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Tomato (Lycopersicon esculentum) is a vegetable rich in antioxidants, such as lycopene, lutein, and zeaxanthin. Their presence is responsible for the characteristic ability of this product to inhibit the formation of reactive oxygen species, including singlet oxygen. The grapes and wines derived from grapes also contain powerful antioxidants. The antioxidant effect is derived from the polyphenols such as resveratrol and proanthocyanidin. Resveratrol is phytoalexin that is synthesized via the activation of the gene, stilbene synthase (STS). We decided to determine if the introduction of this gene into Lycopersicon esculentum Mill could modify its antioxidant activity. Using Electronic Paramagnetic Resonance (EPR) spectroscopy, which permits the detection of antiradical activity, especially •OH (hydroxyl radical), we showed that the antioxidant activity of the products, into which the gene STS had been introduced, was almost double than that of natural products and that their activity was especially pronounced due to ripening. Moreover, resveratrol concentrations in modified tomatoes were much higher than that found in the individual fruit. In the isolated hearts subjected to ischemia/reperfusion, the rats fed with modified tomato exhibited better cardiac performance, reduced myocardial infarct size and decreased number of apoptotic cardiomyocytes, and reduced oxidative stress compared to unmodified tomato or resveratrol alone indicating superior cardioprotective abilities of modified tomatoes. [ABSTRACT FROM AUTHOR]

Seed phytic acid reduces mineral bioavailability by chelating minerals. Consumption of common bean seeds with the low phytic acid 1 (lpa1) mutation improved iron status in human trials but caused adverse gastrointestinal effects, presumably due to increased stability of lectin phytohemagglutinin L (PHA-L) compared to the wild type (wt). A hard-to-cook (HTC) defect observed in lpa1 seeds intensified this problem. We quantified the HTC phenotype of lpa1 common beans with three genetic backgrounds. The HTC phenotype in the lpa1 black bean line correlated with the redistribution of calcium particularly in the cell walls, providing support for the "phytase-phytate-pectin" theory of the HTC mechanism. Furthermore, the excess of free cations in the lpa1 mutation in combination with different PHA alleles affected the stability of PHA-L lectin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This study dealt with the effect of sourdough fermentation on antinutrients, phytochemicals, and antioxidant activities of flours from three Phaseoulus vulgaris L. genotypes with differing composition of lectins. Specifically, cultivar Lady Joy (LJ) devoid of phytohemagglutinin (PHA) and enriched in alfa-amylase inhibitor (αAI), breeding line P500 low in PHA and devoid of αAI, and Taylor's horticultivar, containing normal levels of both proteins. Sourdough fermentation positively affects the nutritional values of all bean flours by reducing some antinutrients, for example, phytic acid while preserving αAI activity. It significantly increased total polyphenols, flavonols, and ascorbic acid content, while reducing flavonoids. No significant differences in antioxidant activity, measured by in vitro and ex vivo assays on human erythrocytes, were found. The kinetic profiles of conjugated dienes analysis showed a strong inhibitory effect on low-density lipoproteins oxidation of all tested powders, with unfermented flours displaying the best antioxidant activity. Among bean powders, unfermented and fermented LJ showed the highest polyphenols level (4.21 ± 0.18 and 4.96 ± 0.15 mg GAE/g dw, respectively), oxygen radical absorbance capacity (ORAC) values (24.17 ± 0.14 and 24.02 ± 0.93 µmol TE/100g dw, respectively) and cellular antioxidant activity (71.6 ± 7.05 and 62.7 ± 3.3 units, respectively). Finally, since fermentation drastically reduces phytic acid content while preserving αAI activity, fermented LJ represents an important natural slimming supplement., (© 2019 Institute of Food Technologists®.)

Consumption of legumes is associated with a number of physiological and health benefits. Legume proteins complement very well those of cereals and are often used to produce gluten-free products. However, legume seeds often contain antinutritional compounds, such as phytate, galactooligosaccharides, phenolic compounds, lectins, enzyme inhibitors, whose presence could affect their nutritional value. Screening natural and induced biodiversity for useful traits, followed by breeding, is a way to remove undesirable components. We used the common bean cv. Lady Joy and the lpa1 mutant line, having different seed composition for absence/presence of lectins,α-amylase inhibitor, (α-AI) and phytic acid, to verify the advantage of their use to make biscuits with improved nutritional properties. We showed that use of unprocessed flour from normal beans (Taylor's Horticulture and Billò) must be avoided, since lectin activity is still present after baking, and demonstrated the advantage of using the cv. Lady Joy, lacking active lectins and having active α-AI. To assess the contribution of bean flour to biscuit quality traits, different formulations of composite flours (B12, B14, B22, B24, B29) were used in combinations with wheat (B14), maize (gluten-free B22 and B29), or with both (B12 and B24). These biscuits were nutritionally better than the control, having a better amino acid score, higher fiber amount, lower predicted glycemic index (pGI) and starch content. Replacement of cv. Lady Joy bean flour with that of lpa1, having a 90% reduction of phytic acid and devoid of α-AI, contributed to about a 50% reduction of phytic acid content. We also showed that baking did not fully inactivate α-AI, further contributing to lowering the pGI of the biscuits. Finally, data from a blind taste test using consumers indicated that the B14 biscuit was accepted by consumers and comparable in terms of liking to the control biscuit, although the acceptability of these products decreased with the increase of bean content. The B22 gluten-free biscuits, although received liking scores that were just above the middle point of the hedonic scale, might represent a good compromise between health benefits (absence of gluten and lower pGI), expectations of celiac consumers and likeness.

