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.

alpha-Defensin levels in whole saliva of totally edentulous subjects.

Salivary levels of alpha-defensins 1-4 and histatins 1, 3 and 5 were determined in 11 totally edentulous patients, 11 younger healthy adults with normal gingival mucosa (Control group I) and 8 subjects, age-matched with edentulous patients, having a minimum of 25 teeth (Control group II). Whole saliva was treated with trifluoroacetic acid and the acidic soluble fraction analyzed by High Performance Liquid Chromatography-Mass Spectrometry. The area of the extracted ion current peaks was used for peptide quantification. Levels of alpha-defensins1-4, but not of histatins, were significantly lower in totally edentulous patients with respect to both Control group I and Control group II. The two control groups did not show significant differences. The reduced level of oral alpha-defensins, which are mainly of crevicular origin, is most likely due to the absence of the gingival sulcus in the edentulous subjects. The near absence of alpha- defensins might be in part responsible for the higher vulnerability of the oral cavity to oral pathogen infections observed in totally edentulous patients.

Salivary biomarkers and proteomics: future diagnostic and clinical utilities.

Saliva testing is a non-invasive and inexpensive test that can serve as a source of information useful for diagnosis of disease. As we enter the era of genomic technologies and -omic research, collection of saliva has increased. Recent proteomic platforms have analysed the human salivary proteome and characterised about 3000 differentially expressed proteins and peptides: in saliva, more than 90% of proteins in weight are derived from the secretion of three couples of "major" glands; all the other components are derived from minor glands, gingival crevicular fluid, mucosal exudates and oral microflora. The most common aim of proteomic analysis is to discriminate between physiological and pathological conditions. A proteomic protocol to analyze the whole saliva proteome is not currently available. It is possible distinguish two type of proteomic platforms: top-down proteomics investigates intact naturally-occurring structure of a protein under examination; bottom-up proteomics analyses peptide fragments after pre-digestion (typically with trypsin). Because of this heterogeneity, many different biomarkers may be proposed for the same pathology. The salivary proteome has been characterised in several diseases: oral squamous cell carcinoma and oral leukoplakia, chronic graft-versus-host disease Sjögren's syndrome and other autoimmune disorders such as SAPHO, schizophrenia and bipolar disorder, and genetic diseases like Down's Syndrome and Wilson disease. The results of research reported herein suggest that in the near future human saliva will be a relevant diagnostic fluid for clinical diagnosis and prognosis.

Two proline-rich peptides from pig (Sus scrofa) salivary glands generated by pre-secretory pathway underlying the action of a proteinase cleaving ProAla bonds.

The primary structures of two salivary proline-rich peptides (PRP-SP-A, M 6156.0 amu and PRP-SP-B, M 1905.0 amu), from pig (Sus scrofa) were determined. The PRP-SP-B peptide, 21 residues long, overlaps with a sequence repeated 43 times in three deposited cDNAs coding for PRP proteins cloned from porcine parotid glands (Swiss-Prot codes: Q95JC9, Q95JD1, Q95JD0). PRP-SP-A peptide, 56 amino acid residues long, overlaps with the N-terminus repeats of Q95JC9 and Q95JD1 and it is phosphorylated at Ser 12 and 14. The two peptides were found both in whole saliva and in granules from pig parotid glands. The biosynthesis of the two peptides implies the action of a proteinase responsible for Pro downward arrow Ala cleavage in the pre-secretory process.

Proteomic characterization of the qualitative and quantitative differences in cervical mucus composition during the menstrual cycle.

