Thymosin ß4 and ß10 are highly expressed at the deep infiltrative margins of colorectal cancer - A mass spectrometry analysis.

OBJECTIVE: Colorectal cancer (CRC) is a complicated tumor, involving several oncogenic signaling pathways, and with a molecular mechanism not fully understood yet. The implication of thymosin ß4 (Tß4) with tumor insurgence and in migration of CRC cells was evidenced in the past with different methodologies, while Tß10 connection with CRC has been sporadically investigated. This study focused on the implication of both types of thymosin in CRC progression and invasion by analyzing the changes in their levels according to different zones of the tumor, and to Dukes stage and budding index. PATIENTS AND METHODS: Tß4 and Tß10 were analyzed in deep and superficial tumor samples, and normal mucosa from 18 patients. Concentrations of Tß4 and Tß10 have been measured by high-pressure liquid chromatography (HPLC) coupled to electrospray-ion trap mass spectrometry (ESI-IT-MS). MS data were compared by t-test and ANOVA statistical analysis. Identification of thymosin and their proteoforms has been performed by HPLC-high resolution-ESI-IT-MSMS. RESULTS: Both Tß4 and Tß10, exhibited intra-tumoral quantitative differences, being upregulated in the deep part of the CRC. They exhibited, moreover, strong association with the Dukes stage and the budding grade, being more concentrated in patients at Dukes stage B and with budding index "2". CONCLUSIONS: The results obtained in the present investigation encouraged the hypothesis that the two thymosin are involved in colorectal cancer progression, and in promoting cancer invasion. Thus, they are good candidates to be diagnostic/prognostic biomarkers and therapy targets.

A dyed substrate for the assay of endo-1,4-beta-glucanases.

A new dyed substrate was prepared for the rapid determination of endo-1,4-beta-glucanases. Carboxymethylcellulose was coupled with Ruthenium red to obtain a violet powder, which was stable enough to allow for reproducible assays. The spontaneous reaction is based on ionic interactions between the negatively charged carboxymethylcellulose and the complex cation of the dye. The enzymic assay is based on spectrophotometric measurement at 535 nm of the enzyme-released dyed fragments with low-molecular-weight filterable through a 0.22 microns filter. The release of coloured fragments from this dyed substrate was proportional to its solubilization. The absorbance was directly proportional to the enzyme amount in the range 0.5-5.5 mU enzyme (A535 = 0.1-0.95). This enzymic assay is advantageous for both rapid time of analysis and sensitivity.

A comparative study on the functional properties of the wild European mouflon and domestic sheep hemoglobins.

The functional properties of Hb B of the wild European mouflon (Ovis gmelini musimon), Hb B of domestic sheep (Ovis aries), and Hb C isolated from anemic mouflon were investigated. Mouflon and sheep Hbs appear to be very similar in their response to organic anions and protons, whereas sheep Hb B displays an oxygen affinity lower than that of mouflon Hb B and sheep Hb A. Mouflon Hb B and Hb C, like sheep Hb A and Hb C, have similar efficiencies in transporting oxygen to the tissues. As in other ruminant Hbs, the effect of temperature on the oxygen affinity is slight. Data suggest that mouflon Hb B is not only structurally, but even functionally, more similar to sheep Hb A than to sheep Hb B.

The hemoglobin polymorphism of the Sardinian wild dwarf horse and the oxygen binding properties of the four different horse hemoglobins.

A study was made of the Hb phenotype of the Sardinian dwarfhorse (Equus caballus jara), one of the last surviving wild horse species in Europe. Hb haplotypes and their frequencies were found to be similar to those described in the Arabian horse (BI = 0.551, BII = 0.389, A = 0.036, V = 0.015), which suggests possible introduction onto the island from North Africa. The oxygen binding properties of the whole hemolysates and of the four different horse Hbs, separated by ion-exchange chromatography, were considered with regard to the effect of chloride, 2,3-bisphosphoglycerate and lactate. Results indicate that no differences exist in the four components that characterize horse Hb. The molecular basis of the intrinsically low oxygen affinity and of the weak interaction of horse Hb with 2,3-bisphosphoglycerate is discussed in the light of the primary structure of the molecule.

Modifications of the acidic soluble salivary proteome in human children from birth to the age of 48months investigated by a top-down HPLC-ESI-MS platform.

