Molecular extinction coefficients of lead sulfide and polymerized diaminobenzidine as final reaction products of histochemical phosphatase reactions.

Molar extinction coefficients of precipitated lead sulfide (PbS) and polymerized diaminobenzidine (polyDAB) have been determined at wavelengths of 450 nm and 480 nm, respectively, for quantitative histochemical analysis of phosphatase reactions. These values are essential for the conversion of cytophotometric (mean integrated) absorbance values to absolute units of substrate converted per unit time and volume of tissue. This conversion allows direct comparison of histochemical and biochemical data. The molar extinction coefficient of PbS at 450 nm was found to be 3,800 and therefore, per mole phosphate liberated, the molar extinction coefficient is 5,700 because 3 moles phosphate are captured by 2 moles lead at neutral or alkaline pH. Parallel experiments with the cerium-DAB method revealed that the molar extinction coefficient of polyDAB at 480 nm is 5,500 with respect to liberated phosphate. The molar extinction coefficients were applied for comparison of data from biochemical and histochemical assays of glucose-6-phosphatase activity in rat livers. A significant correlation was found between both sets of data. The values were in the same order of magnitude with histochemical values approximately 1.4 times higher than biochemical values.     

Quantitative histochemical analysis of glucose-6-phosphatase activity in rat liver using an optimized cerium-diaminobenzidine method

We have optimized a cerium-diaminobenzidine-based method for histochemical analysis of glucose-6-phosphatase (G6Pase) activity and have determined quantitative data on the zonal distribution pattern in the liver acinus of fasted male rats. In the cerium-diaminobenzidine technique, cerium instead of lead ions is used as capturing reagent for the enzymatically liberated phosphate. For light microscopy, the primary reaction product, cerium phosphate, is then visualized by conversion into cerium perhydroxide using hydrogen peroxide and subsequent oxidative polymerization of diaminobenzidine to diaminobenzidine brown as the final reaction product. Variation of the substrate (glucose-6-phosphate) concentration in the incubation medium yielded in periportal zones a KM value of 2.3 +/- 0.7 mM and a Vmax value of 0.96 +/- 0.18 (expressed as mean integrated absorbance). In perivenous zones a KM value of 1.1 +/- 0.4 mM and a Vmax value of 0.51 +/- 0.08 were calculated. The cytophotometric analysis performed in this study demonstrated for the first time that a functional difference of G6Pase, the key enzyme for gluconeogenesis, exists in the periportal and perivenous zones of the liver acinus. Periportal zones contain twice as many enzyme molecules (high Vmax) as perivenous zones, but the affinity for the substrate is twice as low. This may have important implications for the concept of metabolic zonation of the liver and also for glucose homeostasis in the blood.     

Multiple xenobiotic-inducible P450s are involved in alkoxycoumarin and alkoxyresorufin metabolism in higher plants

O-Dealkylation of two series of fluorescent 7-alkoxy-coumarins and 7-alkoxyphenoxazones by plant cytochrome P450s was investigated in Helianthus tuberosus tuber tissues treated with prototype P450 inducers, environmental pollutants or agrochemicals. Methoxy-, ethoxy-, propoxy-and butoxycoumarins and methoxy- and ethoxyresorufins were metabolized by fplant microsomes. Dealkylation of pentoxy- and benzyloxyresorufins was not detected. All dealkylating activities were enhanced by aging plant tissues in the presence of xenobiotics, in some cases up to 20-fold relative to the activities detected in control tissues. Increases in total P450 in the same tissues never exceeded 3-fold. The isozymes induced by prototype P450 inducers clearly differed from those in mammalian liver. That multiple P450s with overlapping substrate specificities were involved in the metabolism of both alkoxycoumarins and alkoxyresorufins was demonstrated by (1) the differential induction of the activities in response to exposure to xenobiotics, (2) the differential inhibition of the activities by clotrimazole, paclobutrazole and tetcyclacis in aminopyrine and benzo(a)pyrene-treated tissues, and(3) the selective inhibition observed with antibodies raised against purified ethoxycoumarin deethylase fractions. Our results suggest that the measurement of the dealkylation of such fluorescent substrates in plants might be useful to monitor environmental pollution.     

Temporal persistence and spatial distribution of an American inoculant strain of the ectomycorrhizal basidiomycete Laccaria bicolor in a French forest plantation

Selected strains of ectomycorrhizal fungi, such as the basidiomycete Laccaria bicolor , are currently being used as inoculants in nurseries to improve growth of forest trees after outplanting. Information is needed on the survival of these introduced strains in forests and their impact on indigenous biodiversity. Dissemination and persistence of an American strain, L. bicolor S238N, were studied 10 years after outplanting in a Douglas fir plantation located at Saint-Brisson (Morvan, France). About 430 Laccaria spp. sporophores were collected over 3 years. Inheritance of nuclear ribosomal DNA, as well as RAPD markers, was characterized in L. bicolor S238N, using a haploid progeny set of 91 monokaryons. More than 50 markers were identified (19 heterozygous and 33 homozygous or cytoplasmic markers), which unambiguously confirmed that the introduced strain was still present in the inoculated plots. Neither selfing ( P < 0.0008) nor introgression with indigenous strains was detected although in vitro interfertility between the American strain and indigenous L. bicolor was identified. No ingress of the introduced genet into adjacent uninoculated plots colonized by various local Laccaria genets was detected. It is proposed that the spatial distributions identified have developed through mycelial propagation of the introduced strain and intraspecific competition with native genets. Although longer-term data is still lacking, the stability of the inoculant strain and the limited disturbance to indigenous populations described support large-scale nursery production of this host-fungal combination.     

