Kappa Opioid Signaling at the Crossroads of Chronic Pain and Opioid Addiction.

Pain is complex and is a unique experience for individuals in that no two people will have exactly the same physiological and emotional response to the same noxious stimulus or injury. Pain is composed of two essential processes: a sensory component that allows for discrimination of the intensity and location of a painful stimulus and an emotional component that underlies the affective, motivational, unpleasant, and aversive response to a painful stimulus. Kappa opioid receptor (KOR) activation in the periphery and throughout the neuroaxis modulates both of these components of the pain experience. In this chapter we focus on recent findings that KORs contribute to the emotional, aversive nature of chronic pain, including how expression in the limbic circuitry contributes to anhedonic states and components of opioid misuse disorder. While the primary focus is on preclinical pain models, we also highlight clinical or human research where there is strong evidence for KOR involvement in negative affective states associated with chronic pain and opioid misuse.

Early maladaptive schemas and depression in adulthood: A systematic review and meta-analysis.

BACKGROUND: Improved understanding of the specific cognitive risk factors associated with depression is needed to inform prevention and treatment approaches. Recent research has examined the relationship between early maladaptive schemas (EMSs) and depression, but the findings were yet to be integrated using meta-analytic methods. The aim of this review was to synthesize the evidence on the relationship between depression and EMS. METHOD: A systematic review and meta-analysis was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, by searching the PsycINFO, PubMed and CINAHL databases. Included studies were peer-reviewed journal articles that examined the relationship between one or more EMS and depression in adulthood in participants aged 18 years or older. RESULTS: A total of 51 studies were included (k = 743; pooled N = 17,830). All 18 EMSs were positively correlated with depression, with effect sizes ranging from small (r = .23 [.17, .29]; Entitlement) to large (r = .53 [.46, .60]; Social Isolation; r = .50, 95% CI [.45, .54]; Defectiveness/Shame). CONCLUSION: The evidence suggests that individuals who feel like they do not belong, or that they are flawed, bad or unlovable, report higher levels of depression. However, most studies used cross-sectional designs, and further longitudinal research is needed to establish the direction of the relationship between EMS and depression. These findings can guide preventative and treatment approaches. Focusing treatment on the Social Isolation and Defectiveness/Shame EMS may aid in relieving depressive symptoms.

Adverse childhood experiences and early maladaptive schemas in adulthood: A systematic review and meta-analysis.

BACKGROUND: Schema Therapy is based on the theory that trauma and neglect in childhood lead to early maladaptive schemas and psychopathology in adulthood. The aim of this review was to evaluate support for this theory by synthesizing the literature on childhood adversity and schemas. METHODS: A systematic review and meta-analysis were completed in compliance with PRISMA. PsycInfo, CINAHL and PubMed were searched to identify eligible studies that reported unadjusted association(s) between adverse childhood events and schema scores when participants were 18 years or older. Meta-analyses were conducted to estimate the pooled effect size of associations between schemas and experiences of childhood adversity. RESULTS: A total of 33 studies met inclusion criteria and provided sufficient data for meta-analyses on childhood experiences relating to toxic frustration of needs (emotional neglect and physical neglect) and trauma and victimization (emotional abuse, physical abuse and sexual abuse). Of the 124 meta-analyses, 65 indicated that schemas show small to large correlations with emotional neglect (range: r = .16 [Failure] to r = .51 [Emotional Deprivation]); small to moderate correlations with emotional abuse (range: r = .20 [Vulnerability to Harm] to r = .44 [Emotional Deprivation]); and small correlations with physical neglect, physical abuse and sexual abuse (range: r = .16 [Vulnerability to Harm] to .26 [Emotional Deprivation and Social Isolation]). CONCLUSIONS: Of the 33 included studies, only one used a longitudinal design. However, based on the correlational studies available, early maladaptive schemas in adulthood are associated with a history of childhood abuse and neglect.

Hyphal growth in Candida albicans requires the phosphorylation of Sec2 by the Cdc28-Ccn1/Hgc1 kinase.

