Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L.

In contrast to undifferentiated cell suspension cultures of Digitalis lanata, photomixotrophic shoot cultures of Digitalis purpurea accumulate cardiac glycosides in substantial concentrations. They are used to investigate enzymes of the cardenolide pathway. All cardenolides are 5-configurated. The progesterone 5-reductase and the 3-hydroxysteroid-5-oxidoreductase are present in shoot cultures but not in undifferentiated cell cultures. These enzymes provide precursors for cardenolides, whereas the presence of the progesterone 5-reductase, also present in shoot cultures, is discussed with regard to its role in phytosterol biosynthesis and may be attributed to the general steroid pathway. The progesterone 5-reductase had an activity maximum during the early growth period seven days after onset of cultivation, whereas the corresponding progesterone 5-reductase activity was highest on day 11. The maximum cardenolide accumulation was after 24 days. The enzyme activities present in crude extracts from shoot cultures were characterized with regard to their requirements for NADPH and NADH, pH-optimum, temperature optimum, affinity to their substrates and their localization in the cell. The progesterone 5-reductase was purified 769-fold.Abbreviations DW dry weight - FW fresh weight - PVP polyvinylpyrrolidone     

Association of the Decline of Nashi Pears with an MLO

Mycoplasma-like organisms (MLOs) were constantly detected by the DAPI technique and by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified DNA in trees of Pyrus pyrifolia cvs Hosui and Kosui grafted on P. communis, seedlings rootstock with symptoms similar to the slow form of pear decline. These symptoms included upward curling of the leaves along the midrib. Leaves were abnormally thick and later turned reddish while major veins became swollen and brown. Trees with symptoms were usually 4–5 years old and were growing in the major pear areas of central Italy. The incidence of affected trees was particularly high in one orchard adjacent to a pear orchard strongly affected with the slow form of pear decline. In this case the distribution pattern of affected Nashi trees suggests that the causal agent was introduced from the adjacent pear orchard by an aerial vector. Although oriental pears are well-known hosts of the pear-decline agent when used as rootstocks of French cultivars, this is the first report of pear decline in P. pyrifolia varieties.     

Restriction fragment length polymorphisms of the phytohemagglutinin genes inPhaseolus andVigna (Leguminosae)

Restriction fragment length polymorphisms at the phytohemagglutinin (PHA) locus were determined among 21 genotypes ofPhaseolus vulgaris, P. coccineus, P. acutifolius, P. lunatus, and threeVigna species, using five restriction enzymes and one double digestion, in order to provide molecular evidence for their genetic relatedness. The dissimilarity between genotypes was estimated from binary RFLP data. The dissimilarity was high among species (from 0.75 to 0.95), and of variable extent among genotypes of the same species (0.33–0.89). InP. vulgaris, two different DNA hybridization patterns were found, giving further evidence for two major gene pools in that species. The restriction patterns ofP. vulgaris var.aborigineus, the putative ancestral form ofP. vulgaris, exhibit clear homology toP. vulgaris genotypes. An undefined landrace from Taiwan could be identified as aP. vulgaris genotype. RFLP-based trees for the phytohemagglutinin genes of the species studied were computed with several distance matrix and parsimony methods.     

A novel isotope labeling protocol for bacterially expressed proteins

Summary A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).     

Scarless skin wound repair in the fetus.

The ability of a fetus to heal without scar formation depends on its gestational age at the time of injury and the size of the wound defect. In general, linear incisions heal without scar until late in gestation whereas excisional wounds heal with scar at an earlier gestational age. The profiles of fetal proteoglycans, collagens, and growth factors are different from those in adult wounds. The less-differentiated state of fetal skin is probably an important characteristic responsible for scarless repair. There is minimal inflammation in fetal wounds. Fetal wounds are characterized by high levels of hyaluronic acid and its stimulator(s) with more rapid, highly organized collagen deposition. The roles of peptide growth factors such as transforming growth factor-beta and basic fibroblast growth factor are less prominent in fetal than in adult wound healing. Platelet-derived growth factor has been detected in scarless fetal skin wounds, but its role is unknown. An understanding of scarless tissue repair has possible clinical application in the modulation of adult fibrotic diseases and abnormal scar-forming conditions.     

Use of polymerase chain reaction and electroporation of Escherichia coli to monitor the persistence of extracellular plasmid DNA introduced into natural soils.

A modified protocol for DNA amplification by polymerase chain reaction (PCR) coupled with laser densitometric determination of the amount of PCR products, which allowed quantitation of target sequence numbers in soil extracts, was developed. The method was applied to monitor target loss during incubation of purified plasmid DNA in natural nonsterile soils. It revealed soil-specific kinetics of target loss. After 60 days, 0.2, 0.05, and 0.01% of the initially added nahA genes on plasmids were detectable by PCR in a loamy sand soil, a clay soil, and a silty clay soil, respectively. Electroporation of Escherichia coli was used in parallel to quantitate plasmid molecules in soil extracts by their transforming activity. It was found that transformation by electroporation was about 20 times more efficient and much less inhibited by constituents of soil extracts than transformation of Ca(2+)-treated cells (G. Romanowski, M.G. Lorenz, G. Sayler, and W. Wackernagel, Appl. Environ. Microbiol. 58:3012-3019, 1992). By electroporation, greater than 10,000-fold plasmid loss was monitored in nonsterile soils. Transforming activity was found up to 60 days after inoculation of the soils. The studies indicate that PCR and electroporation are sensitive methods for monitoring the persistence of extracellular plasmid DNA in soil. It is proposed that plasmid transformation by electroporation can be used for the monitoring in soil and other environments of genetically engineered organisms with recombinant plasmids. The data suggest that genetic material may persist in soil for weeks and even for months after its release from cells.     