The chemical composition of the cervical mucus (CM), its physical characteristics and the volume of secretion change cyclically throughout the menstrual cycle. The aim of this study was to identify the constitutive protein composition of CM of fertile women and the changes in the CM proteome throughout the menstrual cycle. Five fertile women who had a term delivery within 1 year before the study were enrolled. Proteomic analysis was performed using an Ultimate 3000 Nano/Micro-HPLC apparatus equipped with an FLM-3000-Flow manager module and coupled with an LTQ Orbitrap XL hybrid mass spectrometer; bioinformatic software was used for functional and quantitative analysis. 59, 81 and 43 proteins (mean) were respectively identified in the pre-ovulatory, ovulatory and post-ovulatory samples. 38 common proteins were identified. 42, 38 and 17 exclusive proteins were respectively identified in pre-ovulatory, ovulatory and post-ovulatory CM. The main part of CM constituents has a catalytic activity, which is mainly related to hydrolase activity. The label-free quantitative analysis of the common proteins revealed a significant reduction in the protein abundance index for antileukoproteinase, after the ovulation, and a peak of haptoglobin at ovulation. This is the first application of high-resolution MS-based proteomics for the identification of protein constituents of CM. This approach may contribute to the identification of putative biomarkers of the female reproductive tract.

Human erythrocyte metabolism is modulated by the O2-linked transition of hemoglobin.

The metabolic behaviour of human erythrocytes has been investigated with particular regard to the effect of their oxygenation state. Experiments performed at high phosphate concentration (80 mM) within the pH range 7.0-7.8 on erythrocytes at high (HOS) and low (LOS) oxygen saturation showed that at any pH value: (1) glucose consumption was independent of the oxygenation state; (2) pentose phosphate pathway (PPP) flux was about 2 times higher in the HOS than in the LOS state. At low phosphate concentration (1.0 mM) the PPP flux doubled in HOS as well as in LOS erythrocytes, whereas the decrease in glucose consumption was more marked in the HOS state. Metabolism of LOS erythrocytes approached that of HOS erythrocytes under the following conditions: (1) erythrocytes having band 3 modified by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid; (2) CO-saturated erythrocytes. These data support the hypothesis of a modulation of the relative rates of PPP and glycolysis achieved through competition between deoxy-hemoglobin (deoxy-Hb) and glycolytic enzymes for the cytoplasmic domain of band 3.

An anthraquinone and three naphthopyrone derivatives from Cassia pudibunda.

Chemical examination of the methanolic extract of the roots of Cassia pudibunda led to isolation of the new rubrofusarin-6-O-beta-D-glucopyranoside, quinquangulin-6-O-beta- D-apiofuranosyl-(1----6)-O-beta-D-glucopyranoside, quinquangulin-6-O-beta-D-glucopyranoside and chrysophanol dimethyl ether. Moreover the known chrysophanol, physcion, cis-3,3',5,5'-tetrahydroxy-4-methoxystilbene, trans-3,3',5,5' -tetrahydroxy-4-methoxystilbene, and cassiaside B were identified. The antimicrobial activity of some of these compounds is also reported.

1H NMR spectra of normal urines: reference ranges of the major metabolites.

Serial urine samples from 50 normal subjects were studied by 1H NMR spectroscopy operating at 300 MHz. Analyses of the spectra have shown the presence of the following metabolites in 100% of the normal subjects: Creatinine, lactate, alanine, citrate, dimethylamine, trimethylamine-N-oxide, glycine and hippurate. Other analytes, such as creatine, valine, betaine, leucine and isoleucine, were sometimes found. All metabolites were quantified on the basis of peak heights and were expressed as mmol/mol of creatinine. The study of metabolic profiles in serial samples allowed us to evaluate intra-individual variability and physiological changes due to feeding. The aim of our report is to define standard conditions for this analytical technique and to calculate confidence intervals for the major metabolites in normal urine samples, such as preliminary and mandatory stages for clinical diagnostic 1H NMR utilization.

Influence of feeding on metabolite excretion evidenced by urine 1H NMR spectral profiles: a comparison between subjects living in Rome and subjects living at arctic latitudes (Svaldbard).

Urines from 25 normal subjects living in Rome and 25 normal subjects living in Ny-Alesund (Svaldbard) were analysed by 1HNMR spectroscopy. The observed differences in the concentration of the major metabolites were correlated to the composition of the diet. It was found that a diet rich of carbohydrates, such as the Italian diet, is responsible for an increased excretion of citrate, lactate, alanine, and glycine. Thus, a correct diagnostical interpretation of urinary metabolites needs to consider feeding habits.

Glycated human hemoglobin (HbA1c): functional characteristics and molecular modeling studies.

