Effect of high frequency ultrasound pre-treatment on nutritional and technological properties of tomato paste.

This study investigates the separate effect of sonication (US, carried out for 5 and 15 min) and thermal treatment (90 °C for 5 min) on nutritional and technological properties of tomato paste. US treatments did not affect the colour parameters and decreased the level of total acidity. Ascorbic acid content was slightly reduced after 5 min US but halved by pasteurization, while total carotenoids (TCC) and lycopene (LC) decreased in non-pasteurized samples after 15 min US. Neither the TCC nor the LC significantly changed in US-pasteurized samples if compared to controls. Microscopic analyses suggested a possible increased bioaccessibility of lycopene in US treated samples due to an enhancement of free lycopene clusters. Viscosity decreased as a consequence of thermal stress, although sonication contributed as well. The present findings suggest that 5 min sonication before pasteurization can enhance the nutritional characteristics of tomato paste, besides improving its texture.

Effect of temperature on the microstructure of fat globules and the immunoglobulin-mediated interactions between fat and bacteria in natural raw milk creaming.

Natural creaming of raw milk is the first step in production of Grana Padano and Parmigiano Reggiano Protected Denomination of Origin cheeses. This process decreases the fat content and plays an important role in the removal of clostridia species that may cause late-blowing defects in ripened cheeses. Partial coalescence of fat globules-that may influence fat behavior in cheese making and affect the microstructure of fat in the final cheese product-was observed at creaming temperatures higher than 22°C by confocal laser scanning microscopy. The widespread practice of heating of milk at 37°C before creaming at 8°C resulted in important changes in the size distribution of fat globules in raw milk, potentially altering the ability of fat to entrap clostridia spores. We investigated the role of immunoglobulin classes in both the clustering of fat globules and the agglutination of Clostridium tyrobutyricum to fat globules during creaming. Immunogold labeling and transmission electron microscopy showed that IgA and IgM but not IgG were involved in both clustering and agglutination. Both vegetative cells and spores were clearly shown to agglutinate to fat droplets, a process that was suppressed by thermal denaturation of the immunoglobulins. The debacterization of raw milk through natural creaming was improved by the addition of purified immunoglobulins. Overall, these findings provide not only a better understanding of the phenomena occurring during the natural creaming but also practical insights into how the process of creaming may be optimized in cheese production plants.

New insight on crystal and spot development in hard and extra-hard cheeses: Association of spots with incomplete aggregation of curd granules.

Chemical composition and structure of different types of macroparticles (specks, spots) and microparticles (microcrystals) present in hard and extra-hard cheeses were investigated. Light microscopy revealed that the small hard specks had the structure of crystalline tyrosine, as confirmed by amino acid analysis. Spots showed a complex structure, including several curd granules, cavities, and microcrystals, and were delimited by a dense protein layer. Spots contained less moisture and ash than the adjacent cheese area, and more protein, including significantly higher contents of valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine. Microcrystals were observed by light and electron microscopy and analyzed by confocal micro-Raman. Among others, calcium phosphate crystals appeared to consist of a central star-shaped structure immersed in a matrix of free fatty acids plus leucine and phenylalanine in free form or in small peptides. A hypothetical mechanism for the formation of these structures has been formulated.

Mechanisms of Clostridium tyrobutyricum removal through natural creaming of milk: A microscopy study.

Clostridium tyrobutyricum is the main spoilage agent of late blowing defect (LBD) in Grana Padano and Parmigiano-Reggiano cheeses; LBD is characterized by openings and holes and is sometimes accompanied by cracks and an undesirable flavor. Even a very few spores remaining in the cheese curd may cause LBD; thus, it is essential to eradicate them during milk natural creaming. By this process, most of the bacteria, somatic cells, and spores rise to the top of the milk, together with the fat globules, and are removed with the cream. Previous studies suggested that milk immunoglobulins mediate the interactions between fat globules and bacteria that occur upon creaming but no direct evidence for this has been found. Moreover, other physical chemical interactions could be involved; for example, physical entrapment of spores among globule clusters. To maximize the efficiency of the natural creaming step in removing Cl. tyrobutyricum, it is essential to understand the nature of spore-globule interactions. With this aim, raw milk was contaminated with spores of Cl. tyrobutyricum before going to creaming overnight at 8°C, after which spore and bacteria removal was >90%. The obtained cream was analyzed by light interference contrast and fluorescence microscopy and by transmission electron microscopy (TEM). Results showed that most of the vegetative cells and spores, which were stained with malachite green before addition to milk, adhered tightly to the surface of single fat globules, the membranes of which appeared heterogeneous when stained with the fluorescent dye DilC18(3)-DS. Using the same dye, we observed transient and persistent interactions among globules, with formation of clusters of different sizes and partial coalescence of adhering membranes. Transmission electron microscopy examination of replicates of freeze-fractured cream allowed us to observe tight adhesion of spores to fat globules. Ultrathin sections revealed that this adhesion is mediated by an amorphous, slightly electron-opaque material, sometimes granular in appearance. Bacteria also adhered to different fat globules, linking them together, which suggests that adhesion was strong enough to maintain a stable contact. Although we cannot exclude physical entrapment of bacteria among fat globule clusters, we show for the first time that most of the bacteria are adhered to fat globules by an electron-opaque material whose nature has yet to be determined. Immunoglobulins are certainly the best candidates for adhesion but other compounds may be involved.

