Nuclear poly(ADP-ribose) activity is a therapeutic target in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating and fatal motor neuron disease. Diagnosis typically occurs in the fifth decade of life and the disease progresses rapidly leading to death within ~ 2-5 years of symptomatic onset. There is no cure, and the few available treatments offer only a modest extension in patient survival. A protein central to ALS is the nuclear RNA/DNA-binding protein, TDP-43. In > 95% of ALS patients, TDP-43 is cleared from the nucleus and forms phosphorylated protein aggregates in the cytoplasm of affected neurons and glia. We recently defined that poly(ADP-ribose) (PAR) activity regulates TDP-43-associated toxicity. PAR is a posttranslational modification that is attached to target proteins by PAR polymerases (PARPs). PARP-1 and PARP-2 are the major enzymes that are active in the nucleus. Here, we uncovered that the motor neurons of the ALS spinal cord were associated with elevated nuclear PAR, suggesting elevated PARP activity. Veliparib, a small-molecule inhibitor of nuclear PARP-1/2, mitigated the formation of cytoplasmic TDP-43 aggregates in mammalian cells. In primary spinal-cord cultures from rat, Veliparib also inhibited TDP-43-associated neuronal death. These studies uncover that PAR activity is misregulated in the ALS spinal cord, and a small-molecular inhibitor of PARP-1/2 activity may have therapeutic potential in the treatment of ALS and related disorders associated with abnormal TDP-43 homeostasis.

Reduced activity of AMP-activated protein kinase protects against genetic models of motor neuron disease.

A growing body of research indicates that amyotrophic lateral sclerosis (ALS) patients and mouse models of ALS exhibit metabolic dysfunction. A subpopulation of ALS patients possesses higher levels of resting energy expenditure and lower fat-free mass compared to healthy controls. Similarly, two mutant copper zinc superoxide dismutase 1 (mSOD1) mouse models of familial ALS possess a hypermetabolic phenotype. The pathophysiological relevance of the bioenergetic defects observed in ALS remains largely elusive. AMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and thus might be activated in various models of ALS. Here, we report that AMPK activity is increased in spinal cord cultures expressing mSOD1, as well as in spinal cord lysates from mSOD1 mice. Reducing AMPK activity either pharmacologically or genetically prevents mSOD1-induced motor neuron death in vitro. To investigate the role of AMPK in vivo, we used Caenorhabditis elegans models of motor neuron disease. C. elegans engineered to express human mSOD1 (G85R) in neurons develops locomotor dysfunction and severe fecundity defects when compared to transgenic worms expressing human wild-type SOD1. Genetic reduction of aak-2, the ortholog of the AMPK α2 catalytic subunit in nematodes, improved locomotor behavior and fecundity in G85R animals. Similar observations were made with nematodes engineered to express mutant tat-activating regulatory (TAR) DNA-binding protein of 43 kDa molecular weight. Altogether, these data suggest that bioenergetic abnormalities are likely to be pathophysiologically relevant to motor neuron disease.

Gadolinium-induced nephrogenic systemic fibrosis in a patient with an acute and transient kidney injury.

Nephrogenic systemic fibrosis (NSF) describes a characteristic fibrosing disorder which typically presents with indurated plaques on the trunk and extremities of patients with advanced renal disease. We present a case of biopsy-confirmed NSF in a patient with severe acute kidney injury with no prior history of renal disease. A 64-year-old man with an acute and severe decrease in glomerular filtration rate underwent magnetic resonance imaging studies with gadolinium contrast (Omniscan) and subsequently developed NSF. His renal disease had normalized at the time his skin disease developed. Skin biopsies revealed findings of NSF and scanning electron microscopy with energy-dispersive X-ray spectroscopy confirmed insoluble gadolinium within lesional tissue.

Disruption of the FA/BRCA pathway in bladder cancer.

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression.

Fanconi anemia: causes and consequences of genetic instability.

Fanconi anemia (FA) is a rare recessive disease that reflects the cellular and phenotypic consequences of genetic instability: growth retardation, congenital malformations, bone marrow failure, high risk of neoplasia, and premature aging. At the cellular level, manifestations of genetic instability include chromosomal breakage, cell cycle disturbance, and increased somatic mutation rates. FA cells are exquisitely sensitive towards oxygen and alkylating drugs such as mitomycin C or diepoxybutane, pointing to a function of FA genes in the defense against reactive oxygen species and other DNA damaging agents. FA is caused by biallelic mutations in at least 12 different genes which appear to function in the maintenance of genomic stability. Eight of the FA proteins form a nuclear core complex with a catalytic function involving ubiquitination of the central FANCD2 protein. The posttranslational modification of FANCD2 promotes its accumulation in nuclear foci, together with known DNA maintenance proteins such as BRCA1, BRCA2, and the RAD51 recombinase. Biallelic mutations in BRCA2 cause a severe FA-like phenotype, as do biallelic mutations in FANCD2. In fact, only leaky or hypomorphic mutations in this central group of FA genes appear to be compatible with life birth and survival. The newly discovered FANCJ (= BRIP1) and FANCM (= Hef ) genes correspond to known DNA-maintenance genes (helicase resp. helicase-associated endonuclease for fork-structured DNA). These genes provide the most convincing evidence to date of a direct involvement of FA genes in DNA repair functions associated with the resolution of DNA crosslinks and stalled replication forks. Even though genetic instability caused by mutational inactivation of the FANC genes has detrimental effects for the majority of FA patients, around 20% of patients appear to benefit from genetic instability since genetic instability also increases the chance of somatic reversion of their constitutional mutations. Intragenic crossover, gene conversion, back mutation and compensating mutations in cis have all been observed in revertant, and, consequently, mosaic FA-patients, leading to improved bone marrow function. There probably is no other experiment of nature in our species in which causes and consequences of genetic instability, including the role of reactive oxygen species, can be better documented and explored than in FA.

