12 research outputs found
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cGMP modulates stem cells differentiation to neurons in brain in vivo pathological implications
Mechanism and Kinetics of Inducible Nitric Oxide Synthase Auto-<i>S</i>-nitrosation and Inactivation
Nitric oxide (NO), the product of the nitric oxide synthase
(NOS) reaction, was previously shown to result in <i>S</i>-nitrosation of the NOS Zn<sup>2+</sup>-tetrathiolate and inactivation
of the enzyme. To probe the potential physiological significance of
NOS <i>S</i>-nitrosation, we determined the inactivation
time
scale of the inducible NOS isoform (iNOS) and found it directly correlates
with an increase in the level of iNOS <i>S</i>-nitrosation.
A kinetic model of NOS inactivation in which arginine is treated as
a suicide substrate was developed. In this model, NO synthesized at
the heme cofactor is partitioned between release into solution (NO
release pathway) and NOS <i>S-</i>nitrosation followed by
NOS inactivation (inactivation pathway). Experimentally determined
progress curves of NO formation were fit to the model. The NO release
pathway was perturbed through addition of the NO traps oxymyoglobin
(MbO<sub>2</sub>) and Ī²2 H-NOX, which yielded partition ratios
between NO release and inactivation of ā¼100 at 4 Ī¼M MbO<sub>2</sub> and ā¼22000 at saturating trap concentrations. The
results suggest that a portion of the NO synthesized at the heme cofactor
reacts with the Zn<sup>2+</sup>-tetrathiolate without being released
into solution. Perturbation of the inactivation pathway through addition
of the reducing agent GSH or TCEP resulted in a concentration-dependent
decrease in the level of iNOS <i>S</i>-nitrosation that
directly correlated with protection from iNOS inactivation. iNOS inactivation
was most responsive to physiological concentrations of GSH with an
apparent <i>K</i><sub>m</sub> value of 13 mM. NOS turnover
that leads to NOS <i>S</i>-nitrosation might be a mechanism
for controlling NOS activity, and NOS <i>S-</i>nitrosation
could play a role in the physiological generation of nitrosothiols
Upregulation of CD11A on hematopoietic stem cells denotes the loss of long-term reconstitution potential.
Small numbers of hematopoietic stem cells (HSCs) generate large numbers of mature effector cells through the successive amplification of transiently proliferating progenitor cells. HSCs and their downstream progenitors have been extensively characterized based on their cell-surface phenotype and functional activities during transplantation assays. These cells dynamically lose and acquire specific sets of surface markers during differentiation, leading to the identification of markers that allow for more refined separation of HSCs from early hematopoietic progenitors. Here, we describe a marker, CD11A, which allows for the enhanced purification of mouse HSCs. We show through in vivo transplantations that upregulation of CD11A on HSCs denotes the loss of their long-term reconstitution potential. Surprisingly, nearly half of phenotypic HSCs (defined as Lin-KIT(+)SCA-1(+)CD150(+)CD34-) are CD11A(+) and lack long-term self-renewal potential. We propose that CD11A(+)Lin-KIT(+)SCA-1(+)CD150(+)CD34- cells are multipotent progenitors and CD11A-Lin-KIT(+)SCA-1(+)CD150(+)CD34- cells are true HSCs
Upregulation of CD11A on Hematopoietic Stem Cells Denotes the Loss of Long-Term Reconstitution Potential
Small numbers of hematopoietic stem cells (HSCs) generate large numbers of mature effector cells through the successive amplification of transiently proliferating progenitor cells. HSCs and their downstream progenitors have been extensively characterized based on their cell-surface phenotype and functional activities during transplantation assays. These cells dynamically lose and acquire specific sets of surface markers during differentiation, leading to the identification of markers that allow for more refined separation of HSCs from early hematopoietic progenitors. Here, we describe a marker, CD11A, which allows for the enhanced purification of mouse HSCs. We show through inĀ vivo transplantations that upregulation of CD11A on HSCs denotes the loss of their long-term reconstitution potential. Surprisingly, nearly half of phenotypic HSCs (defined as LināKIT+SCA-1+CD150+CD34ā) are CD11A+ and lack long-term self-renewal potential. We propose that CD11A+LināKIT+SCA-1+CD150+CD34ā cells are multipotent progenitors and CD11AāLināKIT+SCA-1+CD150+CD34ā cells are true HSCs
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Upregulation of CD11A on hematopoietic stem cells denotes the loss of long-term reconstitution potential.
Small numbers of hematopoietic stem cells (HSCs) generate large numbers of mature effector cells through the successive amplification of transiently proliferating progenitor cells. HSCs and their downstream progenitors have been extensively characterized based on their cell-surface phenotype and functional activities during transplantation assays. These cells dynamically lose and acquire specific sets of surface markers during differentiation, leading to the identification of markers that allow for more refined separation of HSCs from early hematopoietic progenitors. Here, we describe a marker, CD11A, which allows for the enhanced purification of mouse HSCs. We show through in vivo transplantations that upregulation of CD11A on HSCs denotes the loss of their long-term reconstitution potential. Surprisingly, nearly half of phenotypic HSCs (defined as Lin-KIT(+)SCA-1(+)CD150(+)CD34-) are CD11A(+) and lack long-term self-renewal potential. We propose that CD11A(+)Lin-KIT(+)SCA-1(+)CD150(+)CD34- cells are multipotent progenitors and CD11A-Lin-KIT(+)SCA-1(+)CD150(+)CD34- cells are true HSCs
New tools for studying microglia in the mouse and human CNS
The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease