29. "The mode of lymphoblastoid cell death in response to gas phase cigarette smoke is dose-dependent".
N. D. Sdralia, A. L. Patmanidi, A. D. Velentzas, L. H. Margaritis, G. E. Baltatzis, D. G. Hatzinikolaou and A. Stavridou.
Respiratory Research, vol. 10, Art. No. 82, (2009).  View at publisher's site  Request copy
Abstract: Background: Cigarette smoke (CS) is the main cause in the development of chronic obstructive pulmonary disease (COPD), the pathogenesis of which is related to an extended inflammatory response. In this study, we investigated the effect of low and high doses of gas phase cigarette smoke (GPS) on cultured lymphocyte progenitor cells, using techniques to assess cell viability and to elucidate whether cells die of apoptosis or necrosis upon exposure to different doses of GPS. Methods: In our approach we utilized a newly-established system of exposure of cells to GPS that is highly controlled, accurately reproducible and simulates CS dosage and kinetics that take place in the smokers' lung. This system was used to study the mode of cell death upon exposure to GPS in conjunction with a range of techniques widely used for cell death studies such as Annexin V staining, activation of caspase-3, cytoplasmic release of cytochrome C, loss of mitochondrial membrane potential and DNA fragmentation. Results: Low doses of GPS induced specific apoptotic indexes in CCRF-CEM cells. Specifically, cytochrome C release and cleaved caspase-3 were detected by immunofluorescence, upon treatment with 1-3 puffs GPS. At 4 h post-exposure, caspase-3 activation was observed in western blot analysis, showing a decreasing pattern as GPS doses increased. Concomitant with this behavior, a dose-dependent change in Δψm depolarization was monitored by flow cytometry 2 h post-exposure, while at 4 h Δψm collapse was observed at the higher doses, indicative of a shift to a necrotic demise. A reduction in DNA fragmentation events produced by 5 puffs GPS as compared to those provoked by 3 puffs GPS, also pointed towards a necrotic response at the higher dose of GPS. Conclusion: Collectively, our results support that at low doses gas phase cigarette smoke induces apoptosis in cultured T-lymphocytes, whereas at high doses GPS leads to necrotic death, by-passing the characteristic stage of caspase-3 activation and, thus, the apoptotic route.

28. "Adsorption of major endoglucanase from Thermoascus aurantiacus on cellulosic substrates".
D. Mamma, D. G. Hatzinikolaou, D. Kekos, H. Stamatis and E. Kalogeris.
World Journal of Microbiology and Biotechnology, vol. 25, pages 781-788, (2009).  View at publisher's site  Request copy
Abstract: A thermostable endoglucanase (EndoI) was produced by the thermophilic fungus Thermoascus aurantiacus when grown on cellulosic materials under submerged culture (SC) and solid-state fermentation (SSF). In both cultivation techniques a considerable amount of enzyme activity remained adsorbed onto solid particles, and this was taken into consideration when modeling enzyme production. The results were compatible with the assumption that, following its synthesis, an amount of EndoI was bound on substrate and gradually released into the liquid medium. Adsorption of the enzyme on crystalline cellulose was confirmed in vitro by experiments with purified endoglucanase, which was isolated by anion exchange chromatography. The Langmuir isotherm could efficiently describe the adsorption kinetics, and the estimated A_max and K_ad values compared with those obtained for cellulases bearing a binding domain. EndoI displayed high affinity for crystalline cellulose and low binding capacity, which could be beneficial in textile processing.

27. "Purification and partial characterization of the xanthine-uric acid transporter (UapA) of Aspergillus nidulans".
N. D. Lemuh, G. Diallinas, S. Frillingos, G. Mermelekas, A. D. Karagouni and D. G. Hatzinikolaou.
Protein Expression and Purification, vol. 63, pages 33-39, (2009).  View at publisher's site  Request copy
Abstract: UapA, the uric acid-xanthine permease from the filamentous ascomycete Aspergillus nidulans, is one of the most thoroughly characterized purine/H⁺ transporters in eukaryotes. Detailed studies have addressed its regulation of expression, at both the transcriptional and post-translational levels, in response to physiological and developmental signals. An extensive kinetic profile towards a plethora of purines and mutational analyses have established models on how UapA recognizes the purine ring and revealed specific amino acid residues involved in proper folding, topogenesis, function and specificity. The present work describes for the first time the purification of the UapA transporter of A. nidulans through overexpression via the strong, ethanol-inducible, glucose-repressible, alcA promoter. Purification, almost to homogeneity, was achieved by Ni²⁺ affinity chromatography using a functional His-tagged UapA protein version. It is subsequently shown, by Circular Dichroism (CD) spectroscopy, that the purified protein is structured with a high α-helical content, as expected from the in silico predictions. The result of this work opens the way for further, analytical and biochemical studies on UapA at the protein level.