Polarized growth is a fundamental property of cell growth and development. It requires the delivery of post-Golgi secretory vesicles to the site of polarized growth. This process is mediated by Rab GTPases activated by their guanine exchange factors (GEFs). The human fungal pathogen, Candida albicans, can grow in a budded yeast form or in a highly polarized hyphal form, and thus provides a model to study this phenomenon. During hyphal, but not yeast growth, secretory vesicles accumulate in an apical body called a Spitzenkörper, which acts to focus delivery of the vesicles to the tip. Post-Golgi transport of secretory vesicles is mediated by the Rab GTPase Sec4, activated by its GEF Sec2. Using a combination of deletion mapping, in vitro mutagenesis, an analogue-sensitive allele of Cdc28 and an in vitro kinase assay, we show that localization of Sec2 to the Spitzenkörper and normal hyphal development requires phosphorylation of Serine 584 by the cyclin-dependent kinase Cdc28. Thus, as well as controlling passage through the cell cycle, Cdc28 has an important function in controlling polarized secretion.

Sparse genetically defined neurons refine the canonical role of periaqueductal gray columnar organization.

During threat exposure, survival depends on defensive reactions. Prior works linked large glutamatergic populations in the midbrain periaqueductal gray (PAG) to defensive freezing and flight, and established that the overarching functional organization axis of the PAG is along anatomically-defined columns. Accordingly, broad activation of the dorsolateral column induces flight, while activation of the lateral or ventrolateral (l and vl) columns induces freezing. However, the PAG contains diverse cell types that vary in neurochemistry. How these cell types contribute to defense remains unknown, indicating that targeting sparse, genetically-defined populations may reveal how the PAG generates diverse behaviors. Though prior works showed that broad excitation of the lPAG or vlPAG causes freezing, we found in mice that activation of lateral and ventrolateral PAG (l/vlPAG) cholecystokinin-expressing (CCK) cells selectively caused flight to safer regions within an environment. Furthermore, inhibition of l/vlPAG-CCK cells reduced predator avoidance without altering other defensive behaviors like freezing. Lastly, l/vlPAG-CCK activity decreased when approaching threat and increased during movement to safer locations. These results suggest CCK cells drive threat avoidance states, which are epochs during which mice increase distance from threat and perform evasive escape. Conversely, l/vlPAG pan-neuronal activation promoted freezing, and these cells were activated near threat. Thus, CCK l/vlPAG cells have opposing function and neural activation motifs compared to the broader local ensemble defined solely by columnar boundaries. In addition to the anatomical columnar architecture of the PAG, the molecular identity of PAG cells may confer an additional axis of functional organization, revealing unexplored functional heterogeneity.

Osmoregulation in Streptomyces coelicolor: modulation of SigB activity by OsaC.

As free-living non-motile saprophytes, Streptomyces need to adapt to a wide range of environmental conditions and this is reflected by an enormous diversity of regulatory proteins encoded by, for example, the genome of the model streptomycete Streptomyces coelicolor. In this organism, we have identified a new osmoregulation gene, osaC, encoding a member of a novel family of regulatory proteins. Members of the family have a predicted domain composition consisting of an N-terminal kinase domain related to anti-sigma factors, sensory Pas and Gaf domains, and a C-terminal phosphatase domain. osaC is linked to the response regulator gene osaB; expression analysis of the latter revealed that it is induced after osmotic stress in a sigma(B)-dependent manner. OsaC is required to return osaB and sigB expression back to constitutive levels after osmotic stress. From analysis of the activities of OsaC(DeltaPho), lacking the C-terminal phosphatase domain, and OsaC(N92A), with a substitution of a critical asparagine residue in the kinase domain, we infer that this N-terminal domain functions as a sigma(B) anti-sigma factor. Indeed, co-purification experiments indicate association of OsaC and sigma(B). These results support a model for post-osmotic stress modulation of sigma(B) activity by OsaC.

Mitigation of peroxynitrite-mediated nitric oxide (NO) toxicity as a mechanism of induced adaptive NO resistance in the CNS.

