Chitin-Induced Gene Expression in Secondary Metabolic Pathways of Streptomyces coelicolor A3(2) Grown in Soil

Microarray analyses revealed that the expression of genes for secondary metabolism together with that of primary metabolic genes was induced by chitin in autoclaved soil cultures of Streptomyces coelicolor A3(2). The data also indicated that DasR was involved in the regulation of gene expression for chitin catabolism, secondary metabolism, and stress responses.     

Thermodynamic study of interaction of TSPP,CoTsPc, and FeTsPc with calf thymus DNA

The interaction of two water soluble phthalocyanines, cobalt(II) 4,4′,4″,4‴-tetrasulfo-phthalocyanine (CoTsPc) and iron(II) 4,4′,4″,4‴-tetrasulfo-phthalocyanine (FeTsPc), and one water soluble porphyrin, tetra sodium mesotetrakis(p-sulfophenyl)porphyrin (TSPP), with calf thymus DNA has been studied by UV-Vis spectroscopy at five different temperatures (20, 25, 30, 35, and 40°C). The optical absorption spectra of these materials were analyzed to obtain binding constants and stoichiometries using SQUAD software. The results show that the best fitting corresponds to a 1: 1 complex model between a base pair of DNA and these materials. All of the studied porphyrin and phthalocyanines showed strong electrolyte effect, and increasing NaCl concentration induced self-aggregation of these materials. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 6, pp. 799–804.     

Appendage-Mediated Surface Adherence of Sulfolobus solfataricus

Attachment of microorganisms to surfaces is a prerequisite for colonization and biofilm formation. The hyperthermophilic crenarchaeote Sulfolobus solfataricus was able to attach to a variety of surfaces, such as glass, mica, pyrite, and carbon-coated gold grids. Deletion mutant analysis showed that for initial attachment the presence of flagella and pili is essential. Attached cells produced extracellular polysaccharides containing mannose, galactose, and N-acetylglucosamine. Genes possibly involved in the production of the extracellular polysaccharides were identified.In microbiology, organisms are isolated from their natural habitats and typically cultivated in the laboratory as planktonic species. Though this method has been essential for understanding the concept of life, it remains unclear how microbial ecosystems operate. For bacteria, it is well known that they are able to form large cellular communities with highly complex cellular interactions and symbioses between different microbial or eukaryotic species. Biofilm formation is an essential component of such communities, and studies have shown that bacteria within biofilms are physiologically different from planktonic ones (20, 21). They can exhibit extensive networks of pili on their surfaces and produce and secrete extracellular polysaccharides (EPS), their growth rate is decreased, and cells are much more resistant to physical stresses and antibiotics (19).The study of surface colonization and cellular communities of archaea is crucial for understanding their ecological properties. The only detailed study showed that the hyperthermophilic organism Archaeoglobus fulgidus produced biofilms when challenged with heavy metals and pentachlorophenol (10). Pyrococcus furiosus was able to adhere to different surfaces, such as mica and carbon-coated gold grids, and cells were connected via cable-like bundles of flagella (12). Methanopyrus kandleri was shown to adhere to glass, but P. furiosus could colonize only by attaching to M. kandleri cells, using flagella and direct cell contacts (16).Here we report on the function of cell surface appendages in initial attachment to surfaces of archaea, using directed gene inactivation mutants. The crenarchaeote Sulfolobus solfataricus P2 is a thermoacidophile which grows optimally at 80°C and pH values of 2 to 4 (22). S. solfataricus possesses cell surface structures such as flagella and UV-induced pili (1, 2). The flagellum operon of S. solfataricus encodes, in addition to the structural subunit FlaB, four proteins of unknown function, the ATPase FlaI, and the only integral membrane protein, FlaJ. Previously, we isolated a ΔflaJ mutant which was nonflagellated and had lost its ability for surface motility on Gelrite plates (17). Recently, we described UV-inducible pili in S. solfataricus that directed cellular aggregation after UV stress (8). Deletion of the central ATPase UpsE, responsible for pilus assembly, rendered cells devoid of pili and defective in cellular aggregation after UV treatment (8). In this study, wild-type cells and deletion strains were tested for the ability to attach to a variety of surfaces and the formed structures and extracellular material were analyzed.     