Elevated CO2 changes the interactions between nematode and tomato genotypes differing in the JA pathway

Interactions between the root‐knot nematode Meloidogyne incognita and three isogenic tomato (Lycopersicon esculentum) genotypes were examined when plants were grown under ambient (370 ppm) and elevated (750 ppm) CO₂. We tested the hypothesis that, defence‐recessive genotypes tend to allocate ‘extra’ carbon (relative to nitrogen) to growth under elevated CO₂, whereas defence‐dominated genotypes allocate extra carbon to defence, and thereby increases the defence against nematodes. For all three genotypes, elevated CO₂ increased height, biomass, and root and leaf total non‐structural carbohydrates (TNC):N ratio, and decreased amino acids and proteins in leaves. The activity of anti‐oxidant enzymes (superoxide dismutase and catalase) was enhanced by nematode infection in defence‐recessive genotypes. Furthermore, elevated CO₂ and nematode infection did not qualitatively change the volatile organic compounds (VOC) emitted from plants. Elevated CO₂ increased the VOC emission rate only for defence‐dominated genotypes that were not infected with nematodes. Elevated CO₂ increased the number of nematode‐induced galls on defence‐dominated genotypes but not on wild‐types or defence‐recessive genotypes roots. Our results suggest that CO₂ enrichment may not only increase plant C : N ratio but can disrupt the allocation of plant resources between growth and defence in some genetically modified plants and thereby reduce their resistance to nematodes.     

Experimentally induced colon cancer metastases in rat liver increase the proliferation rate and capacity for purine catabolism in liver cells

Metastases in rat liver were generated experimentally by intraportal injection of colon cancer cells to investigate the effects of cancerous growth on the metabolism of surrounding liver tissue. Maximum activities (capacity) of glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, lactate dehydrogenase, succinate dehydrogenase, alkaline phosphatase, 5-nucleotidase, xanthine oxidoreductase, purine nucleoside phosphorylase and adenosine triphosphatase have been determined. Two types of metastases were found, a small type surrounded by stroma and a larger type in direct contact with hepatocytes. Both types affected the adjacent tissue in a similar way suggesting that the interactions were not mediated by stroma. High capacity of the degradation pathway of extracellular purines released from dead cells of either tumours or host tissue was found in stroma and sinusoidal cells. Metastases induced both an increase in the number of Kupffer cells and proliferation of hepatocytes. The distribution pattern in the liver lobulus of most enzymes investigated did not change distinctly. However, activity of alkaline phosphatase, succinate dehydrogenase and phosphogluconate dehydrogenase was increased in hepatocytes directly surrounding metastases. These data imply that the overall metabolic zonation in liver lobuli is not dramatically disturbed by the presence of cancer cells despite the fact that various metabolic processes in liver cells are affected.In honour of Prof. Dr. Z. Lojda for his 65th birthday     

Long-term survival despite low genetic diversity in the critically endangered Madagascar fish-eagle

The critically endangered Madagascar fish-eagle ( Haliaeetus vociferoides ) is considered to be one of the rarest birds of prey globally and at significant risk of extinction. In the most recent census, only 222 adult individuals were recorded with an estimated total breeding population of no more than 100–120 pairs. Here, levels of Madagascar fish-eagle population genetic diversity based on 47 microsatellite loci were compared with its sister species, the African fish-eagle ( Haliaeetus vocifer ), and 16 of these loci were also characterized in the white-tailed eagle ( Haliaeetus albicilla ) and the bald eagle ( Haliaeetus leucocephalus ). Overall, extremely low genetic diversity was observed in the Madagascar fish-eagle compared to other surveyed Haliaeetus species. Determining whether this low diversity is the result of a recent bottleneck or a more historic event has important implications for their conservation. Using a Bayesian coalescent-based method, we show that Madagascar fish-eagles have maintained a small effective population size for hundreds to thousands of years and that its low level of neutral genetic diversity is not the result of a recent bottleneck. Therefore, efforts made to prevent Madagascar fish-eagle extinction should place high priority on maintenance of habitat requirements and reducing direct and indirect human persecution. Given the current rate of deforestation in Madagascar, we further recommend that the population be expanded to occupy a larger geographical distribution. This will help the population persist when exposed to stochastic factors (e.g. climate and disease) that may threaten a species consisting of only 200 adult individuals while inhabiting a rapidly changing landscape.     

梨分子遗传图谱构建及生长性状的QTL分析

利用鸭梨和京白梨杂交得到的F1(145株)实生苗为作图群体,通过对AFLP和SSR两种分子标记的遗传连锁分析,应用Joinmap 3.0作图软件,368个AFLP标记、34个SSR标记构建了分属18个连锁群的梨分子遗传连锁图谱,各连锁群的LOD值在4.0～7.0范围之间,图谱总长度覆盖梨基因组1395.9cM,平均图距为3.8cM.采用区间作图法,对该群体与生长性状相关的调查数据进行QTL分析,检测到与新梢生长量、新梢茎粗、节间长度、节间数量、树干径、树高及皮孔密度7个农艺性状连锁的QTL位点35个,其中主效QTL位点11个(LOD≥3.5).与生长性状相关的农艺性状QTL位点多集中在LG16连锁群上.