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Background: The prediction of Acute Kidney Injury (AKI)-related outcomes remains challenging. Persistent kidney excretory dysfunction for longer than 7 days has been defined as Acute Kidney Disease (AKD). In this study, we prospectively quantified serum Nostrin, an essential regulator of endothelial NO metabolism, in hospitalized patients with AKI., Design, Setting, Participants, & Measurements: In-hospital subjects with AKI of various etiology were identified through the in-hospital AKI alert system of the Brandenburg University Hospital. Serum Nostrin, and serum NGAL and KIM-1 were measured within a maximum of 48 hours from the timepoint of initial diagnosis of AKI. The following endpoints were defined: in-hospital death, need of kidney replacement therapy (KRT), recovery of kidney function (ROKF) until discharge., Results: AKI patients had significantly higher serum Nostrin levels compared to Controls. The level of serum Nostrin increased significantly with the severity of AKI. Within the group of AKI patients (n = 150) the in-hospital mortality was 16.7%, KRT was performed in 39.3%, no ROKF occurred in 28%. Patients who required KRT had significantly higher levels of serum Nostrin compared to patients who did not require KRT. Significantly higher levels of serum Nostrin were also detected in AKI patients without ROKF compared to patients with ROKF. In addition, low serum Nostrin levels at the timepoint of AKI diagnosis were predictive of in-hospital survival. For comparison, the serum concentrations of NGAL and KIM-1 were determined in parallel to the Nostrin concentrations and the results confirm the prognostic properties of serum Nostrin in AKI., Conclusions: The current study suggests serum Nostrin as novel biomarker of AKI-associated mortality, KRT and Acute Kidney Disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Erfurt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Although transfemoral aortic valve replacement (TAVR) is a safe treatment for elderly patients with severe aortic valve stenosis, postoperative microembolism has been described. In this secondary endpoint analysis of the POST-TAVR trial, we aimed to investigate whether changes in neuron-specific enolase (NSE)-a biomarker of neuronal damage-are associated with changes in memory function or postoperative delirium (POD)., Materials and Methods: This was a prospective single-center study enrolling patients undergoing elective TAVR. Serum NSE was measured before and 24 h after TAVR. POD was diagnosed using CAM-ICU testing. Memory function was assessed before TAVR and before hospital discharge using the "Consortium to Establish a Registry for Alzheimer's Disease" (CERAD) word list and the digit span task (DST) implemented in "∆elta-App"., Results: Subjects' median age was 82 years (25th to 75th percentile: 77.5-85.0), 42.6% of subjects were women. CERAD scores significantly increased from pre- to post-TAVR, with p < 0.001. POD occurred in 4.4% (6/135) of subjects at median 2 days after TAVR. After TAVR, NSE increased from a median of 1.85 ng/mL (1.30-2.53) to 2.37 ng/mL (1.69-3.07), p < 0.001. The median increase in NSE was 40.4% (13.1-138.0) in patients with POD versus 17.3% (3.3-43.4) in those without POD ( p = 0.17)., Conclusions: Memory function improved after TAVR, likely due to learning effects, with no association to change in NSE. Patients with POD appear to have significantly higher postoperative levels of NSE compared to patients without POD after TAVR. This finding suggests that neuronal damage, as indicated by NSE elevation, may not significantly impair assessed memory function after TAVR.

Background: This study aimed to determine whether novel and conventional cardiorenal biomarkers in patients before transcatheter aortic valve implantation may be associated with cardiorenal syndrome (CRS) type 1. Methods: Serum NT-proBNP and urine biomarkers (hepcidin-25, NGAL, IL-6) were measured before and 24 h after transcatheter aortic valve implantation. Results: 16/95 patients had CRS type 1. Those patients had longer length of stay in hospital (12.5 [9.0-16.0] vs 9.0 [8-12] days; p = 0.025) and were more frequently readmitted to hospital within 6 months after discharge (46.7 vs 15.6%; odds ratio: 4.7; 95% CI: 1.5-15.5; p = 0.007). The NT-proBNP/urine hepcidin-25 ratio (odds ratio: 2.89; 95% CI: 1.30-6.41; p = 0.009) was an independent modifier of CRS type 1. Conclusion: The NT-proBNP/urine hepcidin-25 ratio appears to be a modifier of risk of CRS type 1.

