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The filamentous actinobacterium Microbispora sp. ATCC-PTA-5024 produces the lantibiotic NAI-107, which is effective against multidrugresistant Gram-positive pathogens. In actinomycetes, the biosynthesis of antibiotics is elicited as a physiological response that is controlled by a complex regulatory network involving general and pathwayspecific regulators. The ‘omics technologies can be useful to explore molecular physiology in bacterial cells and elucidate molecular and metabolic events associated to antibiotic production in order to develop robust and economically-feasible production processes. To this aim, differential proteomic analyses, based two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry (MS) approaches, combined with differential fluorescence microscopy (DFM) and molecular genetic studies were carried out on M. sp. ATCC-PTA-5024. M. sp. ATCC-PTA-5024 fermentations, showing a first biomass accumulation (A) stage followed by a of biomass yield decline (D) stage, revealed that NAI-107 yield starts at mid A stage and increases up to mid D stage. 2D-DIGE and MS analyses were carried out at A and D stages to reveal patterns of differentially regulated proteins associated to on set and maintenance of NAI-107 production, respectively. Regulatory and metabolic proteins were identified and possible role in physiological differentiation of an unknown regulatory protein was investigated by the construction of an over-expressing strain. Impact of NAI-107 on cell physiology and metabolism was analysed using a non-producing mutant confirming possible role of identified proteins in self-resistance mechanism. Molecular aspect of activation of specific stress response mechanism were confirmed by DFM. These results originally elucidate regulatory networks, biochemical pathways and molecular processes occurring during growth and lantibiotic production, thus providing the first functional picture of a member of the Microbispora genus.

The essential requirement for a successful myocardial tissue engineering is that scaffolds and tissue constructs exhibit biomimetic activity toward cells. Our aim was to investigate the significance of cell-matrix interactions in cardiac tissue microenvironment in vitro. Cardiac fibroblasts from normal (CF-N) and pathological (CF-P) hearts with ischemic cardiopathy deposited extracellular matrix (Mx) and conditioned medium (Cm), which were then characterized by immunohistology, immunoblotting, ELISA-based protein array or mass spectrometry. Normal and pathological cardiac primitive cells CPC-N and -P) were cultured on CF-Mx-N and -P or in CF-Cm-N and -P and their proliferation, apoptosis, migration and maturation were evaluated by BrdU, TdT, scratch wound assays or RT-PCR, respectively. Laminin-1 and tenascin-X were detected in CF-Mx-P. The expression of IGFBP1, Il-6, EGF was higher in CF-Cm-P, while that of SCF, TGF-?3, BMP4 was higher in CF-Cm-N. Migration speed of CPC-P peaked at 21.3 ?m/h on CF-Mx-N. Proliferation of CPCs increased significantly on CF-Mx-P, while apoptosis diminished on both types of matrix. In CPC-N, maturation toward cardiomyocytes was activated by CF Mx-N and toward endothelial cells by CF-Mx-P; in CPC-P the trend toward smooth muscle cell-lineage was evident on both substrates. CF-Cm-N reduced apoptosis of CPC-N (2-fold), while CF-Cm-P reduced apoptosis of CPC-P (3.5-fold) and increased proliferation of CPC-N (1.6-fold, n=3, p

Changes in serum low molecular weight proteins from DL-PCB contaminated heifers

Introduction: Alkanes are biodegraded to generate the corresponding primary alcohol trough alkane hydroxylases (AHs) consisting on an integral membrane alkane monooxygenase (AlkB) and two soluble proteins, rubredoxin and rubredoxin reductase. Recently, an alkB gene was reported to be involved in degradation of long chain n-alkanes in the actinobacterium Gordonia sp. SoCg. This gene was expressed in Streptomyces coelicolor M145 which is unable to degrade n-alkanes. Results: The engineered strain, M145-AH, can biotransform n-hexadecane into the corresponding 1-hexadecanol and it is able to grow on n-hexadecane as sole carbon source. Changes in global protein expression associated with n-hexadecane metabolism in M145-AH were studied using a differential proteomic approach. M145-AH was incubated in three different conditions using a mineral medium supplemented with hexadecane or glucose as the sole carbon source or without any carbon source, respectively. Total proteins, extracted from samples collected after 6, 24 and 48 h of incubation in the three conditions, were analyzed by 2D-Differential Gel Electrophoresis (2D-DIGE). Differentially abundant protein spots were identified by mass spectrometry. The expression profile of proteins involved in central carbon metabolism, amino acid and protein biosynthesis, fatty acid metabolism and respiration revealed a gradual metabolic adaptation to n-hexadecane utilization, which is similar to that of specialized alkane-degraders. Conclusion: Thus, the addition of the alkB gene confers to Streptomyces coelicolor the ability to use an insoluble recalcitrant contaminant as an usual carbon source. These data, expanding the knowledge on n-alkane bioconversion mechanisms in Gram-positive bacteria, provide new technological platforms for bioremediation studies and strategies.

