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In-plane isotropic crossed anisotropy multilayer for high-frequency magnetics was fabricated using in-plane uniaxial anisotropic nanogranular film owing to its high permeability, ferromagnetic resonance (FMR) frequency, and electrical dc resistivity. The multilayer exhibited in-plane isotropy and enabled us to combine permeability of approximately 100, FMR frequency of approximately 3 GHz, and resistivity of approximately $5 ~\mu \Omega \cdot \text {m}$ . Moreover, a low damping factor of 0.0148 was obtained; hence the multilayer is available for a low loss of up to 1 GHz. Meanwhile, the underlayer and cap layer were necessary for improving the isotropy of the multilayer. The results can be attributed to the interfacial stress.

The Stroke Preclinical Assessment Network (SPAN) is a multicenter preclinical trial platform using rodent models of transient focal cerebral ischemia to address translational failure in experimental stroke. In addition to centralized randomization and blinding and large samples, SPAN aimed to introduce heterogeneity to simulate the heterogeneity embodied in clinical trials for robust conclusions. Here, we report the heterogeneity introduced by allowing the 6 SPAN laboratories to vary most of the biological and experimental model variables and the impact of this heterogeneity on middle cerebral artery occlusion (MCAo) performance. We included the modified intention-to-treat population of the control mouse cohort of the first SPAN trial (n=421) and examined the biological and procedural independent variables and their covariance. We then determined their impact on the dependent variables cerebral blood flow drop during MCAo, time to achieve MCAo, and total anesthesia duration using multivariable analyses. We found heterogeneity in biological and procedural independent variables introduced mainly by the site. Consequently, all dependent variables also showed heterogeneity among the sites. Multivariable analyses with the site as a random effect variable revealed filament choice as an independent predictor of cerebral blood flow drop after MCAo. Comorbidity, sex, use of laser Doppler flow to monitor cerebral blood flow, days after trial onset, and maintaining anesthesia throughout the MCAo emerged as independent predictors of time to MCAo. Total anesthesia duration was predicted by most independent variables. We present with high granularity the heterogeneity introduced by the biological and model selections by the testing sites in the first trial of cerebroprotection in rodent transient filament MCAo by SPAN. Rather than trying to homogenize all variables across all sites, we embraced the heterogeneity to better approximate clinical trials. Awareness of the heterogeneity, its sources, and how it impacts the study performance may further improve the study design and statistical modeling for future multicenter preclinical trials.

* Objective: To identify predictors of a long-term response to interferon-[alpha] therapy in chronic hepatitis C and to determine whether hepatitis C virus (HCV) was eradicated in patients with chronic hepatitis C who had a long-term response to therapy. * Design: A retrospective analysis. * Setting: In- and outpatient liver clinic of a municipal hospital in Japan. * Patients: 47 patients with chronic hepatitis C who responded to interferon-[alpha] were divided into two groups: 22 patients with a long-term response (serum aminotransferase levels remained normal for > 1 year after therapy) and 25 patients with a short-term response (serum aminotransferase levels increased again after therapy). * Measurements: Genotyping of HCV, titers of HCV RNA in liver and serum samples (using the reverse transcriptase-polymerase chain reaction), histologic activity index, and liver histologic tests during and 1 year after therapy. * Results: Among the 22 long-term responders, HCV RNA was no longer detectable in liver and serum samples of 21 (95%) at the end of therapy and remained undetectable in the serum of 20 (91%) and in the liver of 19 (86%) 1 year after therapy. Liver histologic tests improved substantially immediately after therapy and 1 year after therapy in the long-term responders; however, 18 (82%) of these patients still had mild, chronic hepatitis. Among the 25 short-term responders, HCV RNA was still detected in the liver of 19 (76%) and in the serum of 9 (36%) at the end of therapy. Multivariate logistic regression analysis showed that the persistent presence of hepatic HCV RNA at the end of therapy was the strongest predictor of relapse. * Conclusion: These findings suggest that HCV infection was eradicated in most of the long-term responders to interferon-[alpha] therapy because HCV RNA could no longer be detected in their serum and liver samples and because a significant improvement gradually occurred in their liver histologic results. The persistent presence of hepatic HCV RNA at the end of therapy was the most important predictor of relapse., The eradication of hepatitis C virus (HCV) after interferon-alpha treatment may be determined better by the absence of HCV RNA in liver tissue rather than in blood serum. Of 47 patients with chronic hepatitis C, 22 had a long-term response to interferon-alpha treatment and 25 had a short-term response. Patients were classified as long-term responders if serum levels of the liver enzyme aminotransferase were normal for more than a year after treatment, and as short-term responders if aminotransferase levels rose again after treatment. HCV RNA was not detected in serum or liver samples from 95% of long-term responders after treatment. In contrast, HCV RNA was detected in liver samples from 76% of short-term responders. Analysis of eight prognostic variables among the two groups determined that the presence of HCV RNA in liver tissue was most significantly associated with hepatitis C relapse.

Despite considerable research on exercise-induced neuroplasticity in the brain, a major ongoing challenge in translating findings from animal studies to humans is that clinical and preclinical settings employ very different techniques. Here we aim to bridge this divide by using diffusion tensor imaging MRI (DTI), an advanced imaging technique commonly applied in human studies, in a longitudinal exercise study with mice. Wild-type mice were exercised using voluntary free-wheel running, and MRI scans were at baseline and after four weeks and nine weeks of running. Both hippocampal volume and fractional anisotropy, a surrogate for microstructural directionality, significantly increased with exercise. In addition, exercise levels correlated with effect size. Histological analysis showed more PDGFRα+ oligodendrocyte precursor cells in the corpus callosum of running mice. These results provide compelling in vivosupport for the concept that similar adaptive changes occur in the brains of mice and humans in response to exercise.