During CNS injury and diseases, nitric oxide (NO) is released at a high flux rate leading to formation of peroxynitrite (ONOO(*)) and other reactive nitrogenous species, which nitrate tyrosines of proteins to form 3-nitrotyrosine (3NY), leading to cell death. Previously, we have found that motor neurons exposed to low levels of NO become resistant to subsequent cytotoxic NO challenge; an effect dubbed induced adaptive resistance (IAR). Here, we report IAR mitigates, not only cell death, but 3NY formation in response to cytotoxic NO. Addition of an NO scavenger before NO challenge duplicates IAR, implicating reactive nitrogenous species in cell death. Addition of uric acid (a peroxynitrite scavenger) before cytotoxic NO challenge, duplicates IAR, implicating peroxynitrite, with subsequent 3NY formation, in cell death, and abrogation of this pathway as a mechanism of IAR. IAR is dependent on the heme-metabolizing enzyme, heme oxygenase-1 (HO1), as indicated by the elimination of IAR by a specific HO1 inhibitor, and by the finding that neurons isolated from HO1 null mice have increased NO sensitivity with concomitant increased 3NY formation. This data indicate that IAR is an HO1-dependent mechanism that prevents peroxynitrite-mediated NO toxicity in motor neurons, thereby elucidating therapeutic targets for the mitigation of CNS disease and injury.

Differential sensitivity of oligodendrocytes and motor neurons to reactive nitrogen species: implications for multiple sclerosis.

Depending on its concentration, nitric oxide (NO) has beneficial or toxic effects. In pathological conditions, NO reacts with superoxide to form peroxynitrite, which nitrates proteins forming nitrotyrosine residues (3NY), leading to loss of protein function, perturbation of signal transduction, and cell death. 3NY immunoreactivity is present in many CNS diseases, particularly multiple sclerosis. Here, using the high flux NO donor, spermine-NONOate, we report that oligodendrocytes are resistant to NO, while motor neurons are NO sensitive. Motor neuron sensitivity correlates with the NO-dependent formation of 3NY, which is significantly more pronounced in motor neurons when compared with oligodendrocytes, suggesting peroxynitrite as the toxic molecule. The heme-metabolizing enzyme, heme-oxygenase-1 (HO1), is necessary for oligodendrocyte NO resistance, as demonstrated by loss of resistance after HO1 inhibition. Resistance is reinstated by peroxynitrite scavenging with uric acid further implicating peroxynitrite as responsible for NO sensitivity. Most importantly, differential sensitivity to NO is also present in cultures of primary oligodendrocytes and motor neurons. Finally, motor neurons cocultured with oligodendrocytes, or oligodendrocyte-conditioned media, become resistant to NO toxicity. Preliminary studies suggest oligodendrocytes release a soluble factor that protects motor neurons. Our findings challenge the current paradigm that oligodendrocytes are the exclusive target of multiple sclerosis pathology.

Transcript-specific translational regulation in the unfolded protein response of Saccharomyces cerevisiae.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes stress and induces the unfolded protein response (UPR). Genome-wide analysis of translational regulation in response to the UPR-inducing agent dithiothreitol in Saccharomyces cerevisiae is reported. Microarray analysis, confirmed using qRT-PCR, identified transcript-specific translational regulation. Transcripts with functions in ribosomal biogenesis and assembly were translationally repressed. In contrast, mRNAs from known UPR genes, encoding the UPR transcription factor Hac1p, the ER-oxidoreductase Ero1p and the ER-associated protein degradation (ERAD) protein Der1p, were enriched in polysomal fractions, indicating translational up-regulation. Splicing of HAC1 mRNA is shown to be required for efficient ribosomal loading.

NO signaling in the CNS: from the physiological to the pathological.

Nitric oxide (NO) is a free radical gas that has a Janus nature. As indicated by the literature and by our studies, in the cell, NO can either function as a beneficial physiological agent utilized for essential functions such as differentiation or neurotransmission, or as a pathological agent that causes or exacerbates central nervous system (CNS) disease and injury. Whether NO is helpful or harmful depends on a variety of factors, such as the cellular environment in which NO is released, the rate of NO flux, as determined by which NOS isozyme is activated, and what array of second messenger cascades are available for utilization by NO for beneficial or toxic cell signalling. Understanding the mechanisms by which NO is salutary in one set of circumstances and toxic in another is critical and will offer therapeutic targets for the mitigation of NO-mediated damage seen during CNS disease and injury. In fact, we have utilized the duality the NO to, in motor neurons, induce adaptive resistance (IAR) to toxic doses of NO. Understanding how the actions of NO are transduced in the cell will lead us to more targeted application of therapies such as IAR.

Induced adaptive resistance to oxidative stress in the CNS: a discussion on possible mechanisms and their therapeutic potential.