Background: Acute kidney injury (AKI) affects increasing numbers of hospitalized patients; the prognosis remains poor. The diagnosis is still based on the 2012 published KDIGO criteria. Numerous new AKI biomarkers have been identified in recent years; they either reflect impaired excretory function or structural damage. The majority of markers are useful for AKI recognition under certain circumstances. Fewer data are available on the role of biomarkers in the prediction of in-hospital survival and renal recovery post-AKI. The current article is intended to provide information about these two aspects., Summary: The following databases were screened: PubMed, Web of Science, Cochrane Library, Scopus. The period lasted from 2000 until 2022. The following terms were applied: "AKI" AND "biomarker" AND "survival" OR "mortality" OR "recovery of kidney function" OR "renal recovery" OR "kidney recovery". The following terms were used for additional literature search: "TIMP-2" AND "IGFBP7" and "RNA biomarker" AND "hematology". Regarding mortality, exclusively those studies were selected that addressed the in-hospital mortality. Nine (9) studies were identified that evaluated biomarker-based prediction of in-hospital mortality and/or of recovery of kidney function (ROKF). A homogenous definition of ROKF is however missing yet. Currently, some biomarkers, measured early during the course of the disease, are associated with increased mortality risk and/or with a higher chance of renal recovery., Key Messages: The literature provides only a few biomarker-related studies that address the issues of mortality and recovery. The definition of ROKF needs to be homogenized., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Background: At the end of November 2020, four years after the adoption of the Masterplan Medizinstudium 2020, the Federal Ministry of Health presented the draft bill for the revision of the medical licensing regulations. This was adapted in August 2021 and stipulates, among other things, the completion of a mandatory scientific project up to the second stage of the medical examination, which is to be conducted within twelve weeks and based on the structured teaching of scientific skills using longitudinal courses. This study examines to what extent the mentioned aspects of the draft bill are already included in the current compulsory curricula in German medical study programs., Methods: In a cross-sectional study, we conducted a document analysis and an online survey of the student deaneries of state and private, state-recognized faculties of medicine in Germany. The objective was to assess the integration of curricular science projects and longitudinal science modules. The data was analyzed descriptively., Results: In total, 40 (93%) of 43 faculties were included in the document analysis. 26 (60.5%) of 43 academic deans responded to the online questionnaire. Only twelve (30%) of the faculties offer a mandatory science project according to the document analysis and eight (30.8%) according to the evaluation of the online survey. In relative terms, a mandatory science project is already a curricular component in the majority of model and reform degree programs (document analysis: 83.3%, n=10 / N=12; questionnaire: 87.5%, n=7 / N=8). This contrasts with a significantly smaller number of regular degree programs that currently offer a science project as a mandatory component (document analysis: 16.7%, n=2 / N=12; questionnaire: 12.5%, n=1 / N=8). In the majority of the model and reform courses, a compulsory (longitudinal) science module has already been integrated into the curriculum (document analysis: 75%, n=12 / N=16; questionnaire: 55.5%, n=6 / N=11). This is only true for 25% (n=4 / N=12; document analysis) and 44.5% (n=5 / N=11; questionnaire) of the regular courses., Discussion: The results of the surveys indicate that the regular study programs, in particular, need to be restructured in order to integrate the intended time frame of twelve weeks for an obligatory science project into the current curriculum. How this is to be achieved without reducing the existing study content seems to be still unclear. Regardless of the current draft bill, however, this step appears to be necessary in order to strengthen the acquisition of scientific competencies in medical studies and to adapt it to international medical education., Conclusion: The majority of the current model and reformed medical study courses already fulfill the requirements of the draft bill for the new licensing regulations with regard to the curricular integration of an obligatory scientific project and longitudinal strand on scientific work, which offer hints on design possibilities for further faculties., (Copyright © 2022. Published by Elsevier GmbH.)

Competing Interests: Conflict of interest: The authors declare that they have no conflict(s) of interest.