This third in a series of vascular cognitive impairment (VCI) workshops, supported by “The Leo and Anne Albert Charitable Trust,” was held from February 8 to 12 at the Omni Resort in Carlsbad, CA. This workshop followed the information gathered from the earlier two workshops suggesting that we focus more specifically on brain white matter in age-related cognitive impairment. The Scientific Program Committee (Frank Barone, Shawn Whitehead, Eric Smith, and Rod Corriveau) assembled translational, clinical, and basic scientists with unique expertise in acute and chronic white matter injury at the intersection of cerebrovascular and neurodegenerative etiologies. As in previous Albert Trust workshops, invited participants addressed key topics related to mechanisms of white matter injury, biomarkers of white matter injury, and interventions to prevent white matter injury and age-related cognitive decline. This report provides a synopsis of the presentations and discussions by the participants, including the existing knowledge gaps and the delineation of the next steps towards advancing our understanding of white matter injury and age-related cognitive decline. Workshop discussions and consensus resulted in action by The Albert Trust to (1) increase support from biannual to annual “White Matter and Cognition” workshops; (2) provide funding for two collaborative, novel research grants annually submitted by meeting participants; and (3) coordinate the formation of the “Albert Research Institute for White Matter and Cognition.” This institute will fill a gap in white matter science, providing white matter and cognition communications, including annual updates from workshops and the literature and interconnecting with other Albert Trust scientific endeavors in cognition and dementia, and providing support for newly established collaborations between seasoned investigators and to the development of talented young investigators in the VCI-dementia (VCID) and white matter cognition arena.

Biomaterials provide novel platforms to deliver stem cell and growth factor therapies for central nervous system (CNS) repair. The majority of these approaches have focused on the promotion of neural progenitor cells and neurogenesis. However, it is now increasingly recognized that glial responses are critical for recovery in the entire neurovascular unit. In this study, we investigated the cellular effects of epidermal growth factor (EGF) containing hydrogels on primary astrocyte cultures. Both EGF alone and EGF‐hydrogel equally promoted astrocyte proliferation, but EGF‐hydrogels further enhanced astrocyte activation, as evidenced by a significantly elevated Glial fibrillary acidic protein (GFAP) gene expression. Thereafter, conditioned media from astrocytes activated by EGF‐hydrogel protected neurons against injury and promoted synaptic plasticity after oxygen–glucose deprivation. Taken together, these findings suggest that EGF‐hydrogels can shift astrocytes into neuro‐supportive phenotypes. Consistent with this idea, quantitative‐polymerase chain reaction (qPCR) demonstrated that EGF‐hydrogels shifted astrocytes in part by downregulating potentially negative A1‐like genes (Fbln5 and Rt1‐S3) and upregulating potentially beneficial A2‐like genes (Clcf1, Tgm1, and Ptgs2). Further studies are warranted to explore the idea of using biomaterials to modify astrocyte behavior and thus indirectly augment neuroprotection and neuroplasticity in the context of stem cell and growth factor therapies for the CNS. Stem Cells Translational Medicine2019;8:1242&1248 Biomaterials provide novel platforms to deliver stem cell and growth factor therapies for central nervous system repair. Our data suggest that epidermal growth factor‐containing hydrogels can shift astrocytes into potentially beneficial A2‐like phenotypes that may augment neuroprotection and neuroplasticity during the recovery phase after brain injury.

JAK-STAT signaling cascade has emerged as an ideal target for the treatment of myeloproliferative diseases, autoimmune diseases, and neurological disorders. Ruxolitinib (Rux), is an orally bioavailable, potent and selective Janus-associated kinase (JAK) inhibitor, proven to be effective to target activated JAK-STAT pathway in the diseases previously described. Unfortunately, limited studies have investigated the potential cytotoxic profile of Rux on other cell populations within the heterogenous CNS microenvironment. Two stem and progenitor cell populations, namely the oligodendrocyte precursor cells (OPCs) and neural stem/progenitor cells (NSPCs), are important for long-term maintenance and post-injury recovery response of the CNS. In light of the limited evidence, this study sought to investigate further the effect of Rux on proliferating and differentiating OPCs and NSPCs populations. In the present study, cultured rat OPCs and NSPCs were treated with various concentrations of Rux, ranging from 2μM to 20μM. The effect of Rux on proliferating OPCs (PDGF-R-α+) and proliferating NSPCs (nestin+) was assessed via a 3-day Rux treatment, whereas its effect on differentiating OPCs (MBP+/PDGF-R-α+) and differentiating NSPCs (neurofilament+) was assessed after a 7-day treatment. Cytotoxicity of Rux was also assessed on OPC populations by examining its influence on cell death and DNA synthesis via YO-PRO-1/PI dual-staining and BrdU assay, respectively. The results suggest that Rux at a dosage above 10μM reduces the number proliferating OPCs, likely via the induction of apoptosis. On the other hand, Rux treatment from 2.5μM to 20μM significantly reduces the number of differentiating OPCs by inducing necrosis. Meanwhile, Rux treatment has no observable untoward impact on NSPC cultures within the dosage range tested. Taken together, OPCs appears to be more vulnerable to the dosage effect of Rux, whereas NSPCs are not significantly impacted by Rux, suggesting a differential mechanism of actions of Rux on the cell types.

