Identification and characterization of the key enzyme in the biosynthesis of the neurotoxin β-ODAP in grass pea

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with β-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme—β-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for β-ODAP production in vivo. The identification of BOS may pave the way toward engineering β-ODAP–free grass pea cultivars, which are safe for human and animal consumption.     

Long-distance trafficking of macromolecules in the phloem

The fact that macromolecules such as proteins and mRNAs overcome the symplastic barriers between various tissue domains was first evidenced by the movement of plant viruses. We have recently demonstrated that viral infection disengages the symplastic restriction present between the sieve element-companion cell complex and neighboring cells in tobacco plants. As a result, green fluorescent protein, which was produced in mesophyll and bundle sheath cells, could traffic into the sieve tube and travel long distances within the vascular system. In this addendum we discuss the likely existence of a novel plant communication network in which macromolecules also act as long-distance trafficking signals. Plasmodesmata interconnecting sieve elements and companion cells as well as plasmodesmata connecting the sieve tube with neighboring cells may play a central role in establishing this communication network.Key words: companion cells, cucumber mosaic virus, Cucumis melo, plasmodesmata, movement protein, sieve-elementsTranslocation of photoassimilates from the source (site of synthesis) to various sink organs is governed, in part, by short-distance intercellular transfer of assimilates to the loading region of the phloem and long-distance transport within the plant vascular system. Sucrose, which is synthesized in the leaf mesophyll, moves cell-to-cell symplastically through plasmodesmata until it reaches the boundary of the sieve element (SE)-companion cell (CC) complex. In many plant species, the connection between phloem parenchyma (PP)/bundle sheath (BS) cells and CCs is characterized by a sparseness of plasmodesmata (e.g., Solanaceae), and sucrose is exported out of the cells to the apoplast. This type of plants (apoplastic loaders) uses sucrose proton symporters to load the sucrose into the vasculature.¹ Cucurbits are considered one of the model plants for symplastic phloem loading.² This type of plant is characterized by abundant plasmodesmata interconnecting the intermediary cells, which are specialized CCs, with the neighboring BS cells. It is generally accepted that in these plants, phloem loading includes intercellular movement of sucrose through the plasmodesmata, along the entire pathway from the mesophyll cell to the SE-CC complex.Interestingly, the existence of plasmodesmata interconnecting the SE-CC complex and neighboring cells is evident in all plant species that are characterized by an apoplastic phloem-loading mechanism. Moreover, microinjection experiments have indicated that plasmodesmata interconnecting the PP-CC are functional, in that they allow the exchange of small membrane-impermeable fluorescent probes.³ Virus movement through plasmodesmata from the mesophyll into the SEs further supports the notion that the symplastic communication between the CC-SE complex and the neighboring cells is functional.⁴One can assume that in apoplastic-loading plants, it would be an advantage to maintain the SE-CC complex as an isolated domain, with no functional plasmodesmata interconnecting it to the neighboring tissue. Symplastic continuity between the two domains could result in leakage of sucrose out of the vasculature and a significant reduction in the efficacy of sucrose loading. The fact that the two domains are interconnected suggests that any back-leakage of sucrose that might occur is insignificant relative to the likely efficacy of this communication route.What might the advantage be for symplastic communication between the SE-CC complex and the neighboring tissue? Accumulated evidence suggests that at the tissue/organ level, cell-to-cell trafficking of information molecules allows for noncell-autonomous control over a range of processes, whereas at the organismal level, the phloem serves as an information superhighway, delivering a wide range of macromolecules to enable the plant to function as a whole organism.^5–8 We advanced the hypothesis that plasmodesmata interconnecting the CCs and PP/BS cells play a pivotal role in controlling the long-distance trafficking of putative signaling molecules.     

Malic acid accumulation by Aspergillus flavus

Summary ¹³C Nuclear magnetic resonance and fumarase and NAD-malate dehydrogenase isoenzyme studies were carried out in a strain of A. flavus which produces relatively high levels of l-malic acid from glucose. The results of the ¹³C NMR showed that the ¹³C label from [1-¹³C] glucose was incorporated only to C-3 (-CH₂-) of l-malic acid and indicated that this acid must be synthesized from pyruvate mainly via oxaloacetate. Electrophoretic analysis has established the presence of unique mitochondrial and cytosolic isoenzymes for fumarase and malate dehydrogenase. Changes in the isoenzyme pattern were observed for malate dehydrogenase but not for fumarase during acid production. Cycloheximide inhibited profoundly both l-malic acid production and the increase in the major isoenzyme of malate dehydrogenase, without affecting either the total activity of fumarase or its isoenzyme pattern. The results suggested that de novo protein synthesis is involved in the increase in the activity of the major isoenzyme of malate dehydrogenase and that this isoenzyme is essential for l-malic acid production and accumulation.     