Competing Interests: Conflict of interest: The authors declare no conflict of interest.

Background: The nitric oxide synthase interacting protein (Nosip) has been associated with diverse human diseases including psychological disorders. In line, early neurogenesis of mouse and Xenopus is impaired upon Nosip deficiency. Nosip knockout mice show craniofacial defects and the down-regulation of Nosip in the mouse and Xenopus leads to microcephaly. Until now, the exact underlying molecular mechanisms of these malformations were still unknown., Results: Here, we show that nosip is expressed in the developing ocular system as well as the anterior neural crest cells of Xenopus laevis. Furthermore, Nosip inhibition causes severe defects in eye formation in the mouse and Xenopus. Retinal lamination as well as dorso-ventral patterning of the retina were affected in Nosip-depleted Xenopus embryos. Marker gene analysis using rax, pax6 and otx2 reveals an interference with the eye field induction and differentiation. A closer look on Nosip-deficient Xenopus embryos furthermore reveals disrupted cranial cartilage structures and an inhibition of anterior neural crest cell induction and migration shown by twist, snai2, and egr2. Moreover, foxc1 as downstream factor of retinoic acid signalling is affected upon Nosip deficiency., Conclusions: Nosip is a crucial factor for the development of anterior neural tissue such the eyes and neural crest cells. Developmental Dynamics 247:1070-1082, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Background: Genetic deletion of Nosip in mice causes holoprosencephaly, however, the function of Nosip in neurogenesis is currently unknown., Results: We combined two vertebrate model organisms, the mouse and the South African clawed frog, Xenopus laevis, to study the function of Nosip in neurogenesis. We found, that size and cortical thickness of the developing brain of Nosip knockout mice were reduced. Accordingly, the formation of postmitotic neurons was greatly diminished, concomitant with a reduced number of apical and basal neural progenitor cells in vivo. Neurospheres derived from Nosip knockout embryos exhibited reduced growth and the differentiation capability into neurons in vitro was almost completely abolished. Mass spectrometry analysis of the neurospheres proteome revealed a reduced expression of Rbp1, a regulator of retinoic acid synthesis, when Nosip was absent. We identified the homologous nosip gene to be expressed in differentiated neurons in the developing brain of Xenopus embryos. Knockdown of Nosip in Xenopus resulted in a reduction of brain size that could be rescued by reintroducing human NOSIP mRNA. Furthermore, the expression of pro-neurogenic transcription factors was reduced and the differentiation of neuronal cells was impaired upon Nosip knockdown. In Xenopus as well as in mouse we identified reduced proliferation and increased apoptosis as underlying cause of microcephaly upon Nosip depletion. In Xenopus Nosip and Rbp1 are similarly expressed and knockdown of Nosip resulted in down regulation of Rbp1. Knockdown of Rbp1 caused a similar microcephaly phenotype as the depletion of Nosip and synergy experiments indicated that both proteins act in the same signalling pathway., Conclusions: Nosip is a novel factor critical for neural stem cell/progenitor self-renewal and neurogenesis during mouse and Xenopus development and functions upstream of Rbp1 during early neurogenesis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Endothelial dysfunction is an early event in cardiovascular disease and characterized by reduced production of nitric oxide (NO). The F-BAR protein NO synthase traffic inducer (NOSTRIN) is an interaction partner of endothelial NO synthase and modulates its subcellular localization, but the role of NOSTRIN in pathophysiology in vivo is unclear., Objective: We analyzed the consequences of deleting the NOSTRIN gene in endothelial cells on NO production and cardiovascular function in vivo using NOSTRIN knockout mice., Methods and Results: The levels of NO and cGMP were significantly reduced in mice with endothelial cell-specific deletion of the NOSTRIN gene resulting in diastolic heart dysfunction. In addition, systemic blood pressure was increased, and myograph measurements indicated an impaired acetylcholine-induced relaxation of isolated aortic rings and resistance arteries. We found that the muscarinic acetylcholine receptor subtype M3 (M3R) interacted directly with NOSTRIN, and the latter was necessary for correct localization of the M3R at the plasma membrane in murine aorta. In the absence of NOSTRIN, the acetylcholine-induced increase in intracellular Ca(2+) in primary endothelial cells was abolished. Moreover, the activating phosphorylation and Golgi translocation of endothelial NO synthase in response to the M3R agonist carbachol were diminished., Conclusions: NOSTRIN is crucial for the localization and function of the M3R and NO production. The loss of NOSTRIN in mice leads to endothelial dysfunction, increased blood pressure, and diastolic heart failure., (© 2015 American Heart Association, Inc.)