26. "Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus".
C. Meintanis, K. I. Chalkou, K. A. Kormas, D. S. Lymperopoulou, E. A. Katsifas, D. G. Hatzinikolaou and A. D. Karagouni.
Letters in Applied Microbiology, vol. 46, pages 395-401, (2008).  View at publisher's site  Request copy
Abstract: AIM: To investigate the applicability of rpoB gene, which encodes the β-subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP- and BOX-polymerase chain reaction) were also used. METHODS AND RESULTS: rpoB DNA (458 bp) were amplified from 21 Geobacillus- and Bacillus type strains, producing different BOX- and REP-PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90–100% (16S rRNA) and 74–100% (rpoB) similarity among examined bacteria. CONCLUSION: BOX- and REP-PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.

25. "Application of Different Processes for the Biodegradation of 1,3-dichloro-2-propanol by the Bacterium Pseudomonas putida DSM437".
E. Kalogeris, O. Antzoulatos, D. Mamma, D. G. Hatzinikolaou, P. Christakopoulos and D. Kekos.
Chemical & Biochemical Engineering Quarterly, vol. 21, pages 297 - 305, (2007).  View at publisher's site  Request copy
Abstract: 1,3-Dichloro-2-propanol (1,3-DCP), is a highly toxic compound used in many industrial processes. Biodegradation of 1,3-DCP, by the bacterial strain Pseudomonas putida DSM 347, was studied applying three different processes. A number of combinations, with respect to glucose and 1,3-DCP concentration were examined during batch process. When the initial concentration of 1,3-DCP was 600 mg·L^–1 in the presence of 400 mg·L^-1 glucose, the biodegradation degree and rate were 10.8 % and 0.68 mg·L^-1h^-1 respectively. 1,3-DCP biodegradation by the resting cells of P. putida DSM 347 was tested at mass concentrations from γ = 200 to 1,000 mg·L^-1 using biomass concentration of 5 g dry cell mass L^-1. Biodegradation of 1,3-DCP ranged from 84 to 90 %, initial biodegradation rates ranged from r = 2.36 to 10.55 mg·L^-1·h^-1, while dependence of both parameters from the initial concentration of halohydrin was observed. A system of two Continuous Stirred Tank Reactors (CSTRs) in series was developed for the biodegradation of a highly toxic stream of 1,3-DCP (2000 mg·L^-1). The overall biodegradation degree of the system was 68 %, while biodegradation rates of the first and second bioreactor were r = 2.88 and 5.21 mg·L^-1·h^-1 respectively.

24. "Cell Bound and Extracellular Glucose Oxidases from Aspergillus niger BTL: Evidence for a Secondary Glycosylation Mechanism".
D. G. Hatzinikolaou, D. Mamma, P. Christakopoulos and D. Kekos.
Applied Biochemistry and Biotechnology, vol. 142, pages 29 - 43, (2007).  View at publisher's site  Request copy
Abstract: Two glucose oxidase (GOX) isoforms where purified to electrophoretic homogeneity from the mycelium extract (GOXI) and the extracellular medium (GOXII) of Aspergillus niger BTL cultures. Both enzymes were found to be homodimers with nonreduced molecular masses of 148 and 159 kDa and pI values of 3.7 and 3.6 for GOXI and GOXII, respectively. The substrate specificity and the kinetic characteristics of the two GOX forms, as expressed through their apparent Km values on glucose, as well as pH and T activity optima, were almost identical. The only structural difference between the two enzymes was in their degrees of glycosylation, which were determined equal to 14.1 and 20.8% (w/w) of their molecular masses for GOXI and GOXII, respectively. The above difference in the carbohydrate content between the two enzymes seems to influence their pH and thermal stabilities. GOXII proved to be more stable than GOXI at pH values 2.5, 3.0, 8.0, and 9.0. Half-lives of GOXI at pH 3.0 and 8.0 were 8.9 and 17.5 h, respectively, whereas the corresponding values for GOXII were 13.5 and 28.1 h. As far as the thermal stability is concerned, GOXII was also more thermostable than GOXI as judged by the deactivation constants determined at various temperatures. More specifically, the half-lives of GOXI and GOXII, at 45°C, were 12 and 49 h, respectively. These results suggest A. niger BTL probably possesses a secondary glycosylation mechanism that increases the stability of the excreted GOX.

