Albumin and mammalian cell culture: implications for biotechnology applications

Albumin has a long historical involvement in design of media for the successful culture of mammalian cells, in both the research and commercial fields. The potential application of albumins, bovine or human serum albumin, for cell culture is a by-product of the physico-chemical, biochemical and cell-specific properties of the molecule. In this review an analysis of these features of albumin leads to a consideration of the extracellular and intracellular actions of the molecule, and importantly the role of its interactions with numerous ligands or bioactive factors that influence the growth of cells in culture: these include hormones, growth factors, lipids, amino acids, metal ions, reactive oxygen and nitrogen species to name a few. The interaction of albumin with the cell in relation to these co-factors has a potential impact on metabolic and biosynthetic activity, cell proliferation and survival. Application of this knowledge to improve the performance in manufacturing biotechnology and in the emerging uses of cell culture for tissue engineering and stem cell derived therapies is an important prospect

Comparative genomics of Pseudomonas fluorescens subclade III strains from human lungs

Abstract Background While the taxonomy and genomics of environmental strains from the P. fluorescens species-complex has been reported, little is known about P. fluorescens strains from clinical samples. In this report, we provide the first genomic analysis of P. fluorescens strains in which human vs. environmental isolates are compared. Results Seven P. fluorescens strains were isolated from respiratory samples from cystic fibrosis (CF) patients. The clinical strains could grow at a higher temperature (>34 °C) than has been reported for environmental strains. Draft genomes were generated for all of the clinical strains, and multi-locus sequence analysis placed them within subclade III of the P. fluorescens species-complex. All strains encoded type- II, −III, −IV, and -VI secretion systems, as well as the widespread colonization island (WCI). This is the first description of a WCI in P. fluorescens strains. All strains also encoded a complete I2/PfiT locus and showed evidence of horizontal gene transfer. The clinical strains were found to differ from the environmental strains in the number of genes involved in metal resistance, which may be a possible adaptation to chronic antibiotic exposure in the CF lung. Conclusions This is the largest comparative genomics analysis of P. fluorescens subclade III strains to date and includes the first clinical isolates. At a global level, the clinical P. fluorescens subclade III strains were largely indistinguishable from environmental P. fluorescens subclade III strains, supporting the idea that identifying strains as ‘environmental’ vs ‘clinical’ is not a phenotypic trait. Rather, strains within P. fluorescens subclade III will colonize and persist in any niche that provides the requirements necessary for growth.http://deepblue.lib.umich.edu/bitstream/2027.42/116129/1/12864_2015_Article_2261.pd

Zinc homeostasis and signaling in health and diseases: Zinc signaling

The essential trace element zinc (Zn) is widely required in cellular functions, and abnormal Zn homeostasis causes a variety of health problems that include growth retardation, immunodeficiency, hypogonadism, and neuronal and sensory dysfunctions. Zn homeostasis is regulated through Zn transporters, permeable channels, and metallothioneins. Recent studies highlight Zn’s dynamic activity and its role as a signaling mediator. Zn acts as an intracellular signaling molecule, capable of communicating between cells, converting extracellular stimuli to intracellular signals, and controlling intracellular events. We have proposed that intracellular Zn signaling falls into two classes, early and late Zn signaling. This review addresses recent findings regarding Zn signaling and its role in physiological processes and pathogenesis

Ion homeostasis in the Chloroplast

peer reviewedThe chloroplast is an organelle of high demand for macro- and micro-nutrient ions, which are required for the maintenance of the photosynthetic process. To avoid deficiency while preventing excess, homeostasis mechanisms must be tightly regulated. Here, we describe the needs for nutrient ions in the chloroplast and briefly highlight their functions in the chloroplastidial metabolism. We further discuss the impact of nutrient deficiency on chloroplasts and the acclimation mechanisms that evolved to preserve the photosynthetic apparatus. We finally present what is known about import and export mechanisms for these ions. Whenever possible, a comparison between cyanobacteria, algae and plants is provided to add an evolutionary perspective to the description of ion homeostasis mechanisms in photosynthesis

Comparative modelling of human PHOSPHO1 reveals a new group of phosphatases within the haloacid dehalogenase superfamily