Holoprosencephaly is a common developmental disorder in humans characterised by incomplete brain hemisphere separation and midface anomalies. The etiology of holoprosencephaly is heterogeneous with environmental and genetic causes, but for a majority of holoprosencephaly cases the genes associated with the pathogenesis could not be identified so far. Here we report the generation of knockout mice for the ubiquitin E3 ligase NOSIP. The loss of NOSIP in mice causes holoprosencephaly and facial anomalies including cleft lip/palate, cyclopia and facial midline clefting. By a mass spectrometry based protein interaction screen we identified NOSIP as a novel interaction partner of protein phosphatase PP2A. NOSIP mediates the monoubiquitination of the PP2A catalytic subunit and the loss of NOSIP results in an increase in PP2A activity in craniofacial tissue in NOSIP knockout mice. We conclude, that NOSIP is a critical modulator of brain and craniofacial development in mice and a candidate gene for holoprosencephaly in humans.

F-BAR proteins are multivalent adaptors that link plasma membrane and cytoskeleton and coordinate cellular processes such as membrane protrusion and migration. Yet, little is known about the function of F-BAR proteins in vivo. Here we report, that the F-BAR protein NOSTRIN is necessary for proper vascular development in zebrafish and postnatal retinal angiogenesis in mice. The loss of NOSTRIN impacts on the migration of endothelial tip cells and leads to a reduction of tip cell filopodia number and length. NOSTRIN forms a complex with the GTPase Rac1 and its exchange factor Sos1 and overexpression of NOSTRIN in cells induces Rac1 activation. Furthermore, NOSTRIN is required for fibroblast growth factor 2 dependent activation of Rac1 in primary endothelial cells and the angiogenic response to fibroblast growth factor 2 in the in vivo matrigel plug assay. We propose a novel regulatory circuit, in which NOSTRIN assembles a signalling complex containing FGFR1, Rac1 and Sos1 thereby facilitating the activation of Rac1 in endothelial cells during developmental angiogenesis.

Background: NO produced by the endothelial NO synthase (eNOS) is an important regulator of cardiovascular physiological and pathological features. eNOS is activated by numerous stimuli, and its activity is tightly regulated. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) has been implicated in regulating eNOS activity in response to shear stress. The current study was conducted to determine the role of PECAM-1 in the regulation of basal eNOS activity., Methods and Results: We demonstrate that PECAM-1-knockout ECs have increased basal eNOS activity and NO production. Mechanistically, increased eNOS activity is associated with a decrease in the inhibitory interaction of eNOS with caveolin-1, impaired subcellular localization of eNOS, and decreased eNOS traffic inducer (NOSTRIN) expression in the absence of PECAM-1. Furthermore, we demonstrate that activation of blunted signal transducers and activators of transcription 3 (STAT3) in the absence of PECAM-1 results in decreased NOSTRIN expression via direct binding of the signal transducers and activators of transcription 3 to the NOSTRIN promoter., Conclusions: Our results reveal an elegant mechanism of eNOS regulation by PECAM-1 through signal transducers and activators of transcription 3-mediated transcriptional control of NOSTRIN.

The secreted epidermal growth factor-like protein 7 (EGFL7) plays an important role in angiogenesis, especially in the recruitment of endothelial and smooth muscle cells to the site of the nascent vessel and their ordered assembly into functional vasculature. However, progress in the understanding of the underlying mechanisms is to date greatly hindered by the lack of recombinant EGFL7 protein in a stable, soluble, native state, thus preventing e.g. the characterization of the proposed functional receptor as well as investigation of additional biological effects of EGFL7. So far all attempts to produce sufficient amounts of recombinant EGFL7 protein by various groups have failed. In this study we describe a procedure for the expression and purification of human EGFL7 from Sf9 cells and for the first time provide means to isolate biologically functional EGFL7 protein in sufficient quantities for its further biological characterization. We believe that the availability of EGFL7 will greatly accelerate our understanding of the precise role of EGFL7 and the underlying molecular mechanisms of EGFL7 action in the fundamentally important process of angiogenesis.