A minor hemoglobin component of human red blood cell hemolysate, HbA1c, is the result of the non-enzymatic reaction of glucose with the alpha-amino groups of the valine residues at the N-terminus of the beta-chains of human hemoglobin. In this paper, the effect of protons, chloride and 2,3-diphosphoglycerate (DPG) on the functional properties of HbA1c has been investigated in some details. Moreover, the structural modifications induced on the native molecule by the sugar moieties, studied by computer modeling, do agree with the observed functional alterations. In particular, the functional results indicate that: (a) the low-affinity conformation (or T-state) of HbA1c is destabilized by the chemical modification per se; (b) the Bohr effect is reduced with respect to that of native HbA0; (c) the affinity of the T-state of HbA1c for 2,3-diphosphoglycerate is about 2.6 x lower than that of the corresponding conformational state of HbA0, while the R-state is less affected with, the affinity being 1.7 x lower. At the structural level, computer modeling studies show that the two sugar moieties are asymmetrically disposed within the 2,3-diphosphoglycerate binding site. In addition, molecular mechanics and dynamics calculations concerning the interaction with 2,3-diphosphoglycerate indicate that while in HbA0 the effector can assume two different stable orientations, in glycated Hb only one orientation is possible. All together, the results show that glycation of the Val 1 residues of both beta-chains does not impair the binding of DPG but imposes a different mode of binding by changing the internal geometry of the complex and the surface distribution of the positive electrostatic potential within the binding pocket.

Effect of 2,2,2-trifluoroethanol on capillary zone electrophoretic peptide separations.

The use of 2,2,2-trifluoroethanol-water mixtures for peptide separations by capillary zone electrophoresis (CZE) displays some advantages over aqueous solutions. First, the increase in viscosity reduces and stabilizes the running current and facilitates heat dispersion, with a consequent improvement in the number of theoretical plates. Second, the decrease in the dielectric constant leads to a modification of the dissociation constants of the ionizable groups. The consequence is a change in selectivity that, for several favourable peptide pairs, provides an increase in resolution. Third, the interaction trifluoroethanol with the peptide modifies the Stokes radius in a manner strongly dependent on the peptide sequence. This can also be utilized for an increase in CZE performance. Fourth, the structural properties of 2,2,2-trifluoroethanol are particularly useful for an improvement in the separation of large apolar peptides. Finally, the use of trifluoroethanol strongly stabilizes the capillary coating.

On the alkaloids of Strychnos. XXXV. The occurrence of akagerine in South American Strychnos.

Akagerine (3) was isolated from three different species of South American Strychnos: from S. gardneri A. D.C. (Rio de Janeiro, Brazil), together with 11-methoxydiaboline (2); from S. jobertiana Baillon (Brazil), together with diaboline (1): and from S. parvifolia D.C. (Bahia, Brazil).

Quantitative determination of the main glucose metabolic fluxes in human erythrocytes by 13C- and 1H-MR spectroscopy.

Information displayed by homonuclear and heteronuclear spin-coupling patterns in 13C- and 1H-MR spectra allowed us to identify the major lactate isotopomers produced either from [1-(13)C]-glucose or from [2-(13)C]-glucose by human erythrocytes. Relative concentrations of detectable isotopomers were determined by integrating the corresponding MR signals. The interpretation of these data in terms of the fractional glucose metabolised through glycolysis and pentose phosphate pathway was performed by a computer simulation of the metabolism that took into account metabolic schemes pertaining to glycolysis and to the F-type of pentose phosphate pathway. The simulation was organised in a way to anticipate the populations of the isotopomers produced from any precursor at a priori established metabolic steady state. By the simulation, isotopomer populations were determined according to different values of pentose cycle, defined as the flux of glyceraldehyde 3-phosphate originating from pentose phosphate pathway at unitary glucose uptake. The populations of the isotopomers originating from [2-(13)C]-glucose were described by polynomials, and ratios between the polynomials were used in conjunction with 13C- and 1H-MR data to determine pentose cycle values. The knowledge of glucose uptake and of pentose cycle value allowed us to perform accurate measurement of the pentose phosphate pathway flux, of the hexokinase and phosphofructokinase fluxes as well as, indirectly, of the carbon dioxide production.

