Role of Tyrosine-103 in Myoglobin Peroxidase Activity:  Kinetic and Steady-State Studies on the Reaction of Wild-Type and Variant Recombinant Human Myoglobins with H2O2†

Biochemistry ◽  
2002 ◽  
Vol 41 (38) ◽  
pp. 11495-11503 ◽  
Author(s):  
Paul K. Witting ◽  
A. Grant Mauk ◽  
Peter A. Lay
Keyword(s):  
Steady State ◽  
Peroxidase Activity ◽  
Wild Type

scholarly journals The critical role of T cells in glucocorticoid-induced osteoporosis

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Lin Song ◽  
Lijuan Cao ◽  
Rui Liu ◽  
Hui Ma ◽  
Yanan Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Steady State ◽  
Side Effects ◽  
Critical Role ◽  
Wild Type ◽  
Receptor Activator ◽  
Steady State Levels ◽  
Induced Apoptosis

AbstractGlucocorticoids (GC) are widely used clinically, despite the presence of significant side effects, including glucocorticoid-induced osteoporosis (GIOP). While GC are believed to act directly on osteoblasts and osteoclasts to promote osteoporosis, the detailed underlying molecular mechanism of GC-induced osteoporosis is still not fully elucidated. Here, we show that lymphocytes play a pivotal role in regulating GC-induced osteoporosis. We show that GIOP could not be induced in SCID mice that lack T cells, but it could be re-established by adoptive transfer of splenic T cells from wild-type mice. As expected, T cells in the periphery are greatly reduced by GC; instead, they accumulate in the bone marrow where they are protected from GC-induced apoptosis. These bone marrow T cells in GC-treated mice express high steady-state levels of NF-κB receptor activator ligand (RANKL), which promotes the formation and maturation of osteoclasts and induces osteoporosis. Taken together, these findings reveal a critical role for T cells in GIOP.

scholarly journals Role of CheB and CheR in the Complex Chemotactic and Aerotactic Pathway of Azospirillum brasilense

2006 ◽  
Vol 188 (13) ◽  
pp. 4759-4768 ◽  
Author(s):  
Bonnie B. Stephens ◽  
Star N. Loar ◽  
Gladys Alexandre
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Azospirillum Brasilense ◽  
Model Organism ◽  
Spatial Gradient ◽  
Wild Type ◽  
Temporal Gradient ◽  
E Coli ◽  
Significant Difference

ABSTRACT It has previously been reported that the alpha-proteobacterium Azospirillum brasilense undergoes methylation-independent chemotaxis; however, a recent study revealed cheB and cheR genes in this organism. We have constructed cheB, cheR, and cheBR mutants of A. brasilense and determined that the CheB and CheR proteins under study significantly influence chemotaxis and aerotaxis but are not essential for these behaviors to occur. First, we found that although cells lacking CheB, CheR, or both were no longer capable of responding to the addition of most chemoattractants in a temporal gradient assay, they did show a chemotactic response (albeit reduced) in a spatial gradient assay. Second, in comparison to the wild type, cheB and cheR mutants under steady-state conditions exhibited an altered swimming bias, whereas the cheBR mutant and the che operon mutant did not. Third, cheB and cheR mutants were null for aerotaxis, whereas the cheBR mutant showed reduced aerotaxis. In contrast to the swimming bias for the model organism Escherichia coli, the swimming bias in A. brasilense cells was dependent on the carbon source present and cells released methanol upon addition of some attractants and upon removal of other attractants. In comparison to the wild type, the cheB, cheR, and cheBR mutants showed various altered patterns of methanol release upon exposure to attractants. This study reveals a significant difference between the chemotaxis adaptation system of A. brasilense and that of the model organism E. coli and suggests that multiple chemotaxis systems are present and contribute to chemotaxis and aerotaxis in A. brasilense.

