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.

Insulin inhibits platelet-derived growth factor-induced cell proliferation.

Cellular behavior can be considered to be the result of a very complex spatial and temporal integration of intracellular and extracellular signals. These signals arise from serum-soluble factors as well as from cell-substrate or cell-cell interactions. The current approach in mitogenesis studies is generally to analyze the effect of a single growth factor on serum-starved cells. In this context, a metabolic hormone such as insulin is found to be a mitogenic agent in many cellular types. In the present study, we have considered the effect of insulin stimulation in platelet-derived growth factor (PDGF)-activated NIH-3T3 and C2C12 cells. Our results show that insulin is able to inhibit strongly both NIH-3T3 and C2C12 cell growth induced by PDGF, one of the most powerful mitotic agents for these cell types. This inhibitory effect of insulin is due primarily to a premature down-regulation of the PDGF receptor. Thus, when NIH-3T3 or C2C12 cells are stimulated with both PDGF and insulin, we observe a decrease in PDGF receptor phosphorylation with respect to cells treated with PDGF alone. In particular, we find that costimulation with insulin leads to a reduced production of H2O2 with respect to cell stimulation with PDGF alone. The relative low concentration of H2O2 in PDGF/insulin-costimulated cell leads to a limited down-regulation of protein tyrosine phosphatases, and, consequently, to a reduced PDGF receptor phosphorylation efficiency. The latter is very likely to be responsible for the insulin-dependent inhibition of PDGF-receptor mitogenic signaling.

Validation of the nerve axon reflex for the assessment of small nerve fibre dysfunction.

OBJECTIVE: To validate nerve-axon reflex-related vasodilatation as an objective method to evaluate C-nociceptive fibre function by comparing it with the standard diagnostic criteria. METHODS: Neuropathy was evaluated in 41 patients with diabetes (26 men and 15 women) without peripheral vascular disease by assessing the Neuropathy Symptom Score, the Neuropathy Disability Score (NDS), the vibration perception threshold (VPT), the heat detection threshold (HDT), nerve conduction parameters and standard cardiovascular tests. The neurovascular response to 1% acetylcholine (Ach) iontophoresis was measured at the forearm and at both feet by laser flowmetry. An age-matched and sex-matched control group of 10 healthy people was also included. RESULTS: Significant correlations were observed between the neurovascular response at the foot and HDT (r(s) = -0.658; p<0.0001), NDS (r(s) = -0.665; p<0.0001), VPT (r(s) = -0.548; p = 0.0005), tibial nerve conduction velocity (r(s) = 0.631; p = 0.0002), sural nerve amplitude (r(s) = 0.581; p = 0.0002) and autonomic function tests. According to the NDS, in patients with diabetes who had mild, moderate or severe neuropathy, a significantly lower neurovascular response was seen at the foot than in patients without neuropathy and controls. A neurovascular response <50% was found to be highly sensitive (90%), with a good specificity (74%), in identifying patients with diabetic neuropathy. CONCLUSION: Small-fibre dysfunction can be diagnosed reliably with neurovascular response assessment. This response is already reduced in the early stages of peripheral neuropathy, supporting the hypothesis that small-fibre impairment is an early event in the natural history of diabetic neuropathy.

Consideraciones sobre la formación basada en competencias y el enfoque Una Salud / Considerations on competency-based training and the One Health approach

Resumen El enfoque Una Salud requiere de una estrecha relación entre los profesionales veterinarios con otros actores y factores del medio donde desarrollan sus actividades. Este trabajo aborda los aspectos que deberían considerarse al incluir los dominios de competencias del enfoque Una salud en el contexto de la formación veterinaria. Se tratan rasgos de los diseños curriculares y analizan posturas en torno a la formación basada en competencias, para proponer la opción del modelo sistémico complejo. Para que los veterinarios logren incrementar su desempeño efectivo e impactar positivamente en sus territorios laborales, es necesario fortalecer su pensamiento sistémico sobre la interdependencia de la salud humana, animal y ambiental, así como su capacidad de moverse en diferentes entornos sociales, políticos, legales y culturales. Los profesionales de la medicina veterinaria deben ser competentes para diseñar, gestionar y evaluar las interacciones entre los seres humanos, los animales y su medio y conformar equipos multidisciplinarios. Las estrategias educativas para mejorar la comprensión de los estudiantes de los determinantes sociológicos y ecológicos de la salud, así como sus responsabilidades profesionales, no deberían ser un agregado a planes de estudio por demás disciplinares y fragmentados.