Patient specific quantitative analysis of fracture fixation in the proximal femur implementing principal strain ratios. Method and experimental validation

Computational patient-specific modeling has the potential to yield powerful information for selection and planning of fracture treatments if it can be developed to yield results that are rapid, focused and coherent from a clinical perspective. In this study we introduce the utilization of a principal strain fixation ratio measure (SR) defined as the ratio of principal strains that develop in a fixated bone relative to the principal strains that develop in the same bone in an intact state. The SR field output variable is theoretically independent of load amplitude and also has a direct clinical interpretation with SR<1?a representing stress shielding and SR>1+b representing overstressed bone. A combined experimental and numerical study was performed with cadaveric proximal femora (n=6) intact and following fracture fixation to quantify the performance of the SR variable in terms of accuracy and sensitivity to uncertainties in density–elasticity relationships and load amplitude as model input variables. For a given axial compressive force the SR field output variable was found to be less sensitive to changes in density–elasticity relationships and the response function to be more accurate than strain values themselves; errors were reduced by 44% on comparing SR with strain in the fixated model. In addition, the experimental data confirmed the assumption that the SR values behave independent of load amplitude. The load independent behavior of SR and its direct clinical interpretation may ultimately provide an appropriate and easily understood comparative computational measure to choose between patient specific fracture fixation alternatives.     

Helpers for efficient encapsidation of SV40 pseudovirions

Plasmid DNA that carries the simian virus 40 (SV40) ori can be packaged as SV40 pseudovirions. The pseudovirions are very efficient in gene transmission into a variety of cell types, including human hemopoietic cells. They are routinely prepared with wild-type (wt) SV40 as a helper. In the present study, several parameters required for the helper function were investigated. Plasmids that carry pBR322 sequences in addition to the late genes of SV40 were inefficient in providing helper functions, presumably because the prokaryotic sequences interfered with expression of the SV40 late genes. Efficient helpers were plasmid pSVPiC [Villarreal and Soo, Mol. Appl. Genet. 3 (1985) 62-71] and an SV40 defective virus SLT3 (presently constructed). Plasmid pSVPiC carries a duplication of the SV40 ori and enhancer regions, and pi AN7 sequences. Because of its large size it was not packaged into virion particles. However, it underwent extensive recombination generating infective SV40 particles. Almost no prokaryotic sequences are included in SLT3, that carries the SV40 late gene. In spite of its small size (3.5 kb) it was packaged efficiently, creating defective (T-antigen-negative) SV40 virions. The availability of T-antigen positive and negative pseudovirion mixtures enabled us to suggest that T-antigen drives gene amplification in the target human hemopoietic cells.     

Origin of life: protoribosome forms peptide bonds and links RNA and protein dominated worlds

Although the mode of action of the ribosomes, the multi-component universal effective protein-synthesis organelles, has been thoroughly explored, their mere appearance remained elusive. Our earlier comparative structural studies suggested that a universal internal small RNA pocket-like segment called by us the protoribosome, which is still embedded in the contemporary ribosome, is a vestige of the primordial ribosome. Herein, after constructing such pockets, we show using the "fragment reaction" and its analyses by MALDI-TOF and LC–MS mass spectrometry techniques, that several protoribosome constructs are indeed capable of mediating peptide-bond formation. These findings present strong evidence supporting our hypothesis on origin of life and on ribosome''s construction, thus suggesting that the protoribosome may be the missing link between the RNA dominated world and the contemporary nucleic acids/proteins life.     

The irreversible ischaemic lesion of human myocardium. Comparison with the experimental animal model

The autolytic ultrastructural changes of the human myocardium were studied in 25 specimens taken 30 min to 18 h post mortem. Damage to the mitochondrial membranes and the appearance of mitochondrial armorphous densities characterized the irreversible lesions of the autolytic myocardium. The development of these changes was observed in consecutive samples. At 30 min, few small densities were noticed inconsistently, and they progressively increased in size and number over the next 90 min. The development of these changes was unrelated to the cause of death. Comparison of the ischaemic heart lesions of animals with those of humans shows a similarity of the mitochondrial amorphous densities. The best analogy was found to exist between the autolytic changes of the human myocardium and the irreversible ischaemic heart lesions of dogs.