Holmfeldt et al. perform a transplant-based screen to identify regulators of HSPC engraftment and report that Foxa3 is critical for optimal HSC function after transplant.

Sullivan JC, Pardieck JL, Hyndman KA, Pollock JS. Renal NOS activity, expression, and localization in male and female spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 298: R61-R69, 2010. First published November 4, 2009 doi:10.1152/ajpregu.00526.2009.-The goal of this study was to examine the status of the renal nitric oxide (NO) system by determining NO synthase (NOS) isoform activity and expression within the three regions of the kidney in 14-wk-old male and female spontaneously hypertensive rats (SHR). NOS activity, and NOS1 and NOS3 protein expressions and localization were comparable in the renal cortex and outer medulla of male and female SHR. In contrast, male SHR had significantly less NOS1 and NOS3 enzymatic activity (0 Ϯ 5 and 53 Ϯ 7 pmol ⅐ mg Ϫ1 ⅐ 30 min Ϫ1 , respectively) compared with female SHR (37 Ϯ 16 and 172 Ϯ 40 pmol ⅐ mg Ϫ1 ⅐ 30 min Ϫ1 , respectively). Lower levels of inner medullary NOS1 activity in male SHR were associated with less NOS1 protein expression [45 Ϯ 7 relative densitometric units (RDU)] and fewer NOS1-positive cells in the renal inner medulla compared with female SHR (79 Ϯ 12 RDU). Phosphorylation of NOS3 is an important determinant of NOS activity. Male SHR had significantly greater phosphorylation of NOS3 on threonine 495 in the renal cortex compared with females (0.25 Ϯ 0.05 vs. 0.15 Ϯ 0.06 RDU). NOS3 phosphorylation was comparable in males and females in the other regions of the kidney. cGMP levels were measured as an indirect index of NO production. cGMP levels were significantly lower in the renal cortex (0.08 Ϯ 0.01 pmol/mg) and inner medulla (0.43 Ϯ 0.02 pmol/mg) of male SHR compared with females (cortex: 0.14 Ϯ 0.02 pmol/mg; inner medulla: 0.56 Ϯ 0.02 pmol/mg). Our data suggest that the effect of the sex of the animal on NOS activity and expression is different in the three regions of the SHR kidney and supports the hypothesis that male SHR have lower NO bioavailability compared with females. nitric oxide synthase; kidney; sex; gender; cGMP NITRIC OXIDE (NO) is an important regulator of blood pressure and kidney function (3,15,43). Therefore, it is not surprising that NO deficiencies have been linked with hypertension and the progression of chronic renal disease in both patients and experimental animals (2, 3, 45). All three NO synthase (NOS) isoforms have been localized to the kidney (1), and intrarenal inhibition of NOS has been shown to increase blood pressure (32). NOS1 (neuronal NOS) is predominantly localized in the macula densa, neurons, Bowman's capsule, and collecting duct, where it participates in the control of glomerular hemodynamics, renin release, and sodium excretion (1,24,46). Expression of NOS2 (inducible NOS) protein has been difficult to reproducibly detect in the normal kidney; however, there appears to be NOS2 mRNA in the medullary thick ascending limb (34). NOS3 (endothelial NOS) is localized in vascular endothelial cells and tubules, where it is important in the maintenance of glomerular filtration rate, vascular tone, and...

Background Dysregulation of voltage-gated cardiac Na+ channels (NaV1.5) by inherited mutations, disease-linked remodeling, and drugs causes arrhythmias. The molecular mechanisms whereby the NaV1.5 voltage-sensing domains (VSDs) are perturbed to pathologically or therapeutically modulate Na+ current (INa) have not been specified. Our aim was to correlate INa kinetics with conformational changes within the four (DI-DIV) VSDs to define molecular mechanisms of NaV1.5 modulation. Method and Results Four NaV1.5 constructs were created to track the voltage-dependent kinetics of conformational changes within each VSD, using voltage-clamp fluorometry (VCF). Each VSD displayed unique kinetics, consistent with distinct roles in determining INa. In particular, DIII-VSD deactivation kinetics were modulated by depolarizing pulses with durations in the intermediate time domain that modulates late INa. We then used the DII-VSD construct to probe the molecular pathology of two Brugada Syndrome (BrS) mutations (A735V and G752R). A735V shifted DII-VSD voltage-dependence to depolarized potentials, while G752R significantly slowed DII-VSD kinetics. Both mutations slowed INa activation, even though DII-VSD activation occurred at higher potentials (A735V) or at later times (G752R) than ionic current activation, indicating that the DII-VSD allosterically regulates the rate ofINa activation and myocyte excitability. Conclusions Our results reveal novel mechanisms whereby the NaV1.5 VSDs regulate its activation and inactivation. The ability to distinguish distinct molecular mechanisms of proximal BrS mutations demonstrates the potential of these methods to reveal how inherited mutations, post-translational modifications and anti-arrhythmic drugs alter NaV1.5 at the molecular level.

Key Points HSPCs fail to persist in the bone marrow of lethally irradiated recipients in the absence of Nfix. Nfix-deficient HSPCs display increased apoptosis during ex vivo culture and in recipient marrow.