Chemical and pharmacological studies of Phyllanthus caroliniensis in mice.

The aim of this study was to isolate and characterize the constituents of the hydroalcoholic extract (HE) of the leaves, stems and roots from P. caroliniensis, and also to evaluate the preliminary antinociceptive action of the HE and purified compounds in mice. Phytosterols, quercetin, gallic acid ethyl ester and geraniin were identified in P. caroliniensis on the basis of 1H and 13C NMR spectral data and by mixed co-TLC and co-HPLC injection with authentic samples. The HE of P. caroliniensis (10-100 mg kg-1, i.p.) inhibited, in a dose-related manner, acetic acid-induced abdominal constrictions in mice, with a mean ID50 value of 23.7 mg kg-1. In the formalin test, the HE given intraperitoneally (1-30 mg kg-1) or orally (25-600 mg kg-1) caused graded inhibitions of both the neurogenic (first phase) and the inflammatory response (late phase) of formalin-induced licking. The HE was 54-fold more effective in inhibiting the late phase than it was in inhibiting the first phase of the formalin test, with mean ID50 values of 3.6 and 196.4 mg kg-1, respectively. The HE failed, however, to affect the oedematogenic response associated with the late phase of formalin-induced pain. In addition, the reference drug, aspirin, given intraperitoneally (1-100 mg kg-1) or orally (100-600 mg kg-1), caused significant inhibition of the late but not the first phase of the formalin test. Pharmacological analysis also revealed that quercetin, gallic acid ethyl ester and a semi-purified fraction of flavonoids (1-100 mg kg-1, i.p.) exhibited graded and significant antinociception against acetic acid-induced abdominal constriction. The mean ID50 values (mg kg-1) for these effects were: 18.8, 34.7 and 5.3, respectively. It is concluded that quercetin, gallic acid ethyl ester and some as yet unidentified flavonoids might account for the antinociceptive action reported for the HE of P. caroliniensis.

Hypoxidaceae. Medicinal uses and the norlignan constituents.

Glycosides of uncommon aglucones, characterized by the Ph-C5-Ph skeleton, were isolated from the rhizomes of some African Hypoxis species. HPLC analysis indicated the occurrence of these compounds in other Hypoxidaceae. On the basis of structural evidence and biogenetic considerations, the aglucones of these glycosides can be considered as norlignans, derived from the union of two phenylpropanoid units in positions alpha, beta' or beta, gamma' and with the loss of the terminal carbon of a chain. Several Hypoxis species are used in traditional medicine as antiinflammatory and antitumor drugs and, recently, preparations based on lipophilic extracts of H. rooperi have been introduced into the market for the treatment of prostatic hypertrophy.

Antibacterial activity and phytochemical analysis of Vochysia divergens (Vochysiaceae).

Vochysia divergens Pohl (Vochysiaceae) is a tree commonly found in wet soils of 'Pantanal' of Mato Grosso, Brazil, and used in folk medicine against infections and asthma. We have studied different extracts and some isolated compounds from this plant for antibacterial activity. From the extracts of the stem bark beta-sitosterol, betulinic acid and sericic acid were isolated. The minimal inhibitory concentration (MIC) for Staphylococcus aureus were: ethanolic extract (MIC = 1.5 mg/ml); ethyl acetate extract (MIC = 2.0 mg/ml); and sericic acid (MIC = 1.0 mg/ml). Escherichia coli was resistant until 5 mg/ml.

The coupling of RP-HPLC and ESI-MS in the study of small peptides and proteins secreted in vitro by human salivary glands that are soluble in acidic solution.

