Flavio Giavarini

Molecular Models of Acidic Peptides from Pea Bud Chromatin and Seminal Plasma. Divalent Cations-Mediated Interaction with DNA

Abstract Small acidic peptides have been isolated from biological fluids (blood and seminal plasma) and from chromatin of several tissues. Their biological activity is related to the control of cell growth and gene expression. This work is an approach to the study of peptide structure-function relationship. Purified fractions from seminal plasma and pea bud chromatin were subjected to fast ion bombardment mass spectrometry. The results obtained were analyzed according to biochemical characteristics of the peptides studied and some possible molecular models have been designed. Two of the proposed sequences were synthesized and their biological activity assayed in cells and cell-free systems. The results demonstrate that the synthetic peptides are able to bind to DNA in the presence of divalent cations (Mg2+, Fe2+, Cu2+) with consequent inhibition of DNA transcription.

Short acidic peptides isolated from wheat sprout chromatin and involved in the control of cell proliferation. Characterization by infrared spectroscopy and mass spectrometry.

Low molecular weight peptides were isolated from the chromatin of wheat sprouts. Following gel filtration the peptide fraction shows a sharp inhibiting activity on the growth of HeLa cancer cells. Infrared (IR) spectroscopy and mass spectrometry have been utilized to characterize the wheat sprout peptides in an attempt to recognize the peptide sequence involved in the control of cell growth. The quantitative presence of a peptide with MH+=572 appears proportional to the cell growth inhibition activity. This compound has been subjected to extensive mass spectrometry analysis. The automatic computational analysis of the ions of second, third and fourth generations indicate a peptide sequence, AcHis-Asp-Ser-Glu-, that binds at the C-terminal a molecule of ethanolamine. Moreover, the results show that some sequences of the wheat sprout peptide family are present in the peptide fractions isolated from several other tissues, thus supporting the hypothesis of ubiquitous regulatory peptides.

Analysis by high-performance liquid chromatography of teucrin A in beverages flavoured with an extract of Teucrium chamaedrys L.

Due to its liver toxicity, the medicinal use of germander (Teucrium chamaedrys L.) was banned in some countries. Nevertheless, alcoholic extracts are still permitted as flavour ingredients since they are fundamental in providing a bitter aromatic taste. Teucrin A represents the substance of major concern regarding the potential toxicity of germander. Hence, teucrin A represents the best analytical and toxicological marker of alcoholic extracts of T. chamaedrys. A sensitive high-performance liquid chromatography method to detect teucrin A in beverages is reported. Teucrin A was prepared by isolation from the plant extract using column chromatography and crystallization. The identity and purity (99%) were established by melting point, nuclear magnetic resonance and liquid chromatography-mass spectrometry. The high-performance liquid chromatography procedure was validated and its intra- and interday performance was established (relative standard deviation ≤13% and error ≤10%). In-house validation was carried out by analysing samples of beverages not containing T. chamaedrys spiked with a range of concentrations of teucrin A. The limit of detection was 0.1 ppm and the limit of quantification was 0.3 ppm. Teucrin A accounted for about 70% of the neo-clerodane diterpenoids found in the total extract of a specimen of T. chamaedrys. The content (± standard deviation) in 18 batches of different geographical origin was 2338 ± 740 ppm, per cent coefficient of variation = 32, minimum–maximum = 999−3445 ppm. The mean level of teucrin A in 10 bottles of the same brand was 6.1 ± 0.8 ppm, per cent coefficient of variation = 12. In 10 different brands found on the Italian market, the content of teucrin A ranged from not detectable to 10 ppm.

Characterization of Phospholipid Molecular Species and Peptide Molecules in Wheat Sprout Hydroalcoholic Extract

The phospholipid molecular species and the main peptide molecules of wheat sprout hydroalcoholic extract have been fully characterized by normal-phase high performance liquid chromatography coupled online with positive electrospray ionization tandem mass spectrometry. The extract that resulted was rich in phospholipid molecular species formed by the combination of the two essential fatty acids (α-linoleic and α-linolenic). These species accounted for 51.7% of total phosphatidic acid, 47.3% of total phosphatidylethanolamine, 37.7% of total phosphatidylcholine, and 14.1% of total phosphatidylinositol. The last one showed the highest amounts of species containing palmitic acid, thus representing the most saturated phospholipid class. The extract was also shown to contain several peptide sequences with both potential antioxidant domains and interaction sites for phospholipids (i.e., H-Ala-Gly-Ser-Met-Met-Cys-NH2, H-Tyr-Met-Thr-Val-Val-Ala-Cys-NH2, etc.); this latter finding can have a highly positive impact on the poor peptides bioavailability. Because of the presence of essential fatty acids-rich phospholipids and bioactive peptides, wheat sprout hydroalcoholic extract can be considered a potential functional food ingredient.

