Mesenchymal stromal cells are present in the heart and promote growth of adult stem cells in vitro

Background aimsFor many years the human heart has been considered a terminally differentiated organ with no regenerative potential after injury. Recent studies, however, have cast doubt on this long-standing dogma. The objective of this study was to investigate the presence of and characterize mesenchymal stromal cells (MSC) in the adult mouse heart. The impact of MSC on growth and differentiation of adult cardiac stem cells (CSC) was also analyzed.MethodsA combination of lineage-negative/c-kit-negative (Lin^?/c-kit^?) immunoselection with a plastic-adhesion technique was used to isolate cardiac-derived MSC. The differentiation capacity and expression of surface markers were analyzed. To investigate the impact of MSC on growth and differentiation of adult CSC, Green Fluorescent Protein (GFP⁺) adult CSC were co-cultured with GFP^? cardiac-derived MSCResultsMSC were present in the adult mouse heart and they met the criteria established to define mouse MSC. They expressed surface markers and were able to differentiate, in a controlled manner, into multiple lineages. In addition, cardiac-derived MSC promoted the survival and expansion of adult CSC in vitroConclusionsMSC can be isolated from the mouse heart and they promote growth and differentiation of adult CSC. The findings from this study could have a significant beneficial impact on future heart failure treatment. Co-culture and co-implantation of cardiac-derived MSC with adult CSC could provide extensive cardiac regeneration and maintenance of the CSC population after implanted into the heart.     

Mechanistic analysis of a DNA damage-induced, PTEN-dependent size checkpoint in human cells

Following DNA damage, human cells undergo arrests in the G(1) and G(2) phases of the cell cycle and a simultaneous arrest in cell size. We previously demonstrated that the cell size arrest can be uncoupled from the cell cycle arrest by mutational inactivation of the PTEN tumor suppressor gene. Here we show that the cell size checkpoint is inducible by DNA-damaging chemotherapeutic agents as well as by ionizing radiation and is effectively regulated by PTEN but not by its oncogenic counterpart, PIK3CA. Mutational analysis of PTEN and pharmacological inhibition of Akt revealed that modulation of Akt phosphorylation is unnecessary for cell size checkpoint control. To discover putative PTEN regulators and/or effectors involved in size checkpoint control, we employed a novel endogenous epitope tagging (EET) approach, which revealed that endogenous PTEN interacts at the membrane with an actin-remodeling complex that includes actin, gelsolin, and EPLIN. Pharmacological inhibition of actin remodeling in PTEN(+/+) cells recapitulated the lack of size checkpoint control seen in PTEN(-/-) cells. Taken together, these results provide further support for the existence of a DNA damage-inducible size checkpoint that is regulated by a major tumor suppressor, and they provide a novel Akt-independent mechanism by which PTEN controls cell size.     

Synthesis of the transferrin receptor by cultures of embryonic chicken spinal neurons

We have purified a glycoprotein from chicken sciatic nerves, sciatin, which has pronounced trophic effects on avian skeletal muscle cells in culture. Recent studies have shown that sciatin is identical to the iron-transport protein, transferrin, in terms of its physicochemical structure, immunological reactivity, and biological activity. To determine whether transferrin is synthesized and released by neuronal tissue, we incubated cultures of dissociated chicken spinal neurons in a medium free of L-leucine containing either L-3H-amino acids or L-[14C]leucine and immunoprecipitated transferrin with highly specific antibodies. The radiolabeled protein precipitated by rabbit heteroclonal, goat heteroclonal, or mouse monoclonal antitransferrin antibodies increased in specific activity in a linear manner for at least 30 min. Synthesis of this protein was abolished by the presence of puromycin (20 micrograms/ml) or cycloheximide (10(-5) M). The disappearance of the radiolabeled protein from cells was linear with a half-life (t 1/2) of 8-10 h. When immunoprecipitates were separated by SDS gel electrophoresis, a prominent band corresponding to transferrin (Mr 84,000) was visualized by staining with Coomassie Blue. However, when such gels were fluorographed, no radioactivity was apparent in the transferrin region of the gel although a prominent radioactive band was visualized at an Mr of 56,000. The protein of Mr 56,000 was not simply a degradation product of transferrin because this particular protein band was not generated by incubating radiolabeled transferrin with unlabeled neuronal homogenates. The protein of Mr 56,000 was purified from embryonic chicken brain and spinal cord by immunoabsorption chromatography on mouse monoclonal antitransferrin IgG conjugated to Sepharose 4B followed by affinity chromatography on immobilized transferrin. The purified protein bound radioiodinated transferrin and was precipitated by rabbit anti-chicken transferrin-receptor antibodies. Furthermore, this receptor protein was found to be localized on the plasma membrane of dorsal root ganglion neurons by immunocytochemistry using the peroxidase-antiperoxidase technique, and by blocking experiments, which showed that antitransferrin receptor IgG could inhibit the binding of fluorescein-conjugated transferrin at 4 degrees C to cultured neurons in vitro. From these data, we conclude that transferrin is not synthesized by cultures of chicken spinal cord neurons, but that the receptor for transferrin is synthesized by these cultures and is precipitated by antitransferrin antibodies as an antigen-receptor complex.     