A series of genetically related lines of common bean (Phaseolus vulgaris L.) integrate a progressive deficiency in major storage proteins, the 7S globulin phaseolin and lectins. SARC1 integrates a lectin-like protein, arcelin-1 from a wild common bean accession. SMARC1N-PN1 is deficient in major lectins, including erythroagglutinating phytohemagglutinin (PHA-E) but not α-amylase inhibitor, and incorporates also a deficiency in phaseolin. SMARC1-PN1 is intermediate and shares the phaseolin deficiency. Sanilac is the parental background. To understand the genomic basis for variations in protein profiles previously determined by proteomics, the genotypes were submitted to short-fragment genome sequencing using an Illumina HiSeq 2000/2500 platform. Reads were aligned to reference sequences and subjected to de novo assembly. The results of the analyses identified polymorphisms responsible for the lack of specific storage proteins, as well as those associated with large differences in storage protein expression. SMARC1N-PN1 lacks the lectin genes pha-E and lec4-B17, and has the pseudogene pdlec1 in place of the functional pha-L gene. While the α-phaseolin gene appears absent, an approximately 20-fold decrease in β-phaseolin accumulation is associated with a single nucleotide polymorphism converting a G-box to an ACGT motif in the proximal promoter. Among residual lectins compensating for storage protein deficiency, mannose lectin FRIL and α-amylase inhibitor 1 genes are uniquely present in SMARC1N-PN1. An approximately 50-fold increase in α-amylase inhibitor like protein accumulation is associated with multiple polymorphisms introducing up to eight potential positive cis-regulatory elements in the proximal promoter specific to SMARC1N-PN1. An approximately 7-fold increase in accumulation of 11S globulin legumin is not associated with variation in proximal promoter sequence, suggesting that the identity of individual proteins involved in proteome rebalancing might also be determined at the translational level.

The role and beneficial effects of plant and food extracts against various diseases induced by oxidative stress have received much attention in recent years. Legumes are rich in bioactive compounds, and some studies suggest a correlation between their consumption and a reduced incidence of diseases. Primary cultures of rat hepatocytes were used to investigate whether and how an extract obtained from a fermented powder of bean named Lady Joy (Phaseolus vulgaris L.) is able to regulate antioxidant and detoxifying enzymes through the NRF2 pathway, inhibit NF-kB activation, and reduce H2O2-induced endoplasmic reticulum (ER) stress. All of the antioxidant and detoxifying enzymes studied were significantly up-regulated by Lady Joy treatment. Western blot showed that Nrf2 was activated by Lady Joy treatment. Also, cells treated with this fermented bean were partially protected against NF-kB activation resulting from H2O2 stress. As a link between oxidative stress and ER dysfunction is hypothesized, we verified whether Lady Joy was able to protect cells from H2O2-induced ER stress, by studying the response of the proteins CHOP, BiP and caspase 12. The results of this study show that Lady Joy can induce the Nrf2 pathway, inhibit NF-kB, and protect ER from stress induced by H2O2.

In common bean (Phaseolus vulgaris L.), the most abundant seed proteins are the storage protein phaseolin and the family of closely related APA proteins (arcelin, phytohemagglutinin and α-amylase inhibitor). High variation in APA protein composition has been described and the presence of arcelin (Arc) has been associated with bean resistance against two bruchid beetles, the bean weevil (Acanthoscelides obtectus Say) and the Mexican bean weevil (Zabrotes subfasciatus Bohemian). So far, seven Arc variants have been identified, all in wild accessions, however, only those containing Arc-4 were reported to be resistant to both species. Although many efforts have been made, a successful breeding of this genetic trait into cultivated genotypes has not yet been achieved. Here, we describe a newly collected wild accession (named QUES) and demonstrate its resistance to both A. obtectus and Z. subfasciatus. Immunological and proteomic analyses of QUES seed protein composition indicated the presence of new Arc and arcelin-like (ARL) polypeptides of about 30 and 27 kDa, respectively. Sequencing of cDNAs coding for QUES APA proteins confirmed that this accession contains new APA variants, here referred to as Arc-8 and ARL-8. Moreover, bioinformatic analysis showed the two proteins are closely related to APA components present in the G12949 wild bean accession, which contains the Arc-4 variant. The presence of these new APA components, combined with the observations that they are poorly digested and remain very abundant in A. obtectus feces, so-called frass, suggest that the QUES APA locus is involved in the bruchid resistance. Moreover, molecular analysis indicated a lower complexity of the locus compared to that of G12949, suggesting that QUES should be considered a valuable source of resistance for further breeding purposes.

• We previously identified the lpa1 (low phytic acid) 280-10 line that carries a mutation conferring a 90% reduction in phytic acid (InsP(6) ) content. In contrast to other lpa mutants, lpa1(280-10) does not display negative pleiotropic effects. In the present paper, we have identified the mutated gene and analysed its impact on the phytic acid pathway. • Here, we mapped the lpa1(280-10) mutation by bulk analysis on a segregating F(2) population, an then, by comparison with the soybean genome, we identified and sequenced a candidate gene. The InsP(6) pathway was analysed by gene expression and quantification of metabolites. • The mutated Pvmrp1(280-10) cosegregates with the lpa1(280-10) mutation, and the expression level of several genes of the InsP(6) pathway are reduced in the lpa1(280-10) mutant as well as the inositol and raffinosaccharide content. PvMrp2, a very similar paralogue of PvMrp1 was also mapped and sequenced. • The lpa1 mutation in beans is likely the result of a defective Mrp1 gene (orthologous to the lpa genes AtMRP5 and ZmMRP4), while its Mrp2 paralog is not able to complement the mutant phenotype in the seed. This mutation appears to down-regulate the InsP(6) pathway at the transcriptional level, as well as altering inositol-related metabolism and affecting ABA sensitivity., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Phytic acid is considered as one of the major antinutritional compounds in cereal and legume seeds. The development of lpa (low phytic acid) grains, resulting in increased mineral cation availability, is considered a major goal in the improvement of the nutritional quality of seed crops, especially those largely consumed in developing countries. From a mutagenized population of common bean we isolated a homozygous lpa mutant line (lpa-280-10) showing, compared to wild type, a 90% reduction of phytic acid, a 25% reduction of raffinosaccharides and a much higher amount of free or weakly bound iron cations in the seed. Genetic analysis showed that the lpa character is due to a recessive mutation that segregates in a monogenic, Mendelian fashion. Germination tests performed using varying ageing or stress conditions, clearly showed that the bean line lpa-280-10 has a better germination response than the wild type. These data, together with those obtained from 2 years of agronomic trials showing that the mutant seed yield is close to that of its parents and other evidence, indicate that the new lpa-280-10 mutation might be the first devoid of visible macroscopic negative effects in plants, pods and seeds.

