Search

Recent studies showed that neuronal nitric oxide synthase (nNOS) plays a role in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity. In the present study we examined the effects of striatal injection of 1-methyl-4-phenylpyridinium (MPP⁺) on substantia nigra degeneration in mutant mice lacking the nNOS gene or the endothelial nitric oxide synthase (eNOS) gene. Both striatal lesion volume and substantia nigra degeneration were significantly attenuated in the nNOS mutant mice but not in the eNOS mutant mice. The mice lacking nNOS showed a significant attenuation of MPP⁺-induced increases of 3-nitrotyrosine concentrations in the striatum. In a separate experiment administration of 7-nitroindazole for 48 h after MPP⁺injections significantly attenuated substantia nigra degeneration in rats. Immunohistochemical studies showed apposition of nNOS-positive neuronal processes on tyrosine hydroxylase-positive neurons. These results provide further evidence that neuronally derived NO and peroxynitrite play a role in MPP⁺neurotoxicity.

Three inhibitors of human immunodeficiency virus (HIV) have been isolated and purified to homogeneity from Euphorbiaceae himalaya seeds ( Gelonium multiflorum) and carnation leaves ( Dianthus caryophytlus). These proteins. GAP 31 (Gelonium Anti-HIV Protein 31 kDa) and DAPs 30 and 32 (dianthus anti-HIV proteins, 30 and 32 kDa), inhibit HIV-1 infection and replication in a dosc-dependent manner with little toxicity to target cells. The therapeutic indices of these compounds are in the order 10 4, suggesting that they may be clinically important agents in the treatment of AIDS. The N-terminal amino acid sequences of these proteins show little homology to those of previously described anti-HIV proteins. The structure-function features of these HIV inhibitors, based on the 40–60 amino acid residues of N-terminal sequences, are examined.

A new inhibitor of human immunodeficiency virus (HIV) has been isolated and purified to homogeneity from the seeds and fruits of the Momordica charantia. This compound, MAP 30 (Momordica Anti-HIV Protein), is a basic protein of about 30 kDa. It exhibits dose-dependent inhibition of cell-free HIV-1 infection and replication as measured by: (i) quantitative focal syncytium formation on CEM-ss monolayers; (II) viral core protein p24 expression; and (iii) viral-associated reverse transcriptase (RT) activity in HIV-1 infected H9 cells. The doses required for 50% inhibition (ID 50) in these assays were 0.83, 0.22 and 0.33 nM, respectively. No cytotoxic or cytostatic effects were found under the assay conditions. These data suggest that MAP 30 may be a useful therapeutic agent in the treatment of HIV-1 infections. The sequence of the N-terminal 44 amino acids of MAP 30 has been determined.

Diffusion‐weighted MRI at 2 T was used to monitor and assess tissue damage after permanent middle cerebral artery occlusion (MCAO) in wild‐type (WT) and mice deficient in nitric oxide synthase gene expression (nNOS−). The ischemic lesion was evaluated 3 h after occlusion and subdivided into the lesion core and peri‐infarct zone based on the magnitude of the apparent diffusion coefficient (ADC) change. Infarct volume, measured by using histochemical staining 24 h after MCA occlusion, correlated best with MRI infarct volume as assessed by an ADC threshold of 25% decrease from baseline at 3 h. For ADC thresholds of greater than 25% decrease, lesion size was not significantly different in nNOS−and WT mice. However, brain tissue showing ADC decreases of 10–25% was significantly smaller in the ipsilateral hemisphere of mutants (27 · 2% and 21 · 2% in WT and nNOS−, respectively; P< 0.05). These findings occurred independently of infarct volume and are consistent with a smaller peri‐infarct zone in nNOS−mice. We postulate that the smaller peri‐infarct zone is a reflection of less severe metabolic disturbance after ischemia in nNOS−mice, possibly related to diminished production of nitric oxide (NO) or a related product. We conclude that magnetic resonance techniques previously used to assess ischemic damage in larger animals can be extended to the mouse, raising the possibility that the molecular mechanisms leading to ischemic damage can be examined by using genetically engineered mice.