Structural properties of periplasmic SodCI that correlate with virulence in Salmonella enterica serovar Typhimurium

Salmonella enterica strains survive and propagate in macrophages by both circumventing and resisting the antibacterial effectors normally delivered to the phagosome. An important aspect of Salmonella resistance is the production of periplasmic superoxide dismutase to combat phagocytic superoxide. S. enterica serovar Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Both enzymes are produced during infection, but only SodCI contributes to virulence in the animal. Although 60% identical to SodCII at the amino acid level with very similar enzymatic properties, SodCI is dimeric, protease resistant, and tethered within the periplasm via a noncovalent interaction. In contrast, SodCII is monomeric and protease sensitive and is released from the periplasm normally by osmotic shock. We have constructed an enzymatically active monomeric SodCI enzyme by site-directed mutagenesis. The resulting protein was released by osmotic shock and sensitive to protease and could not complement the loss of wild-type dimeric SodCI during infection. To distinguish which property is most critical during infection, we cloned and characterized related SodC proteins from a variety of bacteria. Brucella abortus SodC was monomeric and released by osmotic shock but was protease resistant and could complement SodCI in the animal. These data suggest that protease resistance is a critical property that allows SodCI to function in the harsh environment of the phagosome to combat phagocytic superoxide. We propose a model to account for the various properties of SodCI and how they contribute to bacterial survival in the phagosome.     

Expansion of symmetric exon-bordering domains does not explain evolution of lineage specific genes in mammals

In order to examine the evolution of lineage specific genes, we analyzed intron phase distributions and exon-bordering domains in primate and rodent specific genes. We found that the expansion of symmetric exon-bordering domains could not explain the evolution of lineage specific genes. Rather internal intron loss of a domain can partially explain the excess of class 1–1 intron phases in the lineage specific genes. We suggest the event that led to excess of symmetric exons in lineage specific genes had little bearing on shaping the phenotypes specific to the individual lineage. Instead, Kruppel-associated box (KRAB) proteins associated with zinc finger C2H2 (zf-C2H2) type are likely to be responsible for the lineage specific function.     

Gold nanoparticle-based colorimetric detection of kanamycin using a DNA aptamer

A selective kanamycin-binding single-strand DNA (ssDNA) aptamer (TGGGGGTTGAGGCTAAGCCGA) was discovered through in vitro selection using affinity chromatography with kanamycin-immobilized sepharose beads. The selected aptamer has a high affinity for kanamycin and also for kanamycin derivatives such as kanamycin B and tobramycin. The dissociation constants (K_d [kanamycin] = 78.8 nM, K_d [kanamycin B] = 84.5 nM, and K_d [tobramycin] = 103 nM) of the new aptamer were determined by fluorescence intensity analysis using 5′-fluorescein amidite (FAM) modification. Using this aptamer, kanamycin was detected down to 25 nM by the gold nanoparticle-based colorimetric method. Because the designed colorimetric method is simple, easy, and visible to the naked eye, it has advantages that make it useful for the detection of kanamycin. Furthermore, the selected new aptamer has many potential applications as a bioprobe for the detection of kanamycin, kanamycin B, and tobramycin in pharmaceutical preparations and food products.     

The complex interactions between host immunity and non-biotrophic fungal pathogens of wheat leaves

Significant progress has been made in elucidating the mechanisms used by plants to recognize pathogens and activate “immune” responses. A “first line” of defense can be triggered through recognition of conserved Pathogen or Microbe Associated Molecular Patterns (PAMPs or MAMPs), resulting in activation of basal (or non-host) plant defenses, referred to as PAMP-triggered immunity (PTI). Disease resistance responses can also subsequently be triggered via gene-for-gene type interactions between pathogen avirulence effector genes and plant disease resistance genes (Avr-R), giving rise to effector triggered immunity (ETI). The majority of the conceptual advances in understanding these systems have been made using model systems, such as Arabidopsis, tobacco, or tomato in combination with biotrophic pathogens that colonize living plant tissues. In contrast, how these disease resistance mechanisms interact with non-biotrophic (hemibiotrophic or necrotrophic) fungal pathogens that thrive on dying host tissue during successful infection, is less clear. Several lines of recent evidence have begun to suggest that these organisms may actually exploit components of plant immunity in order to infect, successfully colonize and reproduce within host tissues. One underlying mechanism for this strategy has been proposed, which has been referred to as effector triggered susceptibility (ETS). This review aims to highlight the complexity of interactions between plant recognition and defense activation towards non-biotrophic pathogens, with particular emphasis on three important fungal diseases of wheat (Triticum aestivum) leaves.     

Obesity-associated mouse adipose stem cell secretion of monocyte chemotactic protein-1

Studies showed that monocyte chemotactic protein-1 (MCP-1) concentrations are increased in obesity. In our current study, we demonstrate that plasma MCP-1 level in leptin-deficient ob/ob mice is significantly higher than in lean mice. Furthermore, we determined that basal adipose tissue MCP-1 mRNA levels are significantly higher in ob/ob mice compared with lean mice. To determine the mechanisms underlying obesity-associated increases in plasma and adipose tissue MCP-1 levels, we determined adipose tissue cell type sources of MCP-1 production. Our data show that adipose tissue stem cells (CD34(+)), macrophages (F4/80(+)), and stromal vascular fraction (SVF) cells express significantly higher levels of MCP-1 compared with adipocytes under both basal and lipopolysaccharide (LPS)-stimulated conditions. Furthermore, basal and LPS-induced MCP-1 secretion levels were the same for both adipose F4/80(+) and CD34(+) cells, whereas adipose CD34(+) cells have twofold higher cell numbers (30% of total SVF cells) compared with F4/80(+) macrophages (15%). Our data also show that CD34(+) cells from visceral adipose tissue depots secrete significantly higher levels of MCP-1 ex vivo when compared with CD34(+) cells from subcutaneous adipose tissue depots. Taken together, our data suggest that adipose CD34(+) stem cells may play an important role in obesity-associated increases in plasma MCP-1 levels.