Protective Role of Connexin 32 in Experimental Leukemogenesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2239-2239
Author(s):  
Yoko Hirabayashi ◽  
Byung-Il Yoon ◽  
Isao Tsuboi ◽  
Yan Huo ◽  
Yukio Kodama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Bone Marrow Cells ◽  
Pcr Analysis ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Immunomagnetic Bead ◽  
Competitive Assay ◽  
Marrow Cells

Abstract Connexin (Cx) functions in the organization of cell-cell communication in multicellular organisms. Gap junctions have been implicated in the homeostatic regulation of various cellular functions, including growth control and differentiation, apoptosis, and the synchronization of electrotonic and metabolic functions. Primitive hemopoietic progenitor cells form a multicellular system, but a previous report describes that Cx32 is not expressed in the bone marrow. Thus, a question arises as to why Cx molecules are not detected in the hematopoietic tissue other than stromal cells. Based on our preliminary study that suggested a potential impairment of hematopoiesis in Cx32-knockout (KO) mice, the objectives of the present study were to determine whether Cx32 functions in the bone marrow during steady-state hematopoiesis and further to examine its possible protective roles during regeneration after chemical abrasions and during leukemogenesis after the administration of a genotoxic chemical, methyl nitrosourea (MNU). As results, the Cx32 molecule functioning in the hematopoietic stem cell (HSC) compartment during steady-state hematopoiesis was observed for the first time; the expression of Cx32 at the mRNA level determined by PCR analysis and that at the protein level determined using an anti-Cx32 antibody were observed only in the lin−c-kit+ HSC fraction using a combination of immunobead-density gradient and immunomagnetic-bead separation. Hematopoiesis was impaired in the absence of Cx32; it was delayed during regeneration after chemical abrasion with 5-fluorouracil at 150 mg/kg body weight in Cx32-KO mice. Cx32-KO mice also showed increased leukemogenicity compared with wild-type mice after MNU injection; furthermore, in a competitive assay for leukemogenicity in mice that had been lethally irradiated and repopulated with a mixed population of equal amount of bone marrow cells from Cx32-KO mice and wild-type mice, the resulting leukemias were originated predominantly from Cx32-KO bone marrow cells. The present competitive assay clearly showed that Cx32-KO bone marrow cells have a higher risk of becoming leukemogenic. The above-mentioned findings in this study imply that Cxs play an essential role in maintaining the steady-state hematopoiesis and suppressing the neoplastic change. In summary, the role of Cx32 in hematopoiesis was not previously recognized and Cx32 was expressed only in HSCs and their progenitors. The results indicate that Cx32 in wild-type mice protects HSCs from chemical abrasion and leukemogenic impacts. Our results indicate that the risk of developing leukemia in patients with X-chromosome-linked Cx32 deficiency, called Charcot-Marie-Tooth syndrome, may not be incidental.

Mir-199b, a Candidate Effector of Stress Myelopoiesis and Is Differentially Regulated in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1541-1541
Author(s):  
Aldona Karaczyn ◽  
Rose McGlauflin ◽  
Liam Dow ◽  
Amanda Lessard ◽  
Pradeep Sathyanarayana
Keyword(s):  
Gene Expression ◽  
Steady State ◽  
Knockout Mice ◽  
Functional Role ◽  
Leukemic Cells ◽  
Upstream Region ◽  
Data Sets ◽  
H3k9 Methylation ◽  
Wild Type

