Rumen microbial community and milk quality in Holstein lactating cows fed olive oil pomace as part in a sustainable feeding strategy.

The use of alternative feed ingredients from the Agro-industry could be an efficient tool to improve the sustainability of dairy cow production. Since the richness in polyphenols, olive oil pomace (OOP), produced during olive oil milling, seems a promising by-product to ameliorate milk's nutritional value. The aim of this study was to test the use of OOP produced by means of a new technology (biphasic with stone deprivation) in dairy cow feeding strategy to evaluate the effect on animal performances, rumen microbiota, biohydrogenation processes and milk quality by a multidisciplinary approach. Forty multiparous Italian-Friesian dairy cows, at middle lactation, were randomly allotted into two homogenous groups and fed respectively a commercial diet (CON) and the experimental diet (OOPD) obtained by adding OOP to CON as partial replacement of maize silage. The two diets were formulated to be isoproteic and isoenergetic. The same diets were tested also in an in vitro trial aimed to evaluate their rumen degradability (% DEG). The dietary supplementation with OOP did not affect DM intake, rumen % DEG and milk production. The milk's nutritional quality was improved by increasing several important functional fatty acids (FAs; i.e., linoleic acid, conjugated linoleic acid, oleic acid, vaccenic acid). This finding was related to a decrease in rumen liquor biohydrogenation rate of unsaturated FAs. The stochiometric relation between volatile FA production in the rumen and methanogenesis suggested that OOP lowers the methane potential production (CON = 0.050 mol/L vs OOPD = 0.024 mol/L, SEM = 0.005, P = 0.0011). Rumen microbiota and fungi community did not be strongly altered by OOP dietary inclusion because few bacteria were affected at the genus level only. Particularly, Acetobacter, Prevotellaceae_UCG-004, Prevotellaceae_UCG-001, Eubacterium coprostanoligenes, Lachnospira, Acetitomaulatum, Lachnospiraceae_NK3A20 group were more abundant with OOPD condition (P < 0.05). Data reported in this study confirm that the use of OOP in dairy cow feeding can be an interesting strategy to improve milk nutritional quality increasing functional FA content without compromising the rumen degradability of the diet or causing strong perturbation of rumen ecosystem and maintaining animal performances.

Progressive pearl necklace collapse mechanism for cerato-ulmin aggregation film.

Cerato-ulmin (CU) is a fungal toxin class II hydrophobin, involved in Dutch elm disease. The formation of hydrophobin films at the air-water interface is a key mechanism which plays a role of paramount importance at different stages of the fungal development. We present a study on the precursor stages of growth towards the self-assembly aggregation film of CU. Atomic force microscopy images of CU dropped on mica substrates indicate that the system self-organizes in almost one-dimensional pearl-necklace-like chains, which subsequently collapse and possibly merge to form extended and rather compact planar films. We propose and verify a simple model to describe the self-aggregation mechanism in terms of progressive thickening of the pearl chains due to the successive merging and collapse of the elementary constitutive units.

Partitioning the structural features that underlie expansin-like and elicitor activities of cerato-platanin.

Cerato-platanin family (CPF) proteins are produced by fungi and elicit defences when applied to plants, behaving as PAMPs/MAMPs. CPF proteins share structural similarity to plant and bacterial expansins, and have been demonstrated, in some cases, to possess expansin-like loosening activity on cellulose. This is the case of cerato-platanin (CP), the founder of the CPF, which shows both eliciting and cellulose-loosening activities, raising the question as to whether the expansin-like activity may be responsible for defence activation. To pinpoint structural and thermodynamic features underlying eliciting and expansin-like activity of CP, we carried out site-directed mutagenesis targeting separately net charge (N84D mutation), conformational stability (V63A mutation), or conserved position previously shown to affect expansin-like activity in CP (D77A mutation), and characterized wild-type protein and its variants. Removing or adding negative charges on the protein surface led to reducing or increasing, respectively, the expansin-like activity. The activity was instead not affected by mutations affecting protein fold and stability. In contrast, all the mutants showed reduced capacity to elicit defences in plants. We conclude that the expansin-like activity of CP depends on net charge and ability to (weakly) bind cellulose, whereas the eliciting activity on plants does not depend on the cellulose-loosening activity.

Honey extracts inhibit PTP1B, upregulate insulin receptor expression, and enhance glucose uptake in human HepG2 cells.