H assignment and secondary structure determination of human melanoma growth stimulating activity (MGSA) by NMR spectroscopy

The solution structure of melanoma growth stimulating activity (MGSA) has been investigated using proton NMR spectroscopy. Sequential resonance assignments have been carried out, and elements of secondary structure have been identified on the basis of NOE, coupling constant, chemical shift, and amide proton exchange data. Long-range NOEs have established that MGSA is a dimer in solution. The secondary structure and dimer interface of MGSA appear to be similar to those found previously for the homologous chemokine interleukin-8 [Clore et al. (1990) Biochemistry 29, 1689-1696]. The MGSA monomer contains a three stranded anti-parallel β-sheet arranged in a ‘Greek-key’ conformation, and a C-terininal -helix (residues 58 69).     

Complete ammonia oxidization in agricultural soils: High ammonia fertilizer loss but low N2O production

The contribution of agriculture to the sustainable development goals requires climate-smart and profitable farm innovations. Increasing the ammonia fertilizer applications to meet the global food demands results in high agricultural costs, environmental quality deterioration, and global warming, without a significant increase in crop yield. Here, we reported that a third microbial ammonia oxidation process, complete ammonia oxidation (comammox), is contributing to a significant ammonia fertilizer loss (41.9 ± 4.8%) at the rate of 3.53 ± 0.55 mg N kg⁻¹ day⁻¹ in agricultural soils around the world. The contribution of comammox to ammonia fertilizer loss, occurring mainly in surface agricultural soil profiles (0–0.2 m), was equivalent to that of bacterial ammonia oxidation (48.6 ± 4.5%); both processes were significantly more important than archaeal ammonia oxidation (9.5 ± 3.6%). In contrast, comammox produced less N₂O (0.98 ± 0.44 μg N kg⁻¹ day⁻¹, 11.7 ± 3.1%), comparable to that produced by archaeal ammonia oxidation (16.4 ± 4.4%) but significantly lower than that of bacterial ammonia oxidation (72.0 ± 5.1%). The efficiency of ammonia conversion to N₂O by comammox (0.02 ± 0.01%) was evidently lower than that of bacterial (0.24 ± 0.06%) and archaeal (0.16 ± 0.04%) ammonia oxidation. The comammox rate increased with increasing soil pH values, which is the only physicochemical characteristic that significantly influenced both comammox bacterial abundance and rates. Ammonia fertilizer loss, dominated by comammox and bacterial ammonia oxidation, was more intense in soils with pH >6.5 than in soils with pH <6.5. Our results revealed that comammox plays a vital role in ammonia fertilizer loss and sustainable development in agroecosystems that have been previously overlooked for a long term.     

Constitutive expression of small heat shock proteins in vegetative tissues of the resurrection plant Craterostigma plantagineum

Using antibodies raised against two sunflower small heat shock proteins (sHSPs), we have detected immunologically related proteins in unstressed vegetative tissues from the resurrection plant Craterostigma plantagineum. In whole plants, further accumulation of these polypeptides was induced by heat-shock or water-stress. In desiccation-intolerant Craterostigma callus tissue, we failed to detect sHSP-related polypeptides, but their expression, and the concurrent acquisition of desiccation tolerance was induced by exogenous abscisic acid (ABA) treatment. In untressed plants, the cross-reacting polypeptides were abundant in the roots and lower part of the shoots, where they showed homogeneous tissue-distributions. This constitutive expression is novel for vegetative tissues of higher plants, and resembles the expression patterns of sHSPs in desiccation-tolerant zygotic embryos and germinating seeds.J.A. and C.A. contributed equally to this work and are both considered to be first author     

Zolpidem Displays Heterogeneity in Its Binding to the Nonhuman Primate Benzodiazepine Receptor In Vivo

Abstract: The distinctive pharmacological activity of zolpidem in rats compared with classical benzodiazepines has been related to its differential affinity for benzodiazepine receptor (BZR) subtypes. By contrast, in nonhuman primates the pharmacological activity of zolpidem was found to be quite similar to that of classical BZR agonists. In an attempt to explain this discrepancy, we examined the ability of zolpidem to differentiate BZR subtypes in vivo in primate brain using positron emission tomography. The BZRs were specifically labeled with [¹¹C]flumazenil. Radiotracer displacement by zolpidem was monophasic in cerebellum and neocortex, with in vivo Hill coefficients close to 1. Conversely, displacement of [¹¹C]flumazenil was biphasic in hippocampus, amygdala, septum, insula, striatum, and pons, with Hill coefficients significantly smaller than 1, suggesting two different binding sites for zolpidem. In these cerebral regions, the half-maximal inhibitory doses for the high-affinity binding site were similar to those found in cerebellum and neocortex and ～100-fold higher for the low-affinity binding site. The low-affinity binding site accounted for <32% of the specific [¹¹C]-flumazenil binding. Such zolpidem binding characteristics contrast with those reported for rodents, where three different binding sites were found. Species differences in binding characteristics may explain why zolpidem has a distinctive pharmacological activity in rodents, whereas its pharmacological activity in primates is quite similar to that of classical BZR agonists, except for the absence of severe effects on memory functions, which may be due to the lack of substantial zolpidem affinity for a distinct BZR subtype in cerebral structures belonging to the limbic system.