PHOSPHO1 is a recently identified phosphatase whose expression is upregulated in mineralizing cells and is implicated in the generation of inorganic phosphate for matrix mineralization, a process central to skeletal development. The enzyme is a member of the haloacid dehalogenase (HAD) superfamily of magnesium-dependent hydrolases. However, the natural substrate(s) is as yet unidentified and to date no structural information is known. We have identified homologous proteins in a number of species and have modelled human PHOSPHO1 based upon the crystal structure of phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The model includes the catalytic Mg2+ atom bound via three conserved Asp residues (Asp32, Asp34 and Asp203); O-ligands are also provided by a phosphate anion and two water molecules. Additional residues involved in PSP-catalysed hydrolysis are conserved and are located nearby, suggesting both enzymes share a similar reaction mechanism. In PHOSPHO1, none of the PSP residues that confer the enzyme’s substrate specificity (Arg56, Glu20, Met43 and Phe49) are conserved. Instead, we propose that two fully conserved Asp residues (Asp43 and Asp123), not present in PSPs contribute to substrate specificity in PHOSPHO1. Our findings show that PHOSPHO1 is not a member of the subfamily of PSPs but belongs to a novel, closely related enzyme group within the HAD superfamily

Quantification of isomeric equilibria formed by metal ion complexes of 8-[2-(phosphonomethoxy)ethyl]-8-azaadenine (8,8aPMEA) and 9-[2-(phosphonomethoxy)ethyl]-8-azaadenine (9,8aPMEA) : derivatives of the antiviral nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA)

The acidity constants of the two-fold protonated acyclic 9-[2-(phosphonomethoxy)ethyl]-8-azaadenine, H2(9,8aPMEA)(+)(-), and its 8-isomer, 8-[2-(phosphonomethoxy)ethyl]-8-azaadenine, H2(8,8aPMEA)(+)(-), both abbreviated as H2(PA)(+)(-), as well as the stability constants of their M(H;PA)+ and M(PA) complexes with the metal ions M2+=Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+, have been determined by potentiometric pH titrations in aqueous solution at I=0.1 M (NaNO3) and 25 degrees C. Application of previously determined straight-line plots of log K(M)M(R-PO3) versus pK(H)H(R-PO3)for simple phosph(on)ate ligands, R-PO3(2-), where R represents a residue without an affinity for metal ions, proves that for all M(PA) complexes a larger stability is observed than is expected for a sole phosphonate coordination of the metal ion. This increased stability is attributed to the formation of five-membered chelates involving the ether oxygen present in the aliphatic residue (-CH2-O-CH2-PO3(2-)) of the ligands. The formation degrees of these chelates were calculated; they vary between about 13% for Ca(8,8aPMEA) and 71% for Cu(8,8aPMEA). The adenine residue has no influence on complex stability except in the Cu(9,8aPMEA) and Zn(9,8aPMEA) systems, where an additional stability increase attributable to the adenine residue is observed and equilibria between four different isomers exist. This means (1) an open isomer with a sole phosphonate coordination, M(PA)op, where PA(2-)=9,8aPMEA2-, (2) an isomer with a five-membered chelate involving the ether oxygen, M(PA)cl/O, (3) an isomer which contains five- and seven-membered chelates formed by coordination of the phosphonate group, the ether oxygen and the N3 site of the adenine residue, M(PA)cl/O/N3, and finally (4) a macrochelated isomer involving N7, M(PA)cl/N7. For Cu(9,8aPMEA) the formation degrees are 15, 30, 48 and 7% for Cu(PA)op, Cu(PA)cl/O, Cu(PA)cl/O/N3 and Cu(PA)cl/N7, respectively; this proves that the macrochelate involving N7 is a minority species. The situation for the Cu(PMEA) system, where PMEA2- represents the parent compound, i.e. the dianion of 9-[2-(phosphonomethoxy)ethyl]adenine, is quite similar. The relationship between the antiviral activity of acyclic nucleoside phosphonates and the structures of the various complexes is discussed and an explanation is offered why 9,8aPMEA is biologically active but 8,8aPMEA is not