Improving pulse crops as a source of protein, starch and micronutrients.

Pulse crops have been known for a long time to have beneficial nutritional profiles for human diets but have been neglected in terms of cultivation, consumption and scientific research in many parts of the world. Broad dietary shifts will be required if anthropogenic climate change is to be mitigated in the future, and pulse crops should be an important component of this change by providing an environmentally sustainable source of protein, resistant starch and micronutrients. Further enhancement of the nutritional composition of pulse crops could benefit human health, helping to alleviate micronutrient deficiencies and reduce risk of chronic diseases such as type 2 diabetes. This paper reviews current knowledge regarding the nutritional content of pea (Pisum sativum L.) and faba bean (Vicia faba L.), two major UK pulse crops, and discusses the potential for their genetic improvement.

Improving wheat as a source of iron and zinc for global nutrition.

Wheat is the staple food crop in temperate countries and increasingly consumed in developing countries, displacing traditional foods. However, wheat products are typically low in bioavailable iron and zinc, contributing to deficiencies in these micronutrients in countries where wheat is consumed as a staple food. Two factors contribute to the low contents of bioavailable iron and zinc in wheat: the low concentrations of these minerals in white flour, which is most widely consumed, and the presence of phytates in mineral-rich bran fractions. Although high zinc types of wheat have been developed by conventional plant breeding (biofortification), this approach has failed for iron. However, studies in wheat and other cereals have shown that transgenic (also known as genetically modified; GM) strategies can be used to increase the contents of iron and zinc in white flour, by converting the starchy endosperm tissue into a 'sink' for minerals. Although such strategies currently have low acceptability, greater understanding of the mechanisms which control the transport and deposition of iron and zinc in the developing grain should allow similar effects to be achieved by exploiting naturally induced genetic variation. When combined with conventional biofortification and innovative processing, this approach should provide increased mineral bioavailability in a range of wheat products, from white flour to wholemeal.

Directed evolution and biophysical characterization of a full-length, soluble, human caveolin-1 variant.

Protein engineering by directed evolution can alter proteins' structures, properties, and functions. However, membrane proteins, despite their importance to living organisms, remain relatively unexplored as targets for protein engineering and directed evolution. This gap in capabilities likely results from the tendency of membrane proteins to aggregate and fail to overexpress in bacteria cells. For example, the membrane protein caveolin-1 has been implicated in many cell signaling pathways and diseases, yet the full-length protein is too aggregation-prone for detailed mutagenesis, directed evolution, and biophysical characterization. Using a phage-displayed library of full-length caveolin-1 variants, directed evolution with alternating subtractive and functional selections isolated a full-length, soluble variant, termed cavsol, for expression in E. coli. Cavsol folds correctly and binds to its known protein ligands HIV gp41, the catalytic domain of cAMP-dependent protein kinase A, and the polymerase I and transcript release factor. As expected, cavsol does not bind off-target proteins. Cellular studies show that cavsol retains the parent protein's ability to localize at the cellular membrane. Unlike truncated versions of caveolin, cavsol forms large, oligomeric complexes consisting of approximately >50 monomeric units without requiring additional cellular components. Cavsol's secondary structure is a mixture of α-helices and ß-strands. Isothermal titration calorimetry experiments reveal that cavsol binds to gp41 and PKA with low micromolar binding affinity (KD). In addition to the insights into caveolin structure and function, the approach applied here could be generalized to other membrane proteins.

Chemical characteristics for optimizing CYP2E1 inhibition.