The free radical, nitric oxide (NO), is synthesized by mammalian cells and is utilized for normal cellular functions. High levels of NO are released during disease, injury and inflammation. NO at high concentrations more readily combines with other oxidants to form reactive nitrogenous species (RNS), which can wreak havoc on the cell by damaging a variety of cellular targets, such as DNA and proteins, ultimately leading to apoptosis, mutagenesis or carcinogenesis. Cells have natural resistance mechanisms to nitrooxidative stress that are either defective (as can occur in disease), or overwhelmed (as can occur in injury and inflammation). It has been found recently in the CNS that resistance to normally toxic levels of NO can be induced by nontoxic levels of NO and that this induction is correlated with and dependent upon increased levels and activity of the heme-metabolizing enzyme, heme oxygenase-1 (HO-1). HO1-mediated metabolism of heme groups released from NO-damaged proteins leads to a change in the levels of redox-active iron and a release of carbon monoxide (CO) and bilirubin, all of which have been implicated in cellular resistance to oxidative stress. Perhaps one or more of the products of HO1 heme metabolism is involved in induced adaptive resistance or perhaps a heme-independent mechanism is involved. In fact, a variety of possible mechanisms may be involved in induced resistance to NO in the CNS. Ultimately elucidating these mechanisms will enable us to modulate them for therapeutic potential.

Thoracoscopic lobectomy and segmentectomy for infectious lung disease.

BACKGROUND: The potential benefits of thoracoscopic lobectomy and segmentectomy for early stage non-small cell lung cancer have been well documented in the literature. However, little is known about the use of these techniques in patients requiring resection for infectious or inflammatory lung disease. METHODS: Using a prospectively collected database, we performed a retrospective review of consecutive operations from July 2004 to June 2010. All patients who underwent elective thoracoscopic lobectomy or segmentectomy for focal bronchiectasis or cavitary lung disease associated with active pulmonary infection were included. RESULTS: In all, 212 resections were performed in 171 patients. The average age was 59 years (range, 26 to 82 years). Patients were predominately white (93%) and female (93%). Indications for surgery included recurrent active infection, hemoptysis, or antibiotic intolerance associated with focal bronchiectasis (86%), cavitary disease (7%), or both (7%). Operations included 126 lobectomies, 73 segmentectomies, 10 lobe plus segmental resections, and 3 bilobectomies. Conversion to thoracotomy occurred in 10 patients. The operative mortality rate was zero. Complications occurred in 9%, consisting largely of prolonged air leak and atrial fibrillation. The mean hospital length of stay was 3.7 days. CONCLUSIONS: Thoracoscopic lobectomy and segmentectomy for individuals with infectious lung disease can be accomplished safely with minimal morbidity and mortality. These techniques may provide the optimal surgical approach for patients with focal bronchiectasis or cavitary lung disease requiring resection.

Lady Windermere revisited: treatment with thoracoscopic lobectomy/segmentectomy for right middle lobe and lingular bronchiectasis associated with non-tuberculous mycobacterial disease.

OBJECTIVE: Lady Windermere syndrome is a well-known but poorly understood female predominant phenotype of isolated right middle lobe and lingular bronchiectasis associated with non-tuberculous mycobacterial (NTM) infection. Despite lengthy multidrug antibiotic treatment, the presence of damaged parenchymal tissue leads to symptomatic disease recurrence, often with resistant organisms. The use of surgical resection as an adjunct to medical therapy may alter this cycle, although little is known about the use of thoracoscopic lung resection in this patient population. METHODS: This is a retrospective review of a prospectively collected database of patients with pulmonary NTM disease from July 2004 to December 2009. All patients had focal bronchiectasis of the right middle lobe and lingula, treated with targeted antimicrobial therapy for several months prior to resection. RESULTS: A total of 134 patients underwent 172 operations, with 38 patients having staged bilateral resections. The cohort was predominately female (96%) and Caucasian (95%), with a mean age of 59 years (range 34-81 years). Using a thoracoscopic approach in all patients, 102 middle lobectomies and 70 lingulectomies were performed. Conversion to open thoracotomy occurred in five cases (3%). Secondary procedures were performed in 20 cases (12%). There was no operative mortality. Postoperative morbidity was noted following 12 operations (7%), primarily consisting of prolonged air leak. The mean length of stay was 3.3 days (range 1-15 days). CONCLUSIONS: Although medical therapy remains the primary treatment modality for patients with pulmonary NTM disease, the selective use of pulmonary resection may reduce the incidence of symptomatic disease recurrence. The addition of thoracoscopic resection to treatment regimens for patients with Lady Windermere syndrome can be accomplished with minimal morbidity and mortality.