1The role of endogenous nitric oxide (NO) generated by neuronal nitric oxide synthase (NOS‐1) in the control of respiration during hypoxia and hypercapnia was assessed using mutant mice deficient in NOS‐1.2Experiments were performed on awake and anaesthetized mutant and wild‐type control mice. Respiratory responses to varying levels of inspired oxygen (100, 21 and 12 % O2) and carbon dioxide (3 and 5 % CO2balanced oxygen) were analysed. In awake animals, respiration was monitored by body plethysmograph along with oxygen consumption (VO2), CO2production (VCO2) and body temperature. In anaesthetized, spontaneously breathing mice, integrated efferent phrenic nerve activity was monitored as an index of neural respiration along with arterial blood pressure and blood gases. Cyclic 3′,5′‐guanosine monophosphate (cGMP) levels in the brainstem were analysed by radioimmunoassay as an index of nitric oxide generation.3Unanaesthetizedmutant mice exhibited greater respiratory responses during 21 and 12 % O2than the wild‐type controls. Respiratory responses were associated with significant decreases in oxygen consumption in both groups of mice, and the magnitude of change was greater in mutant than wild‐type mice. Changes in CO2production and body temperature, however, were comparable between both groups of mice.4Similar augmentation of respiratory responses during hypoxia was also observed in anaesthetizedmutant mice. In addition, five of the fourteen mutant mice displayed periodic oscillations in respiration (brief episodes of increases in respiratory rate and tidal phrenic nerve activity) while breathing 21 and 12 % O2, but not during 100 % O2. The time interval between the episodes decreased by reducing inspired oxygen from 21 to 12 % O2.5Changes in arterial blood pressure and arterial blood gases were comparable at any given level of inspired oxygen between both groups of mice, indicating that changes in these variables do not account for the differences in the response to hypoxia.6Respiratory responses to brief hyperoxia (Dejours test) and to cyanide, a potent chemoreceptor stimulant, were more pronounced in mutant mice, suggesting augmented peripheral chemoreceptor sensitivity.7cGMP levels were elevated in the brainstem during 21 and 12 % O2in wild‐type but not in mutant mice, indicating decreased formation of nitric oxide in mutant mice.8The magnitude of respiratory responses to hypercapnia (3 and 5 % CO2balanced oxygen) was comparable in both groups of mice in the awake and anaesthetized conditions.9These observations suggest that the hypoxic responses were selectively augmented in mutant mice deficient in NOS‐1. Peripheral as well as central mechanisms contributed to the altered responses to hypoxia. These results support the idea that nitric oxide generated by NOS‐1 is an important physiological modulator of respiration during hypoxia.

Infarct volume and edema were assessed after transient focal ischemia in mice lacking neuronal nitric oxide synthase (NOS) gene expression. With use of an 8–0 coated monofilament, the middle cerebral artery (MCA) of mutant (n = 32) and wild-type mice [SV-129 (n = 31), C57Black/6 (n = 18)] were occluded for 3 h and reperfused for up to 24 h. Regional CBF (rCBF), neurological deficits, water content, and infarct volume were examined in all three strains. rCBF, blood pressure, and heart rate did not differ between groups when measured for 1 h after reperfusion. Neurological deficits were less severe in mutant mice after MCA occlusion. Brain water content at 3 h after reperfusion and infarct volume at 24 h after reperfusion were greater in wild-type mice. These data indicate that genetic deletion of neuronal NOS confers resistance to focal ischemic injury in a reperfusion model. The findings agree with previous studies showing that tissue injury is less extensive after both permanent MCA occlusion and global ischemia in mice lacking neuronal NOS gene expression. Hence, NO may play a pivotal role in the pathogenesis of ischemic brain damage.

Infarct size and vascular hemodynamics were measured 24 h after middle cerebral artery (MCA) occlusion in mice genetically deficient in the endothelial nitric oxide synthase (eNOS) isoform. eNOS mutant mice developed larger infarcts (21%) than the wild-type strain when assessed 24 h after intraluminal filament occlusion. Moreover, regional CBF values recorded in the MCA territory by laser-Doppler flowmetry were more severely reduced after occlusion and were disproportionately reduced during controlled hemorrhagic hypotension in autoregulation experiments. Unlike the situation in wild-type mice, nitro-L-arginine superfusion (1 mM) dilated pial arterioles of eNOS knockout mice in a closed cranial window preparation. As noted previously, eNOS mutant mice were hypertensive. However, infarct size remained increased despite lowering blood pressure to normotensive levels by hydralazine treatment. Systemic administration of nitro-L-arginine decreased infarct size in eNOS mutant mice (24%) but not in the wild-type strain. This finding complements published data showing that nitro-L-arginine increases infarct size in knockout mice expressing the eNOS but not the neuronal NOS isoform (i.e., neuronal NOS knockout mice). We conclude that NO production within endothelium may protect brain tissue, perhaps by hemodynamic mechanisms, whereas neuronal NO overproduction may lead to neurotoxicity.