Gordonia sp. SoCg, a Gram positive strain able to grow on long chain n-alkanes1, possess a single copy of alkB2 gene, whose product is required for n-alkane hydroxylation3. An analysis of alkB flanking regions revealed five ORFs which were designed as orf1, rubA3, rubA4, rubB and alkU, according to the sequence 14 homology with that of known alk clusters3. In G. sp. SoCg the transcription of these genes was induced by long-chain and solid n-alkanes as revealed by quantitative RT-PCR, and the essential role of alkB in nalkane degradation was demonstrated by the construction of an alkB disruption mutant strain3. The SoCg alkB gene was successfully expressed in Streptomyces coelicolor M145 (M145-AH), and the production of 1-hexadecanol from n-hexadecane oxidation was observed3. A differential study of global gene expression of M145-AH cultures was performed, where n-hexadecane (C16) glucose (GLU) and none (NC) were provided as only carbon source, respectively. Proteomic analysis, based on 2D-DIGE and MS procedures, revealed a gradual metabolic adaptation to n-hexadecane utilization, not dissimilar from that one revealed in specialized alkane-degraders4. In addition, expression profiles of central carbon metabolism enzymes revealed that the addition of a single gene confers the ability to use recalcitrant pollutants as simple sugars in Streptomyces. Altogether these data, expanding the knowledge on n-alkane bioconversion mechanisms in Gram positive bacteria, could provide new technological platforms for bioremediation studies and strategies. Quatrini, P., Scaglione, G., De Pasquale, C., Riela, S., & Puglia, A. M. (2008). Isolation of gram-positive n-alkane degraders from a hydrocarbon- contaminated mediterranean shoreline. Journal of Applied Microbiology, 104(1), 251-259. Lo Piccolo, L., De Pasquale, C., Fodale, R., Puglia, A. M. & Quatrini, P. . An alkane hydroxylase system of Gordonia sp. Strain SoCg is involved in degradation of solid n-alkanes. Applied and Environmental Microbiology. In revision. van Beilen, J. B., & Funhoff, E. G. (2007). Alkane hydroxylases involved in microbial alkane degradation. Applied Microbiology and Biotechnology, 74(1), 13-21. Sabirova, J. S., Ferrer, M., Regenhardt, D., Timmis, K. N., & Golyshin, P. N. (2006). Proteomic insights into metabolic adaptations in Alcanivorax borkumensis induced by alkane utilization. Journal of Bacteriology, 188(11), 3763-3773.

The Maillard reaction includes a complex network of processes affecting food and biopharmaceutical products; it also occurs in living organisms and has been strictly related to cell aging, to the pathogenesis of several (chronic) diseases, such as diabetes, uremia, cataract, liver cirrhosis and various neurodegenerative pathologies, as well as to peritoneal dialysis treatment. Dozens of compounds are involved in this process, among which a number of protein-adducted derivatives that have been simplistically defined as early, intermediate and advanced glycation end-products. In the last decade, various bottom-up proteomic approaches have been successfully used for the identification of glycation/glycoxidation protein targets as well as for the characterization of the corresponding adducts, including assignment of the modified amino acids. This article provides an updated overview of the mass spectrometry-based procedures developed to this purpose, emphasizing their partial limits with respect to current proteomic approaches for the analysis of other post-translational modifications. These limitations are mainly related to the concomitant sheer diversity, chemical complexity, and variable abundance of the various derivatives to be characterized. Some challenges to scientists are finally proposed for future proteomic investigations to solve main drawbacks in this research field.

Cladosporols, purified and characterized as secondary metabolites from Cladosporium tenuissimum, display an antifungal activity. In this study, we tested the antiproliferative properties of cladosporol A, the main isoform of this metabolite family, against human cancer cell lines. By assessing cell viability, we found that cladosporol A inhibits the growth of various human colon cancers derived cell lines (HT-29, SW480, and CaCo-2) in a time- and concentration-dependent manner, specifically of HT-29cells. The reduced cell proliferation was due to a G1-phase arrest, as assessed by fluorescence activated cell sorting analysis on synchronized HT-29cells, and was associated with an early and robust over-expression of p21(waf1/cip1) , the well-known cyclin-dependent kinases inhibitor. This suggests that the drug may play a role in the control of cancer cell proliferation. Consistently, cyclin D1, cyclin E, CDK2, and CDK4 proteins were reduced and histone H1-associated CDK2 kinase activity inhibited. In addition to p21(waf1/cip1) , exposure to 20µM cladosporol A caused a simultaneous increase of pERK and pJNK, suggesting that this drug activates a circuit that integrates cell cycle regulation and the signaling pathways both involved in the inhibition of cell proliferation. Finally, we showed that the increase of p21(waf1/cip1) expression was generated by a Sp1-dependent p53-independent stimulation of its gene transcription as mutagenesis of the Sp1 binding sites located in the p21 proximal promoter abolished induction. To our knowledge, this is the first report showing that cladosporol A inhibits colon cancer cell proliferation by modulating p21(waf1/cip1) expression. © 2011 Wiley Periodicals, Inc.