Establishment and characterization of two human breast carcinoma cell lines by spontaneous immortalization: Discordance between Estrogen, Progesterone and HER2/neu receptors of breast carcinoma tissues with derived cell lines

Background

Breast cancer is one of the most common cancers among women throughout the world. Therefore, established cell lines are widely used as in vitro experimental models in cancer research.

Methods

Two continuous human breast cell lines, designated MBC1 and MBC2, were successfully established and characterized from invasive ductal breast carcinoma tissues of Malaysian patients. MBC1 and MBC2 have been characterized in terms of morphology analysis, population doubling time, clonogenic formation, wound healing assay, invasion assay, cell cycle, DNA profiling, fluorescence immunocytochemistry, Western blotting and karyotyping.

Results

MBC1 and MBC2 exhibited adherent monolayer epithelial morphology at a passage number of 150. Receptor status of MBC1 and MBC2 show (ER⁺, PR⁺, HER2⁺) and (ER⁺, PR^-, HER2⁺), respectively. These results are in discordance with histopathological studies of the tumoral tissues, which were triple negative and (ER^-, PR^-, HER2⁺) for MBC1 and MBC2, respectively. Both cell lines were capable of growing in soft agar culture, which suggests their metastatic potential. The MBC1 and MBC2 metaphase spreads showed an abnormal karyotype, including hyperdiploidy and complex rearrangements with modes of 52–58 chromosomes per cell.

Conclusions

Loss or gain in secondary properties, deregulation and specific genetic changes possibly conferred receptor changes during the culturing of tumoral cells. Thus, we hypothesize that, among heterogenous tumoral cells, only a small minority of ER⁺/PR⁺/HER2⁺ and ER⁺/PR^-/HER2⁺ cells with lower energy metabolism might survive and adjust easily to in vitro conditions. These cell lines will pave the way for new perspectives in genetic and biological investigations, drug resistance and chemotherapy studies, and would serve as prototype models in Malaysian breast carcinogenesis investigations.     

Release profiles of tricalcium phosphate nanoparticles from poly(L-lactic acid) electrospun scaffolds with single component, core-sheath, or porous fiber morphologies: effects on hASC viability and osteogenic differentiation

Functional PLA scaffolds are created with single component, core-sheath, or porous fiber morphology and doped with TCP nanoparticles to study the release profiles for use in bone tissue engineering applications. Pharmacokinetic analyses are performed for the three different nanofibrous structures after doping with TCP. Results indicate that single component and porous fiber scaffolds exhibit an initial-burst release profile whereas core-sheath fibers show a steady release. All scaffolds are then seeded with human adipose-derived stem cells (hASC), which remain viable and continue proliferation on all nanofibrous morphologies for up to 21 d. Osteogenic differentiation of hASC and cell-mediated calcium accretion are largest on porous fibers.     

cAMP-mediated induction of cyclin E sensitizes growth-arrested adipose stem cells to DNA damage-induced apoptosis

The differentiation capacity of mesenchymal stem cells has been extensively studied, but little is known on cell cycle–related events in the proliferation and differentiation phases of these cells. Here, we demonstrate that exposure to cAMP-increasing agents inhibits proliferation of adipose stem cells (ASCs). This antiproliferative effect is associated with both reduced cdk2 activity and pRB phosphorylation. Concomitantly, however, the level of cyclin E markedly increases upon cAMP induction, indicating that cyclin E may have cdk2-independent functions in these cells besides its role as a cdk2 activator. Indeed, we found indications of a cdk2-independent role of cyclin E in DNA damage–induced apoptosis. 8-CPT-cAMP sensitizes ASCs to γ-irradiation–induced apoptosis, an effect abolished by knockdown of cyclin E. Moreover, cAMP induces early activation of ERK, leading to reduced degradation of cyclin E. The cAMP-mediated up-regulation of cyclin E was blocked by knockdown of ERK or by an inhibitor of the ERK kinase MEK. We conclude that cAMP inhibits cdk2 activity and pRB phosphorylation, leading to reduced ASC proliferation. Concomitant with this growth inhibition, however, cyclin E levels are increased in a MEK/ERK-dependent manner. Our results suggest that cyclin E plays an important, cdk2-independent role in genotoxic stress–induced apoptosis in mesenchymal stem cells.