Involvement of Coenzyme M during Aerobic Biodegradation of Vinyl Chloride and Ethene by Pseudomonas putida Strain AJ and Ochrobactrum sp. Strain TD

The involvement of coenzyme M in aerobic biodegradation of vinyl chloride and ethene in Pseudomonas putida strain AJ and Ochrobactrum sp. strain TD was demonstrated using PCR, hybridization, and enzyme assays. The results of this study extend the range of eubacteria known to use epoxyalkane:coenzyme M transferase.     

Direct semi-synthesis of the anticancer lead-drug protoapigenone from apigenin, and synthesis of further new cytotoxic protoflavone derivatives

Protoapigenone, a natural flavonoid possessing an unusual p-quinol moiety on its B-ring, is a novel prospective anticancer agent with low toxicity that is currently in development. The first economical, one-step synthesis of protoapigenone from apigenin is described on up to gram scale. 13 new 1'-O-alkylflavone analogs were also synthesized, either from apigenin or β-naphthoflavone. The in vitro cytotoxic activity of each compound was tested on six human cancer cell lines (HepG2, Hep3B, Ca9-22, A549, MCF-7 and MDA-MB-231). In the case of 1'-O-alkyl-protoapigenone derivatives, structure-activity relationships were found depending on the side-chain, and protoapigenone 1'-O-butyl ether was found to exert significantly stronger activity against three of the cell lines (Hep3B, MCF-7 and MDA-MB-231) than its non-substituted analog, protoapigenone itself. In contrast to this, all β-naphthoflavone derivatives bearing the same pharmacophore on their B-ring showed decreased cytotoxic activities when substituted with an O-alkyl side-chain at position 1', comparing to that of the non-substituted compound.     

Molecular chaperone, HSP60, and cytochrome P450 2E1 co-expression in dilated cardiomyopathy

Physiological stresses (heat, hemodynamics, genetic mutations, oxidative injury and myocardial ischemia) produce pathological states in which protein damage and misfolded protein structures are a common denominator. The specialized proteins family of antistress proteins - molecular chaperons (HSPs) - are responsible for correct protein folding, dissociating protein aggregates and transport of newly synthesized polypeptides to the target organelles for final packaging, degradation or repair. They are inducible at different cell processes such as cell division, apoptosis, signal transduction, cell differentiation and hormonal stimulation. HSPs are involved in numerous diseases including cardiovascular pathologies, revealing changes of expression and cell localization. We studied the possible changes in expression level of abundant mitochondrial chaperon Hsp60 and main human cytochrome P450 monooxygenase (2E1 isoform) at dilated cardiomyopathy (DCM) progression at the end stage of heart failure using Western blot analysis. The ischemic and normal humans' hearts were studied as control samples. We observed the decrease of Hsp60 level in cytoplasmic fraction of DCM- and ischemia-affected hearts' left ventricular and significant increase of Hsp60 in mitochondrial fractions of all hearts investigated. At the same time we detected the increase of P450 2E1 expression level in ischemic and dilated hearts' cytoplasmic fractions in comparison with normal myocardium and no detectable changes in microsomal fractions of hearts investigated which could be linked with increased level of oxidative injury for DCM heart muscle. In addition, all the changes described are accompanied by significant decrease of ATPase activity of myosin purified from DCM-affected heart in comparison with normal and ischemic myocardia as well. The data obtained allow us to propose a working hypothesis of functional link between antistress (HSPs) and antioxidative (cytochromes) systems at DCM progression.     

Canine distemper virus and measles virus fusion glycoprotein trimers: partial membrane-proximal ectodomain cleavage enhances function