Aims: The prerequisite and the rationale for the benefit of cardiac resynchronization therapy (CRT) is that it is able to resynchronize left ventricular (LV) walls that have a delayed activation., Methods and Results: In 69 consecutive patients who underwent biventricular (BIV) pacemaker implantation, we assessed the magnitude of intraventricular resynchronization achieved by means of simultaneous (BIV 0) and sequential BIV pacing (with an individually optimized VV interval value among +80 ms and -80 ms) using pulsed-wave tissue Doppler imaging techniques and in particular the measurement of the intra-LV electromechanical delay. The intra-LV delay was defined as the difference between the longest and the shortest activation time in the six basal segments of the LV. An abnormal intra-LV delay was defined as a value >41 ms. The intra-LV delay was 63 +/- 28 ms baseline, decreased to 44 +/- 26 ms with BIV 0 and to 26 +/- 15 ms with optimized BIV (P = 0.001). BIV 0 determined the shortest delay in 28 (41%) patients (23 +/- 12 ms). In 41 (59%) patients, a better resynchronization was achieved with optimized VV intervals (LV first in 32 and RV first in 5) or single-chamber pacing (LV in 3 and RV in 1). With BIV 0, the intra-LV delay remained abnormal in 41% and was longer than baseline in 30% of patients compared with 9 and 12% with optimized BIV, respectively (P = 0.001)., Conclusion: A sub-optimal resynchronization is achieved with simultaneous BIV pacing in most patients. A tailored programming of the relative contribution of RV and LV pacing forms the prerequisite for improving CRT results.

The APA (Arcelin/Phytohemagglutinin/alpha-Amylase inhibitor) gene family is composed of various members, present in Phaseolus species and coding for lectin and lectin-related seed proteins having the double role of storage and defense proteins. Here members of the APA family have been identified by immunological, functional, and molecular analyses and representative genes were sequenced in nine wild species of Phaseolus. All taxa possessed at least one member of the true lectin gene. No arcelin type sequences have been isolated from the species examined. Among the wild species studied, only P. costaricensis contained an alpha-amylase inhibitor (alpha-AI). In addition P. augusti, P. maculatus, P. microcarpus, and P. oligospermus showed the presence of the lectin-related alpha-amylase inhibitor-like (AIL) genes and alpha-AI activity. Data from Southern blot analysis indicated the presence of only one lectin gene in P. parvulus and P. filiformis, while an extensive gene duplication of the APA locus was found in the other Phaseolus species. Phylogenetic analysis carried out on the nucleotide sequences showed the existence of two main clusters and clearly indicated that lectin-related genes originated from a paralogous duplication event preceding the development of the ancestor to the Phaseolus genus. The finding of detectable alpha-AI activity in species containing AIL genes suggests that exploiting APA genes variability in the Phaseolus genus may represent a valuable tool to find new members that may have acquired insecticidal activities.

Tomato (Lycopersicon esculentum) is a vegetable rich in antioxidants, such as lycopene, lutein, and zeaxanthin. Their presence is responsible for the characteristic ability of this product to inhibit the formation of reactive oxygen species, including singlet oxygen. The grapes and wines derived from grapes also contain powerful antioxidants. The antioxidant effect is derived from the polyphenols such as resveratrol and proanthocyanidin. Resveratrol is phytoalexin that is synthesized via the activation of the gene, stilbene synthase (STS). We decided to determine if the introduction of this gene into Lycopersicon esculentum Mill could modify its antioxidant activity. Using Electronic Paramagnetic Resonance (EPR) spectroscopy, which permits the detection of antiradical activity, especially *OH (hydroxyl radical), we showed that the antioxidant activity of the products, into which the gene STS had been introduced, was almost double than that of natural products and that their activity was especially pronounced due to ripening. Moreover, resveratrol concentrations in modified tomatoes were much higher than that found in the individual fruit. In the isolated hearts subjected to ischemia/reperfusion, the rats fed with modified tomato exhibited better cardiac performance, reduced myocardial infarct size and decreased number of apoptotic cardiomyocytes, and reduced oxidative stress compared to unmodified tomato or resveratrol alone indicating superior cardioprotective abilities of modified tomatoes.

Tomato (Lycopersicon esculentum Mill.) tissues were transformed with a grape (Vitis vinifera L.) stilbene synthase cDNA, transcriptionally regulated by the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic plants accumulated new compounds, not present in either wild-type or vector-transformed plants. These were identified, by high-pressure liquid chromatography, as trans-resveratrol and trans-resveratrol-glucopyranoside. The amounts of trans-resveratrol and its piceid form were evaluated in the transgenic fruit. It was found that the content of the metabolite varied during fruit maturation to up to 53 microg/g fresh weight of total trans-resveratrol at the red stage of ripening. This metabolite accumulation was possibly dependent on a combination of sufficiently high levels of stilbene synthase and the availability of substrates. With the aim of verifing the metabolic impairment, the amounts of chlorogenic acid and naringenin in both transgenic and wild-type ripening fruit were compared and no dramatic variation in the synthesis profile of the two metabolites was noted. To our knowledge, no data are available on the assessment of the effects of the expression of the StSy gene on other antioxidant compounds present in tomato fruit. To establish whether the presence of a novel antioxidant molecule affected the redox regulation in transgenic tomato fruit cells, the effect of resveratrol accumulation on the naturally present antioxidant pool was analysed. We showed that, in transgenic fruit which accumulate trans-resveratrol, there is an increase in the levels of ascorbate and glutathione, the soluble antioxidants of primary metabolism, as well as in the total antioxidant activity. Conversely, the content of tocopherol and lycopene, which are membrane-located antioxidants, is not affected. Consistent with the increased antioxidant properties, the lipid peroxidation was lower in transformed than in wild-type fruit.

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.