23. "Differential physiological and developmental expression of the UapA and AzgA purine transporters in Aspergillus nidulans".
A. Pantazopoulou, N. D. Lemuh, D. G. Hatzinikolaou, C. Drevet, G. Cecchetto, C. Scazzocchio and G. Diallinas.
Fungal Genetics and Biology, vol. 44, pages 627 - 640, (2007).  View at publisher's site  Request copy
Abstract: In this article we study the cellular expression of UapA and AzgA, the two major purine transporters of Aspergillus nidulans, by constructing strains expressing, from their native promoters, fully functional fluorescent (UapA-sGFP, AzgA-sGFP) or immunological (UapA-His) chimeric transporters. Epifluorescence microscopy and immunodetection showed that under different physiological conditions and during Aspergillus development: (i) UapA and AzgA expression in the plasma membrane becomes evident early during germination and remains at a significant basal level in mycelium, (ii) Neither of the two transporters is expressed in the stalk, the vesicle, the phialides and the conidiospores, but surprisingly, UapA is specifically and strongly expressed in the periphery of metulae, (iii) Both transporters are expressed in ascogenous hyphae and in hülle cells but not in cleistothecia or ascospores, (iv) Purine induction leads to not, vert, similar 4-fold increase in UapA and AzgA protein content in mycelium, compatible with an analogous increase at the transcriptional level, (v) Ammonium leads to removal of UapA, but not of AzgA, from the plasma membrane by sorting of the protein to the vacuole.

22. "Cellulose, hemicelluloses, lignin and ash content of some organic materials and their suitability for use as paper pulp supplements".
C. Ververis, K. Georghiou, D. Danielidis, D. G. Hatzinikolaou, P. Santas, R. Santas and V. Corleti.
Bioresource Technology, vol. 98, pages 296 - 301, (2007).  View at publisher's site  Request copy
Abstract: Freshwater algal biomass and orange and lemon peels were assessed as tissue paper pulp supplements. Cellulose and hemicellulose contents of algal biomass were 7.1% and 16.3%, respectively, whereas for citrus peels cellulose content ranged from 12.7% to 13.6% and hemicellulose from 5.3% to 6.1%. For all materials, lignin and ash content was 2% or lower, rendering them suitable for use as paper pulp supplements. The addition of algal biomass to paper pulp increased its mechanical strength significantly. However, brightness was adversely affected by chlorophyll. The addition of citrus peels in paper pulp had no effect on breaking length, increased bursting strength and decreased tearing resistance. Brightness was negatively affected at proportions of 10%, because citrus peel particles behave as coloured pigments. The cost of both materials is about 45% lower than that of conventional pulp, resulting in a 0.9–4.5% reduction in final paper price upon their addition to the pulp.

21. "Analysis of the Gas Phase of Cigarette Smoke by Gas Chromatography Coupled with UV-Diode Array Detection".
D. G. Hatzinikolaou, V. Lagesson, A. J. Stavridou, A. E. Pouli, L. Lagesson-Andrasko and J. C. Stavrides.
Analytical Chemistry, vol. 78, pages 4509 - 4516, (2006).  View at publisher's site  Request copy
Abstract: A gas chromatography method, coupled with diode array photometric spectral detection in the ultraviolet region (167−330 nm), was developed for the analysis of the gas phase of cigarette smoke. The method enabled us to identify more than 20 volatiles present in the vapor phase of cigarette smoke. In that way, all major volatile organic compounds (including aldehydes, conjugated dienes, ketones, sulfides, furans, and single-ring aromatics), as well as nitric oxide (NO) and hydrogen sulfide (H₂S), can be analyzed in a straightforward manner through a single chromatographic run of <50-min duration. The method can easily be applied by the introduction of a small volume of the gas-phase stream into the GC injection loop directly through the smoking apparatus exhaust circuit, thus providing an excellent alternative to available methods, which usually require extraction or concentration steps prior to any chromatographic analysis. Furthermore, all problems concerning aging of the gas phase are eliminated. Twelve compounds (including NO) chosen for quantification through the use of appropriate calibration standards. Comparison of the vapor phase yields of these compounds for the reference cigarette Kentucky 1R4F with already reported data indicates that this method is very reliable as far as accuracy and reproducibility of the results are concerned. Finally, the proposed methodology was used to compare the concentration of these cigarette smoke gas-phase constituents among individual puffs.