Early events in Populus hybrid and Fagus sylvatica leaves exposed to ozone.

This paper aims to investigate early responses to ozone in leaves of Fagus sylvatica (beech) and Populus maximowiczii x Populus berolinensis (poplar). The experimental setup consisted of four open-air (OA) plots, four charcoal-filtered (CF) open-top chambers (OTCs), and four nonfiltered (NF) OTCs. Qualitative and quantitative analyses were carried out on nonsymptomatic (CF) and symptomatic (NF and OA) leaves of both species. Qualitative analyses were performed applying microscopic techniques: Evans blue staining for detection of cell viability, CeCl3 staining of transmission electron microscope (TEM) samples to detect the accumulation of H2O2, and multispectral fluorescence microimaging and microspectrofluorometry to investigate the accumulation of fluorescent phenolic compounds in the walls of the damaged cells. Quantitative analyses consisted of the analysis of the chlorophyll a fluorescence transients (fast kinetics). The early responses to ozone were demonstrated by the Evans blue and CeCl3 staining techniques that provided evidence of plant responses in both species 1 month before foliar symptoms became visible. The fluorescence transients analysis, too, demonstrated the breakdown of the oxygen evolving system and the inactivation of the end receptors of electrons at a very early stage, both in poplar and in beech. The accumulation of phenolic compounds in the cell walls, on the other hand, was a species-specific response detected in poplar, but not in beech. Evans blue and CeCl3 staining, as well as the multispectral fluorescence microimaging and microspectrofluorometry, can be used to support the field diagnosis of ozone injury, whereas the fast kinetics of chlorophyll fluorescence provides evidence of early physiological responses.

Responsiveness of Lycopersicon pimpinellifolium to acute UV-C exposure: histo-cytochemistry of the injury and DNA damage.

The in vivo and in vitro effects of UV-C (254 nm) exposure (0.039 watt . m(-2) . s for 2 h) of currant tomato (Lycopersicon pimpinellifolium), indigenous to Peru and Ecuador, were assayed. H(2)O(2) deposits, dead cells and DNA damage were localized, 12/24 h after irradiation, mainly in periveinal parenchyma of the 1st and 2nd order veins of the leaves, and before the appearance of visible symptoms, which occurred 48 h after irradiation. Cell death index was of 43.5 +/- 12% in exposed leaf tissues, 24 h after treatment. In currant tomato protoplasts, the percentage of viable cells dropped 1 h after UV-C irradiation from 97.42 +/- 2.1% to 43.38 +/- 4.2%. Afterwards, the protoplast viability progressively decreased to 40.16 +/- 7.25% at 2 h, to 38.31 +/- 6.9% at 4 h, and to 36.46 +/- 1.84% at 6 h after the exposure. The genotoxic impact of UV-C radiation on protoplasts was assessed with single cell gel electrophoresis (SCGE, or comet assay). UV-C treatment greatly enhanced DNA migration, with 75.37 +/- 3.7% of DNA in the tail versus 7.88 +/- 5.5% in the case of untreated nuclei. Oxidative stress by H(2)O(2) used as a positive control, induced a similar damage on non-irradiated protoplasts, with 71.59 +/- 5.5% of DNA in the tail, whereas oxidative stress imposed on UV-C irradiated protoplasts slightly increased the DNA damage (85.13 +/- 4.1%). According to these results, SCGE of protoplasts could be an alternative to nuclei extraction directly from leaf tissues.

Formation of structured polymers upon controlled denaturation of beta-lactoglobulin with different chaotropes.