Endothelial progenitor cells (EPCs) have been pursued as a potential cellular therapy for stroke and central nervous system injury. However, their underlying mechanisms remain to be fully defined. Recent experimental studies suggest that mitochondria may be released and transferred between cells. In this proof‐of‐concept study, we asked whether beneficial effects of EPCs may partly involve a mitochondrial phenomenon as well. First, EPC‐derived conditioned medium was collected and divided into supernatant and particle fractions after centrifugation. Electron microscopy, Western blots, and flow cytometry showed that EPCs were able to release mitochondria. ATP and oxygen consumption assays suggested that these extracellular mitochondria may still be functionally viable. Confocal microscopy confirmed that EPC‐derived extracellular mitochondria can be incorporated into normal brain endothelial cells. Adding EPC particles to brain endothelial cells promoted angiogenesis and decreased the permeability of brain endothelial cells. Next, we asked whether EPC‐derived mitochondria may be protective. As expected, oxygen–glucose deprivation (OGD) increased brain endothelial permeability. Adding EPC‐derived mitochondria particles to the damaged brain endothelium increased levels of mitochondrial protein TOM40, mitochondrial DNA copy number, and intracellular ATP. Along with these indirect markers of mitochondrial transfer, endothelial tightness was also restored after OGD. Taken together, these findings suggest that EPCs may support brain endothelial energetics, barrier integrity, and angiogenic function partly through extracellular mitochondrial transfer. StemCells2018;36:1404–1410 Endothelial progenitor cell (EPC)‐derived mitochondria particles to damaged brain endothelium after oxygen–glucose deprivation (OGD) increased levels of mitochondrial protein TOM40, mitochondrial DNA copy number, and intracellular ATP along with restoring endothelial tightness after OGD. These findings suggest that EPCs may support brain endothelial energetics, barrier integrity, and angiogenic function partly through extracellular mitochondrial transfer.

Oligodendrocyte precursor cells (OPCs) give rise to oligodendrocytes in cerebral white matter. However, the underlying mechanisms that regulate this process remain to be fully defined, especially in adult brains. Recently, it has been suggested that signaling via A‐kinase anchor protein 12 (AKAP12), a scaffolding protein that associates with intracellular molecules such as protein kinase A, may be involved in Schwann cell homeostasis and peripheral myelination. Here, we asked whether AKAP12 also regulates the mechanisms of myelination in the CNS. AKAP12 knockout mice were compared against wild‐type (WT) mice in a series of neurochemical and behavioral assays. Compared with WTs, 2‐months old AKAP12 knockout mice exhibited loss of myelin in white matter of the corpus callosum, along with perturbations in working memory as measured by a standard Y‐maze test. Unexpectedly, very few OPCs expressed AKAP12 in the corpus callosum region. Instead, pericytes appeared to be one of the major AKAP12‐expressing cells. In a cell culture model system, conditioned culture media from normal pericytes promoted in‐vitro OPC maturation. However, conditioned media from AKAP12‐deficient pericytes did not support the OPC function. These findings suggest that AKAP12 signaling in pericytes may be required for OPC‐to‐oligodendrocyte renewal to maintain the white matter homeostasis in adult brain. StemCells2018;36:751–760 In adult white matter, AKAP12 in pericytes regulates the production of growth factors, which promote oligodendrocyte differentiation for maintaining white matter homeostasis.

Supplemental Digital Content is available in the text.

In a mouse model of traumatic brain injury, treatment with a carbon-monoxide-releasing molecule is shown to reduce pericyte cell death and promote neurogenesis, leading to an amelioration of neurological deficits.

Aging and vascular comorbidities such as hypertension comprise critical cofactors that influence how the brain responds to stroke. Ischemic stress induces neurogenesis and oligodendrogenesis in younger brains. However, it remains unclear whether these compensatory mechanisms can be maintained even under pathologically hypertensive and aged states. To clarify the age-related remodeling capacity after stroke under hypertensive conditions, we assessed infarct volume, behavioral outcomes, and surrogate markers of neurogenesis and oligodendrogenesis in acute and subacute phases after transient focal cerebral ischemia in 3- and 12-month-old spontaneously hypertensive rats (SHRs). Hematoxylin and eosin staining showed that 3- and 12-month-old SHRs exhibited similar infarction volumes at both 3 and 14 days after focal cerebral ischemia. However, recovery of behavioral deficits (neurological score assessment and adhesive removal test) was significantly less in 12-month-old SHRs compared to 3-month-old SHRs. Concomitantly, numbers of nestin+neural stem/progenitor cells (NSPCs) near the infarct border area or subventricular zone in 12-month-old SHRs were lower than 3-month-old SHRs at day 3. Similarly, numbers of PDGFR-α+oligodendrocyte precursor cells (OPCs) in the corpus callosum were lower in 12-month-old SHRs at day 3. Lower levels of NSPC and OPC numbers were accompanied by lower expression levels of phosphorylated CREB. By day 14 postischemia, NSPC and OPC numbers in 12-month-old SHRs recovered to similar levels as in 3-month-old SHRs, but the numbers of proliferating NSPCs (Ki-67+nestin+cells) and proliferating OPCs (Ki-67+PDGFR-α+cells) remained lower in the older brains even at day 14. Taken together, these findings suggest that aging may also decrease poststroke compensatory responses for neurogenesis and oligodendrogenesis even under hypertensive conditions.