The aim of this study was the development of a method based on the coupling of RP-HPLC and ESI-MS for identifying and quantifying proteins and peptides secreted by human salivary glands in vitro. Salivary gland specimens, obtained from informed patients undergoing surgical resection, were incubated in an optimized medium. Incubation media of glandular specimens, selected on the basis of cytomorphological and ultrastructural analysis, were investigated by HPLC-MS. Several salivary peptides/proteins, previously recognized in human whole saliva, were searched for along the chromatogram by the selected ion monitoring (SIM) strategy. Analysis of the incubation media of parotid glands revealed the presence of basic PRPs PC, PD, PH, IB-1, II-2, and acidic PRP-1 and PRP-3 in all of the investigated samples. Basic PRPs PB and PA, acidic PRPs, and cystatins SN and S1 were detected in all of the incubation media of submandibular glands, whereas histatin 1 was detected in only one sample. Moreover, the method allowed detection of some post-translational derivatives of known salivary proteins, as well as of several previously unidentified small peptides. The present method represents a sensitive and powerful instrument to detect peptides and proteins secreted by human salivary glands in vitro.

Human salivary peptides derived from histatins.

Human saliva from a healthy donor was subjected to fractionation by gel chromatography and six pools were collected and analysed by MALDI-TOF-MS and HPLC-ESI-MS. Three peptides, corresponding to 888.3, 687.3, and 524.1 amu and SNYLYDN, YLYDN, and LYDN sequences (determined by automated Edman sequencing), were isolated from pool 4. YLYDN was not previously described in human saliva. The peptides show the common C-terminal sequence of histatin 3 and histatin 1. To exclude the possibility that the three peptides were an artifact of the purification procedure, nine samples of human saliva were collected from healthy donors, immediately acidified with 0.2% TFA, and analysed by RP-HPLC-ESI-MS. The three peptides were detected in all the analyzed samples. SNYLYDN was always found at a concentration higher than that of YLYDN and LYDN. A correlation analysis performed on quantitative data indicated that the three peptides derive only from histatin 3. Other already known histatins also were searched for in the chromatogram. Histatins 1, 2, 3, 5, 6, 7, 8, and 10 were observed, although not in all samples analyzed, whereas other minor histatins were not detected.

Antinociceptive properties of morusin, a prenylflavonoid isolated from Morus nigra root bark.

The antinociceptive effects of morusin (1), the main prenylflavonoid present in the Morus nigra root barks have been investigated in classical models of pain in mice. The results showed that 1 exhibits a promising antinociceptive or analgesic profile by the intraperitoneal route, being more potent than some standard drugs used as reference. The mechanism by which the morusin exerts antinociceptive activity still remains undetermined, but our results strongly suggest that it involves the participation of the opioid system.

Thymosin beta 4 and thymosin beta 10 expression in hepatocellular carcinoma.

Thymosin beta 4 (Tß4) and thymosin beta 10 (Tß10) are two members of the beta-thymosin family involved in many cellular processes such as cellular motility, angiogenesis, inflammation, cell survival and wound healing. Recently, a role for beta-thymosins has been proposed in the process of carcinogenesis as both peptides were detected in several types of cancer. The aim of the present study was to investigate the expression pattern of Tß4 and Tß10 in hepatocellular carcinoma (HCC). To this end, the expression pattern of both peptides was analyzed in liver samples obtained from 23 subjects diagnosed with HCC. Routinely formalin-fixed and paraffin-embedded liver samples were immunostained by indirect immunohistochemistry with polyclonal antibodies to Tß4 and Tß10. Immunoreactivity for Tß4 and Tß10 was detected in the liver parenchyma of the surrounding tumor area. Both peptides showed an increase in granular reactivity from the periportal to the periterminal hepatocytes. Regarding HCC, Tß4 reactivity was detected in 7/23 cases (30%) and Tß10 reactivity in 22/23 (97%) cases analyzed, adding HCC to human cancers that express these beta-thymosins. Intriguing finding was seen looking at the reactivity of both peptides in tumor cells infiltrating the surrounding liver. Where Tß10 showed a strong homogeneous expression, was Tß4 completely absent in cells undergoing stromal invasion. The current study shows expression of both beta-thymosins in HCC with marked differences in their degree of expression and frequency of immunoreactivity. The higher incidence of Tß10 expression and its higher reactivity in tumor cells involved in stromal invasion indicate a possible major role for Tß10 in HCC progression.