Honey is a food known for its medical properties. In this work, we have studied the impact of different types of honey on insulin signalling pathway. We found that honey extracts inhibit the enzyme PTP1B, one of the main negative regulators of insulin receptor signalling. HPLC-MS analysis allowed us to confirm the presence of several natural PTP1B inhibitors in the honey extracts analysed. Statistical analysis methods show a correlation between specific 1H-NMR resonance frequencies/HPLC peaks and the inhibitory power of the samples. This finding will allow the prediction of the biological properties of honey samples applying relative simple analytical methods. Finally, we demonstrated that the treatment of HepG2 cells with honey extracts enhances the expression of insulin receptor, and stimulates glucose uptake. For the first time, our results demonstrate that bioactive components of honey could improve glycaemic control by both inhibiting PTP1B and stimulating the expression of insulin receptor in liver cells.

The amino acid sequences of two acylphosphatase isoforms from fish muscle (Lamna nasus).

Two acylphosphatase isoenzymes have been purified from Lamna nasus muscle, and their complete amino acid sequences have been determined. The former (E1) consists of 99 amino acid residues, while the latter (E2) consists of 102 residues. Both are acetylated at their N termini. E1 has the FFRK active site motif characteristic of all common-type acylphosphatase isoenzymes, whereas E2 contains the CFRM active site motif characteristic of all muscle-type acylphosphatase isoenzymes. They have quite similar kinetic properties. The comparison of sequences of fish E1 and E2 isoenzymes with other known mammalian and bird acylphosphatases reveals that the E2 isoenzyme has an N terminus tail, four residues long, similar to those previously found in all known bird species muscle-type isoenzymes. Among organ-common-type acylphosphatases about 50% of residues are completely conserved, whereas about 60% of muscle-type acylphosphatase residues are completely conserved, indicating that the latter type of isoenzyme has a slower evolutionary rate than the former. The sequences of E1 and E2 acylphosphatases from L. nasus represent the first primary structures of this kind of enzyme determined among fish species.

The role of Cys-17 in the pyridoxal 5'-phosphate inhibition of the bovine liver low M(r) phosphotyrosine protein phosphatase.

Mammalian tissues contain two low M(r) phosphotyrosine protein phosphatase isoforms (type-1 and type-2) that differ in the 40-73 amino-acid sequence. Only one isoform (type-2) is strongly inhibited by pyridoxal 5'-phosphate, whereas the other is poorly inhibited by this compound. The mechanism of pyridoxal 5'-phosphate inhibition of the bovine liver enzyme (a type-2 isoform) has been studied by kinetic methods using a series of pyridoxal 5'-phosphate analogues. These studies indicate that pyridoxal 5'-phosphate interacts with the enzyme in both the phosphate and aldehyde groups. Active site-directed mutagenesis has been used to investigate the sites of pyridoxal 5'-phosphate binding. Our results indicate that Cys-17, essential for enzyme activity, interacts with the phosphate moiety of pyridoxal 5'-phosphate. On the other hand, Cys-12, which is also involved in the catalytic mechanism, does not participate in pyridoxal 5'-phosphate binding.

Dephosphorylation of tyrosine phosphorylated synthetic peptides by rat liver phosphotyrosine protein phosphatase isoenzymes.

Five phosphotyrosine-containing peptides have been synthesized by FMOC solid-phase peptide synthesis. These peptides correspond to the 411-419 sequence of the Xenopus src oncogene, to the 1191-1220 sequence of the human EGF receptor precursor, to the 1146-1158 sequence of the human insulin receptor, to the 856-865 sequence of the human beta-PDGF receptor, and to the 5-16 sequence of the erythrocyte human band 3. The peptides were used as substrates for activity assay of two isoforms (AcP1 and AcP2) of a low molecular weight cytosolic PTPase. The assay, performed in microtiter EIA plates using Malachite green to determine the released phosphate, was rapid, reproducible, and sensitive. Both PTPase isoforms were able to hydrolyze all synthesized peptides, though with different affinity and rate. The main kinetic parameters were compared and discussed with respect to the role of the two enzymes in the cell.

A peptide fraction from factor VIII reduces PKC activity in cultured endothelial cells.

A peptide fraction of low molecular weight (Vueffe) prepared from bovine Factor VIII by enzymatic hydrolysis with trypsin, reduces significantly (p<0.05) membrane bound protein kinase C (PKC) activity in cultured bovine pulmonary artery endothelial cells grown with enhanced glucose levels (22.2 mM) or stimulated by phorbol 12-myristate 13-acetate (PMA). The activation of PKC is a common pathway by which mediators increase transendothelial permeability during tissue inflammation and in the development of diabetic vascular complications. Our results suggest that the antihaemorrhagic properties of Vueffe could be related to a decrease in endothelial permeability mediated by PKC.

Purification, kinetic properties and primary structure of bovine erythrocyte acylphosphatase.