Persistent mitochondrial hyperpolarization (MHP) and enhanced calcium fluxing underlie aberrant T cell activation and death pathway selection in systemic lupus erythematosus. Treatment with rapamycin, which effectively controls disease activity, normalizes CD3/CD28-induced calcium fluxing but fails to influence MHP, suggesting that altered calcium fluxing is downstream or independent of mitochondrial dysfunction. In this article, we show that activity of the mammalian target of rapamycin (mTOR), which is a sensor of the mitochondrial transmembrane potential, is increased in lupus T cells. Activation of mTOR was inducible by NO, a key trigger of MHP, which in turn enhanced the expression of HRES-1/Rab4, a small GTPase that regulates recycling of surface receptors through early endosomes. Expression of HRES-1/Rab4 was increased in CD4(+) lupus T cells, and in accordance with its dominant impact on the endocytic recycling of CD4, it was inversely correlated with diminished CD4 expression. HRES-1/Rab4 overexpression was also inversely correlated with diminished TCRzeta protein levels. Pull-down studies revealed a direct interaction of HRES-1/Rab4 with CD4 and TCRzeta. Importantly, the deficiency of the TCRzeta chain and of Lck and the compensatory up-regulation of FcepsilonRIgamma and Syk, which mediate enhanced calcium fluxing in lupus T cells, were reversed in patients treated with rapamcyin in vivo. Knockdown of HRES-1/Rab4 by small interfering RNA and inhibitors of lysosomal function augmented TCRzeta protein levels in vitro. The results suggest that activation of mTOR causes the loss of TCRzeta in lupus T cells through HRES-1/Rab4-dependent lysosomal degradation.

We recently observed that a novel, shortened variant of eNOS trafficking inducer (NOSTRIN) is expressed in cirrhotic liver. This shortened variant (NOSTRINbeta) lacks the first 78 amino acids of full-length NOSTRIN (NOSTRINalpha) and thus a substantial part of its F-BAR domain. In contrast to NOSTRINalpha, NOSTRINbeta mainly localizes to the cell nucleus. In this study, we show that nuclear import of NOSTRINbeta depends on two nuclear localization signals (aa 32-36: KKRK and aa 57-61: KAKKK). Each of the sequences is independently functional, but both are required to sustain nuclear localization of NOSTRINbeta. Export of NOSTRINbeta from the nucleus is facilitated by a CRM1-dependent mechanism relying on the nuclear export sequence LELEKERIQL (aa 135-145). Unlike NOSTRINbeta, the full-length variant NOSTRINalpha was conspicuously absent from the nucleus. This is most likely because of the fact that its N-terminal F-BAR domain, which is truncated in NOSTRINbeta, facilitates association with cellular membranes. NOSTRINbeta directly binds to the 5'-regulatory region of the NOSTRIN gene (bp -200 to -1), and overexpression of NOSTRINbeta strongly decreases transcription of a reporter gene under control of this DNA region. Taken together, our results suggest that nuclear NOSTRINbeta may negatively regulate transcription of the NOSTRIN gene.

The iromycins A-D are members of a new family of rare alpha-pyridone metabolites. The isolation and structure elucidation of these microbial secondary metabolites from Streptomyces sp. Dra 17 revealed a N-heterocyclic core structure with two unusual side chains. Iromycins act as inhibitors of nitric oxide synthases (NOS), a protein family, which produces the crucial second messenger nitric oxide (NO). Importantly, these compounds inhibit selectively endothelial NOS rather than neuronal NOS and thus set prospects for both medical therapy and basic research. Feeding experiments with 13C- and 15N -labeled precursors indicated an uncommon type of polyketide biosynthesis and clearly ruled out an isoprenoid origin. A detoxification pathway of a particular secondary metabolite in the host strain is a rare observation and here we demonstrate it with the iromycin family.