Supplemental Digital Content is available in the text.

White matter injury caused by cerebral hypoperfusion may contribute to the pathophysiology of vascular dementia and stroke, but the underlying mechanisms remain to be fully defined. Here, we test the hypothesis that oxidative stress interferes with endogenous white matter repair by disrupting renewal processes mediated by oligodendrocyte precursor cells (OPCs).

Aging is one of the major risk factors for white matter injury in cerebrovascular disease. However, the effects of age on the mechanisms of injuryrepair in white matter remain to be fully elucidated. Here, we ask whether, compared with young brains, white matter regions in older brains may be more vulnerable in part because of decreased rates of compensatory oligodendrogenesis after injury.

The sphingosine 1-phosphate receptor agonist fingolimod reduces infarct size in rodent models of stroke and enhances blood–brain barrier integrity. Based on these observations, we hypothesized that combination of fingolimod with tissue plasminogen activator (tPA) would reduce the risk of hemorrhagic transformation associated with delayed administration of tPA.

Subarachnoid hemorrhage (SAH) pathophysiology involves neurovascular proteolysis and inflammation. How these 2 phenomena are related remains unclear. We hypothesize that matrix metalloproteinases (MMPs) mediate the depletion of anti-inflammatory plasma-type gelsolin (pGSN).

Normobaric oxygen (NBO) therapy may be neuroprotective in acute ischemic stroke. However, how NBO may affect intracerebral hemorrhage is unclear. We tested NBO in a rat model of striatal intracerebral hemorrhage.

Stroke remains a major health problem worldwide, and is the leading cause of serious long-term disability. Recent findings now suggest that strategies to enhance angiogenesis after focal cerebral ischemia may provide unique opportunities to improve clinical outcomes during stroke recovery. In this mini-review, we survey emerging mechanisms and potential targets for angiogenic therapies in brain after stroke. Multiple elements may be involved, including growth factors, adhesion molecules and progenitor cells. Furthermore, cross talk between angiogenesis and neurogenesis may also provide additional substrates for plasticity and remodeling in the recovering brain. A better understanding of the molecular interplay between all these complex pathways may lead to novel therapeutic avenues for tackling this difficult disease.

We recently reported that delayed lithium therapy can improve stroke recovery in rats by augmenting neurovascular remodeling. We tested the hypothesis that lithium can promote the expression of growth factors in brain endothelial cells and astrocytes.

Future demographic changes predict an increase in the number of patients with atrial fibrillation. As long-term anticoagulation for the prevention of ischemic strokes becomes more prevalent, the burden of warfarin-associated intracerebral hemorrhage (W-ICH) is likely to grow. However, little is known about the clinical aspects and pathophysiologic mechanisms of W-ICH. This study describes the development of a mouse model of W-ICH in which hematoma growth and outcomes can be correlated with anticoagulation parameters.

The concept of the neurovascular unit suggests that effects on brain vasculature must be considered if neuroprotection is to be achieved in stroke. We previously reported that 12/15-lipoxygenase (12/15-LOX) is upregulated in the peri-infarct area after middle cerebral artery occlusion in mice, and 12/15-LOX contributes to brain damage after ischemia–reperfusion. The current study was designed to investigate 12/15-LOX involvement in vascular injury in the ischemic brain.

The natural product baicalein is a specific inhibitor of 12/15-lipoxygenase, but it also has antioxidant properties. The current study was designed to test if the neuroprotective properties of baicalein are related to its lipoxygenase inhibition.

Previously, we reported generation of erythropoiesis from acetyl low-density lipoprotein (Ac-LDL)- incorporating endothelial cells (ECs) in the mouse embryo. However, it is still unclear whether the other types of definitive hematopoietic cells (HCs) can be generated from these cells. In this study, ECs at 10 days post coitum (dpc) were tagged with Ac-LDL-DiI and were shown to release DiI+ HCs into the circulation after 12 h of whole embryo culture. The hematopoietic clusters in the main arteries were also stained with DiI. The circulating DiI+ HCs expressed c-Kit and half of these cells displayed blastic morphology. In vitro culture and in vivo reconstitution experiments demonstrated that the circulating DiI+ HCs contained definitive multipotent progenitors, including hematopoietic stem cells (HSCs). Furthermore, the sorted DiI+ HCs were able to colonize the fetal liver (FL) when introduced back into the blood stream of a secondary recipient embryo. These results suggest that Ac-LDL incorporating ECs can produce definitive HSCs and HCs colonizing FL in the mouse embryo.

Plasmacytoid dendritic cells (pDCs) play an important primary role for antiviral innate immunity by rapidly producing large amounts of type 1 interferon (IFN) upon viral infection. To study pDC biology, we generated a monoclonal antibody, termed 2E6, that recognizes pDCs. Molecular cloning of a cDNA encoding the 2E6 antigen revealed that it is a type II C-type lectin, Ly49Q, that consists of 247 amino acids with high homology to the natural killer (NK) receptor family Ly49, with an immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic domain. Ly49Q is expressed on pDCs but not on NK cells or myeloid dendritic cells. B220+, CD11c+, CD11b–pDCs in bone marrow were divided into Ly49Q+and Ly49Q–subsets. While both subsets produced IFN-α upon cytosine-phosphate-guanosine (CpG) and herpes simplex virus stimulation, Ly49Q–pDCs responded poorly to influenza virus. In addition, Ly49Q–pDCs produced inflammatory cytokines such as interleukin 6 (IL-6), IL-12, and tumor necrosis factor α (TNF-α) upon stimulation at lower levels than those produced by Ly49Q+pDCs. In contrast to bone marrow, Ly49Q+pDCs were only found in peripheral blood, lymph nodes, and spleen. These results indicate that Ly49Q is a specific marker for peripheral pDCs and that expression of Ly49Q defines 2 subsets of pDCs in bone marrow.