Abstract The One health approach requires a close relationship between veterinary professionals and other actors and factors in the environment where they carry out their activities. This paper addresses the aspects that should be considered when including the domains of the One Health approach competencies in the context of a veterinary study program. Features of the curricular designs are discussed and positions on competency-based training are analyzed in order to propose the option of the complex-systemic model. In order for veterinarians to be able to enhance their effective performance and positively impact on their working environments, it is necessary to strengthen their systemic thinking about the interdependence of human, animal and environmental health, as well as their ability to move in different social, political, legal and cultural environments. Veterinary medicine professionals must be competent to design, manage and evaluate interactions between humans, animals and their environment and to be part of multidisciplinary teams. Educational strategies to enhance students' understanding of the sociological and ecological determinants of health, as well as their professional responsibilities, should not be an add-on to too fragmented and disciplinary curricula.

Kinetic studies on rat liver low M(r) phosphotyrosine protein phosphatases. The activation mechanism of the isoenzyme AcP2 by cGMP.

The reaction mechanisms of p-nitrophenyl phosphate hydrolysis catalyzed by two rat liver isoenzymes of the low M(r) phosphotyrosine protein phosphatase (AcP1 and AcP2) were compared. Furthermore, the effect of some heterocyclic compounds on their activities were tested. Cyclic GMP and guanosine causes a particularly high activation of the isoenzyme AcP2, whereas its effect on AcP1 is very poor. A study on the mechanism of cyclic GMP activation was carried out. The results suggest that cyclic GMP activates the AcP2 isoenzyme by increasing the rate of the step that leads to the hydrolysis of the covalent enzyme-substrate phosphorylated complex formed during the catalytic process. The physiological significance of cyclic GMP activation of only one of the two isoenzymes (AcP2) remains uncertain.

Identity of zinc ion-dependent acid phosphatase from bovine brain and myo-inositol 1-phosphatase.

A 62 kDa Zn(2+)-dependent acid phosphatase has been purified from bovine brain. The protein was carboxymethylated and then cleaved by endoproteinase Glu-C, trypsin and CNBr. Several fragments were subjected to structural analysis either by using mass spectrometry or automated peptide sequencing. The four sequenced peptides were compared with the known protein sequences contained in the EMBL Data Bank. All four peptide sequences were identical to the corresponding amino-acid sequences present in myo-inositol 1-phosphatase from bovine brain. Furthermore we found that the amino-acid composition of Zn(2+)-dependent acid phosphatase purified in our laboratory is very similar to that of myo-inositol 1-phosphatase, and that several peptide fragments have molecular weights (measured by mass spectrometry techniques) identical to those expected for cleavage-fragments originated from the authentic myo-inositol 1-phosphatase. This is one of the key enzymes in the receptor-stimulated inositol phospholipid metabolism and it has been considered as the probable target of Li+ ion during LiCl therapy in manic-depressive patients. The comparison of the Zn(2+)-dependent acid phosphatase and the Mg(2+)-dependent myo-inositol-1-phosphatase activities, measured at different purification steps, shows that the ratio between the two activities was remarkably constant during enzyme purification. We also demonstrated that in the presence of Mg2+ this enzyme efficiently catalyses the hydrolysis of myo-inositol 1-phosphate, and that the Li+ ion inhibits this activity. Furthermore, the thermal treatment of the enzyme causes a time-dependent parallel decrease of both Zn-dependent p-nitrophenyl phosphatase (assayed at pH 5.5) and Mg(2+)-dependent myo-inositol-1-phosphatase (assayed at pH 8.0) activities, suggesting the hypothesis that the same protein possesses both these activities.

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.

In vivo inactivation of phosphotyrosine protein phosphatases by nitric oxide.