Prolonged exposure (>90 days) of bovine beta-lactoglobulin (BLG) to subdenaturing concentrations of either urea or potassium thiocyanate resulted in the formation of ordered polymers in the form of fibrils. The fibrils obtained with each chaotrope showed major differences in morphology, surface properties, thiol accessibility, and stability to dissociating agents as a consequence of the different chemical bonds involved in their stabilization. Hydrophobic interactions between BLG monomers are predominant in thiocyanate-formed fibrils, whereas urea-formed fibrils are stabilized by intermolecular disulfides generated through a thiol-disulfide exchange reaction. The different features of fibrils obtained with each chaotrope relate to the peculiar structural features and chemical properties of the "active" monomers generated by subdenaturing chaotrope concentrations in the early phases of the polymerization process, as detected by spectroscopic and limited proteolysis/mass spectrometry studies in the earliest stages of the action of individual chaotropes. The chaotrope-specific features of these early intermediates in turn affect the polymerization mechanism, whose intermediates were studied by size-exclusion chromatography on the soluble fraction at different times of fibril formation. The potential of these findings for the production of protein-derived nanostructures having different and controlled geometries and chemical properties is also discussed.

Cell death-mediated antiviral effect of chitosan in tobacco.

The antiviral activity induced by chitosan (CHT), and the mechanisms underlying it, were studied in a tobacco-tobacco necrosis necrovirus (TNV) pathosystem. Treatments with 0.1% CHT enhanced tobacco inducible defenses against TNV, reducing significantly the virus-induced necrotic lesions (in a range from 32% to 83%). In planta, this resistance was associated with a network of callose deposits, micro-oxidative bursts and micro-hypersensitive responses (micro-HRs), as assessed, respectively, by aniline blue, 3,3'-diaminobenzidine (DAB) and Evans blue staining. In order to verify if CHT-elicited cell death could be regarded as an apoptotic process, tobacco bright yellow 2 (BY2) cell cultures were treated with different CHT concentrations, ranging from 0.01% to 0.1%. After 6 h about half of the cultured cells incubated in 0.05% CHT were Evans blue positive, showing some typical morphological features of apoptosis, such as cytoplasm shrinkage and nuclear chromatin condensation. The latter was checked by 4',6-diamino-2-phenylindole (DAPI) and ethidium bromide nuclear staining and was visible already at 2 h after treatment. Moreover, the cell death kinetic induced by CHT was delayed by Verapamil(R), a calcium channel blocker. Finally, electrophoresis of genomic DNA extracted from cultured cell after 48 h treatment showed internucleosomal fragmentation, visualized as a distinct ladder of DNA bands corresponding to oligonucleosomal units.

Lepidium meyenii (Maca) does not exert direct androgenic activities.

Maca is the edible root of the Peruvian plant Lepidum meyenii, traditionally employed for its purported aphrodisiac and fertility-enhancing properties. This study aimed at testing the hypothesis that Maca contains testosterone-like compounds, able to bind the human androgen receptor and promote transcription pathways regulated by steroid hormone signaling. Maca extracts (obtained with different solvents: methanol, ethanol, hexane and chloroform) are not able to regulate GRE (glucocorticoid response element) activation. Further experiments are needed to assess which compound, of the several Maca's components, is responsible of the observed in vivo effects.

The product of the rice myb7 unspliced mRNA dimerizes with the maize leucine zipper Opaque2 and stimulates its activity in a transient expression assay.

myb7 mRNA is present in rice in spliced and unspliced forms, splicing being enhanced by anoxia. The protein (Mybleu) encoded by the unspliced mRNA is composed of an incomplete Myb domain followed by a leucine zipper; however, it lacks canonical sequences for DNA binding, transcriptional activation, and nuclear localization. We show here that in transiently transformed tobacco protoplasts, Mybleu is able to enhance the transcriptional activity of the maize leucine zipper Opaque2 on its target b32 promoter. The Mybleu transactivation effect is strictly dependent on the presence of Opaque2 and is driven by Mybleu-Opaque2 heterodimers. Mybleu is located in the nucleus, both in rice and in transformed tobacco protoplasts. In rice, the protein is expressed in regions corresponding to undifferentiated cells of roots and coleoptiles. Therefore, myb7 mRNA encodes, depending on its splicing, two transcription factors belonging to separate classes. One of them, Mybleu, has novel structural characteristics, suggesting the existence of new mechanisms acting in the activation of transcription.

Misfolding and aggregation of vacuolar glycoproteins in plant cells.

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.

In rice, Oryzalin and abscisic acid differentially affect tubulin mRNA and protein levels.

The effect of the anti-microtubular drug Oryzalin (3,5-dinitro-N4,N4-dipropylsulfanilamide) on growth and elongation of rice (Oryza sativa L. cv. Arborio) roots and coleoptiles was investigated. At 100 nM, Oryzalin strongly reduced primary root elongation, caused loss of cell anisotropy and the disappearance of the cortical microtubule array. Under these conditions the amounts of alpha- and beta-tubulin protein, but not mRNA, were heavily reduced. Similar data were also obtained in coleoptile segments treated with different concentrations of Oryzalin. However, when coleoptile elongation was inhibited by cis-abscisic acid, remarkable decreases in alpha- and beta-tubulin accumulation were observed to occur at the mRNA level but not at the protein level. The transcriptional decreases could be reversed by re-addition of 3-indole acetic acid. Altogether, these data indicate that rice tubulin accumulation can be controlled at different levels, mRNA or protein, in response to Oryzalin or abscisic acid treatments.