Erythropoiesis is characterized by 2 waves of production during mouse embryogenesis: a primitive one originating from the yolk sac (YS) and a definitive one produced from both the YS and the embryo proper. How the latter wave is generated remains unclear. To investigate our hypothesis that endothelial cells (ECs) could generate erythroid cells, we designed a method to label ECs at 10 days after coitus. This labeling method associates 2 techniques: an intracardiac inoculation that allows molecules to be delivered into the bloodstream followed by a whole-embryo culture period. DiI-conjugated acetylated low-density lipoproteins (Ac-LDL-DiI) were used to specifically tag ECs from the inside. One hour after inoculation, DiI staining was found along the entire endothelial tree. Fluorescence-activated cell sorter (FACS) analysis revealed that DiI+ cells were CD31+, CD34+, and CD45–, an antigen makeup characteristic of the endothelial lineage. Twelve hours after inoculation, 43% of DiI+ circulating cells belonged to the erythroid lineage. These cells expressed Ter119 and displayed an adult globin chain arrangement; thus they belonged to the definitive lineage as confirmed in erythroid colony formation. The remaining cells likely represent committed white blood cells or multipotent progenitors, as revealed by a mixed-colony formation. Beyond the 29-somite stage, the proportion of DiI+ erythroid cells gradually decreased. These results demonstrate the generation of hematopoietic cells from an endothelial intermediate, using in vivo tracing. We provide evidence for a release of these cells into the circulation and hypothesize that these cells are able to colonize the fetal liver and generate definitive erythrocytes in vivo.

PriA protein is essential for RecA-dependent DNA replication induced by stalled replication forks in Escherichia coli. PriA is a DEXH-type DNA helicase, ATPase activity of which depends on its binding to structured DNA including a D-loop-like structure. Here, we show that the N-terminal 181-amino acid polypeptide can form a complex with D-loop in gel shift assays and have identified a unique motif present in the N-terminal segment of PriA that plays a role in its DNA binding. We have also identified residues in the C terminus proximal helicase domain essential for D-loop binding. PriA proteins mutated in this domain do not bind to D-loop, despite the presence of the N-terminal DNA-binding motif. Those mutants that cannot bind to D-loop in vitrodo not support a recombination-dependent mode of DNA replication in vivo, indicating that binding to a D-loop-like structure is essential for the ability of PriA to initiate DNA replication and repair from stalled replication forks. We propose that binding of the PriA protein to stalled replication forks requires proper configuration of the N-terminal fork-recognition and C-terminal helicase domains and that the latter may stabilize binding and increase binding specificity.

Cdc7-Dbf4 kinase complexes, conserved widely in eukaryotes, play essential roles in initiation and progression of the S phase. Cdc7 kinase activity fluctuates during cell cycle, and this is mainly the result of oscillation of expression of the Dbf4 subunit. Therefore, it is crucial to understand the mechanisms of regulation of Dbf4 expression. We have isolated and characterized the promoter region of the human ASKgene encoding Dbf4-related regulatory subunit for human Cdc7 kinase. We have identified a 63-base pair ASKpromoter segment, which is sufficient for mediating growth stimulation. This minimal promoter segment (MP), containing an Sp1 site but no canonical E2F site, can be activated by ectopic E2F expression as well. Within the 63-base pair region, the Sp1 site as well as other elements are essential for stimulation by growth signals and by E2F, whereas an AT-rich sequence proximal to the coding region may serve as an element required for suppression in quiescence. Gel shift assays in the presence of an antibody demonstrate the presence of E2F1 in the protein-DNA complexes generated on the MP segment. However, the complex formation on MP was not competed by aDHFRpromoter fragment, known to bind to E2F, nor by a consensus E2F binding oligonucleotide. Gel shift assays with point mutant MP fragments indicate that a non-canonical E2F site in the middle of this segment is critical for generation of the E2F complex. Our results suggest that E2F regulates theASKpromoter through an atypical mode of recognition of the target site.

The Ca2+signal facilitates nuclear translocation of NFAT through the dephosphorylation of clustered serine residues in the calcium regulatory domain by the Ca2+/calmodulin-dependent phosphatase calcineurin. The conformation of dephosphorylated NFAT exposes the nuclear localization signal for translocation into the nucleus and masks the nuclear export sequence to keep the protein in the nucleus. It has been reported that deletion of some serine-rich motifs masking the nuclear localization signal results in the translocation of NFAT into the nucleus, but that the nuclear export sequence located at the N terminus also needs to be deleted for NFATx (NFAT4/NFATc3) to exert efficient transactivation function. Here, we report that deletion of the critical serine-rich motifs of NFATx leads to a conformation that efficiently exposes the nuclear localization signal and that has stronger transcription activity compared with the fully activated wild-type protein in the presence of the nuclear export sequence. This also suggests that the regulation of the transactivation domain by phosphorylation observed in NFAT1 may not contribute significantly to the transcription activity of NFATx. The expression of this constitutively nuclear form of NFATx in the CD4+CD8+T cell line facilitates differentiation into the CD4 single-positive stage upon stimulation with phorbol ester. Our data suggest that NFATx is involved in the regulation of co-receptor expression during differentiation into the CD4 single-positive stage.