Genes for lectin, a component of legume storage proteins, were identified and characterised in two lentil cultivars ( Lens culinaris ssp. culinaris) and six wild relatives. In each taxon no differences were found among the two or three lectin clones sequenced, while differences were observed among lectin genes isolated from the different taxa. All of the clones analysed contained an insert of 828 bp and showed a high similarity with the nucleotide sequence of Pisum sativum seed lectin, PSL1. The deduced amino acid lectin sequences in all taxa were 275 amino acids long, and their multiple alignment showed that most of the variation among them occurred in regions which are not important for metal- and sugar-binding. The data from Southern blot analysis indicated the presence of only one lectin gene in all Lens taxa except L. tomentosus. Phylogenetic analyses carried out on the lectin sequences showed the existence of two main clusters and clearly indicated that L. nigricans falls outside the two groups.

Abundant lectin-related proteins found in common beans ( Phaseolus vulgaris L.) have been shown to confer resistance against the larvae of a number of bruchid species. Genes encoding for these proteins are members of the lectin multigene family, the most representative components being arcelins, phytohemagglutinins and alpha-amylase inhibitors. Arcelins have been described in seven variants, some of which are resistance factors against the Mexican bean weevil ( Zabrotes subfasciatus), a major bean predator. In this study the isolation and sequencing of arcelin genes from wild P. vulgaris genotypes, containing Arc3 and Arc7 variants, is reported, and similarities and evolutionary relationships among the seven known arcelins are described. The evolutionary analysis shows that arcelins 3 and 4 cluster together and are the most-ancient variants. A duplication event gave rise to two additional clusters, one comprising arcelins 1, 2 and 6 and separated from the cluster of arcelins 5 and 7. A multiple number of arcelin genes were found in arcelin 3 and 4 genotypes indicating that more than one type of arcelin gene may be present in the same locus. Some of these sequences are reminiscent of ancient duplication events in arcelin evolution demonstrating that arcelins have evolved through multiple duplications. A further aim of this paper was to better understand and describe the evolution of the entire lectin multigene family. Beside arcelins, a number of other types of sequences, such as putative lectins and sequences not easily classifiable, were found in genotypes containing Arc3 and Arc4. These results, together with the evolutionary analysis, indicate that lectin loci are quite complex and confirm their origin by multiple duplication events.

Lectin-related polypeptides are a class of defence proteins found in seeds of Phaseolus species. In Lima bean (P. lunatus), these proteins and their genes have been well characterized in the Andean morphotype, which represents one of the two gene pools of this species. To study the molecular evolution of the lectin family in Lima bean we characterized the polypeptides belonging to this multigene family and cloned the genes belonging to the Mesoamerican gene pool. The latter gene pool contains components similar to those of the Andean pool, namely: an amylase inhibitor-like (AIL), an arcelin-like (ARL) lectin and the less abundant Lima bean lectin (LBL). These proteins originate from an ancestor gene of the lectin type which duplicated to yield the lectin gene and the progenitor of ARL and AIL. In this species. ARL represents an evolutionary intermediate form that precedes AIL. Phylogenetic analysis supports an Andean origin for Lima bean. The molecular evolutionary studies were extended to the genes of common bean and demonstrated that true lectin genes and the ancestor of lectin-related genes are the result of a duplication event that occurred before speciation. Lima and common bean followed different evolutionary pathways and in the latter species a second duplication event occurred that gave rise, in Mesoamerican wild genotypes, to arcelin genes.

Phaseolin and lectin-related polypeptides, the abundant oligomeric glycoproteins of bean seeds, are synthesized on the endoplasmic reticulum (ER) and then transported to the storage vacuole via the Golgi apparatus. Glycosylation and folding are among the major modifications these proteins undergo in the ER. Although a recurrent role of N-glycosylation is on protein folding, in previous studies on common bean (Phaseolus vulgaris) seeds we demonstrated that the oligosaccharide side-chains are not required for folding, intracellular transport and activity of storage glycoproteins. We show here that in lima bean (Phaseolus lunatus), incubation of the developing cotyledon with tunicamycin to prevent glycosylation has a dramatic effect on the intracellular transport of the storage glycoproteins. When lacking their glycans, phaseolin and lectin-related polypeptides misfold and are retained in the ER as mixed aggregates to which the chaperone BiP irreversibly associates. The lumen of the ER becomes enlarged to accommodate the aggregated polypeptides. Intracellular transport of legumin, a naturally unglycosylated storage protein, is mostly unaffected by the inhibitor, indicating that the observed phenomenon specifically occurs on glycoproteins. Furthermore, recombinant lima bean phaseolin synthesized in tobacco protoplasts is also correctly folded and matured in the presence of tunicamycin. To our knowledge, this is the first report that describes in detail the block of intracellular transport of vacuolar glycoproteins in plant cells due to aggregation following glycosylation inhibition.

We assessed the effects of left ventricular pacing on echocardiographic and clinical parameters in 13 consecutive patients with heart failure and bundle branch block by means of a controlled acute and medium-term evaluation. Left ventricular pacing induced a significant improvement in left ventricular ejection fraction, Minnesota Living with Heart Failure Questionnaire score, New York Heart Association class, and 6-minute walking test compared with sinus rhythm or right ventricular pacing.

The only component of the lectin-related protein family so far reported in Lima bean (Phaseolus lunatus L.) seeds is the minor seed lectin (LBL). In the morphotype Big Lima, we have isolated and characterised two abundant lectin-related seed proteins and the corresponding cDNA clones. The clones show 93.7% nucleotide identity and encode an arcelin-like (ARL) and an alpha-amylase inhibitor-like (AIL) protein. Not considering the signal peptides, ARL and AIL polypeptides contain 239 and 233 amino acids, respectively. Each polypeptide is present in the mature protein as two glycoforms. ARL subunits (43 and 46 kDa) make up oligomers of about 125 to 130 kDa whereas AIL subunits (40 and 42 kDa) oligomerise in dimers of about 88 to 100 kDa. cDNA clones encoding two isoforms of the less abundant Lima bean lectin were also isolated. In common bean (P. vulgaris) the lectin locus encodes the lectin and the lectin-related proteins alpha-amylase inhibitor and arcelin, all plant defence proteins. Our data indicate extensive evolution of the locus also in Lima bean.

