Influence of Tween-80 on the production and structure of water-insoluble curdlan from Agrobacterium sp.

In order to explore the mechanism by which Tween-80 enhances the production of curdlan produced by Agrobacterium sp., the effects of Tween-80 on the production and structure of curdlan and Agrobacterium sp. were evaluated. Maximum curdlan production (51.94g/L) was achieved when 16g/L Tween-80 was added at the beginning of the cell growth stage. The addition of Tween-80 at higher concentration inhibited cell growth. However, the addition of 16g/L Tween-80 enhanced the production of curdlan with a looser ultrastructure, significantly weakened the envelopment of curdlan on Agrobacterium sp., altered the fine structure of cell membrane, and increased the cell membrane permeability. Moreover, the efficiency of oxygen and mass transport, respiration intensity, UTP regeneration, ATP regeneration, activity of curdlan synthetase, capacity of stress response and energy supply of Agrobacterium sp. were all greatly improved by the addition of Tween-80. These findings demonstrate the mechanisms by which Tween-80 enhances curdlan production and provide a cheap and feasible approach to weaken the envelopment of water-insoluble polysaccharides on bacteria.

Emerging Strategies to Enhance Homing and Engraftment of Hematopoietic Stem Cells.

Successful clinical outcomes from transplantation of hematopoietic stem cells (HSCs) depend upon efficient HSC homing to bone marrow (BM), subsequent engraftment, and, finally, BM repopulation. Homing of intravenously administered HSCs from peripheral blood (PB) through the circulation to the BM stem cell niches, which is the first critical step that precedes their engraftment, is enforced by chemotactic factors released in the BM microenvironment that chemoattract HSCs. These chemotactic factors include α-chemokine stromal-derived factor 1 (SDF-1), the bioactive phosphosphingolipids sphingosine-1-phosphate (S1P) and ceramid-1-phosphate (C1P), and the extracellular nucleotides ATP and UTP. Stem cells may also respond to a Ca(2+) or H(+) gradient by employing calcium- or proton-sensing receptors, respectively. In this review, we will present emerging strategies based on ex vivo manipulation of graft HSCs that are aimed at enhancing the responsiveness of HSCs to BM-secreted chemoattractants and/or promoting HSC adhesion and seeding efficiency in the BM microenvironment.

Differential agonist-induced desensitization of P2Y2 nucleotide receptors by ATP and UTP.

The equal potency and efficacy of the agonists, ATP and UTP, pharmacologically distinguish the P2Y2 receptor from other nucleotide receptors. Investigation of the desensitization of the P2Y2 receptors is complicated by the simultaneous expression of different P2 nucleotide receptor subtypes. The co-expression of multiple P2 receptor subtypes in mammalian cells may have led to contradictory reports on the efficacy of the natural agonists of the P2Y2 receptor to induce desensitization. We decided to investigate the desensitization of human and murine isoforms of the P2Y2 receptor, and to rigorously examine their signaling and desensitization properties. For these purposes, we used 1321N1 astrocytoma cells stably transfected with the human or murine P2Y2 receptor cDNA, as well as human A431 cells that endogenously express the receptor. The mobilization of intracellular calcium by extracellular nucleotides was used as a functional assay for the P2Y2 receptors. While ATP and UTP activated the murine and human P2Y2 receptors with similar potencies (EC50 values were 1.5-5.8 microM), ATP was approximately 10-fold less potent (IC50 = 9.1-21.2 microM) than UTP (IC50 = 0.7-2.9 microM) inducing homologous receptor desensitization in the cell systems examined. Individual cell analyses of the rate and dose dependency of agonist-induced desensitization demonstrated that the murine receptor was slightly more resistant to desensitization than its human counterpart. To our knowledge, this is the first individual cell study that has compared the cellular heterogeneity of the desensitized states of recombinant and endogenously expressed receptors. This comparison demonstrated that the recombinant system conserved the cellular regulatory elements needed to attenuate receptor signaling by desensitization.

Evidence that P2Y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP.

1. Previous studies have shown that ATP and UTP are able to stimulate phospholipase C (PLC) and proliferation in cultured aortic smooth muscle cells. Here we set out to characterize the receptor responsible, and investigate a possible role for p42 and p44 mitogen activated protein kinase (MAPK) in the proliferative response. 2. The phospholipase C response of spontaneously hypertensive rat (SHR) derived aortic smooth muscle cells in culture showed that the response to ATP was partial compared to the response to UTP. 3. Further studies characterized the responses of the SHR derived cells. UTP was the only full agonist with the SHR cells; UDP gave a partial response while ADP, 2-methythio-ATP and alpha,beta-methylene ATP were essentially ineffective. The response to UDP was almost lost in the presence of hexokinase, consistent with this being due to extracellular conversion to UTP. These observations are inconsistent with the response being mediated by either P2Y1 or P2Y6 receptors. 4. When increasing concentrations of ATP were present with a maximally effective concentration of UTP, the size of the response diminished, consistent with UTP and ATP acting at a single population of receptors for which ATP was a partial agonist. This is inconsistent with a response mainly at P2Y2 receptors. 5. 1321N1 cells transfected with human P2Y4 receptors gave a similar agonist response profile, with ATP being partial compared to UTP, loss of response to UDP with hexokinase treatment, and with the response to UTP diminishing in the presence of increasing concentrations of ATP. 6. Use of the reverse transcriptase-polymerase chain reaction confirmed the presence of mRNA encoding P2Y4 receptors in SHR derived vascular smooth muscle cells. Transcripts for P2Y2, P2Y4 and P2Y6 receptors, but not P2Y1 receptors, were detected. 7. Stimulation of SHR derived cells with UTP enhanced the tyrosine phosphorylation of both p42 and p44 MAPK, and the incorporation of [3H]-thymidine into DNA. Both these responses were diminished in the presence of an inhibitor of activation of MAPK. 8 These results lead to the conclusion that in SHR derived cultured aortic smooth muscle cells, PLC responses to extracellular UTP and ATP are predominantly at P2Y4 receptors, and suggest that these receptors are coupled to mitogenesis via p42/p44 MAPK.