DNA replication results from the action of a staged set of highly regulated processes. Among the stages of DNA replication, initiation is the key point at which all the G1 regulatory signals culminate. Cdc7 kinase is the critical regulator for the ultimate firing of the origins of initiation. Cdc7, originally identified in budding yeast and later in higher eukaryotes, forms a complex with a Dbf4-related regulatory subunit to generate an active kinase. Genetic evidence in mammals demonstrates essential roles for Cdc7 in mammalian DNA replication. Mini-chromosome maintenance protein (MCM) is the major physiological target of Cdc7. Genetic studies in yeasts indicate additional roles of Cdc7 in meiosis, checkpoint responses, maintenance of chromosome structures, and repair. The interplay between Cdc7 and Cdk, another kinase essential for the S phase, is also discussed. J. Cell. Physiol. 190: 287–296, 2002. © 2002 Wiley-Liss, Inc.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by synovial pannus formation, leukocyte infiltration, and angiogenesis. Adenoviral production of interleukin-13 (IL-13) reduces levels of proinflammatory mediators in an explant model of RA synovial tissue in vitro. To assess this approach in an animal model of arthritis, we compared intra-articular injections of an adenovirus producing rat IL-13 (AxCArIL-13), a control virus, and rat ankles receiving phosphate-buffered saline (PBS) in rat adjuvant-induced arthritis (AIA). We demonstrate that IL-13 levels are normally low in ankles throughout the course of rat AIA. We show that administration of AxCArIL-13 before arthritis onset significantly reduces ankle circumference, paw volume, bony destruction, the number of polymorphonuclear cells (PMNs), the quantity of blood vessels, and levels of monocyte chemoattractant protein (MCP)-1 in ankles. When administered as a treatment to inflamed ankles, AxCArIL-13 decreases articular index scores, paw volumes, bony destruction, vascularization, tumor necrosis factor-α (TNF-α) levels, and the quantity of monocytes, lymphocytes, and PMNs. Thus, increasing IL-13 levels significantly ameliorates the course of rat AIA, suggesting that similar strategies for the treatment of human RA are worthy of further study.

Although erythropoietin (EPO) and its receptor (EPOR) are crucial for the proliferation, survival, and terminal differentiation of erythroid progenitors, it remains to be elucidated whether EPOR-unique signaling is required for erythropoiesis. To address this issue, human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor (hGMR)–transgenic mice and heterozygous EPOR mutant mice were crossed by in vitro fertilization. In methylcellulose clonal culture of fetal liver (FL) cells of generated hGMR-expressing EPOR−/−embryos at embryonic day (E) 12.5 of gestation, hGM-CSF stimulated erythroid colony formation under serum-containing and serum-free conditions. Analysis of globin expression in individual erythrocyte-containing colonies formed from E12.5 FL cells showed that hGM-CSF supports primitive and definitive erythropoiesis even in EPOR−/−embryos. In comparison of activities between hGM-CSF and EPO in hGMR-expressing EPOR+/+embryos, the 2 substances supported the formation of similar numbers of erythroid colonies in clonal culture of E12.5 FL cells; enhanced adult, but not embryonic, globin synthesis; and induced increase of GATA-1 expression and decrease of erythroid Kruppel-like factor and cMyb expression in the FL cells. On the other hand, in E8.0 yolk sac erythropoiesis, both substances had a similar effect on erythroid colony formation, but hGM-CSF induced an increase of β-major globin expression, while EPO did not. All together, the results of the present study demonstrated that hGM-CSF can stimulate the proliferation and differentiation of primitive and definitive erythroid cells independently of EPOR signal if they express hGMR, and the activity is comparable to that of EPO in definitive, but not primitive, erythropoiesis.

Box1 and 2 (box1/2) are conserved cytoplasmic motifs located in the membrane proximal region of cytokine receptors, including the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor common βc. Deletion of box1/2 abrogated all the examined activities of GM-CSF, and this phenomenon is explained by the loss of binding by Jak2. To test if a molecule other than Jak2 interacting with the box1/2 region plays a role in GM-CSF receptor signal transduction, we screened for molecules interacting with the box1/2 region by a pull-down assay using recombinant purified protein of GST fused with the βc box1/2 region and a Ba/F3 cell lysate. The mouse homologue of Mad2 protein, which plays an important role in the M phase of the cell cycle, was revealed to associate with the box1/2 region specifically. Peptides corresponding to the box1 sequence also bound to Mad2, and mutation of the box1 decreased the Mad2 interaction. Deletion analysis indicated that interaction with box1/2 occurred through the C-terminal portion of Mad2. Mad2 is known to change affinity for binding partners cell cycle dependently. Binding affinity of Mad2 to box1/2 increased in the late M phase, suggesting the possibility that GM-CSF participates in regulation of the M phase check point through interaction with Mad2.