To acquire information on the relationships between structural maturation of proteins in the endoplasmic reticulum (ER) and their transport along the secretory pathway, we have analyzed the destiny of an assembly-defective form of the trimeric vacuolar storage glycoprotein phaseolin. In leaves of transgenic tobacco, where assembly-competent phaseolin is correctly targeted to the vacuole, defective phaseolin remains located in the ER or a closely related compartment where it represents a major ligand of the chaperone BiP. Defective phaseolin maintained susceptibility to endoglycosidase H and was slowly degraded by a process that is not inhibited by heat shock or brefeldin A, indicating that degradation does not involve transport along the secretory pathway. These results provide evidence for the presence of a quality control mechanism in the ER of plant cells that avoids intracellular trafficking of severely defective proteins and eventually leads to their degradation.

A suitable protocol for the transient expression of seed protein genes in protoplasts derived from cell suspension cultures of common bean has been established. Preliminary analyses of cultures to verify the synthesis of phaseolin - actively accumulated by the starting tissue, the developing cotyledon - showed that the protein was no longer synthesised after 5 days of culture. Transient expression of a phaseolin sequence, driven by a constitutive promoter, resulted in the accumulation of the correctly glycosylated and assembled protein. This system, when compared to tobacco protoplasts, largely avoids phaseolin fragmentation and the presence of contaminant polypeptides in the immunoprecipitates. Therefore, bean protoplasts are a good system to study the expression of wild-type as well as in-vitro-modified bean seed proteins.

Many of the proteins that are translocated into the endoplasmic reticulum are glycosylated with the addition of a 14-saccharide core unit (Glc3Man9GlcNAc2) to specific asparagine residues of the nascent polypeptide. Glucose residues are then removed by endoplasmic reticulum-located glucosidases, with diglucosylated and monoglucosylated intermediates being formed. In this study, we used a cell-free system constituted of wheat germ extract and bean microsomes to examine the role of glucose trimming in the structural maturation of phaseolin, a trimeric glycoprotein that accumulates in the protein storage vacuoles of bean seeds. Removal of glucose residues from the N-linked chains of phaseolin was blocked by the glucosidase inhibitors castanospermine and N-methyldeoxynojirimycin. If glucose trimming was not allowed to occur, the assembly of phaseolin was accelerated. Conversely, polypeptides bearing partially trimmed glycans were unable to form trimers. The effect of castanospermine on the rate of assembly was much more pronounced for phaseolin polypeptides that have two glycans but was also evident when a single glycan chain was present, indicating that glycan clustering can modulate the effect of glucose trimming on the rate of trimer formation. Therefore, the position of glycan chains and their accessibility to the action of glucosidases can be fundamental elements in the control of the structural maturation of plant glycoproteins.

Phaseolin is the most abundant storage protein of bean seeds. To modify its amino-acidic composition by protein engineering, for the improvement of its nutritional value, regions which could be modified without detrimental effects on structural features of the protein must be identified. Data presented here, on the characterisation of the major storage protein of lima bean (Phaseolus lunatus L.) seeds, a phaseolin-like glycoprotein, provide good indications on one of such region. Phaseolus lunatus phaseolin consists of four major oligomers containing two subunit classes. Polypeptides of one class show a molecular mass ranging from 38.5 kDa to 32 kDa, while the molecular mass of polypeptides belonging to the other class ranges from 27 kDa to 21 kDa. The subunits originate from the cleavage of precursor forms, with molecular masses of 58 kDa and 54 kDa, which are still present - in residual amounts - in the nature protein. Comparison of their N-terminal sequences with those of the subunits demonstrate that cleavage occurs in a region of the molecule that instead remains uncleaved in phaseolins of the other species. Since this region can accommodate such a drastic modification, we suggest it could be a good candidate for in vitro manipulation.

Atrial fibrillation is a relative contraindication to atrial synchronous pacing because of the risk of the tracking of rapid atrial rhythms by the pacemaker. In this study, we describe the clinical results of an AV synchronous rate responsive pacemaker with an original algorithm, which is able to sense pathological increments in atrial rate and automatically to switch into a non-AV synchronous mode of pacing. This pacemaker was implanted in 12 patients who had undergone radiofrequency ablation of the AV junction in order to cure severely symptomatic, drug refractory, paroxysmal atrial fibrillation. In an acute, intrapatient comparison between the standard AV synchronous mode and the automatic switching mode, ventricular tracking of atrial fibrillation occurred in 35% and 4% of total beats at rest and in 24% and 2% of total beats during exercise, respectively (P < 0.001). During 5 +/- 4 months of follow-up, no further tachyarrhythmia related symptoms occurred. In conclusion, the standard DDDR mode is unable to eliminate ventricular tracking of atrial fibrillation, thus undermining the efficacy of AV junction ablation therapy. The automatic switching mode eliminates this adverse effect of dual chamber pacing.

The purpose of this study was to evaluate the effects of atrioventricular junction radiofrequency ablation on the quality of life, exercise performance, and echocardiographic parameters in 23 patients with chronic, severely symptomatic, drug-refractory atrial fibrillation or flutter. Initially, patients were randomized to receive ablation plus pacemaker therapy (n = 12) or pacemaker therapy alone (n = 11). After 15 days, palpitations decreased by 92% and 37% (p = 0.004), rest dyspnea by 79% and 40% (p = NS), effort dyspnea by 65% and 30% (p = 0.03), exercise intolerance by 54% and 17% (p = 0.005), and asthenia by 67% and 31% (p = 0.02) in the 2 groups, respectively. At the end of this short-term study, control patients also underwent ablation therapy, and a 3-month intrapatient follow-up study was performed in 22 patients. New York Heart Association functional class > or = 3 was present in 14 patients (64%) before, but in only 3 patients (14%) after ablation therapy (p = 0.002); specific activity scale functional class > or = 3 was present in 9 patients (41%) before, but in only 5 (23%) after ablation therapy (p = NS). Exercise duration during standardized stress testing increased by a mean of 63 +/- 93 seconds (15% increase) (p = 0.001). In the 9 patients with depressed left ventricular systolic function, echocardiographic fractional shortening increased by 34% (from 23 +/- 5% to 31 +/- 9%) (p = 0.003). In the remaining 13 patients with normal systolic function, fractional shortening decreased by 10% (from 40 +/- 5% to 36 +/- 6%) (p = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

An in vitro culture of Alkanna tinctoria Tausch cells was set up in order to investigate the possibility of producing alkannin, a red naphthoquinone naturally present in the root bark of this plant. Furthermore, an in vitro culture of callusderived roots was established and the production of alkannin evaluated. In the different experimental conditions investigated, differences in the production of alkannin derivatives as well as in the type of pigments produced, were observed. The potential use of this technology is discussed.