The effect of NO on phosphotyrosine protein phosphatases (PTPases) has been investigated in vivo. NO production is induced in interferon-gamma and lipopolysaccharide stimulated RAW-264.7 macrophages as indicated by the increase of NO2- in the medium. Our results demonstrate an inhibition of p-nitrophenylphosphatase activity as a consequence of macrophages activation. Under the described experimental conditions, most of the hydrolysis of p-nitrophenylphosphate can be ascribed to the action of cellular PTPases. The presence of NG-mono-methyl-L-arginine, a specific inhibitor of NO synthase decreases the inactivation rate of both membrane-bound and soluble PTPases. This evidence further confirms the ability of NO to inactivate PTPases and suggests a possible role of NO in the regulation of cellular processes involving this class of phosphatases.

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.

Counterimmunoelectrophoresis (CIE) for the detection of anti-liver-kidney microsome (LKM) antibodies in the sera of patients with chronic liver disease.

A counterimmunoelectrophoresis (CIE) test for the detection of liver-kidney microsome specific antibodies in human sera is described. By testing different subcellular preparations the LKM antigen was found in the membranes of the smooth endoplasmic reticulum subfraction. The antigen was sensitive to trypsin digestion and behaved as an anionic protein in the experimental conditions used in the test. All sera positive for LKM in immunofluorescence gave a precipitin line of identity while none of the control sera gave a positive reaction. The CIE titers ranged between neat and 1/4096. A significant correlation was observed between the LKM titers obtained in immunofluorescence and those obtained in CIE. Moreover, by absorption experiments, it was concluded that the antigen preparation reactive in CIE was able to abolish the immunofluorescence pattern of LKM positive sera on rat liver and kidney sections. The LKM target antigen, although previously considered a structural protein of microsomal membranes, was shown to solubilize spontaneously during the isolation of microsomal membranes. Counterimmunoelectrophoresis appears to be an appropriate test for anti-LKM antibodies in human sera.

Conformational analysis and active site modelling of angiotensin-converting enzyme inhibitors.

The discovery of captopril as a potent, orally active inhibitor of angiotensin-converting enzyme (ACE) led to the recent development of many series of novel structures with similar biological activity. To date, however, all of these inhibitors are flexible or semiflexible molecules, and there is therefore no clear definition of the conformational requirements for ACE inhibition. In an effort to solve this problem, we have carried out conformational energy calculations on a series of eight structurally diverse ACE inhibitors. Comparison of the low-energy conformations available to these molecules leads to the conclusion that there is a common low-energy conformation throughout the series. The calculations thus define the structural and conformational requirements for ACE inhibition. Expansion of this model to the receptor level has been achieved by considering possible alternative receptor sites for each of the molecules in its proposed biologically active conformation and leads to an active-site model for ACE which may be useful for the design of further inhibitors.

Synthesis and biological activity of some transition-state inhibitors of human renin.

A series of renin inhibitors containing the dipeptide transition state mimics (2S,4S,5S)-5-amino-4-hydroxy-2-isopropyl-7-methyloctanoic acid (Leu (OH)/Val) and (2S,4S,5S)-5-amino-4-hydroxy-2-isopropyl-6-cyclohexylhexanoic acid (CHa /(OH)/Val) was prepared. A structure-activity study with Boc-Phe-His-Leu (OH)/Val-Ile-His-NH2 (8a) as starting material led to N-[(2S)-2-[(tert-butylsulfonyl)methyl]-3-phenylpropionyl]-His-Cha (OH)/ Val- NHC4H9-n (8i) which has the length of a tetrapeptide and contains only one natural amino acid. Compound 8i had an IC50 of 2 x 10(-9) M against human renin and showed high enzyme specificity; IC50 values against the related aspartic proteinases pepsin and cathepsin D were (8 x 10(-6) and 3 x 10(-6) M, respectively). In salt-depleted marmosets, 8i inhibited plasma renin activity PRA and lowered blood pressure for up to 2 h after oral administration of a dose of 10 mg/kg.

Pattern of abnormal tangential forces in the diabetic neuropathic foot.