An erythrocyte isoenzyme of acylphosphatase was purified from bovine red cells. The protein was characterized as regards the kinetic parameters and amino acid sequence. A simple and rapid sequencing strategy, based on a few experiments, was used for reconstructing the primary structure of the enzyme, since the purification procedure gave a very low yield. The length of the polypeptide chain is 100 residues. Comparison with the analogous human isoenzyme indicates that the primary structure is about 90% conserved. The presence of two additional residues at the acetylated N-terminus confirms the hypervariability for this region found in other acylphosphatases.

Atomic force microscopy images suggest aggregation mechanism in cerato-platanin.

Cerato-platanin (CP), the first member of the "cerato-platanin family", is a moderately hydrophobic protein produced by Ceratocystis fimbriata, the causal agent of a severe plant disease called "canker stain". The protein is localized in the cell wall of the fungus and it seems to be involved in the host-plane interaction and induces both cell necrosis and phytoalexin synthesis (one of the first plant defence-related events). Recently, it has been determined that CP, like other fungal surface protein, is able to self assemble in vitro. In this paper we characterize the aggregates of CP by Atomic Force Microscopy (AFM) images. We observe that CP tends to form early annular-shaped oligomers that seem to constitute the fundamental bricks of a hierarchical aggregation process, eventually resulting in large macrofibrillar assemblies. A simple model, based on the hypothesis that the aggregation is energetically favourable when the exposed surface is reduced, is compatible with the measured aggregates' shape and size. The proposed model can help to understand the mechanism by which CP and many other fungal surface proteins exert their effects.

Photosensitivity reaction to fluoxetine and alprazolam.

We report the case of a male individual who developed a severe photosensitive reaction after sun exposure during therapy with fluoxetin 20 mg bid plus alprazolam 0.25 mg bid due to depressive disturbances. The patients, of North-African origin, was not taking other prescription medications nor over the counter products; in the past he had never shown any reaction to sunlight nor had ever used sun protective agents because of its phenotypical skin mark. Three months later, after a second photosensitive reaction, the pharmacological therapy was finally discontinued.

Bovine testis acylphosphatase: purification and amino acid sequence.

Two acylphosphatase molecular forms have been isolated from bovine testis. Their amino acid sequence was determined. One (ACY1) consists of 98 amino acid residues, while the other one (ACY2) consists of 100 amino acid residues. Both molecular forms are N-acetylated and differ only in the amino terminus. ACY2 has an additional Ser-Met tail with respect to ACY1. Both ACY1 and ACY2 are organ-common type isoenzymes and thus differ for about half of the amino acid positions from the previously sequenced bovine muscle isoenzyme.

Metabolic responses of the limpet Patella caerulea (L.) to anoxia and dehydration.

This study examined the metabolic responses of the limpet Patella caerulea (L.) to anoxia and dehydration, attempting to tease apart the effect of these two stressful conditions, which are often not clearly distinguished in experiments. Specimens were exposed to: (a) oxygen-free sea water; (b) oxygen-saturated water (controls); (c) low-humidity air (55% RH); and (d) high-humidity air (100% RH). For each of the treatments, we took samples of five specimens after 6 and 18 h of exposure to the experimental conditions and determined the concentrations in the foot muscle of succinate, acetate, propionate, aspartate and alanine. Exposure to anoxia caused an increase in the levels of succinate (6 and 18 h) and acetate and propionate (18 h) with respect to control specimens. Anoxia also induced a decrease of aspartate and an increase of alanine after both 6 and 18 h. Exposure to both moist and dry air generally had negligible effects on the organic acid levels. Aspartate content increased after 18 h of exposure to moist air. Alanine levels also increased with respect to control values after exposure to air, with dry air having the more pronounced effect. In conclusion, the results of this study suggest that one should be cautious when inferring anaerobic conditions from the simple exposure of intertidal species to air, without strict control of the experimental conditions and actual respiration rates.

Porcine liver low M(r) phosphotyrosine protein phosphatase: the amino acid sequence.

Porcine low M(r) phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH2-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH2-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low M(r) PTPases. This PTPase is strongly inhibited by pyridoxal 5'-phosphate (Ki = 21 microM) like the low M(r) PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40-73 sequence with the corresponding sequence of other low M(r) PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low M(r) PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5'-phosphate inhibition has been proposed.

Nitric oxide causes inactivation of the low molecular weight phosphotyrosine protein phosphatase.