Anatomic lung resection for nontuberculous mycobacterial disease.

BACKGROUND: Chronic lung infections involving nontuberculous mycobacteria are often inadequately treated owing to concomitant lung parenchymal damage, leading to persistence of the offending organisms. Little is known about the results of surgical therapy as part of a multimodality approach to these infections. METHODS: A retrospective review was conducted of 236 consecutive patients who underwent anatomic lung resection for nontuberculous mycobacteria disease at our institution as part of a multimodality treatment program. RESULTS: In all, 236 patients underwent 265 operations. The average age was 54 years (range, 23 to 77). Fifty-three patients had prior thoracic procedures. All patients had in-vitro sensitivity testing of cultured organisms, and had several months of guided antibiotic therapy. Special emphasis was placed on nutritional status. Eighty percent of patients had Mycobacterium avium complex disease. Anatomic lung resection was performed in all patients, with 126 lobectomies, 55 segmentectomies, 44 pneumonectomies, and 40 mixed procedures. Sixty-seven patients had either muscle or omental transposition. Mortality rate was 2.6%. The major morbidity rate was 11.7%. Average length of stay was 6.5 days. Presence on postoperative bronchopleural fistula was associated with positive sputum at operation and right pneumonectomy, particularly right completion pneumonectomy. CONCLUSIONS: This series represents the largest cohort of patients in the literature to date who underwent operation for nontuberculous mycobacteria infection. Surgery for nontuberculous mycobacteria disease may be accomplished with minimal morbidity and mortality. A multidisciplinary approach including targeted antimicrobial therapy and complete anatomic resection is the key to success.

Phenotypic heterogeneity can enhance rare-cell survival in 'stress-sensitive' yeast populations.

Individual cells within isogenic microbial cultures exhibit phenotypic heterogeneity, an issue that is attracting intense interest. Heterogeneity could confer benefits, in generating variant subpopulations that may be better equipped to persist during perturbation. We tested this hypothesis by comparing the survival of wild-type Saccharomyces cerevisiae with that of mutants which are considered stress-sensitive but which, we demonstrate, also have increased heterogeneity. The mutants (e.g. vma3, ctr1, sod1) exhibited the anticipated sensitivities to intermediate doses of nickel, copper, alkaline pH, menadione or paraquat. However, enhanced heterogeneity meant that the resistances of individual mutant cells spanned a broad range, and at high stress occasional-cell survival in most of these populations overtook that of the wild type. Green fluorescent protein (GFP) reporter studies showed that this heterogeneity-dependent advantage was not related to perturbation of buffered gene expression. Deletion strain screens combined with other approaches revealed that vacuolar alkalinization resulting from loss of Vma-dependent vacuolar H(+)-ATPase activity was not the cause of vma mutants' net stress sensitivities. An alternative Vma-dependent resistance mechanism was found to suppress an influence of variable vacuolar pH on the metal resistances of individual wild-type cells. In addition to revealing new mechanisms of heterogeneity generation, the results demonstrate experimentally a benefit under adverse conditions that arises specifically from heterogeneity, and in populations conventionally considered to be disadvantaged.

Removal rate model for magnetorheological finishing of glass.

Magnetorheological finishing (MRF) is a deterministic subaperture polishing process. The process uses a magnetorheological (MR) fluid that consists of micrometer-sized, spherical, magnetic carbonyl iron (CI) particles, nonmagnetic polishing abrasives, water, and stabilizers. Material removal occurs when the CI and nonmagnetic polishing abrasives shear material off the surface being polished. We introduce a new MRF material removal rate model for glass. This model contains terms for the near surface mechanical properties of glass, drag force, polishing abrasive size and concentration, chemical durability of the glass, MR fluid pH, and the glass composition. We introduce quantitative chemical predictors for the first time, to the best of our knowledge, into an MRF removal rate model. We validate individual terms in our model separately and then combine all of the terms to show the whole MRF material removal model compared with experimental data. All of our experimental data were obtained using nanodiamond MR fluids and a set of six optical glasses.

Myostatin auto-regulates its expression by feedback loop through Smad7 dependent mechanism.