During the first year of life the infant oral environment undergoes dramatic changes. To investigate how the salivary proteome of human children evolves during infant development we have analyzed whole saliva of 88 children aged between 0 and 48months by a top-down platform based on RP-HPLC-ESI-MS. Children were divided according to their age into five groups (A, 0-6months, N=17; B, 7-12months, N=14; C, 13-24months, N=32; D, 25-36months, N=16; E, 37-48months, N=9). The proteins and peptides analyzed were histatins (histatin-1, histatin-3 1/24), acidic proline-rich proteins, statherin, P-B peptide, and salivary cystatins. Protein and peptide quantification based on the area of the RP-HPLC-ESI-MS extracted ion current peak evidenced that: (i) concentrations of the major salivary proteins/peptides showed a minimum in the 0-6-month-old group and increased with age; (ii) the level of histatin-1 reached a maximum in the 7-12-month-old group, a minimum in the 13-24-month-aged babies and it increased again in the 25-36-month-old group; (iii) S-type cystatins were almost undetectable in the 0-6-month-old group; (iv) P-B peptide concentration greatly increased with age; (v) histatin-3 1/24 and statherin concentrations did not show any age-related variation. BIOLOGICAL SIGNIFICANCE: The top-down proteomic approach undertaken in this work reveals that the salivary proteome of human children from birth to 48months of age shows important quantitative modifications. The concentrations of the major salivary proteins, with the exception of statherin and histatin-3 1/24, showed a minimum in the 0-6-month-old group when the expression in salivary glands is probably not fully activated. Concentrations of the salivary proteins slowly increased with age, with different trends. Only histatin-1 showed the highest concentration in the 7-12-month-old group, followed by a decrease in the 13-24-month-aged children. This particular trend could be related to the phenomenon of eruption of primary dentition. This study gives a contribution to the knowledge on the physiological variability occurring in human saliva during the early childhood. It could represent a strong and reliable basis for further investigation of saliva to develop diagnostic and prognostic biomarkers.

Effect of NAD(P)H:quinone oxidoreductase on tyrosinase-mediated oxidation of opioid neuropeptides Leu-enkephalin and Met-enkephalin.

In vitro experiments are reported showing that NAD(P)H:(quinone acceptor) oxidoreductase (QR), purified from Glycine max seedlings, reduces Leu- and Met-enkephalin-tyrosinase oxidation products, in the presence of NADH or NADPH. QR was not capable to catalyze the reduction of N-acetyl-dopaquinone formed by the cation of mushroom tyrosinase on N-acetyl-L-tyrosine, while it was able to reduce dopachrome. The results support the hypothesis that QR can inhibit the formation of melanin-like compounds, as catalyzed by the action of tyrosinase on Leu-enkephalin and Met-enkephalin. It is proposed that, in the presence of NAD(P)H as the electron donor, the inhibition occurs by the specific conversion of the dopachrome-derivative into the reduced precursor, leucodopachrome-derivative.

Low-temperature sensitivity and enhanced Bohr effect in red deer (Cervus elaphus) haemoglobin: a molecular adaptive strategy to life at high altitude and low temperature.

A study of the functional properties of haemoglobin from red deer (Cervus elaphus) whose habitat varies over a wide range of latitude, was performed. The oxygen-binding properties of the most common haemoglobin phenotype from the species living in Sardinia were examined with particular attention to the effect of pH, chloride, 2, 3-bisphosphoglycerate and temperature. Results indicate that red deer haemoglobin, like all haemoglobins from ruminants so far examined, is characterized by a low intrinsic oxygen affinity, with chloride being its main physiological modulator in vivo. The functional results and the low temperature sensitivity of the oxygen affinity are discussed in the light of the amino acid sequence of closely related ruminant haemoglobins.

Circe's haemoglobins, pig-human hybrids: functional characterization and structural considerations.

We report the isolation and the functional characterization of alpha and beta chains from pig (Sus scropha domesticus) haemoglobin, as well as of the pig-human hybrid haemoglobins, alpha2(h)beta2(p) and alpha2(p)beta2(h) (i.e. Circe's haemoglobins), obtained by mixing the purified alpha and beta pig chains respectively with the corresponding partner human chains. Their functional properties have been compared with those of both parental haemoglobins in order to obtain information on the role of the different subunits and of their inter-relationships, both at the structural and functional levels. The results indicate that the functional properties of both hybrids are closer to those of the parental haemoglobin that provides the beta chains, confirming the major role of the beta chains in determining the oxygen affinity and the modulation mechanisms of the tetrameric molecule. This is supported by the thermodynamic properties, since the very low DeltaH of oxygen binding that characterizes pig haemoglobin and the alpha2(h)beta2(p) hybrid haemoglobin may be taken as the reflection of specific structural properties of pig beta chain.