Recently, we characterized a novel endothelial nitric-oxide synthase (eNOS)-interacting protein, NOSTRIN (for eNOS-trafficking inducer), which decreases eNOS activity upon overexpression and induces translocation of eNOS away from the plasma membrane. Here, we show that NOSTRIN directly binds to caveolin-1, a well-established inhibitor of eNOS. Because this interaction occurs between the N terminus of caveolin (positions 1-61) and the central domain of NOSTRIN (positions 323-434), it allows for independent binding of each of the two proteins to eNOS. Consistently, we were able to demonstrate the existence of a ternary complex of NOSTRIN, eNOS, and caveolin-1 in Chinese hamster ovary (CHO)-eNOS cells. In human umbilical vein endothelial cells (HUVECs), the ternary complex assembles at the plasma membrane upon confluence or thrombin stimulation. In CHO-eNOS cells, NOSTRIN-mediated translocation of eNOS involves caveolin in a process most likely representing caveolar trafficking. Accordingly, trafficking of NOSTRIN/eNOS/caveolin is affected by altering the state of actin filaments or cholesterol levels in the plasma membrane. During caveolar trafficking, NOSTRIN functions as an adaptor to recruit mediators such as dynamin-2 essential for membrane fission. We propose that a ternary complex between NOSTRIN, caveolin-1, and eNOS mediates translocation of eNOS, with important implications for the activity and availability of eNOS in the cell.

Unlike most other endogenous messengers that are deposited in vesicles, processed on demand and/or secreted in a regulated fashion, NO (nitric oxide) is a highly active molecule that readily diffuses through cell membranes and thus cannot be stored inside the producing cell. Rather, its signalling capacity must be controlled at the levels of biosynthesis and local availability. The importance of temporal and spatial control of NO production is highlighted by the finding that differential localization of NO synthases in cardiomyocytes translates into distinct effects of NO in the heart. Thus NO synthases belong to the most tightly controlled enzymes, being regulated at transcriptional and translational levels, through co- and post-translational modifications, by substrate availability and not least via specific sorting to subcellular compartments, where they are in close proximity to their target proteins. Considerable efforts have been made to elucidate the molecular mechanisms that underlie the intracellular targeting and trafficking of NO synthases, to ultimately understand the cellular pathways controlling the formation and function of this powerful signalling molecule. In the present review, we discuss the mechanisms and triggers for subcellular routing and dynamic redistribution of NO synthases and the ensuing consequences for NO production and action.

Infectious entry of hepatitis B viruses (HBV) has nonconventional facets. Here we analyzed whether a cell-permeable peptide [translocation motif (TLM)] identified within the surface protein of human HBV is a general feature of all hepadnaviruses and plays a role in the viral life cycle. Surface proteins of all hepadnaviruses contain conserved functional TLMs. Genetic inactivation of the duck HBV TLMs does not interfere with viral morphogenesis; however, these mutants are noninfectious. TLM mutant viruses bind to cells and are taken up into the endosomal compartment, but they cannot escape from endosomes. Processing of surface protein by endosomal proteases induces their exposure on the virus surface. This unmasking of TLMs mediates translocation of viral particles across the endosomal membrane into the cytosol, a prerequisite for productive infection. The ability of unmasked TLMs to translocate processed HBV particles across cellular membranes was shown by confocal immunofluorescence microscopy and by infection of nonpermissive cell lines with HBV processed in vitro with endosomal lysate. Based on these data, we propose an infectious entry mechanism unique for hepadnaviruses that involves virus internalization by receptor-mediated endocytosis followed by processing of surface protein in endosomes. This processing activates the function of TLMs that are essential for viral particle translocation through the endosomal membrane into the cytosol and productive infection.

