is a direct analytical signal via low-cost instrumentation, a nonisotopic labelling is used, and the membranes can be reused. These characteristics are ideal in implementing the procedure developed in kit form.     

Identification of the Adenosine Uptake Sites in Guinea Pig Brain

Nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport, was employed as a photolabile probe of the adenosine transporter in guinea pig brain membranes. Reversible, high-affinity binding of [3H]NBMPR to a crude preparation of guinea pig brain membranes was demonstrated (apparent KD 0.075 +/- 0.012 nM; Bmax values of 0.24 +/- 0.04 pmol/mg protein). Adenosine, uridine, dipyridamole, and nitrobenzylthioguanosine inhibited high-affinity binding. Low concentrations of cyclohexoadenosine (10-300 nM) had no effect on NBMPR binding. These properties of the high-affinity NBMPR binding sites were consistent with NBMPR binding to the nucleoside transport protein. Exposure of brain membranes in the presence of [3H]NBMPR and dithiothreitol, a free-radical scavenger, to ultraviolet light resulted in covalent incorporation of 3H into polypeptides of apparent MW 66,000-45,000, a value similar to that for the human erythrocyte nucleoside transporter. Covalent attachment of [3H]NBMPR was inhibited by adenosine, dipyridamole, and nitrobenzylthioguanosine.     

Origin of nucleolus-like bodies found in the nucleoplasm and cytoplasm of Vicia faba meristematic cells

Cytohistochemical staining and RNase-gold labelling have been applied to root-tip meristematic cells of Vicia faba to study the origin and biological significance of 2 types of inclusions: one seen in the nucleoplasm and the other in the cytoplasm of early telophase cells. They have been termed "dense bodies" and "cytoplasmic nucleolus-like bodies" (NLB), respectively. Both types of inclusions respond positively to silver staining and ribonucleoprotein (RNP) staining in a similar fashion to nucleolus. Interestingly, the dense bodies label heavily with the RNase-gold complex, as does the nucleolus, while the cytoplasmic NLB have no affinity with the label. In most cases, the dense bodies label more heavily than the nucleolus. Light microscope surveys reveal that the dense bodies sometimes appear to be released from the surface of the nucleolus. On the other hand, prenucleolar material showing the same silver staining and RNP preferential staining characteristics as the dense bodies begin to accumulate on the surface of chromosomes in mid-anaphase. This material does not label with RNase-gold. These data are discussed in terms of the hypothesis that the dense bodies are derived from the nucleolus by direct budding or fragmentation, and the cytoplasmic NLB are composed of prenucleolar material that failed to attach to chromosomes.     

Non-radioactively labelled DNA probes for the detection of periodontopathogenic Prevotella and Porphyromonas species

Abstract Dioxigenin-labelled synthetic DNA probes directed against the 16S rRNA were used for the direct detection of the periodontopathogenic bacteria Prevotella intermedia and Porphyromonas gingivalis in subgingival plaque by applying a DNA-RNA dot-blot hybridization procedure. The test was evaluated with 134 plaque samples from 26 patients with adult periodontitis or rapidly progressive periodontitis. The lower limit of detection was 10⁴–10⁵ bacteria/specimen. A semiquantitative assessment of the two species in each sample and in the corresponding periodontal site was achieved by this technique. It is possible to examine 80–90 samples within two days with low material costs.     

Fate and dynamics of recently fixed C in pasture plant–soil system under field conditions

The flow of photosynthetically fixed C from plants to selected soil C pools was studied after ¹³CO₂ pulse labeling of pasture plants under field conditions, dynamics of root-derived C in soil was assessed and turnover times of the soil C pools were estimated. The transport of the fixed C from shoots to the roots and into the soil was very fast. During 27 h, net C belowground allocation reached more than 10% of the fixed C and most of the C was already found in soil. Soil microbial biomass (C_MIC) was the major sink of the fixed C within soil C pools (ca 40–70% of soil ¹³C depending on sampling time). Significant amounts of ¹³C were also found in other labile soil C pools connected with microbial activity, in soluble organic C and C associated with microbial biomass (hot-water extract from the soil residue after chloroform fumigation-extraction) and the ¹³C dynamics of all these pools followed that of the shoots. When the labelling (2 h) finished, the fixed ¹³C was exponentially lost from the plant–soil system. The loss had two phases; the first rapid phase corresponded to the immediate respiration of ¹³C during the first 24 h and the second slower loss was attributable to the turnover of ¹³C assimilated in C_MIC. The corresponding turnover times for C_MIC were 1.1 days and 3.4 days respectively. Such short turnover times are comparable to those measured by growth kinetics after the substrate amendment in other studies, which indicates that microbial growth in the rhizosphere is probably not limited by substrate availability. Our results further confirmed the main role of the soil microbial community in the transformation of recently fixed C, short turnover time of the easily degradable C in the rhizosphere, and its negligible contribution to more stable soil C storage.     

地高辛标记探针电镜原位杂交技术探讨

应用ＬｏｗｉｃｒｙｌＫ４Ｍ低温包埋,Ｄｉｇ标记ｃ－ｆｏｓｃＤＮＡ探针对人肝癌组织进行原位杂交,用金标抗Ｄｉｇ抗体进行检测,银增强放大信号处理。电镜观察表明,免疫胶体金颗粒特异性地标记在肝癌细胞胞浆及核内,呈散在分布,金颗粒大小基本一致,背景清晰。