The pH dependence of predictive models relating electrophoretic mobility to peptide chemico-physical properties in capillary zone electrophoresis.

We applied best fitting procedures to capillary electrophoresis (CE) mobility values, measured at varying acidic pH, of a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da. This method allowed the contemporary measurements of C-terminus and carboxylic group of the side-chain of aspartic and glutamic acid dissociation constants and of peptide Stokes radius at different protonation stages. Stokes radius was related to peptide molecular mass M at the power of a fractional coefficient, and best correlation was found at pH 2.25, the fractional coefficient being equal to 0.68. This value is close to that proposed by R. E. Offord (Nature 1966, 211, 591-593), who suggested a proportionality between the polymer Stokes radius and M(2/3). The coefficient value decreases at higher pH, reaching a value of 0.58 at pH 4.25, corresponding to a mean peptide conformational transition towards more compact structures as a consequence of C-terminus dissociation. The measurement of the dissociation constants of each peptide allowed us to determine the percentage error on peptide charge predictions performed utilizing mean dissociation constants. Even for the charge, the best predictive performance is obtained at the most acidic edge of the range of the pH studied, mainly at pH 2.25. Conclusively, this study shows that the best performance of predictive models for peptide CE mobility is obtainable in the very acidic pH range (2.25-2.50) and in the absence of electroosmotic flow, and that a satisfactory predictive equation of peptide electrophoretic mobility (m2V(-1)s(-1) is given by mu = 85.4(Z/M(0.68))10(-8).

Predictive model for capillary electrophoretic peptide mobility in 2,2,2-trifluoroethanol-water solution.

Using capillary electrophoresis (CE) on a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da, the Stokes radii at different protonation stages and the acidic dissociation constants in water and in a 2,2,2-trifluoroethanol (TFE) water mixture (30% v/v) were determined. These results permitted us to establish separately the reliability of semiempirical models utilized for the prediction of peptide size and charge at different acidic pHapp (pHapp range: 2.00-4.25). The data obtained on size and charge were utilized in order to provide suitable mobility predictions on the basis of the charge-to-size ratio. The best predictive conditions for size and charge were found at the most acidic range of pHapp studied (2.00-2.25), either in water or a TFE-water mixture, and reliable predictive equations for peptide mobility were established at this pHapp.

Structure/function relationships in the hemoglobin components from moray (Muraena helena).

Concerning the number and type of the hemoglobin components, the moray Muraena helena is characterized by three different phenotypes whose frequencies are nearly identical. Thus, the cathodal component is present in all individuals, whereas one or both of two anodal components may be present in the same phenotype. These components have been separated by chromatography. The oxygen binding properties of the purified hemoglobin components have been studied in the absence and presence of saturating concentrations of ATP or GTP and as a function of pH. The cathodal component shows an intrinsic O2 affinity four times higher than that of both anodal components, a very small Bohr effect and a significant decrease in O2 affinity upon addition of ATP and GTP (three and four times respectively with respect to stripped conditions), the latter being more effective than the former over the entire pH range examined. The anodal components do not appear functionally distinguishable and show the presence of an enhanced Bohr effect (Root effect) that is under the strict control of nucleotide triphosphates ATP, GTP, which, unlike in the cathodic component, exert the same effect on oxygen affinity. The complete sequence of the beta chains of the cathodal and of one of the anodal components have been determined. The possible molecular basis of these different functional characteristics are discussed in the light of the globin sequence and of those amino acid residues which are known to be responsible of hemoglobin functional behaviour.

Functional alterations in adult and fetal hemoglobin Sassari Asp-alpha 126(H9)-->His. The role of alpha 1 alpha 2 contact.

The effects of pH, organic phosphates (2,3-diphosphoglycerate), and temperature on the functional properties of both adult and fetal hemoglobin Sassari alpha (Asp-126-->His) have been studied. The functional properties of the adult variant are characterized by the following: (i) an oxygen affinity higher than that of normal HbA in all the experimental conditions used; (ii) a dramatic reduction of homotropic interactions (n50 very close to unity); and (iii) a significant decrease of the effect of 2,3-diphosphoglycerate, which is 35% lower than that observed on HbA. The fetal variant shows an increased oxygen affinity compared with normal HbF and an almost abolished heme-heme interaction. The molecular basis of these functional differences is discussed in terms of the possible role played by the substitution of alpha (Asp-26-->His) on the stability of the R state of the molecule due to a decreased interaction at the level of alpha 1 alpha 2 contact.