OBJECTIVES: The role of tangential stress in neuropathic foot ulceration is yet unknown. The aim of this study was to investigate the tangential forces developed during gait by the whole foot and by selected subareas of it, namely the heel, the metatarsals and the hallux. METHODS: 61 diabetic patients have been evaluated: 27 without neuropathy, 19 with neuropathy and 15 with previous neuropathic ulcer. The patients were compared with 21 healthy volunteers. A piezo-dynamometric platform was used to measure the three components of the ground reaction force under the total foot and the selected subareas. RESULTS: A significant reduction was observed for the forward peak and the backward peak of the anteroposterior ground reaction force component measured under the whole foot. Patients with previous neuropathic ulcer showed a significant increase of the mediolateral stress under the metatarsals. CONCLUSIONS: Tangential stress is altered in diabetic neuropathic patients; the increased mediolateral component suggests that tangential stress could have a role in the high risk of recurrence observed in patients with previous ulceration. RELEVANCE: To assess the effectiveness of a non-invasive methodology for the estimation and the monitoring of significant alterations of the tangential stress with the increase of neuropathy.

[Clinical reflexions on a case of primary achalasia of the esophagus diagnosed late]. / Riflessioni cliniche su di un caso di acalasia primitiva dell'esofago tardivamente diagnosticata.

We describe an unusual case of achalasia. The patient, a 33-year-old woman, presented with a clinical history of esophageal disease verified by gastroscopy. The diagnosis of hysterical anorexia that had been made some years previously did not correspond with the nosological classifications (DSM III-R, DSM IV). This case underscores the importance of the correct use of clinical methodology, particularly when conclusive diagnosis is essential for successful treatment.

[Rare neoplasms of the pancreas: the papillary-cystic tumor. A report of 2 cases]. / Neoplasie rare del pancreas: il tumore papillare-cistico. Osservazioni a proposito di due casi.

The authors report two cases of papillary-cystic tumor of the pancreas in two young women. Making a review of literature, they emphasize the rarity of this neoplasm and analyze the possible histopathologic origin, the difficult diagnosis before surgery, surgical therapy and the good prognosis of this pancreatic "curable" tumor.

[Manual versus mechanical sutures in gastric surgery in cancer]. / Suture manuali versus suture meccaniche nella chirurgia gastrica per cancro.

The Authors, on the basis of their experience in San Camillo Hospital of Rome, compare the results between manual and mechanical sutures in 597 gastric cancers operated in the last 12 years. They confirm that at present staplers are of great reliability.

[Prognostic factors in acute mesenteric ischemia. Experience of 64 cases]. / Fattori prognostici nell'ischemia mesenterica acuta. Esperienza di 64 casi.

A retrospective study of 64 cases of acute mesenteric ischaemia is reported. The patients have been treated, during the past 12 years, at the Department of Surgery "Flaiani", Ospedale San Camillo de Lellis-Rome. In terms of favourable prognosis, the following factors have shown to be statistically significant: enteric resection carried out, thrombosis of superior mesenteric vein, preoperative normal BUN and WBC count. The diagnostic value of CPK-BB is emphasized as well as the prognostic significance of a prompt surgical treatment.

Contribution of volcanic ashes to the regional geochemical balance: the 2008 eruption of Chaitén volcano, Southern Chile.

The environmental geochemical behaviour of the rhyolitic ashes from the 2008 eruption of Chaitén volcano, Southern Chile, has been studied. After the bulk characterisation, the potential contribution to the regional geochemical fluxes was examined using: i) single batch leaching tests to provide a rapid screening of the implied major and trace elements; and ii) column experiments to evaluate the temporal mobility of leached elements. The environmental concerns of these ashes are related to the fine grained component present in each sample (independent of distance from the source), in particular the presence of cristobalite, and the geochemical hazards posed by ash-water interaction. Leaching experiments show the fast dissolution of surface salts and aerosols, which dominate over glass dissolution during the first steps of the ash-water interaction. Chaitén ashes could transfer to the environment more than 1×10(10)g or 10,000 metric tonnes (mt) of Cl, S, Ca, Na, Si, and K; between 1000 and 10,000 mt of F, Mg, and Al; between 100 and 1000 mt of As, Pb, P, Fe, Sr, Zn, Mn, and Br; between 10 and 100 mt of Ba, Li, Ti, Ni, Nb, Cu, Rb, Zr, V, Mo, Co, and Sc; and less than 10 mt of Cr, Sb, Ce, Ga, Cs, and Y. These results show the fertilising potential of the ashes (e.g., providing Ca and Fe) but also the input of potentially toxic trace elements (e.g., F and As) in the regional geochemical mass balance. The Chaitén results evidence lower potentials for poisoning and fertilising than low silica ashes due to the lower contents released of practically all elements.