Osteoarthritis (OA) and osteochondrosis (OC) are two of the main challenges in orthopedics, whose definitive diagnosis is usually based on radiographic/arthroscopic evidences. Their early diagnosis should allow preventive or timely therapeutic actions, which are generally precluded from the poor relationships occurring between symptomatologic and radiographic evidences. These limitations should be overcome by improving the knowledge on articular tissue metabolism and on molecular factors regulating its normal homeostasis, also identifying novel OA and OC biomarkers suitable for their earlier diagnoses, whenever clinical/pathological inflammatory scenarios between these joint diseases seem somewhat related. To identify proteins involved in their aetiology and progression, we undertook a differential proteomic analysis of equine synovial fluid (SF), which compared the protein pattern of OA or OC patients with that of healthy individuals. Deregulated proteins in OA and OC included components related to inflammatory state, coagulation pathways, oxidative stress and matrix damage, which were suggestive of pathological alterations in articular homeostasis, plasma-SF exchange, joint nutritional status and vessel permeability. Some proteins seemed commonly deregulated in both pathologies indicating that, regardless of the stimulus, common pathways are affected and/or the animal joint uses the same molecular mechanisms to restore its homeostasis. On the other hand, the increased number of deregulated proteins observed in OA with respect to OC, together with their nature, confirmed the high inflammatory character of this disease. Some deregulated proteins in OA found a verification by analyzing the SF of injured arthritic joints following autologous conditioned serum treatment, an emergent therapy that provides positive results for both human and equine OA. Being the horse involved in occupational/sporting activities and considered as an excellent animal model for human joint diseases, our data provide suggestive information for tentative biomedical extrapolations, allowing to overcome the limitations in joint size and workload that are typical of other small animal models.

Physical exercise induces various stress responses and metabolic adaptations that have not yet been completely elucidated. Novel biomarkers are needed in sport veterinary medicine to monitor training levels and to detect subclinical conditions that can develop into exercise-related diseases. In this study, protein modifications in horse plasma induced by prolonged, aerobic physical exercise were investigated by using a proteomic approach based on 2-DE and combined mass spectrometry procedures. Thirty-eight protein spots, associated with expression products of 13 genes, showed significant quantitative changes; spots identified as membrane Cu amine oxidase, ?-1 antitrypsin, ?-1 antitrypsin-related protein, caeruloplasmin, ?-2 macroglobulin and complement factor C4 were augmented in relative abundance after the race, while haptoglobin ? chain, apolipoprotein A-I, transthyretin, retinol binding protein 4, fibrinogen ? chain, complement factor B and albumin fragments were reduced. These results indicate that prolonged physical exercise affects plasma proteins involved in pathways related to inflammation, coagulation, immune modulation, oxidant/antioxidant activity and cellular and vascular damage, with consequent effects on whole horse metabolism.

The embryonic rat ventricle H9c2 cells maintain a proliferative state (P condition) in the presence of 10% FCS. However, by reducing serum concentration and in the presence of retinol acetate, proliferation is stopped, myogenic transdifferentiation is inhibited while cardiac differentiation is preserved (D condition). Two-dimensional gel electrophoresis and mass spectrometry analysis was used to define the modifications of the nuclear proteome occurring during the P-to-D transition. Among the proteins observed as modified, a reduced expression of the SM22/transgelin protein was associated with the D state. Also SM22 mRNA levels were reduced during P-to-D transition. Cell transfection experiments indicated that this decrease was partially due to a reduction of the SM22 promoter activity. GATA-4 had a repressive effect on SM22 promoter activity. Thus, since GATA-4 is known as a target of retinoids and may act as a transcriptional repressor, a mechanism to explain the SM22 reduction during the P-to-D transition is tentatively proposed. Immunohistochemical studies on heart cells confirmed the nuclear localization of SM22. Moreover, a differential expression of this protein in different districts of the human heart embryo was detected. Therefore, these data suggest that SM22 expression is regulated during heart development.