Functional and computer modelling studies of haemoglobin from horse. The haemoglobin system of the Sardinian wild dwarf horse.

A study was made of the haemoglobin (Hb) system from the Sardinian dwarf horse (Equus caballus jara), one of the last surviving wild horse species in Europe. The oxygen binding properties of the whole haemolysate and of the four different horse Hbs, separated by ion-exchange chromatography, were studied with special regard to the effect of chloride, 2,3-diphosphoglycerate and lactate. Results indicate that no significant functional differences exist between the four Hb components of horse haemolysate. Moreover, the molecular basis of the intrinsically low oxygen affinity and of the weak interaction of horse Hb with 2,3-diphosphoglycerate is discussed in the light of the primary structure of the molecule and of the results of a computer modelling approach. On these bases, it is suggested that the A1 (Thr-->Ser) and A2 (Pro-->Gly) substitutions observed in the beta chains from horse Hb may be responsible for the displacement of the A helix that is known to be a key structural feature of those Hbs that display an altered interaction with 2,3-diphosphoglycerate as compared with human Hb.

The hemoglobin system of the brown moray Gymnothorax unicolor: structure/function relationships.

The Gymnothorax unicolor hemoglobin system is characterized by two components, called cathodic and anodic on the basis of their isoelectric point, which were separated by ion-exchange chromatography. The oxygen-binding properties of the purified components were studied in the absence and presence of chloride and/or GTP or ATP in the pH range 6.5-8.0. Stripped cathodic hemoglobin showed a small reverse Bohr effect, high oxygen affinity, and low co-operativity; the addition of chloride only caused a small decrease in oxygen affinity. In the presence of GTP or ATP, the oxygen affinity was dramatically reduced, the co-operativity increased, and the reverse Bohr effect abolished. Stripped anodic hemoglobin is characterized by both low oxygen affinity and co-operativity, and displayed a normal Bohr effect; the addition of chloride increased co-operativity, whereas ATP and GTP significantly modulated oxygen affinity at acidic pH values, enhancing the Bohr effect and giving rise to the Root effect. The complete amino-acid sequences of the alpha and beta chains of both hemoglobins were established; the molecular basis of the functional properties of the hemoglobins is discussed in the light of the primary structure and compared with those of other fish hemoglobins.

Determination of the human salivary peptides histatins 1, 3, 5 and statherin by high-performance liquid chromatography and by diode-array detection.

A reversed-phase high-performance liquid chromatography (HPLC) method with diode-array detection for the quantification of several human salivary peptides is described. Sample pretreatment consisted of the acidification of whole saliva by phosphate buffer. This treatment produced precipitation of mucins, alpha-amylases and other high-molecular-mass salivary proteins, simultaneous inhibition of intrinsic protease activities and reduction of sample viscosity. Direct HPLC analysis by diode-array detection of the resulting acidic sample allowed one to quantify histatin 1, histatin 3, histatin 5, statherin, as well as uric acid, in normal subjects. Moreover, the groups of peaks pertaining to proline-rich proteins and cystatins were tentatively identified. The method can be useful in assessing the concentration of salivary peptides from normal subjects and from patients suffering oral and/or periodontal diseases.

Adult and fetal haemoglobin J-Sardegna [alpha50(CE8)His-->Asp]: functional and molecular modelling studies.

Haemoglobin (Hb) J-Sardegna [alpha50(CE8)His-->Asp] is a haemoglobin variant characteristic of subjects from the island of Sardinia. Here we report a study of the functional properties of both fetal and adult Hb J-Sardegna. The results indicate that adult Hb J-Sardegna displays an oxygen affinity that is higher than that of adult Hb only in the presence of 2,3-diphosphoglycerate (2,3-DPG). On the contrary, at 20 degrees C, the oxygen affinity of fetal Hb J-Sardegna is identical to that of normal fetal haemoglobin, both in the presence and in the absence of 2,3-DPG. A significant difference between these two systems (i.e. a higher oxygen affinity of fetal Hb J-Sardegna) shows up very clearly only when temperature is increased to 37 degrees C. Hence in fetal Hb, the main effect of the amino acid substitution is a decrease in the overall enthalpy change of oxygenation. The results outline the role of the alpha(1)-beta(1) interface in assessing the thermodynamics of oxygen binding. The functional properties of both adult and fetal Hb J-Sardegna have been interpreted at the structural level in light of the results obtained by a computational modelling approach performed in comparison with HbA and Hb Aichi, a variant characterized by a different mutation [alpha50(CE8)His-->Arg] at the same position.