SWATH-mass spectrometry (MS) enables accurate and reproducible proteomic profiling in multiple model organisms including the mouse. Here, we present a comprehensive mouse reference spectral library (MouseRefSWATH) that permits quantification of up to 10,597 proteins (62.2% of the mouse proteome) by SWATH-MS. We exploit MouseRefSWATH to develop an analytical pipeline for species-specific deconvolution of proteomic alterations in human tumour xenografts (XenoSWATH). This method overcomes the challenge of high sequence similarity between mouse and human proteins, facilitating the study of host microenvironment-tumour interactions from ‘bulk tumour’ measurements. We apply the XenoSWATH pipeline to characterize an intraductal xenograft model of breast ductal carcinoma in situ and uncover complex regulation consistent with stromal reprogramming, where the modulation of cell migration pathways is not restricted to tumour cells but also operates in the mouse stroma upon progression to invasive disease. MouseRefSWATH and XenoSWATH open new opportunities for in-depth and reproducible proteomic assessment to address wide-ranging biological questions involving this important model organism.

Nitric oxide (NO) has multiple important actions that contribute to the maintenance of vascular homeostasis. NO is synthesized by three different isoforms of NO synthase (NOS), all of which have been reported to be expressed in human atherosclerotic vascular lesions. Although the regulatory roles of endothelial NOS (eNOS) and inducible NOS (iNOS) on the development of atherosclerosis have been described, little is known about the role of neuronal NOS (nNOS). Here, we show that nNOS also exerts important vasculoprotective effects in vivo. In a carotid artery ligation model, nNOS gene‐deficient (nNOS‐KO) mice exhibited accelerated neointimal formation and constrictive vascular remodeling caused by blood flow disruption. In a rat balloon injury model, the selective inhibition of nNOS activity potently enhanced vasoconstrictor responses to a variety of calcium‐mobilizing stimuli, suppressed tissue cGMP concentrations, a marker of vascular NO production, and exacerbated neointimal formation. In both models, nNOS was absent before injury and was up‐regulated only after the injury, and was predominantly expressed in the neointima and medial smooth muscle cells. These results provide the first direct evidence that nNOS plays important roles in suppressing arteriosclerotic vascular lesion formation in vivo.

GAP 31 is an anti-HIV plant protein that we have identified and purified to homogeneity from Gelonium multiflorum. It is the first reported example of an anti-HIV agent capable of acting against multiple stages of the viral life cycle, on viral infection and viral replication. GAP 31 is a unique paragon of multi-functional protein. In addition to anti-HIV activity, it also exhibits anti-tumor action, DNA binding, RNA binding and ribosome inactivation. The present crystals diffract up to 2·0 Å resolution and belong to monoclinic space group P2₁. The cell dimensions are a = 49·30(2) Å, b = 44·57 Å, c = 137·78(7) Å and β = 98·32(3)°. There are two molecules of molecular weight 31 kDa in an asymmetric unit with a solvent content of 49%. Copyright 1994, 1999 Academic Press

OBJECTIVE: The association of human immunodeficiency virus with a clinical picture of motor neuron disease is uncommon, with three cases reported to date. This case represents an additional case of a human immunodeficiency virus—infected patient with apparent motor neuron disease. DESIGN: Single patient case report. SETTING: Large urban public hospital. PATIENT: A 45-year-old human immunodeficiency virus—positive Hispanic man who presented with muscle wasting, fasciculations, areflexia, cranial nerve deficits, and weakness progressing to a complete quadriplegia. RESULTS: Electrophysiologic data showed evidence of diffuse denervation with normal sensory and motor nerve conductions and no evidence of demyelination. Electromyography showed diffuse sharp waves and fibrillation. CONCLUSIONS: This case demonstrates a progressive motor neuron dysfunction in a patient positive for the human immunodeficiency virus and provides additional evidence that infection with the human immunodeficiency virus should be considered in the differential diagnosis of apparent motor neuron disease.