Nitric oxide (NO) plays a key role in vascular function, cell proliferation, and apoptosis. Proper subcellular localization of endothelial NO synthase (eNOS) is crucial for its activity; however, the role of eNOS trafficking for NO biosynthesis remains to be defined. Overexpression of NOS-interacting protein (NOSIP) induces translocation of eNOS from the plasma membrane to intracellular compartments, thereby impairing NO production. Here we report that endogenous NOSIP reduces the enzymatic capacity of eNOS, specifically in the G(2) phase of the cell cycle by targeting eNOS to the actin cytoskeleton. This regulation is critically dependent on the nucleocytoplasmic shuttling of NOSIP and its cytoplasmic accumulation in the G(2) phase. The predominant nuclear localization of NOSIP depends on a bipartite nuclear localization sequence (NLS) mediating interaction with importin alpha. Mutational destruction of the NLS abolishes nuclear import and interaction with importin alpha. Nuclear export is insensitive to leptomycin B and hence different from the CRM1-dependent default mechanism. Inhibition of NOSIP expression by RNA interference completely abolishes G(2)-specific cytoskeletal association and inhibition of eNOS. These findings describe a novel cell cycle-dependent modulation of endogenous NO levels that are critical to the cell cycle-related actions of NO such as apoptosis or cell proliferation.

Endothelial nitric oxide synthase (eNOS), the major nitric oxide (NO)-generating enzyme of the vasculature, is regulated through multiple interactions with proteins, including caveolin-1, Hsp90, Ca2+-calmodulin, and the recently discovered eNOS-interacting protein, NOSIP. Previous studies indicate that NOSIP may contribute to the intricate regulation of eNOS activity and availability. Because eNOS has been shown to be abundantly expressed in the airways, we determined the expression and cellular localization of NOSIP in rat trachea and lung by RT-PCR and immunohistochemistry and examined the interaction of NOSIP with eNOS in lung by coimmunoprecipitation. In tracheal epithelium and lung, NOSIP mRNA expression was prevalent, as shown by RT-PCR, and the corresponding protein interacted with eNOS, as demonstrated by coimmunoprecipitation. Using immunohistochemistry, we found both NOSIP and eNOS immunoreactivity in ciliated epithelial cells of trachea and bronchi, while Clara cells showed immunoreactivity for NOSIP only. NOSIP and eNOS were present in vascular and bronchial smooth muscle cells of large arteries and airways, whereas endothelial cells, as well as bronchiolar and arteriolar smooth muscle cells, exclusively stained for NOSIP. Our results point to functional role(s) of NOSIP in the control of airway and vascular diameter, mucosal secretion, NO synthesis in ciliated epithelium, and, therefore, of mucociliary and bronchial function.

Mechanisms governing the activity of neuronal nitric oxide synthase (nNOS), the major source of nitric oxide (NO) in the nervous system, are not completely understood. We report here a protein-protein interaction between nNOS and NOSIP (nitric oxide synthase-interacting protein) in rat brain in vivo. NOSIP and nNOS are concentrated in neuronal synapses and demonstrate significant colocalization in various regions of the central and peripheral nervous systems. NOSIP produces a significant reduction in nNOS activity in a neuroepithelioma cell line stably expressing nNOS. Furthermore, overexpression of NOSIP in cultured primary neurons reduces the availability of nNOS in terminal dendrites. These results thus suggest that the interaction between NOSIP and nNOS is functionally involved in endogenous mechanisms regulating NO synthesis. Furthermore, we found that the subcellular distribution and expression levels of NOSIP are dynamically regulated by neuronal activity in vitro as well as in vivo, suggesting that NOSIP may contribute to a mechanism via which neuronal activity regulates the synaptic availability and activity of nNOS.

Nitric oxide (NO) is a gaseous radical with unique biological functions essential for the cardiovascular system, host defense and neuro-transmission. For two decades it was thought that NO was able to diffuse freely across relatively long distances and to traverse major parts of the cell, if not multiple cell layers. However, NO has been proven to be extremely reactive: it reacts with other reactive oxygen species, heavy metals, as well as with cysteine and tyrosine residues in proteins. In accordance, it is now widely accepted that once NO is generated, it is very short-lived and diffuses only over a short distance. This urges for the local production of NO and the localization of NO synthases in the proximity of their downstream targets. This review discusses the highly organized localization of NO synthases, with the endothelial isoform (eNOS) as its main focus, since from this synthase most is known about its subcellular localization and regulation.