Cytochrome P450 2E1 (CYP2E1) expression and activity in the liver is associated with the degree of liver damage in patients with alcoholic steatohepatitis (ASH) as well as non-alcoholic steatohepatitis (NASH). CYP2E1 is known to generate reactive oxygen species, which leads to oxidative stress, one of the hallmarks of both diseases. Apart from ROS, toxic metabolites can be formed by CYP2E1 metabolism, further potentiating liver injury. Therefore, CYP2E1 is implicated in the pathogenesis of ASH and NASH. The aim of this study was to determine the chemical characteristics of compounds that are important to inhibit CYP2E1. To this end, structurally related analogs that differed in their lipophilic, steric and electronic properties were tested. In addition, homologues series of aliphatic primary alcohols, secondary alcohols, aldehydes, ketones and carboxylic acids were tested. It was found that inhibition of the CYP2E1 activity is primarily governed by lipophilicity. The optimal log D7.4 (octanol/water distribution coefficient at pH 7.4) value for inhibition of CYP2E1 was approximately 2.4. In the carboxylic acids series the interaction of the carboxylate group with polar residues lining the CYP2E1 active site also has to be considered. This study sketches the basic prerequisites in the search for inhibitors of CYP2E1, which would strengthen our therapeutic armamentarium against CYP2E1 associated diseases, such as ASH and NASH.

Acupuncture for systemic lupus erythematosus: a pilot RCT feasibility and safety study.

The objective of this study was to determine the feasibility of studying acupuncture in patients with systemic lupus erythematosus (SLE), and to pilot test the safety and explore benefits of a standardized acupuncture protocol designed to reduce pain and fatigue. Twenty-four patients with SLE were randomly assigned to receive 10 sessions of either acupuncture, minimal needling or usual care. Pain, fatigue and SLE disease activity were assessed at baseline and following the last sessions. Safety was assessed at each session. Fifty-two patients were screened to enroll 24 eligible and interested persons. Although transient side effects, such as brief needling pain and lightheadedness, were reported, no serious adverse events were associated with either the acupuncture or minimal needling procedures. Twenty-two participants completed the study, and the majority (85%) of acupuncture and minimal needling participants were able to complete their sessions within the specified time period of 5-6 weeks. 40% of patients who received acupuncture or minimal needling had >/=30% improvement on standard measures of pain, but no usual care patients showed improvement in pain. A ten-session course of acupuncture appears feasible and safe for patients with SLE. Benefits were similar for acupuncture and minimal needling.

Nar1p, a conserved eukaryotic protein with similarity to Fe-only hydrogenases, functions in cytosolic iron-sulphur protein biogenesis.

The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases. Previously, we have shown that Nar1p is an Fe-S protein and that assembly of its co-factors depends on the mitochondrial Fe-S cluster biosynthesis apparatus. Using functional studies in vivo, we demonstrated that Nar1p has an essential role in the maturation of cytosolic and nuclear, but not of mitochondrial, Fe-S proteins. Here we provide further spectroscopic evidence that Nar1p possesses two Fe-S clusters. We also show that Nar1p is required for Fe-S cluster assembly on the P-loop NTPase Nbp35p, another newly identified component of the cytosolic Fe-S protein assembly machinery. These data suggest a complex biochemical pathway of extra-mitochondrial Fe-S protein biogenesis involving unique eukaryotic proteins.

The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release.

In mammals, mitochondria have been shown to play a key intermediary role in apoptosis, a morphologically distinct form of programmed cell death (PCD), for example, through the release of cytochrome c, which activates a proteolytic enzyme cascade, resulting in specific nuclear DNA degradation and cell death. In plants, PCD is a feature of normal development, including the penultimate stage of anther development, leading to dehiscence and pollen release. However, there is little evidence that plant mitochondria are involved in PCD. In a wide range of plant species, anther and/or pollen development is disrupted in a class of mutants termed CMS (for cytoplasmic male sterility), which is associated with mutations in the mitochondrial genome. On the basis of the manifestation of a number of morphological and biochemical markers of apoptosis, we have shown that the PET1-CMS cytoplasm in sunflower causes premature PCD of the tapetal cells, which then extends to other anther tissues. These features included cell condensation, oligonucleosomal cleavage of nuclear DNA, separation of chromatin into delineated masses, and initial persistence of mitochondria. In addition, immunocytochemical analysis revealed that cytochrome c was released partially from the mitochondria into the cytosol of tapetal cells before the gross morphological changes associated with PCD. The decrease in cytochrome c content in mitochondria isolated from male sterile florets preceded a decrease in the integrity of the outer mitochondrial membrane and respiratory control ratio. Our data suggest that plant mitochondria, like mammalian mitochondria, play a key role in the induction of PCD. The tissue-specific nature of the CMS phenotype is discussed with regard to cellular respiratory demand and PCD during normal anther development.