Regional cerebral blood flow (rCBF) was studied in type III nitric oxide (NO) synthase (endothelial, eNOS) mutant and wild type mice during mechanical whisker stimulation before and after nitro-L-arginine (L-NA) superfusion using the closed cranial window technique. rCBF increased equally in cortical barrel fields in both strains during stimulation, as measured by laser Doppler-flowmetry, and was inhibited by L-NA superfusion (1 mM) in both groups. Hence, coupling of blood flow and metabolism appears neuronal NOS- (nNOS) but not eNOS-dependent in cortical barrel fields of the mouse.

Mid-ocean ridges with well developed median valleys are commonly the sites of large normal-faulting earthquakes1,2. A recent global study3–6of the centroid depths and source mechanisms of such earthquakes has yielded two important conclusions: first, that all the largest mid-ocean ridge earthquakes occur beneath the inner floor of the median valley, and second, that the maximum thickness of the seismogenic layer increases with decreasing spreading rate. These results lend support to the hypothesis that the median valley arises from the necking of a strong, brittle lithosphere under horizontal extension7–9. Here we compare the global rate of seismic moment release along slowly spreading mid-ocean ridges with the rate predicted if widening of the median valley occurs entirely by seismogenic extension of the brittle lithosphere beneath the inner floor. We find that earthquakes account for no more than about 10–20% of the rate of plate separation across the central median valley.

Lymph nodes (LNs) act as filters, constantly sampling peripheral cues. This is facilitated by the conduit network, a tubular structure of aligned extracellular matrix (ECM) fibrils ensheathed by fibroblastic reticular cells (FRCs). LNs undergo rapid 3- to 5-fold expansion during adaptive immune responses, but these ECM-rich structures are not permanently damaged. Whether conduit flow or filtering function is affected during LN expansion is unknown. Here, we show that conduits are partially disrupted during acute LN expansion, but FRC-FRC contacts remain connected. We reveal that polarized FRCs deposit ECM basolaterally using LL5-β and that ECM production is regulated at transcriptional and secretory levels by the C-type lectin CLEC-2, expressed by dendritic cells. Inflamed LNs maintain conduit size exclusion, and flow is disrupted but persists, indicating the robustness of this structure despite rapid tissue expansion. We show how dynamic communication between peripheral tissues and LNs provides a mechanism to prevent inflammation-induced fibrosis in lymphoid tissue.

Slow-wave activity (SWA) is an oscillatory neocortical activity occurring in the electroencephalogram delta (d) frequency range (~0.5-4 Hz) during nonrapid eye movement sleep. SWA is a reliable indicator of sleep homeostasis after acute sleep loss and is involved in memory processes. Evidence suggests that cortical neuronal nitric oxide synthase (nNOS) expressing neurons that coexpress somatostatin (SST) play a key role in regulating SWA. However, previous studies lacked selectivity in targeting specific types of neurons that coexpress nNOS-cells which are activated in the cortex after sleep loss. We produced a mouse model that knocks out nNOS expression in neurons that coexpress SST throughout the cortex. Mice lacking nNOS expression in SST positive neurons exhibited significant impairments in both homeostatic low-d frequency range SWA production and a recognition memory task that relies on cortical input. These results highlight that SST+/nNOS+ neurons are involved in the SWA homeostatic response and cortex-dependent recognition memory.

Supplemental Digital Content is available in the text.

To the Editor.—Acute exertional rhabdomyolysis is a syndrome occurring in apparently normal, healthy individuals as a result of performing strenuous exercise. It has been observed in marathon runners1-3 and military recruits4-6 and the activities of mechanical bull riding,7 conga drumming,8 karate kicking,9 and wrestling.10 We now describe a case of rhabdomyolysis occurring after jetskiing—a growing water sport in which the operator rides a motorcycle-like vehicle on the water's surface.Report of a Case.—A previously healthy 31-year-old man was initially seen with the complaint of right arm pain and swelling. Four and five days earlier, he rode a jet ski for three hours daily. He experienced only "ordinary" muscle aches until two days later, when he noted swelling of his right arm from the wrist to shoulder accompanied by paresthesias of the right hand. The patient denied recent trauma or participation in strenuous

EGFR and downstream signaling networks contribute to the hallmark characteristics of glioma.