Abstract Recently, we reported the functional role of miR-199b in AML and its prognostic implications where low miR-199b in AML patients correlated with worse overall survival for the FAB-M5 subtype. Herein, we have attempted to define the role of miR-199b in stress myelopoiesis using knockout mice and examine miR-199b's gene regulation. To define the role of miR-199b and understand its functional role in myeloid proliferation and AML, we generated miR-199b knockout mice via CRISPR approach. The deleted region was confirmed by DNA sequencing and silencing of miR-199b transcripts in nucleated peripheral blood cells was verified via RT-PCR. At steady state, except for significantly elevated neutrophil levels in miR-199b KO mice (n=5) compared to control mice (n=5) at 6 weeks of age, there were no significant differences in peripheral blood profiles. Bone marrow derived HSPCs (hematopoietic stem and progenitor cells) from miR-199b and wild type mice were examined for LT-HSC, KL, LSK, MPP, GMP and MEP populations. At steady state no significant differences were observed in all the cohorts except for KL (Lin-Kit+Sca-) population, where miR-199b KO mice exhibited significantly decreased levels compared to wild type mice (p<0.0093). Next, we examined the role of miR-199b under stress myelopoiesis by using 5-Fluouracil (5FU) induced myeloablation model. miR-199b KO mice exhibited significantly elevated levels of CFU-GEMM and CFU-GM compared to wild type mice. miR-199b levels in C57Bl/6 wild type mice were significantly decreased in HSCs post 5FU treatment. Gene profiling studies in HSCs post 5FU in wild type C57BL/6 mice identified three putative highly conserved miR-199b targets. They were Itga8 (Intergrin alpha 8), Zmpste 24 (metalloproteinase) and Scamp1 (secretory carrier membrane protein 1), whose roles are being investigated using miR-199b KO mice. Though we recently showed silencing of miR-199b in AML, the regulation of miR-199b gene expression in normal and leukemic cells is yet to be understood. Here, for the first time, using bioinformatics approach in integrated Genomics Viewer, we mined genomic database ENCODE (Encyclopedia of DNA elements) to identify both transcriptional and epigenetic landscape for miR-199b. Compared to CD34 and CD14, AML lines such as NB4 and HL-60 had relatively more regions of DNAase hypersensitivity. For DNA methylation, via reverse bisulphite sequencing data sets, no aberrant methylation was present between normal CD34 and HL-60, NB4 leukemic cells, which corroborates with our earlier finding of decitabine's ineffectiveness in inducing miR-199b expression in AML cells. Previously we had demonstrated that HDAC inhibitors significantly induced miR-199b expression in THP-1 cells. Here, for CD34 cells, we observed a significant presence of H3K4 and H3K9 methylation in miR-199b promoter via CHiP-seq data sets, implying an active transcriptional status. In CD14+ monocyte progenitors, a similar pattern for H3K4 methylation was observed and as well as significant levels of H3K9 acetylation. However, in CD15+ cells (neutrophil) H3K9 methylation was absent. Interestingly, in HL-60 and NB4 AML cells, H3K4me3 histone modifications peaked directly above the miR-199b coding region, whose significance is yet to be understood. For transcription factors, in HL-60 cells, via Chip-seq data sets, we found significant levels of GABP, NRSF and PU.1 binding activity in the upstream region of miR-199b. In NB4 (APL) cells, the Chip-seq data revealed a significant co-presence of cMyc and MAX binding in the upstream region of miR-199b. Further, the binding sites for the respective factors were also tested and confirmed using Genomatix-MatInspector. To experimentally validate these genomics derived data, we treated AML lines (HL-60, NB4 and THP-1) with inhibitors for cMyc (10058-F4), Myc-Max heterodimer (10074-G5), Sp1 (Mithramycin A) and NRSF/REST (X5050) for 24 and 48 hours. Exposure to Myc-Max heterodimer and NRSF/REST inhibitors significantly elevated miR-199b levels for HL-60 cells. For THP-1 cells, treatment with Myc-Max and Sp1 inhibitors significantly elevated miR-199b levels. Further functional assays are required to identify the biological roles of miR-199b in AML. In terms of miR-199b gene expression, we observe a differential regulation pattern in normal and leukemic cells, which in part may reflect the molecular heterogeneity in AML. Disclosures No relevant conflicts of interest to declare.

Laminin α4 Deficiency Results in Impaired Hematopoietic Recovery Accompanied with Altered Bone Marrow Niche after Myelosuppression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 27-27
Author(s):  
Makoto Kondo ◽  
Pingnan Xiao ◽  
Lakshmi Sandhow ◽  
Monika Dolinska ◽  
Thibault Bouderlique ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Steady State ◽  
Progenitor Cells ◽  
Wild Type ◽  
Post Irradiation ◽  
Colony Forming Unit ◽  
Hematopoietic Recovery