Light differentially regulates cell division and the mRNA abundance of pea nucleolin during de-etiolation.

The abundance of plant nucleolin mRNA is regulated during de-etiolation by phytochrome. A close correlation between the mRNA abundance of nucleolin and mitosis has also been previously reported. These results raised the question of whether the effects of light on nucleolin mRNA expression were a consequence of light effects on mitosis. To test this we compared the kinetics of light-mediated increases in cell proliferation with that of light-mediated changes in the abundance of nucleolin mRNA using plumules of dark-grown pea (Pisum sativum) seedlings. These experiments show that S-phase increases 9 h after a red light pulse, followed by M-phase increases in the plumule leaves at 12 h post-irradiation, a time course consistent with separately measured kinetics of red light-induced increases in the expression of cell cycle-regulated genes. These increases in cell cycle-regulated genes are photoreversible, implying that the light-induced increases in cell proliferation are, like nucleolin mRNA expression, regulated via phytochrome. Red light stimulates increases in the mRNA for nucleolin at 6 h post-irradiation, prior to any cell proliferation changes and concurrent with the reported timing of phytochrome-mediated increases of rRNA abundance. After a green light pulse, nucleolin mRNA levels increase without increasing S-phase or M-phase. Studies in animals and yeast indicate that nucleolin plays a significant role in ribosome biosynthesis. Consistent with this function, pea nucleolin can rescue nucleolin deletion mutants of yeast that are defective in rRNA synthesis. Our data show that during de-etiolation, the increased expression of nucleolin mRNA is more directly regulated by light than by mitosis.

Translocation of cytochrome c from the mitochondria to the cytosol occurs during heat-induced programmed cell death in cucumber plants.

In mammals mitochondria play a critical role in the activation of programmed cell death (PCD). One mechanism by which mitochondria can commit a cell to death is by translocating cytochrome c into the cytosol where it activates cell death caspases. However, release of cytochrome c does not appear to be a feature of caspase activation in nematodes or insects, similarly, there is no evidence for cytochrome c release during the caspase-independent PCD that can occur in Dictyostelium cells. In an attempt to understand the underlying regulation of PCD in plants we investigated if mitochondrial components were released into the cytosol when plant cells are induced to undergo PCD. PCD was triggered in cucumber cotyledons by subjecting them to a short 55 degrees C heat treatment. This heat treatment has previously been shown to trigger PCD in other plant species and cell death was confirmed in cucumber using morphological (cellular condensation) and molecular (DNA 'laddering') markers of PCD. We present evidence that, unlike Dictyostelium and invertebrate PCDs, cytochrome c release is an early event in plant PCD. The mitochondrial release of cytochrome c following a PCD-inducing stimulus in both plants and mammals suggests the pathways have been conserved during evolution, having been derived from ancestral unicellular death programmes.

Light regulation of the abundance of mRNA encoding a nucleolin-like protein localized in the nucleoli of pea nuclei.

A cDNA encoding a nucleolar protein was selected from a pea (Pisum sativum) plumule library, cloned, and sequenced. The translated sequence of the cDNA has significant percent identity to Xenopus laevis nucleolin (31%), the alfalfa (Medicago sativa) nucleolin homolog (66%), and the yeast (Saccharomyces cerevisiae) nucleolin homolog (NSR1) (28%). It also has sequence patterns in its primary structure that are characteristic of all nucleolins, including an N-terminal acidic motif, RNA recognition motifs, and a C-terminal Gly- and Arg-rich domain. By immunoblot analysis, the polyclonal antibodies used to select the cDNA bind selectively to a 90-kD protein in purified pea nuclei and nucleoli and to an 88-kD protein in extracts of Escherichia coli expressing the cDNA. In immunolocalization assays of pea plumule cells, the antibodies stained primarily a region surrounding the fibrillar center of nucleoli, where animal nucleolins are typically found. Southern analysis indicated that the pea nucleolin-like protein is encoded by a single gene, and northern analysis showed that the labeled cDNA binds to a single band of RNA, approximately the same size and the cDNA. After irradiation of etiolated pea seedlings by red light, the mRNA level in plumules decreased during the 1st hour and then increased to a peak of six times the 0-h level at 12 h. Far-red light reversed this effect of red light, and the mRNA accumulation from red/far-red light irradiation was equal to that found in the dark control. This indicates that phytochrome may regulate the expression of this gene.