The low M(r) phosphotyrosine protein phosphatase (PTPase) and Yersinia enterocolitica PTPase are inactivated by nitric oxide-generating compounds. Inorganic phosphate, a competitive inhibitor, protects the enzymes from inactivation, suggesting that the action of NO is directed to the active sites. Low M(r) PTPase from bovine liver lost two out of eight thiol groups present in the molecule during the inactivation with sodium nitroprusside and with other NO-producing compounds. The mass spectrometric analyses of tryptic fragments of the enzyme, performed after chemical modification of the NO-unreacted thiol groups, demonstrated that NO caused the oxidation of Cys-12 and Cys-17 to form an S-S bond. A similar reaction was described previously for the reaction of NO with N-methyl-D-aspartate receptor. The NO-inactivated low M(r) PTPase was reactivated by treating the inactive enzyme with thiol-containing reagents. Since all members of the PTPase family have the same reaction mechanism and possess a conserved active site motif that contains an essential cysteine residue, the findings on low M(r) and Yersinia PTPases are potentially interesting for all PTPases, an enzyme class that is involved in a number of important biological processes.

Purification, characterization, and amino acid sequence of cerato-platanin, a new phytotoxic protein from Ceratocystis fimbriata f. sp. platani.

A new phytotoxic protein (cerato-platanin) of about 12.4 kDa has been identified in culture filtrates of the Ascomycete Ceratocystis fimbriata f. sp. platani, the causal agent of canker stain disease. The toxicity of the pure protein was bioassayed by detecting the inducing necrosis in tobacco leaves. The pure protein also elicited host synthesis of fluorescent substances in tobacco and plane (Platanus acerifolia) leaves. We purified the protein from culture medium to homogeneity. Its complete amino acid sequence was determined; this protein consists of 120 amino acid residues, contains 4 cysteines (S-S-bridged), and has a high percentage of hydrophobic residues. The molecular weight calculated from the amino acid sequence agrees with that determined by mass spectrometry, suggesting that no post-transnational modification occurs. Searches performed by the BLAST program in data banks (Swiss-Prot, EBI, and GenBank(TM)) revealed that this protein is highly homologous with two proteins produced by other Ascomycete fungi. One, produced during infection of wheat leaves, is codified by the snodprot1 gene of Phaeosphaeria nodorum (the causal agent of glume blotch of wheat), whereas the other is the rAsp f13 allergen from Aspergillus fumigatus. Furthermore, the N terminus of cerato-platanin is homologous with that of cerato-ulmin, a phytotoxic protein belonging to the hydrophobin family and produced by Ophiostoma (Ceratocystis) ulmi, a fungus responsible for Dutch elm disease.

Preparation and properties of des-Tyr98 and des-Arg97-Tyr98 acylphosphatase (muscular isoenzyme).

Previous NMR reports indicated that Tyr98, the C-terminal residue of the muscular form of acylphosphatase, is likely to be part of the enzyme's active site. In addition, there is evidence that an arginine residue participates to the catalyzed reaction, possibly as phosphate binding site. Among all Arg residues present in the muscular forms of acylphosphatase, four, i.e. Arg23, Arg74, Arg77, and Arg97, appear to be conserved in all species checked thus far. We prepared the des-Tyr98 and des-Arg97-Tyr98 derivatives of the native acylphosphatase to investigate the properties of both modified enzymes. The enzyme lacking Tyr98 was found to be catalytically less effective than the native one, whereas the des-Arg97-Tyr98 acylphosphatase was completely inactive. This evidence suggests that Arg97 participates directly to the active site catalytic mechanism. Fluorescence and CD spectra revealed that the latter enzyme could have been undergone some conformational change that could account for the loss of activity; on the other hand, the one-dimensional NMR spectra of either native and des-Arg97-Tyr98 enzymes were strictly similar, thus demonstrating that the removal of the two C-terminal residues does not markedly affect the fold of the enzyme. The results reported are proof of a critical contribution of Arg97 to the acylphosphatase active site; however, we cannot exclude that the function of this residue is merely to stabilize the active site conformation and dynamics.

Rat muscle acylphosphatase: purification, amino sequence, and immunological characterization.

Acylphosphatase was purified from rat skeletal muscle essentially by gel filtration and high-performance ion-exchange chromatography. The complete amino acid sequence was reconstructed by using the sequence data obtained from tryptic, peptic, and S. aureus V8 protease peptides. The protein consists of 96 amino acid residues and is acetylated at the NH2-terminus. The immunological cross-reactivity of acylphosphatase from rat and horse skeletal muscle was examined by ELISA. The reaction with rabbit antiserum revealed the presence of at least five antigenic sites on rat enzyme, two of which are common to horse muscle enzyme. Anti-rat antibodies also recognize the peptide that corresponds to the initial part of the molecule, which varies greatly from equine enzyme. Two completely new antigenic sites are herein described: the first can be considered the main antigenic site and is located within positions 21-36, the second is in the COOH-terminal part of the molecule. A mixture of immunoreactive peptides gives strong antibody-antigen reaction inhibition (94%).

Rat liver low M(r) phosphotyrosine protein phosphatase isoenzymes: purification and amino acid sequences.

Two low M(r) phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as low M(r) acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH2-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40-73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.