Treatment of developing bean cotyledons with the inhibitor of N-glycosylation tunicamycin enhanced the synthesis of at least two polypeptides with molecular mass 78 kDa and 97 kDa. Pulse-chase experiments and subcellular fractionation indicated that these are endoplasmic reticulum (ER) residents. The 78 kDa protein is a major component of the ER protein fraction and, by N-terminal sequencing, was identified as a bean homolog of the mammalian 78 kDa glucose-regulated protein (GRP78). This is a molecular chaperone that is probably involved in the folding and oligomerization of several animal and yeast proteins in the ER. When newly synthesized storage glycoproteins phaseolin, phytohemagglutinin or alpha-amylase inhibitor were immunoprecipitated from an ER preparation of tunicamycin-treated tissue, the GRP78 homolog was always co-precipitated. Bound GRP78 homolog could be released by ATP treatment. These results suggest that, at least when glycosylation is inhibited, this protein plays a role in the early stages of the synthesis of vacuolar storage proteins.

The role played by subunit assembly in the intracellular transport of the bean storage protein phaseolin, a soluble trimeric glycoprotein, was investigated using Xenopus oocytes injected with RNA. We show that phaseolin assembly is dependent upon the level of synthesis of the protein and is required for intracellular transport out of the endoplasmic reticulum. We also show that a fraction of the assembled phaseolin is permanently retained in a post-endoplasmic reticulum compartment. Deletion of the C-terminal alpha-helical domain fully prevents in vivo assembly but not endoplasmic reticulum retention. This indicates that this domain is necessary for trimerization but not for interactions of unassembled subunits with endoplasmic reticulum components. The truncated phaseolin has high in vivo stability. The potential implications of these findings on the possibility to improve the nutritional value of phaseolin through genetic engineering are discussed.

In patients affected by high degree AV block without preexisting congestive heart failure there is no definite demonstration that DDD pacing gives real clinical advantages in respect to VVIR pacing. We performed an intrapatient, long-term study between the two pacing modes in 14 high degree AV block patients, using the Medtronic Synergyst 7027 dual chamber pacemaker, who could be programmed alternatively in DDD or VVIR mode. After a 4-week run-in period following the pacemaker implant, patients completed a randomized, double-blind, cross-over study to compare the effect of 6-week period VVIR and DDD pacing on symptoms and cardiovascular parameters. A semiquantitative score scale was used to quantify the symptoms of general well-being, palpitations, dizziness, pulsating sensation in the neck or abdomen, shortness of breath at rest and during effort, chest pain, and NYHA classification. The sum of symptom scores was 10.4 +/- 6.7 in VVIR period and 4.6 +/- 2.7 in DDD period (P less than 0.001); five patients (36%) crossed over early from VVIR to DDD because of intolerable symptoms; overall, eight patients preferred the DDD mode and no one preferred the VVIR. Cardiac output at rest (echo-Doppler method) was 4.7 +/- 1.4 versus 5.7 +/- 1.6 liter/min (P less than 0.01), body weight was 65.9 +/- 6.6 versus 64.9 +/- 6.1 kg (P less than 0.02), atrial natriuretic peptide was 236 +/- 112 versus 198 +/- 110 pg/mL (P less than 0.01), respectively, during VVIR and DDD modes. Effort tolerance was similar with the two modes of pacing (68 +/- 15 vs 70 +/- 18 watts/min).(ABSTRACT TRUNCATED AT 250 WORDS)

The temperature-sensitive carrot cell variant ts11c, arrested in somatic embryogenesis after the globular stage, was characterized. The sensitivity to a shift from 24 degrees C (permissive temperature) to 32 degrees C (non-permissive temperature) is greatest at the globular stage of embryogenesis, while cells proliferating in unorganized fashion and plantlets are not affected. Embryogenesis in ts11c is also arrested at the permissive temperature by replacement of conditioned culture medium with fresh medium. The timing of sensitivity of ts11c to medium replacement coincides with the sensitivity to temperature shift. Both sensitivities are recessive in somatic hybrids between ts11c and wild-type cells. Extracellular glycoproteins synthesized by ts11c at the non-permissive temperature contain much less fucose than those synthesized by the wild type. The glycoproteins synthesized by the variant under non-permissive conditions do not accumulate at the periphery of the embryo, as their wild-type counterparts do, but instead show a diffuse distribution throughout the embryo. The defect in ts11c can be fully complemented by the addition of extracellular wild-type proteins. A revertant of ts11c was isolated that simultaneously reacquired temperature insensitivity and normal glycosylation ability. Collectively, these observations indicate that ts11c is not able to perform proper glycosylation at the non-permissive temperature and suggest that the activity of certain extracellular proteins, essential for the transition of globular to heart stage somatic embryos, depends on the correct modification of their oligosaccharide side-chains.