Dopamine D2-like sites in schizophrenia, but not in Alzheimer's, Huntington's, or control brains, for [3H]benzquinoline.

Although the basis of schizophrenia is not known, evidence indicates a possible overactivity of dopamine pathways. In order to detect any new dopamine receptor-like sites which may be altered in schizophrenia, the present study used a new radioligand, a [3H]benzo[g]quinoline. The receptors were labelled by this ligand in the presence of other drugs to block the known dopamine D1, D2, D3, or D5 receptors (no D4-selective ligands are available to block D4). Using this method, we found that schizophrenia brain striata had elevated levels of a D2-like site not detected in control human postmortem brains or in Alzheimer's, Huntington's, or Parkinson's disease brains. The ligand acted as an agonist at this D2-like site, because binding was abolished by guanine nucleotide. The binding of the ligand to the D4 receptor, however, was not sensitive to guanine nucleotide. The site differed from D2 itself, because S- and R-sulpiride were equally potent at the D2-like site. The D2-like sites were present in rat and mouse brain but were absent in brain slices from transgenic mice where D2 had been knocked out. The abundance of the receptor was not related to premortem use of antipsychotic drugs. Future research should examine the biochemical differences between the D2 dopamine receptor and these D2-like sites in schizophrenia.

Inhibition of the biosynthesis of uroporphyrinogen and heme in rat liver during obstructive jaundice produced by bile duct ligation.

Altered hepatic microsomal drug metabolism has been reported to occur in afflicted with hyperbilirubinemia. Similarities of the chemical structures of hydroxymethylbilane, an intermediate in the biosynthesis of uroporphyrinogen, to bilirubin prompted investigations of the effect of bilirubin on the activity of uroporphyrinogen I synthase (porphobilinogen deaminase, EC 4.3.1.8) and the biosynthesis of heme. Bilirubin was found to be a reversible, noncompetitive inhibitor of uroporphyrinogen I synthase. The inhibition constant (Ki) for bilirubin was 1.5 microM. Bile acids had no effect on rat hepatic uroporphyrinogen I synthase activity. Hyperbilirubinemia was achieved in rats by biliary ligation in order to investigate whether elevated levels of bilirubin impair the biosynthesis of hepatic heme in vivo. The relative rate of heme biosynthesis, as measured by the rate of incorporation of delta-[4-14C]aminolevulinic acid into heme, was decreased 59% 24 h after biliary obstruction. The levels of hepatic microsomal heme and cytochrome P-450 were decreased by 43 and 40%, respectively, 72 h after biliary obstruction. The activities of hepatic delta-aminolevulinic acid synthase and uroporphyrinogen I synthase were increased by 39 and 46%, respectively, 72 h after biliary obstruction. During the 48- to 72-h period following biliary obstruction, the urinary excretion of porphobilinogen and uroporphyrin was increased 3.0- and 3.5-fold, respectively, whereas, the urinary excretion of delta-aminolevulinic acid was not altered. During this 48-to 72-h time interval following biliary obstruction, 100% of the uroporphyrin was excreted as isomer I. These results indicate that bilirubin is capable of depressing the biosynthesis of rat hepatic heme and thus cytochrome P-450-mediated drug metabolism by inhibition of the formation of uroporphyrinogen. These findings are a plausible mechanism for reports of impaired clearance of various drugs in patients afflicted with hyperbilirubinemic disease states.