β1-Adrenergic receptor (β1AR) shows the resistance to agonist-induced internalization. However, β1AR can internalize as G protein-coupled receptor kinase 2 (GRK2) is fused to its carboxyl terminus. Internalization of the β1AR and GRK2 fusion protein (β1AR/GRK2) is dependent on dynamin but independent of β-arrestin and phosphorylation. The β1AR/GRK2 fusion protein internalizes via clathrin-coated pits and is found to co-localize with the endosome that contains transferrin. The fusion proteins consisting of β1AR and various portions of GRK2 reveal that the residues 498–502 in the carboxyl-terminal domain of GRK2 are critical to promote internalization of the fusion proteins. This domain contains a consensus sequence of a clathrin-binding motif defined as a clathrin box. In vitrobinding assays show that the residues 498–502 of GRK2 bind the amino-terminal domain of clathrin heavy chain to almost the same extent as β-arrestin1. The mutation of the clathrin box in the carboxyl-terminal domain of GRK2 results in the loss of the ability to promote internalization of the fusion protein. GRK2 activity increases and then decreases as the concentration of clathrin heavy chain increases. Taken together, these results imply that GRK2 contains a functional clathrin box and directly interacts with clathrin to modulate its function.

Assembly of replication complexes at the replication origins is strictly regulated. Cdc45p is known to be a part of the active replication complexes. In Xenopusegg extracts, Cdc45p was shown to be required for loading of DNA polymerase α onto chromatin. The fission yeast cdc45 homologue was identified as a suppressor for nda4 and named sna41. Nevertheless, it is not known how Cdc45p facilitates loading of DNA polymerase α onto chromatin, particularly to prereplicative complexes. To gain novel insight into the function of this protein in fission yeast, we characterized the fission yeast Cdc45 homologue, Sna41p. We have constructed C-terminally epitope-tagged Sna41p and Polαp and replaced the endogenous genes with the corresponding tagged genes. Analyses of protein-protein interactions in vivoby the use of these tagged strains revealed the following: Sna41p interacts with Polαp throughout the cell cycle, whereas it interacts with Mis5p/Mcm6p in the chromatin fractions at the G1-S boundary through S phase. In an initiation-defective sna41mutant,sna41goa1, interaction of Polαp with Mis5p is not observed, although Polαp loading onto the chromatin that occurs before G1START is not affected. These results show that fission yeast Sna41p facilitates the loading of Polαp onto minichromosome maintenance proteins. Our results are consistent with a model in which loading of Polαp onto replication origins occurs through two steps, namely, loading onto chromatin at preSTART and association with prereplicative complexes at G1-S through Sna41p, which interacts with minichromosome maintenance proteins in a cell cycle-dependent manner.

Hsk1, Saccharomyces cerevisiaeCdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect inhsk1-89is indicated by accumulation ofcutcells at 30°C. hsk1-89displays synthetic lethality in combination with rad3deletion, indicating that survival of hsk1-89depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling.hsk1-89displays apparent defect in mitosis at 37°C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those ofrad21-K1and are significantly enhanced in ahsk1-89 rad21-K1double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.

Several lines of evidence indicate that transcriptional activation is coupled with DNA replication initiation, but the nature of initiation of DNA replication in mammalian cells is unclear. Polyoma virus replicon is an excellent system to analyze the initiation of DNA replication in murine cells because its replication requires an enhancer, and all components of replication machinery, except for DNA helicase large T antigen, are supplied by host cells. This system was used to examine the role of signal transducer and activator of transcription (STAT5) in replication initiation of polyoma replicon in the mouse lymphoid cell line BA/F3. The plasmid with tandem repeats of consensus STAT5 binding sites followed by polyoma replication origin was replicated by stimulation with human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in the presence of polyoma large T antigen in BA/F3 cells. Mutation analysis of the hGM-CSF receptor β subunit revealed that only the box1 region is essential, and the C-terminal tyrosine residues are dispensable for the activity. Addition of the tyrosine kinase inhibitor genistein suppressed this replication without affecting transcriptional activation of STAT5. Because deletion analysis of STAT5 indicates the importance of the C-terminal transcriptional activation domain of STAT5 for the initiation of replication, the role of this region in the activation of replication was examined with a GAL4–STAT5 fusion protein. GAL4–STAT5 activated replication of the plasmid containing tandem repeats of GAL4 binding sites and polyoma replication origin in BA/F3 cells. Mutation analysis of GAL4–STAT5 indicated that multiple serine residues coordinately have a role in activating replication. This is the first direct evidence indicating the potential involvement of STAT5 in replication.

Dbf4 and its related molecules were originally identified as cyclin-like partners for Cdc7 kinases, essential for S-phase. Recent reports and database search indicate the presence of multiple Dbf4-related molecules with distinct functions. We have identified three stretches of amino acids which are conserved in various Dbf4-related molecules and possibly play distinct functions in binding to and activation of the catalytic subunits as well as in interactions with other proteins. Discovery of conserved motifs for this possible new protein family would serve as a useful framework for future identification of new members of this family as well as for probing their functions.

Purpose:Several different anti-hypertensive regimens have been introduced for the prevention of systemic hyperdynamic responses after electrically induced seizures. In the present study, the effects of anti-hypertensive medications on cerebral circulation were studied.