Akt mediates postnatal angiogenesis through eNOS signaling.

Nitric oxide (NO) plays a central role in the pathogenesis of bacterial meningitis. However, the role of NO produced by endothelial NO synthase (eNOS) in meningitis is still unclear. We investigated the influence of eNOS depletion on the inflammatory host response, intracranial complications, and outcome in experimental pneumococcal meningitis. Leukocyte accumulation in the cerebrospinal fluid was more pronounced in infected eNOS-deficient mice than in infected wild type mice. This effect could be attributed to an increased expression of p-selectin, macrophage inflammatory protein-2, keratinocyte-derived cytokine, and interleukin (IL)-1β in the brain of infected eNOS-deficient mice. However, no differences in the cerebral expression of intercellular adhesion molecule-1, tumor necrosis factor-α, and IL-6 as well as of neuronal NOS and inducible NOS could be detected between infected wild type and mutant mice. In addition to enhanced leukocyte infiltration into the CSF, meningitis-associated intracranial complications including blood-brain barrier disruption and the rise in intracranial pressure were significantly augmented in infected eNOS-deficient mice. The aggravation of intracranial complications was paralleled by a worsening of the disease, as evidenced by a more pronounced hypothermia, an enhanced weight reduction, and an increased death rate. The current data indicate that eNOS deficiency is detrimental in bacterial meningitis. This effect seems to be related to an increased expression of (certain) cytokines/chemokines and adhesion molecules; thus leading to increased meningeal inflammation and, subsequently, to aggravated intracranial complications.

Thiamine deficiency (TD) models the cellular and molecular mechanisms by which chronic oxidative deficits lead to death of select neurons in brain. Region- and cell-specific oxidative stress and vascular changes accompany the TD-induced neurodegeneration. The current studies analyzed the role of oxidative stress in initiating these events by testing the role of intercellular adhesion molecule-1 (ICAM-1) and endothelial nitric oxide synthase (eNOS) in the selective neuronal loss that begins in the submedial thalamic nucleus of mice. Oxidative stress to microvessels is known to induce eNOS and ICAM-1. TD increased ICAM-1 immunoreactivity in microvessels within the submedial nucleus and adjacent regions 1 day prior to the onset of neuronal loss. On subsequent days, the pattern of ICAM-1 induction overlapped that of neuronal loss, and of induction of the oxidative stress marker heme oxygenase-1 (HO-1). The intensity and extent of ICAM-1 and HO-1 induction progressively spread in parallel with the neuronal death in the thalamus. Targeted disruption of ICAM-1 or eNOS gene, but not the neuronal NOS gene, attenuated the TD-induced neurodegeneration and HO-1 induction. TD induced ICAM-1 in eNOS knockout mice, but did not induce eNOS in mice lacking ICAM-1. These results demonstrate that in TD, an ICAM-1-dependent pathway of eNOS induction leads to oxidative stress-mediated death of metabolically compromised neurons. Thus, TD provides a useful model to help elucidate the role of ICAM-1 and eNOS in the selective neuronal death in diseases in which oxidative stress is implicated.

We have investigated the role of nitric oxide as a mediator of the cerebrovascular blood flow response to hypercapnia in wild-type mice and knock-out mice that lack the neuronal nitric oxide synthase (NOS) gene. Mice were anesthetized, intubated, and ventilated. Baseline measurements of arterial blood pressure, end-tidal CO2, and blood gases were monitored, and were comparable in both groups of mice. Relative cerebral blood flow (rCBF) was measured by lased Doppler flowmetry. Hypercapnia induced by inhalation of 5 and 10% CO2 resulted in a 38±15 and 77±34% increase in rCBF in both the mutant and wild-type mice. Superfusion with nitro-L-arginine (LNA) markedly attenuated the rCBF response in wild-type mice, but did not affect the response in the knock-out mice. Brain cGMP levels increased in response to hypercapnia in the wild-type animals, but not in the knock-out animals. Endothelium dependent dilation to topical acetylcholine was completely blocked by topical L-NA in both groups, demonstrating that endothelial NOS is not involved in the preserved hypercapnic response in the knockout animals.