Abstract Myelosuppression is a life-threatening complication of anti-cancer therapy including irradiation. Rapid and complete hematopoietic recovery after therapy-induced myelosuppression is required for a successful treatment outcome. This process relies on efficient regeneration of hematopoietic stem cells (HSCs) and is tightly controlled by bone marrow (BM) microenvironment consisting of mesenchymal stem/progenitor cells, endothelial cells as well as secreted factors including cytokines and extracellular matrix proteins (ECM). However, the extrinsic factors critical for promoting the hematopoietic recovery remain poorly understood. Laminins are heterotrimetric ECM composed of α, β, and γ chains. Laminin α4 chain (LAMA4) is an active component for laminin-411 and -421, which are located in vascular basement membrane. LAMA4 plays an important role for HSC homing after transplantation via interaction with laminin receptor integrin α6 (Qian H et al., Blood 2006). However, the role of LAMA4 in normal hematopoiesis and HSC reconstitution after irradiation-induced myelosuppression is not known. In this study, we first detected Lama4 gene expression in BM endothelial cells (CD31+), mesenchymal stem cells (MSC: CD45-Ter119-CD31-CD44-Sca1+CD51+), and mesenchymal progenitor cells (MPC: CD45-Ter119-CD31-CD44-Sca1-CD51+) in young adult mice. By using Lama4 deficient (Lama4-/-) mice, we analyzed the functional role of LAMA4 on hematopoietic activity at steady state. We found the lower number of platelets (PLTs) (p = 0.03), and neutrophils (Gr1+CD11b+) (p = 0.03) in the peripheral blood (PB) of Lama4-/- mice, but a higher frequency of common myeloid progenitor (Lin-Sca1-Kit+CD34+FcRlow) (p < 0.01) in the Lama4-/- BM at steady state, indicating that LAMA4 plays a role in the maintenance of physiological hematopoiesis. The important role of LAMA4 in hematopoietic recovery was demonstrated by delayed and incomplete recoveries of mature red blood cells, PLTs, and Gr1+CD11b+ cells in PB following sublethal irradiation (7Gy). The impaired recovery of erythropoiesis was also indicated by the higher values of mean corpuscular hemoglobin and mean corpuscular volume in PB as well as the higher frequency of megakaryocyte-erythrocyte progenitor (Lin-Sca1-Kit+CD34-FcR-) (p < 0.01) and colony-forming unit-erythrocyte (CFU-E) (p = 0.03) in the BM of the Lama4-/- mice at 6 weeks after irradiation, suggesting blocked erythrocyte maturation. In keeping with the refractory neutropenia, the frequency of colony-forming unit-granulocyte-macrophage (CFU-GM) was lower in the Lama4-/- BM compared to that in the age- and gender-matched wild type mice (p = 0.04). These data indicate that LAMA4 is critical for multiple hematopoietic lineage reconstitution post irradiation. To investigate the cellular and molecular mechanisms underlying the critical role of LAMA4 in hematopoietic recovery after the irradiation, we characterized the BM niche by colony assay, flow cytometry immunophenotyping, quantitative real time PCR (qPCR), and histological analysis. The number of colony-forming unit-fibroblast (CFU-F) was comparable between wild type and Lama4-/- in steady state. Interestingly, the proportion of BM MPCs, a population containing osteoblast progenitors, was significantly lower in the Lama4-/- mice compared to that in the wild type controls at steady state (p < 0.01). qPCR analysis showed downregulation of Il6 (p < 0.05) in the MSC and Angpt1 (p = 0.02) in the MPC of the Lama4-/-mice post irradiation. These data suggest that Lama4 deficiency alters BM stromal cell composition and gene expressions, which may be related to the impaired hematopoietic reconstitution. The recovery of BM vascular structure is essential for efficient reconstitution of hematopoiesis. We observed uniquely dilated blood vessels in Lama4-/- BM at 6-week post irradiation. This might be caused by the lower Angpt1 expression in Lama4-/- MPC since Angpt1/Tie2 signaling is required for vascular regeneration (Kopp HG et al., Blood 2005, Zhou BO et al., eLife 2015). The functional consequences of this phenotype are still under investigation. Altogether, LAMA4 is required for rapid and complete hematopoietic recovery post irradiation-induced myelosuppression. Therapeutic strategies to upregulate Lama4 may facilitate the recovery of hematopoiesis following HSC transplantation under preconditioning using irradiation. Disclosures No relevant conflicts of interest to declare.

HSP70.1 and -70.3 are required for late-phase protection induced by ischemic preconditioning of mouse hearts

2003 ◽  
Vol 285 (2) ◽  
pp. H866-H874 ◽  
Author(s):  
Craig R. Hampton ◽  
Akira Shimamoto ◽  
Christine L. Rothnie ◽  
Jeaneatte Griscavage-Ennis ◽  
Albert Chong ◽  
...  
Keyword(s):  
Steady State ◽  
Infarct Size ◽  
Ischemic Preconditioning ◽  
Tissue Injury ◽  
Late Phase ◽  
Wild Type ◽  
Hsp70 Protein ◽  
Steady State Levels ◽  
Shock Proteins

We investigated the role of inducible heat shock proteins 70.1 and 70.3 (HSP70.1 and HSP70.3, respectively) in myocardial ischemic preconditioning (IP) in mice. Wild-type (WT) mice and HSP70.1- and HSP70.3-null [HSP70.1/3(–/–)] mice were subjected to IP and examined 24 h later during the late phase of protection. IP significantly increased steady-state levels of HSP70.1 and HSP70.3 mRNA and expression of inducible HSP70 protein in WT myocardium. To assess protection against tissue injury, mice were subjected to 30 min of regional ischemia and 3 h of reperfusion. In WT mice, IP reduced infarct size by 43% compared with sham IP-treated mice. In contrast, IP did not reduce infarct size in HSP70.1/3(–/–) mice. Absence of inducible HSP70.1 and HSP70.3 had no effect, however, on classical or early-phase protection produced by IP, which significantly reduced infarct size in HSP70.1/3(–/–) mice. We conclude that inducible HSP70.1 and HSP70.3 are required for late-phase protection against infarction following IP in mice.