Myostatin, a secreted growth factor, is a member of the TGF-beta superfamily and an inhibitor of myogenesis. Previously, we have shown that myostatin gene expression is regulated at the level of transcription and that myostatin is a downstream target gene of MyoD. Here we show that myostatin gene expression is auto-regulated by a negative feedback mechanism. Northern blot analysis indicated that there are relatively higher levels of myostatin mRNA in the biceps femoris muscle of cattle that express a non- functional myostatin allele (Belgian Blue) as compared to normal cattle. In contrast, addition of exogenous myostatin decreases endogenous myostatin mRNA. Consistent with this result, wild type myostatin protein is able to repress myostatin promoter activity via Activin type IIb receptor (ActRIIB) and ALK5 (P < 0.001). However, non-functional myostatin (Piedmontese) failed to repress the myostatin promoter suggesting that myostatin auto-regulates its promoter by negative feedback inhibition. Auto-regulation by myostatin appears to be signaled through Smad7, since the expression of the inhibitory Smad7 is induced by myostatin and the over-expression of Smad7 in turn inhibits the myostatin promoter activity (P < 0.001). In contrast down regulation of Smad7 by siRNA results in increased myostatin mRNA indicating that Smad7 is a negative regulator of myostatin gene expression. Consistent with these results, a decrease in Smad7 mRNA and concomitant increase in myostatin expression is seen in myotubes that express non functional myostatin. In addition, interference with myostatin signaling prevents the induction of Smad7 promoter activity by myostatin. Based on these results, we propose that myostatin auto-regulates its gene expression through a Smad7 dependent mechanism in myogenic cells.

The therapeutic potential of agents that inactivate myostatin.

Myostatin is a member of the TGF-beta superfamily of secreted growth factors. A lack of functional myostatin or inhibition of the normal myostatin function results in an increased muscling phenotype and, conversely, the systemic administration of myostatin results in muscle wasting. Thus, myostatin is well established as a negative regulator of skeletal muscle mass. Myostatin binds to cell-surface receptors to inhibit both the proliferation and differentiation of myoblasts. Moreover, it functions to regulate both embryonic and post-natal musculature. Thus, potential antagonists to myostatin, whether targeting myostatin synthesis, secretion or receptor binding, show great promise as therapies against muscle-wasting diseases. This review provides an expert opinion on the biology and potential of myostatin antagonists in the treatment of muscle-wasting disorders.

Systematic insertional mutagenesis of a streptomycete genome: a link between osmoadaptation and antibiotic production.

The model organism Streptomyces coelicolor represents a genus that produces a vast range of bioactive secondary metabolites. We describe a versatile procedure for systematic and comprehensive mutagenesis of the S. coelicolor genome. The high-throughput process relies on in vitro transposon mutagenesis of an ordered cosmid library; mutagenized cosmids with fully characterized insertions are then transferred by intergeneric conjugation into Streptomyces, where gene replacement is selected. The procedure can yield insertions in upward of 90% of genes, and its application to the entire genome is underway. The methodology could be applied to many other organisms that can receive DNA via RK2/RP4-mediated intergeneric conjugation. The system permits introduction of mutations into different genetic backgrounds and qualitative measurement of the expression of disrupted genes as demonstrated in the analysis of a hybrid histidine kinase and response regulator gene pair, osaAB, involved in osmoadaptation in Streptomyces. The independently transcribed response regulator gene, osaB, is essential for osmoadaptation; when grown with supplementary osmolyte, an osaB mutant cannot erect aerial hyphae and produces up to fivefold greater antibiotic yields than the wild-type strain.

A key role for heme oxygenase-1 in nitric oxide resistance in murine motor neurons and glia.

Nitric oxide is utilized at low levels for intercellular signaling, and at high levels as a cytotoxic weapon during inflammation. Cellular NO resistance can be increased by prior exposure to sublethal NO levels to induce defense gene expression (adaptive NO resistance), which has been correlated with increased expression of heme oxygenase-1 (HO1) and was blocked by a heme oxygenase inhibitor. However, the possibility remained that other activities were affected by the inhibitor. To address this question, we conducted a genetic study of the HO1 role. We show here that primary cultures of spinal motor neurons and glia from homozygous HO1-null mice are strikingly more sensitive to NO cytotoxicity than are cells expressing HO1. Following an exposure to NO, the HO1-deficient cells were much more prone to apoptosis than were HO1-expressing cells with either one or two copies of a functional HO1 gene. These results confirm the in vivo role of HO1 as a front-line defense against NO toxicity in neuronal cells.