Proteomic insight into the primycin fermentation process of Saccharomonospora azurea.

Saccharomonospora azurea SZMC 14600 is a member of the family Pseudonocardiaceae exclusively used for industrial scale production of primycin a large 36-membered non-polyene macrolide lactone antibiotic belonging to the polyketide class of natural products. Even though maximum antibiotic yield has been achieved by empirically optimized two-step fermentation process, little is known about the molecular components and mechanisms underlying the efficient antibiotic production. In order to identify differentially expressed proteins (DEPs) between the pre- and main-fermentation stages of primycin, comparative 2D-PAGE experiments were performed. In total, 98 DEP spots were reproducibly detected, out of which four spots were excised from gels, and identified through MALDI-TOF/TOF mass spectrometry. Peptide mass fingerprint analysis revealed peptide matches to HicB antitoxin for the HicAB toxin-antitoxin system (EHK86651), to a nucleoside diphosphate kinase regulator ((Ndk; EHK81899) and two other proteins with unknown function (EHK88946 and EHK86777).

Mass spectrometric identification of ancient proteins as potential molecular biomarkers for a 2000-year-old osteogenic sarcoma.

Osteosarcoma is the most common primary malignant tumor of bone usually occurring in young adolescent and children. This disease has a poor prognosis, because of the metastases in the period of tumor progression, which are usually developed previous to the clinical diagnosis. In this paper, a 2000-year-old ancient bone remain with osteogenic sarcoma was analyzed searching for tumor biomarkers which are closely related to this disease. After a specific extraction SDS-PAGE gel electrophoresis followed by tryptic digestion was performed. After the digestion the samples were measured using MALDI TOF/TOF MS. Healthy bone samples from same archaeological site were used as control samples. Our results show that in the pathological skeletal remain several well known tumor biomarkers are detected such as annexin A10, BCL-2-like protein, calgizzarin, rho GTPase-activating protein 7, HSP beta-6 protein, transferrin and vimentin compared to the control samples. The identified protein biomarkers can be useful in the discovery of malignant bone lesions such as osteosarcoma in the very early stage of the disease from paleoanthropological remains.

Preparation and investigation of LC packing made by microwave-assisted solid-phase synthesis.

The preparation of the so-called bonded phase liquid chromatographic packings is usually carried out by heating the silica, the silane, a catalyst, or a scavenger in an appropriate solvent (often toluene.) Due to the longtime of boiling, the procedure is time and energy consuming, and solvent intensive. The goal of this work is to present a simple, environment-friendly preparation method with reduced solvent consumption to synthetize RP liquid chromatographic stationary phases. The effects of reaction conditions (amount of reagents, composition of the reagent, microwave energy, reaction time, reproducibility of the synthesis) are discussed. Pore structure, surface coverage, the change of the pore structure and surface coverage upon reaction are demonstrated, the efficiency of the column (van Deemter plot for different solutes) is presented. A variety of applications (aromatic hydrocarbons, halobenzenes, bioactive peptides, resveratrol from red wine) demonstrates the separation power of the new phase.

Mass spectrometry-based salivary proteomics for the discovery of head and neck squamous cell carcinoma.

The 5-year survival rates for cases of head and neck squamous cell carcinoma (HNSCC) are only some 60%, mainly because 20%-40% of the patients develop a local relapse in the same or an adjacent anatomic region, even when the surgical margins are histologically tumour-free. Tumours are often discovered in an advanced stage because of the lack of specific symptoms and the diagnostic difficulties. The more advanced the stage of the tumour, the more invasive the diagnostic and treatment interventions needed. An early molecular diagnosis is therefore of vital importance in order to increase the survival rate. The aim of this study was to develop an efficient rapid and sensitive mass spectrometric method for the detection of differentially expressed proteins as tumour-specific biomarkers in saliva from HNSCC patients. Whole saliva samples were collected from patients with HNSCC and from healthy subjects. The proteins were profiled by using SDS PAGE, MALDI TOF/TOF mass spectrometry and the Mascot database search engine. Several potential tumour markers were identified, including annexin A1, beta- and gamma-actin, cytokeratin 4 and 13, zinc finger proteins and P53 pathway proteins. All of these proteins play a proven role in tumour genesis, and have not been detected previously in saliva. Salivary proteomics is a non-invasive specific method for cancer diagnosis and follow-up treatment. It provides facilities for the readily reproducible and reliable detection of tumours in early stages.

Comparative salivary proteomics of cleft palate patients.

OBJECTIVES: To investigate salivary proteins with proteomic technologies to evaluate protein composition differences between samples with cleft lip and palate and healthy controls. DESIGN, PARTICIPANTS: Matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI TOF/TOF) mass spectrometry was used as a high-throughput analytical technique for identification of nonsyndromic cleft lip and palate stimulated salivary proteins. The samples consisted of two groups: 31 cleft lip and palate patients and a control group with 20 healthy volunteers. RESULTS: The presence of cleft lip and palate stimulated the expression of several proteins, included adaptor-related protein complex 3, dermokine, nidogen 1 precursor, transforming growth factor-ß3, and a zinc finger RAN-binding domain containing 2. CONCLUSIONS: The salivary proteome of cleft lip and palate patients differs from the protein composition of healthy control saliva samples. Several common secreted proteins such as actins, salivary cystatins, and keratins were upregulated by cleft; increased levels of TGF-ß3 and dermokine were detected in the pathologic samples. The current proteomic results suggest keratinocyte activation among patients with cleft lip and palate. The score of our preliminary results suggests the hypothesis that identified salivary proteins are of vital clinical importance in tissue regeneration and the molecular repair mechanism seen in patients with cleft lip and palate.

The effect of toxins on inorganic phosphate release during actin polymerization.

During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P(i) release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P(i) release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P(i) from F-actin. The products of ATP hydrolysis were released by denaturation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P(i). The effect of both toxins can be sufficiently explained by local and mechanical blockade of P(i) dissociation.