Reduced biliary sterol output with no change in total faecal excretion in mice expressing a human apolipoprotein A-I variant

Apolipoprotein (apo)A‐IMilano, is a molecular variant of apoA‐Iwild‐type, associated with dramatically low HDL‐cholesterol levels, but no increased risk for cardiovascular disease. In view of the present uncertainties on the role of apoA‐I in liver cholesterol removal by way of bile acids and neutral sterols, and of the greater capacity of apoA‐IMilano to remove arterial cholesterol, biliary sterol metabolism was evaluated in transgenic mice expressing apoA‐IMilano.

A HEMOGLOBIN VARIANT FOUND DURING GLYCOHEMOGLOBIN MEASUREMENT, IDENTIFIED AS Hb TOULON [α77(EF6)Pro→His] BY TANDEM MASS SPECTROMETRY

Structural hemoglobin (Hb) variants are typically based on a point mutation in a globin gene that produces a single amino acid substitution in a globin chain. Although most of these are of limited clinical significance because they are asymptomatic or mildly symptomatic, diagnosis may be important for genetic counseling. In this paper we describe the first Italian case of Hb Toulon [a77(EF6)Pro!His], and report the analysis of the variant peptide sequences by electrospray ionization mass-mass spectrometry (ESI-MS=MS). The patient, a 59-year-old woman of Northern Italian descent, suffered from multiple benign pathologies, and venous thrombosis. She was obese and reported to have diabetic brothers. No significantly abnormal data resulted from routine clinical chemistry and hematology tests: Hb 13.1 g=dL; RBC 4.3610=L; MCV 91 fL; WBC 7.9610=L. Because of obesity and familial diabetes, glycohemoglobin (Hb A1c) measurement was ordered. By ion exchange high performance liquid chromatography (HPLC) assay (L-9100; Hitachi, Maidenhead, Kent, UK) using a 3-minute program, the Hb A1c was 3.0% (a rather low value); no additional peak was evident in the short chromatogram, but a marked abnormality of the baseline was shown. Whole blood HPLC analysis with a variant Hb dedicated program

Biochemical and mass spectrometry recognition of phospholipid–peptide complexes in wheat sprouts extract

Total hydroalcoholic extract of wheat sprouts was treated with 90% cold acetone as a preliminary step directed to separate antioxidant peptides from antioxidant polyphenols. Surprisingly, the addition of acetone causes the formation of a yellow buoyant gelatinous drop that prevailingly contains peptides and phospholipids. In this context, evidences have been presented that support the hypothesis that peptides (and perhaps other active molecules) are complexed with phospholipids. In fact, the MS/MS analysis of some main ions, present in RP HPLC fractions of wheat sprout extract, generates several ions that correspond to molecular weight of phospholipids or phospholipid fragments. Moreover, several ions were detected that correspond to lysophosphatidylcholine or phosphatidylcholine–peptide complexes. The possibility that phospholipids can be complexed with peptides has been discussed in the light of potential involvement in the peptide bioavailability. Copyright

Bovine seminal plasma contains factors that enhance lymphocyte transformation in vitro.

The important immunosuppressive properties of seminal plasma have significant functions in the processes of reproduction. They mask the presence of an immunostimulating activity. From bovine seminal plasma two active factors have been isolated and characterized with marked enhancing activity for in vitro PHA-dependent lymphocyte transformation. They have inosine and hypoxanthine structures, as confirmed by UV absorption profiles, TLC, mass spectrometry, HPLC patterns, behavior to enzymatic treatments, and breaking of purine ring after acid treatment. Nevertheless, their biological activities are about two orders of magnitude higher than those of commercially available inosine and hypoxanthine standards. Biological activities became practically identical when these were processed (HPLC) in the same way as native molecules. A study to explain such a discrepancy is in progress.

The First Case of Hb G-Honolulu [α30(B11)Glu→Gln (GAG>CAG); HBA2:c.91G>A] Observed in Association with Hb S [β6(A3)Glu→Val, GAG>GTG] in a Healthy Italian Child

We report the first observation of Hb G-Honolulu [α30(B11)Glu→Gln (GAG>CAG); HBA2:c.91G>A] in a Caucasian family and the first case of this variant to be found in association with Hb S [β6(A3)Glu→Val, GAG>GTG]. The proband was a healthy 4-year-old Italian boy. His chromatographic hemoglobin (Hb) pattern showed an abnormal peak having the typical retention time of Hb S (25.6% ), a second abnormal peak eluted soon after (13.6%) and a third minor peak eluted at the end of the run (6.5%). Identification of Hb variants were performed by peptide mapping using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Two abnormal peptides at m/z 765.1 and 922 were found, corresponding to the αT-4 and βT-1 peptides characteristic for Hb G-Honolulu and Hb S, respectively. The third minor abnormal peak presumably corresponded to the hybrid molecule (αG-Honolulu/βS). The concomitant presence of Hb G-Honolulu and Hb S does not seem to produce any relevant clinical manifestation.