Nitric oxide and nitric oxide synthases are key players in synaptic plasticity events in the spinal cord, which underly the development of chronic pain states. To date, little is known about the molecular mechanisms regulating the activity of nitric oxide synthases in nociceptive systems. The present study was aimed at the immunohistochemical determination of the expression of a nitric oxide synthase-interacting protein (NOSIP) in the rat spinal cord and dorsal root ganglia and studying its regulation in states of nociceptive hypersensitivity in a rat model of post-inflammatory pain. NOSIP is predominantly expressed in nociceptive primary neurons and in neurons of the spinal dorsal horn and the number of NOSIP-positive spinal neurons increases significantly following induction of unilateral intraplantar injection of complete Freund's adjuvant. Thus, NOSIP may modulate nitric oxide homeostasis in physiological and pathological pain conditions.

Activity and localization of endothelial nitric oxide synthase (eNOS) is regulated in a remarkably complex fashion, yet the complex molecular machinery mastering stimulus-induced eNOS translocation and trafficking is poorly understood. In a search by the yeast two-hybrid system using the eNOS oxygenase domain as bait, we have identified a previously uncharacterized eNOS-interacting protein, dubbed NOSTRIN (for eNOS traffic inducer). NOSTRIN contains a single polypeptide chain of 506-aa residues of 58 kDa with an N-terminal cdc15 domain and a C-terminal SH3 domain. NOSTRIN mRNA is abundant in highly vascularized tissues such as placenta, kidney, lung, and heart, and NOSTRIN protein is expressed in vascular endothelial cells. Coimmunoprecipitation experiments demonstrated the eNOS-NOSTRIN interaction in vitro and in vivo, and NOSTRIN's SH3 domain was essential and sufficient for eNOS binding. NOSTRIN colocalized extensively with eNOS at the plasma membrane of confluent human umbilical venous endothelial cells and in punctate cytosolic structures of CHO-eNOS cells. NOSTRIN overexpression induced a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern and at the same time led to a significant inhibition of NO release. We conclude that NOSTRIN contributes to the intricate protein network controlling activity, trafficking, and targeting of eNOS.

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine. Its pleiotropic biological properties are signaled through two distinct cell surface receptors: the TNF receptor type I (TNFR-I) and the TNF receptor type II. Neither of the two receptors possesses tyrosine kinase activity. A large majority of TNF-alpha-dependent activities can be mediated by TNFR-I. Recently, c-Raf-1 kinase was identified as an intracellular target of a signal transduction cascade initiated by binding of TNF-alpha to TNFR-I. However, the mechanism engaged in TNF-alpha-dependent activation of c-Raf-1 kinase is still enigmatic. Here we report that the cytosolic adapter protein Grb2 is a novel binding partner of TNFR-I. Grb2 binds with its COOH-terminal SH3 domain to a PLAP motif within TNFR-I and with its NH2-terminal SH3 domain to SOS (son of sevenless). A PLAP deletion mutant of TNFR-I fails to bind Grb2. The TNFR-I/Grb2 interaction is essential for the TNF-alpha-dependent activation of c-Raf-1 kinase; activation of c-Raf-1 kinase by TNF-alpha can be blocked by coexpression of Grb2 mutants harboring inactivating point mutations in the NH2- or COOH-terminal SH3 domain, cell-permeable peptides that disrupt the Grb2/TNFR-I interaction or transdominant negative Ras. Functionality of the TNFR-I/Grb2/SOS/Ras interaction is a prerequisite but not sufficient for TNF-alpha-dependent activation of c-Raf-1 kinase. Inhibition of the TNFR-I/FAN (factor associated with neutral sphingomyelinase) interaction, which is essential for TNF-alpha-dependent activation of the neutral sphingomyelinase, either by cell-permeable peptides or by deletion of the FAN binding domain, prevents activation of c-Raf-1 kinase. In conclusion, binding of the Grb2 adapter protein via its COOH-terminal SH3 domain to the nontyrosine kinase receptor TNFR-I results in activation of a signaling cascade known so far to be initiated, in the case of the tyrosine kinase receptors, by binding of the SH2 domain of Grb2 to phosphotyrosine.