The p53 protein binds DNA as a tetramer inside the nucleus, but a form of the p53 protein during nuclear transport has not been fully elucidated. To verify whether the human p53 protein passes through the nuclear pore as a monomer or oligomer, two different p53 mutants N1 and C1NLS−with or without a nuclear localization signal (NLS), respectively, were expressed in Xenopus laevisembryos. By the whole-mount immunostaining method, their intracellular distributions were observed to exist in an NLS-dependent manner. In a immunoprecipitation assay system, NLS-defective mutants formed oligomer in the cytoplasm. When coexpressed with NLS-containing N1, C1NLS−still stayed in the cytoplasm and did not inhibit N1 transport into the nucleus. Furthermore, when oligomerization-defective p53 mutant was expressed in Xenopusembryos, efficiency of its nuclear transport was demonstrated to be unchanged compared to that of the wild type. Assuming that NLS-defective p53 mutants have no dominant-negative effect on wild-type p53 in the nucleus of p53 heterozygous cells, we investigated the dominant-negative effect by CAT activity assay using human cell line Saos-2 and NLS-defective mutants. It was found that the NLS-defective p53 mutant did not have a dominant-negative effect on the function of wild-type p53 protein in the nucleus. Data indicate that each monomeric p53 protein independently passes through the nuclear pore; however, the possibility of homooligomeric p53 protein transport into the nucleus is not completely excluded.

Nuclear Factor of Activated T cell (NFAT) is a family of transcription factors that are important for the coordinate expression of various cytokines and immunoregulatory cell surface molecules in T cells and other types of cells in the immune system. In addition, analysis of gene disrupted mice revealed that some members of NFAT family are important for the development of myocardium, myocardial hypertrophy, and mesenchymal stem cells. NFAT family proteins have two conserved domains, the NFAT Homology Domain (NHD) and the Rel Similarity Domain (RSD). The RSD is DNA binding and AP-1 interacting domain which has structural similarity to the Rel Homology Region, the DNA binding domain of Rel family proteins. The NHD is a regulatory domain required for the Ca regulated translocation of NFAT. We report here the isolation and initial characterization of a novel RSD containing protein designated NFATz. NFATz has a RSD but no NHD. NFATz protein is localized in the nucleus without Ca signal. There is no detectable binding to a typical NFAT site even in the presence of AP-1, and it is not capable of activating transcription through the NFAT site. The chromosomal location determined by FISH revealed that NFATz and NFATx genes are in the same region.

Reducing brain blood flow induces toxic but druggable cis P-tau that drives progressive neurodegeneration and memory loss in mice.

The cyclic AMP (cAMP) elevating agent PGE2 and dibutyryl cyclic AMP (dBcAMP) affect T cell functions. Using human helper T cell clones, we examined effects of cAMP on c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), which are assumed to play a role in T cell regulation. When we analyzed the effects of dBcAMP on activities of mitogen–activated protein kinase (MAPK) family members ERK2, JNKp55 and JNKp46, dBcAMP did not inhibit the activities of ERK2 and JNKp55 induced by PMA/A23187 stimulation. JNKp46 activity was, however, inhibited by dBcAMP. JNK phosphorylates c-Jun on Ser-63 and Ser-73, the result being induction of its transcriptional activity. We found that dBcAMP inhibited the phosphorylation of c-Jun Ser-63 induced by PMA/A23187 stimulation. We suggest a different mechanism of regulation of JNKp55 and JNKp46 activities and that JNKp46 is a specific c-Jun kinase by which the activity of c-Jun is regulated in T lymphocytes.

Saccharomyces cerevisiae CDC7encodes a serine/threonine kinase required for G1/S transition, and its related kinases are present in fission yeast as well as in higher eukaryotes, including humans. Kinase activity of Cdc7 protein depends on the regulatory subunit, Dbf4, which also interacts with replication origins. We have identified him1+from two-hybrid screening with Hsk1, a fission yeast homologue of Cdc7 kinase, and showed that it encodes a regulatory subunit of Hsk1. Him1, identical to Dfp1, previously identified as an associated molecule of Hsk1, binds to Hsk1 and stimulates its kinase activity, which phosphorylates both catalytic and regulatory subunits as well as recombinant MCM2 protein in vitro. him1+is essential for DNA replication in fission yeast cells, and its transcription is cell cycle regulated, increasing at middle M to late G1. The protein level is low at START in G1, increases at the G1/S boundary, and is maintained at a high level throughout S phase. Him1 protein is hyperphosphorylated at G1/S through S during the cell cycle as well as in response to early S-phase arrest induced by nucleotide deprivation. Deletion of one of the motifs conserved in regulatory subunits for Cdc7-related kinases as well as alanine substitution of three serine and threonine residues present in the same motif resulted in a defect in checkpoint regulation normally induced by hydroxyurea treatment. The alanine mutant also showed growth retardation after UV irradiation and the addition of methylmethane sulfonate. In keeping with this result, a database search indicates that him1+is identical to rad35+. Our results reveal a novel function of the Cdc7/Dbf4-related kinase complex in S-phase checkpoint control as well as in growth recovery from DNA damage in addition to its predicted essential function in S-phase initiation.

A novel human protein, ASK (activator of S phase kinase), was identified on the basis of its ability to bind to human Cdc7-related kinase (huCdc7). ASK forms an active kinase complex with huCdc7 that is capable of phosphorylating MCM2 protein. ASK appears to be the major activator of huCdc7, since immunodepletion of ASK protein from the extract is accompanied by the loss of huCdc7-dependent kinase activity. Expression of ASK is regulated by growth factor stimulation, and levels oscillate through the cell cycle, reaching a peak during S phase. Concomitantly, the huCdc7-dependent kinase activity significantly increases when cells are in S phase. Furthermore, we have demonstrated that ASK serves an essential function for entry into S phase by showing that microinjection of ASK-specific antibodies into mammalian cells inhibited DNA replication. Our data show that ASK is a novel cyclin-like regulatory subunit of the huCdc7 kinase complex and that it plays a pivotal role in G1/S transition in mammalian cells.