Sorting of phaseolin to the vacuole is saturable and requires a short C-terminal peptide.

Phaseolin, one of the major legume proteins for human nutrition, is a trimeric glycoprotein of the 7S class that accumulates in the protein storage vacuoles of common bean. Phaseolin is cotranslationally introduced into the lumen of the endoplasmic reticulum; from there, it is transported through the Golgi complex to the storage vacuoles. Phaseolin is also transported to the vacuole in vegetative tissues of transgenic plants. By transient and permanent expression in tobacco leaf cells, we show here that vacuolar sorting of phaseolin is saturable and that saturation leads to Golgi-mediated secretion from the cell. A mutated phaseolin, in which the four C-terminal residues (Ala, Phe, Val, and Tyr) were deleted, efficiently formed trimers but was secreted entirely outside of the cells in transgenic tobacco leaves, indicating that the deleted sequence contains information necessary for interactions with the saturable vacuolar sorting machinery. In the apoplast, the secreted phaseolin remained intact; this is similar to what occurs to wild-type phaseolin in bean storage vacuoles, whereas in vegetative vacuoles of transgenic plants, the storage protein is fragmented.

Protein quality control along the route to the plant vacuole.

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.

Heat-induced synthesis and tunicamycin-sensitive secretion of the putative storage glycoprotein conglutin gamma from mature lupin seeds.

SDS/PAGE, immune blotting with specific antibodies and amino acid sequence analyses revealed that 90% of the protein released from Lupinus albus seeds incubated in water at 60 degrees C for about 3 h was conglutin gamma, a putative storage glycoprotein already present in the protein bodies of mature seeds. Incorporation of [14C]leucine into the protein demonstrated that conglutin gamma was newly synthesized during the treatment and the use of protein synthesis inhibitors ruled out the secretion of constitutive conglutin gamma. Synthesis and secretion took place only over a narrow temperature range, 57.5-62.5 degrees C, and in a short time interval, 135-180 min, of incubation of the seed. The amount of secreted conglutin gamma, i.e. 1 mg/seed, was about three times that present inside the treated or untreated seed. Secreted conglutin gamma contained covalently linked carbohydrate as well as the constitutive protein. Inhibition of the glycosylation by tunicamycin did not affect conglutin gamma synthesis, but prevented its secretion from the seed, as indicated by quantifying conglutin gamma remaining in the seed. An accumulation of the protein outside the protein bodies and at the cotyledonary cell periphery was shown in these samples by immunocytochemistry. Peptide mapping of the fragments obtained by incubation of constitutive and secreted conglutin gamma with trypsin and pepsin revealed no difference between the two proteins. Lupin seeds were still viable after the treatment. However no similarities between conglutin gamma and heat-shock proteins were observed either in the amino acid sequence or other molecular features.

Synthetic peptides related to the dermorphins. II. Synthesis and biological activities of new analogues.

A new series of analogues of the potent opiate-like peptides dermorphins (mainly tetra- and pentapeptides) were synthesized in order to better evaluate the structure-activity relationships. Relative potencies were referred to dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), the prototype of this class of frog skin peptides. Peripheral opioid activity (guinea-pig ileum and mouse vas deferens) was determined for all the dermorphin analogues. For a selected number of them also central analgesic (hot plate and tail-flick tests) and cataleptic activities were assayed in the rat by intracerebroventricular administration.

Synthesis of dermorphins, a new class of opiate-like peptides.

H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (dermorphin) and H-Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser-NH2 (Hyp6-dermorphin) are two members of a new class of opiate-like peptides present in the amphibian skin. Their syntheses and that of the L-Ala2-analogue of dermorphin have been accomplished by conventional segment condensation in solution.

Synthetic peptides related to the dermorphins. I. Synthesis and biological activities of the shorter homologues and of analogues of the heptapeptides.

Dermorphins are potent opiate-like peptides isolated from the skin of some species of frogs. They are characterized by the presence of a D-amino acid residue, which is crucial for bioactivity. A number of analogues were prepared in order to evaluate the structure-activity relationships. The syntheses were accomplished either by conventional or solid-phase procedures. In vitro assays included both guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Central analgesic (tail-flick and hot plate tests) and cataleptic activities were determined in the rat by intracerebroventricular route. The potency of dermorphin (H- Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) in the different tests was: GPI: IC50 = 3.3 nM; MVD: IC50 = 29 nM; hot plate: ED50 = 13.3 pmol/rat; tail-flick: ED50 = 23 pmol/rat; catalepsy: ED50 = 130 pmol/rat.