The seeds of Phaseolus vulgaris cv. Pinto III are known to lack detectable amounts of phytohemagglutinin (PHA) and to accumulate very reduced levels of PHA mRNA compared with normal cultivars. Using PHA complementary-DNA clones and monospecific antibodies we analyzed cv. Pinto III genomic DNA and cotyledonary proteins synthesized both in vitro and in vivo. We detected genomic DNA sequences that hybridize with complementary-DNA clones for the two different classes of PHA polypeptides (PHA-E and PHA-L), at levels comparable to a normal bean cultivar. This indicates that the cv. Pinto III phenotype is not the result of a large deletion of the PHA structural genes. Messenger RNA isolated from cv. Pinto III developing cotyledons synthesizes in vitro very small amounts of a protein which is recognized by antibodies specific for PHA, and gives, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single band with molecular weight similar but not identical to that of PHA-L polypeptides. This protein is also synthesized in vivo at a very reduced level, less than 1% compared with PHA in normal cultivars, and has mitogenic activity comparable to that of the PHA-L subunit, while it shows very weak erythroagglutinating activity. The initial steps in the synthesis and processing of this protein are identical to those already identified for PHA polypeptides. The cv. Pinto III protein could be either a PHA-L polypeptide whose synthesis is not affected by the mutation or a PHA-like lectin present normally at low levels in P. vulgaris.

Cytyledons of the common bean, Phaseolus vulgaris L., were incubated with radioactive amino acids at different stages of seed development. The proteins were fractionated by ion-exchange chromatography, sucrose gradients, and sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. From 16 to 28 d after flowering about 40% of the incorporated radioactivity was associated with the polypeptides of vicilin and 10% with those of phytohemagglutinin.Polysomes were isolated from developing cotyledons 20-25 d after flowering and free polysomes were separated from membrane-bound polysomes. Aurintricarboxylic acid, an inhibitor of initiation in cell-free translation systems, did not inhibit the incorporation of amino acids into in-vitro synthesized proteins, indicating that synthesis was limited to the completion of already initiated polypeptides. Autofluorography of SDS-polyacrylamide gels showed that the two classes of polysomes made two different sets of polypeptides and that there was little overlap between these two sets.Four polypeptides similar in size to the 4 polypeptides of vicilin were made by membrane-bound polysomes and not by free polysomes. Antibodies specific for vicilin bound to those 4 polypeptides. Free polysomes made only polypeptides which did not bind to antibodies specific for vicilin. Antibodies against phytohemagglutinin did not bind to any of the invitro synthesized polypeptides.The membranes to which the polysomes were bound were characterized on sucrose gradients and by electron microscopy. Polysomes recovered from membranes which banded on top of 35 and 50% sucrose synthesized the vicilin polypeptides most rapidly. These membrane fractions were rich in vesicles of rough endoplasmic reticulum (ER). The ER marker-enzyme NADH-cytochrome-c reductase banded with an average density of 1.18 g/cm(3) (40% w/w sucrose) on continuous gradients. These experiments demonstrate that the ER is the site of vicilin synthesis in developing bean cotyledons. Quantitative determinations of several ER parameters (RNA and lipid-phosphate content, NADH-cytochrome-c-reductase activity) show that expansion of the cotyledons is accompanied by a 4-6-fold increase in ER.

Elongation factor EF1 was found in a low salt homogenate of wheat embryos, either in the 100 000 X g supernatant or in the ribosome pellet. The ribosome-linked EF1 (EF1R), deteched by high salt washing, was purified to electrophoretical homogenetiy and its molecular and functional properties compared to those of a purified high molecular weight species of EF1 obtained from cytoplasm (EF1H). The two forms are associations of different polypeptides having in common only the polypeptide which can form the ternary complex with aminoacyl-tRNA and GTP. Whereas EF1R is able to fulfill all the EF1 functions, EF1H, incubated with ribosomes completely deprived of elongation factors, can catalyze the aminoacyl-tRNA binding to ribosomes, but, in the presence of EF2, forms only a very small amount of poly(Phe).

The genome of the common bean Phaseolus vulgaris contains a small gene family that encodes lectin and lectin-like proteins (phytohemagglutinin, arcelin, and others). One of these phytohemagglutinin-like genes was cloned by L. M. Hoffman et al. ([1982] Nucleic Acids Res 10: 7819-7828), but its product in bean cells has never been identified. We identified the product of this gene, referred to as lectin-like protein (LLP), as an abundant polypeptide synthesized on the endoplasmic reticulum (ER) of developing bean cotyledons. The gene product was first identified in extracts of Xenopus oocytes injected with either cotyledonary bean RNA or LLP-mRNA obtained by hybrid-selection with an LLP cDNA clone. A tryptic map of this protein was identical with a tryptic map of a polypeptide with the same SDS-PAGE mobility detectable in the ER of bean cotyledons pulse-labeled with either [(3)H]glucosamine or [(3)H]amino acids, both in a normal and in a phytohemagglutinin-deficient cultivar (cultivars Greensleeves and Pinto UI 111). Greensleeves LLP has M(r) 40,000 and most probably has four asparagine-linked glycans. Pinto UI 111 LLP has M(r) 38,500. Unlike phytohemagglutinin which is a tetramer, LLP appears to be a monomer by gel filtration analysis. Incorporation of [(3)H]amino acids indicates that synthesis of LLP accounts for about 3% of the proteins synthesized on the ER, a level similar to that of phytohemagglutinin.

The asparagine-linked oligosaccharide chains of glycoproteins can be processed to form a wide variety of structures. The Golgi complex is the main compartment involved in this processing. In mammalian cells the first enzyme acting along the Golgi processing pathway is mannosidase I, whose action is a prerequisite for any further processing and which is inhibited by the mannose analog 1-deoxymannojirimycin (dMM). To have insights into the processing pathway in plant cells, we have studied the in vivo effect of dMM on the processing of the bean (Phaseolus vulgaris) storage proteins phaseolin and phytohemagglutinin, two well characterized plant glycoproteins. Cotyledons obtained from developing seeds were labeled with radioactive leucine, glucosamine, or fucose in the presence or absence of dMM. Treatment with dMM fully inhibited the acquisition of resistance to endo-beta-N-acetylglucosaminidase H by phaseolin and phytohemagglutinin and the incorporation of fucose into protein. Furthermore, the apparent molecular weight of the polypeptides of phaseolin and phytohemagglutinin synthesized in dMM-treated cotyledons was consistent with the exclusive presence of oligommanose oligosaccharide chains which had not been processed in the Golgi complex. The inhibition of processing did not prevent exit from the Golgi complex, and most probably the storage proteins were correctly targeted to the protein bodies as indicated by the post-translational polypeptide cleavage of phaseolin. These results indicate that the action of a mannosidase is the first obligatory step of Golgi-mediated processing also in a plant cell and, together with data obtained in other laboratories on the in vitro specificity of glycosidases and glycosyltransferases present in the Golgi complex of plant cells, support the hypothesis that the key early reactions in Golgi-mediated processing are similar if not identical in plants and mammals.