Evaluation of role of G-CSF in the production, survival, and release of neutrophils from bone marrow into circulation

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 854-861 ◽  
Author(s):  
Sunanda Basu ◽  
George Hodgson ◽  
Melissa Katz ◽  
Ashley R. Dunn
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Transit Time ◽  
Bone Marrow Cells ◽  
Mean Transit Time ◽  
Wild Type ◽  
Blast Cells ◽  
And Control ◽  
Deficient Mice

Abstract In steady-state hematopoiesis, G-CSF (granulocyte-colony stimulating factor) regulates the level of neutrophils in the bone marrow and blood. In this study, we have exploited the availability of G-CSF–deficient mice to evaluate the role of G-CSF in steady-state granulopoiesis and the release of granulocytes from marrow into circulation. The thymidine analogue bromodeoxyuridine (BrdU) was used to label dividing bone marrow cells, allowing us to follow the release of granulocytes into circulation. Interestingly, the labeling index and the amount of BrdU incorporated by blast cells in bone marrow was greater in G-CSF–deficient mice than in wild-type mice. In blood, 2 different populations of BrdU-positive granulocytes, BrdUbright and BrdUdim, could be detected. The kinetics of release of the BrdUbright granulocytes from bone marrow into blood was similar in wild-type and G-CSF–deficient mice; however, BrdUdim granulocytes peaked earlier in G-CSF–deficient mice. Our findings suggest that the mean transit time of granulocytes through the postmitotic pool is similar in G-CSF–deficient and control mice, although the transit time through the mitotic pool is reduced in G-CSF–deficient mice. Moreover, the reduced numbers of granulocytes that characterize G-CSF–deficient mice is primarily due to increased apoptosis in cells within the granulocytic lineage. Collectively, our data suggest that at steady state, G-CSF is critical for the survival of granulocytic cells; however, it is dispensable for trafficking of granulocytes from bone marrow into circulation.

scholarly journals Modulation of flavocytochrome b2 intraprotein electron transfer via an interdomain hinge region

1996 ◽  
Vol 316 (2) ◽  
pp. 507-513 ◽  
Author(s):  
R. Eryl SHARP ◽  
Stephen K. CHAPMAN ◽  
Graeme A. REID
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Lactate Dehydrogenase ◽  
Structural Integrity ◽  
Hinge Region ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Lactate Dehydrogenase Activity ◽  
Flavocytochrome B2

The two domains of flavocytochrome b2 are connected by a typical hinge peptide. To probe the role of the hinge in modulating the efficiency of intraprotein electron transfer between these two domains, a number of mutant enzymes with truncated hinge regions were previously constructed and characterized [Sharp, Chapman and Reid (1996) Biochemistry 35, 891–899]. In the present study two mutant enzymes with elongated hinge regions have been constructed (HI3 and HI6) to further our understanding of the controlling influence of hinge length and primary structure on intraprotein electron transfer. Modification of the hinge had little effect on the lactate dehydrogenase activity of the enzyme, as was evident from steady-state experiments using ferricyanide as electron acceptor and from pre-steady-state experiments monitoring flavin reduction. However, the hinge insertions lowered the enzyme's effectiveness as a cytochrome c reductase. This effect results from a defect at the first interdomain electron-transfer step (FMNH2 → haem electron transfer), where the rate constants for haem reduction in HI3 and HI6 were 50-and 100-fold lower than the corresponding value for the wild-type enzyme. Preservation of structural integrity within the hinge region is apparently essential for efficient intraprotein electron transfer.

Differential Role of Components of the Fibrinolytic System in the Formation and Removal of Thrombus Induced by Endothelial Injury

1999 ◽  
Vol 81 (04) ◽  
pp. 601-604 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Osamu Kozawa ◽  
Masayuki Niwa ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Thrombus Formation ◽  
Endothelial Injury ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

Sign in / Sign up

Export Citation Format

Share Document