The trimeric fusion (F) glycoproteins of morbilliviruses are activated by furin cleavage of the precursor F(0) into the F(1) and F(2) subunits. Here we show that an additional membrane-proximal cleavage occurs and modulates F protein function. We initially observed that the ectodomain of approximately one in three measles virus (MV) F proteins is cleaved proximal to the membrane. Processing occurs after cleavage activation of the precursor F(0) into the F(1) and F(2) subunits, producing F(1a) and F(1b) fragments that are incorporated in viral particles. We also detected the F(1b) fragment, including the transmembrane domain and cytoplasmic tail, in cells expressing the canine distemper virus (CDV) or mumps virus F protein. Six membrane-proximal amino acids are necessary for efficient CDV F(1a/b) cleavage. These six amino acids can be exchanged with the corresponding MV F protein residues of different sequence without compromising function. Thus, structural elements of different sequence are functionally exchangeable. Finally, we showed that the alteration of a block of membrane-proximal amino acids results in diminished fusion activity in the context of a recombinant CDV. We envisage that selective loss of the membrane anchor in the external subunits of circularly arranged F protein trimers may disengage them from pulling the membrane centrifugally, thereby facilitating fusion pore formation.     

Midbrain-derived neural stem cells: from basic science to therapeutic approaches

Neural stem cells (NSCs) are a subtype of tissue-specific progenitor cells capable of extended self-renewal and the ability to generate all major cell types of nervous tissue, such as neurons, astroglia and oligodendroglial cells. Recent studies suggest that salient patterning in anterior–posterior and dorsal–ventral axes occurs early, concomitantly with neural induction and therefore stem cells and restricted precursors exhibit regionalization. Fetal mesencephalic NSCs can be isolated and expanded in vitro for many months while retaining their potential to differentiate into glia and neurons, with a subset of neurons displaying all the major properties of mature functional dopaminergic neurons. Since Parkinsons disease (PD) is characterized by the loss of a specific type of dopaminergic cells, the prospect of replacing the missing or damaged cells is very attractive in PD. Thus, mesencephalic NSCs might serve as a new and continuous source of dopaminergic neurons for regenerative strategies in this neurodegenerative disorder. This review discusses new data concerning the cell biology and therapeutic potential of NSCs derived from the midbrain region of the central nervous system.The work of the authors was supported in part by the Interdisziplinäres Zentrum für klinische Forschung (IZKF) Ulm (Project D6) to A.S., the BMBF (Polish-German Cooperation in Neuroscience Program) to A.S., the Ministerium für Wissenschaft und Kultur Baden-Württemberg (Landesforschungsschwerpunkt Neurodegeneration und Neuroregeneration) to A.S., and the Landesstiftung Baden-Württemberg (Förderprogramm Adulte Stammzellen) to A.S. M.S. was supported by a fellowship from the IZKF Ulm     

A global metagenomic map of urban microbiomes and antimicrobial resistance

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Low extracellular calcium is sufficient for survival and proliferation of murine mesencephalic neural precursor cells

Various media and Ca²⁺ concentrations are employed to culture neural progenitor cells (NPCs). We have therefore explored the effects of extracellular calcium concentrations on the survival, proliferation, spontaneous apoptosis and self-renewal capacity of mesencephalic NPCs grown adherently and as free-floating neurospheres. We employed EMEM supplemented with various concentrations of extracellular CaCl₂ (0.1–1 mM). Raising the calcium concentration from 0.1 mM to 0.6 mM resulted in an increased number of NPCs growing as a monolayer and increased the protein yield of cells growing in neurospheres (24±3 μg total proteins in 0.1 mM Ca²⁺ medium vs. 316±34 μg proteins in 1 mM Ca²⁺ medium). Concentrations more than 0.6 mM did not result in a further improvement of proliferation or survival. Elimination of calcium from our control medium by 1 mM EGTA resulted in a decrease in cell number from 82±2×10⁴ NPCs/ml observed in control medium to 62±2×10⁴ NPCs/ml observed in calcium-free media. Protein yield dropped significantly in calcium-free media, accompanied by the decreased expression of the proliferation marker PCNA and the pro-survival marker Bcl-2. Two weeks of expansion as neurospheres caused spontaneous cell death in more than 90% of NPCs grown in 0.1 mM CaCl₂ EMEM compared with 42% in 1 mM CaCl₂ EMEM. Although the action of Ca²⁺ on NPCs appears to be complex, the presented data strongly suggest that extracellular calcium plays a crucial role in the maintenance of NPCs in a healthy and proliferating state; physiological concentrations (>1.0 mM) are not required, a concentration of 0.5 mM being adequate for cell maintenance.