We prepared in vitro an mRNA transcript coding for the erythroagglutinating subunit of the kidney bean glycoprotein phytohemagglutinin, E-PHA. The mRNA, injected into Xenopus oocytes, synthesized E-PHA carrying two Asn-linked carbohydrate chains, one of which was processed and acquired resistance to endo-beta-N-acetylglucosaminidase H, as occurs in the native bean cells. When the mannose analog 1-deoxymannojirimycin, an inhibitor of mammalian Golgi mannosidase I, was included in the oocyte culture medium, the acquisition of endo-beta-N-acetylglucosaminidase H resistance was abolished, indicating that also in an amphibian cell the inhibitor blocks a key reaction in Golgi-mediated processing.

The origin of the molecular heterogeneity of phaseolin was investigated by studying, both in vivo and in vitro, the synthesis and processing of four different banding types of phaseolin in five cultivars of Phaseolus vulgaris L. The results demonstrate: I) Newly-synthesized (unprocessed) phaseolin in all cultivars is composed of three major components. These differ between cultivars, both in charge and Mr. II) The processing of these precursors is highly conserved and consists of the co-translational cleavage of a signal peptide, two glycosylation steps in the endoplasmic reticulum and a further modification inside the protein bodies to give the mature form. III) Some of the molecular heterogeneity of each phaseolin banding type is due to a different extent of glycosylation of its polypeptide components.

Cotyledons of developing Phaseolus vulgaris L. cv Greensleeves seeds were labeled for 2 to 3 hours with (3)H-amino acids, and newly synthesized phytohemagglutinin (PHA) was isolated by affinity chromatography with thyroglobulin-Sepharose. The presence of 1% Tween in the homogenate increased the yield of radioactive PHA by 50 to 100%. Isopycnic sucrose gradients were used to show that this detergent-released PHA was associated with the endoplasmic reticulum (ER), and pulse-chase experiments showed that the half-life of the PHA in the ER was 90 to 120 minutes. Since PHA is transiently associated with the ER and accumulates in protein bodies, we postulate that this rapidly turning over pool of PHA in the ER represents protein en route to the protein bodies. The PHA in the ER has the same sedimentation constant as mature PHA and is capable of agglutinating red blood cells. The observations substantiate earlier claims that plant cells contain membrane-bound lectins. However, they also indicate that these lectins are not necessarily functional components of the membranes with which they are associated, but may represent transport pools of lectin normally localized in other cellular compartments.

Phytohemagglutinin (PHA) is a family of tetrameric isolectins which accumulate in the protein bodies of developing Phaseolus vulgaris cotyledons. Each tetramer contains erythroagglutinating (E) or lymphocyte-mitogenic (L) subunits, or a combination of both. The subunits have Mr around 33000, E being slightly larger than L. Phytohemagglutinin is a glycoprotein, and its carbohydrate moiety contains N-acetylglucosamine, mannose, fucose and xylose, indicating that this protein has complex oligosaccharide sidechains. Several steps in the biosynthesis and in the cotranslational and post-translational processing of the glycopolypeptides of PHA have been identified. The polypeptides of PHA are synthesized by polysomes attached to the endoplasmic reticulum. The glycosylation of the polypeptides is a cotranslational process, in which each PHA polypeptide usually acquires two oligosaccharide sidechains. The oligosaccharides of PHA isolated from the endoplasmic reticulum are susceptible to digestion with alpha-mannosidase and endo-beta-N-acetylglucosaminidase H indicating that they are of the high-mannose type. In the presence of tunicamycin two unglycosylated polypeptides of PHA are synthesized, indicating that the differences in Mr between the E and L subunits of PHA are not due to differences in glycosylation alone. Transport of PHA to the protein bodies is mediated by the Golgi apparatus where at least part of the oligosaccharide chains of PHA are modified [ Chrispeels , M. J. (1983) Planta ( Berl .) 157, 454-461, and 158, 140-151]. The modified oligosaccharide chains of PHA are then gradually trimmed to a smaller size when the protein is already in the protein bodies. This processing results in an increase in the mobility of the PHA subunits in denaturing polyacrylamide gels.

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the reserve protein phaseolin. The polypeptides are synthesized on membrane-bound polysomes, pass through the endoplasmic reticulum (ER) and accumulate in protein bodies. For a study of the biosynthesis and processing of phaseolin, developing cotyledons were labeled with radioactive amino acids, glucosamine and mannose, and isolated fractions (polysomal RNA, polysomes, and rough ER) were used for in vitro protein synthesis. Newly synthesized phaseolin present in the ER of developing cotyledons can be fractioned into four glycopolypeptides by SDS PAGE. In vitro synthesis with polysomal RNA results in the formation of two polypeptides by polysome run-off shows that glycosylation is a co-translational event. The two unglycosylated polypeptides formed by polysome run-off are slightly smaller than the two polypeptides formed by in vitro translation of isolated RNA, indicating that a signal peptide may be present on these polypeptides. Run-off synthesis with rough ER produces a pattern of four polypeptides similar to the one obtained by in vivo labeling. The two abundant glycopolypeptides formed by polysome run-off. This result indicates the existence of a second glycosylation event for the abundant polypeptides. Inhibition of glycosylation by Triton X-100 during chain-completion with rough ER was used to show that these two glycosylation steps normally occur sequentially. Both glycosylation steps are inhibited by tunicamycin. Analysis of carhohydrate to protein ratios of the different polypeptides and of trypsin digests of polypeptides labeled with [(3)H]glucosamine confirmed the conclusion that some glycosylated polypeptides contain two oligosaccharide chains, while others contain only one. An analysis of tryptic peptide maps shows that each of the unglycosylated polypeptides is the precursor for one glycosylated polypeptide with one oligosaccharide chain and one with two oligosaccharide chains.