Partial liquid ventilation in acute salt water-induced lung injury.

BACKGROUND AND OBJECTIVES: Salt-water aspiration results in pulmonary oedema and hypoxia. We tested the hypothesis that partial liquid ventilation has beneficial effects on gas exchange and rate of survival in acute and extended salt water-induced lung injury. METHODS: Anaesthetized, ventilated rats (tidal volume 6 mL kg(-1), PEEP 5 cmH2O) received a tracheal salt-water instillation (3%, 8 mL kg(-1) body weight) and were randomly assigned to three groups (n = 10 per group). While lungs of Group 1 were gas-ventilated, lungs of Group 2 received a single perfluorocarbon instillation (30 min after the injury, 5 mL kg(-1) perfluorocarbon) and lungs of Group 3 received an additional continuous perfluorocarbon application into the treachea (5 mL kg(-1) h(-1)) Arterial blood gases were measured with an intravascular blood gas sensor. RESULTS: Salt-water instillation resulted in a marked decrease in PaO2 values within 30 min (from 432 +/- 65 to 83 +/- 40 mmHg, FiO2 = 1.0, P < 0.01). Arterial oxygenation improved in all three groups irrespective of treatment. We observed no significant differences between groups in peak PaO2 and PaCO2 values. CONCLUSIONS: Our results suggest that partial liquid ventilation has no additional beneficial effects on gas exchange after life-threatening salt water-induced lung injury when compared to conventional gas ventilation with positive end-expiratory pressure.

Screening Fanconi anemia lymphoid cell lines of non-A, C, D2, E, F, G subtypes for defects in BRCA2/FANCD1.

Biallelic mutations in BRCA2/FANCD1 were recently recognized as a rare cause of Fanconi anemia (FA). Using immunodetection with an antiserum directed against the carboxyterminus of the BRCA2 protein, we screened 38 lymphoid cell lines from FA patients whom we could not previously assign, via retroviral complementation analysis, to any of six known FA complementation groups (FA-A, -C, -D2, -E, -F, or -G). Three of these 38 cell lines lacked the 380-kDa BRCA2 signal on immunoblots. DNA sequencing showed biallelic compound and truncating mutations in two of the immuno-negative cell lines, whereas a monoallelic frameshift mutation and an amino acid substitution were detected in the third cell line. Our data show that less than 10% of unassigned FA cell lines harbor truncating mutations in BRCA2/FANCD1. This finding strongly suggests the existence of (an) additional, as yet unknown FA gene(s).

Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway.

A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin-1 (Ang-1) is an endothelium-specific ligand essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serine-threonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo.

Requirement for nitric oxide activation of p21(ras)/extracellular regulated kinase in neuronal ischemic preconditioning.

The mechanisms underlying neuronal ischemic preconditioning, a phenomenon in which brief episodes of ischemia protect against the lethal effects of subsequent periods of prolonged ischemia, are poorly understood. Ischemia can be modeled in vitro by oxygen-glucose deprivation (OGD). We report here that OGD preconditioning induces p21(ras) (Ras) activation in an N-methyl-D-aspartate receptor- and NO-dependent, but cGMP-independent, manner. We demonstrate that Ras activity is necessary and sufficient for OGD tolerance in neurons. Pharmacological inhibition of Ras, as well as a dominant negative mutant Ras, block OGD preconditioning whereas a constitutively active form of Ras promotes neuroprotection against lethal OGD insults. In contrast, the activity of phosphatidyl inositol 3-kinase is not required for OGD preconditioning because inhibition of phosphatidyl inositol 3-kinase with a chemical inhibitor or with a dominant negative mutant does not have any effect on the development of OGD tolerance. Furthermore, using recombinant adenoviruses and pharmacological inhibitors, we show that downstream of Ras the extracellular regulated kinase cascade is required for OGD preconditioning. Our observations indicate that activation of the Ras/extracellular regulated kinase cascade by NO is a critical mechanism for the development of OGD tolerance in cortical neurons, which may also play an important role in ischemic preconditioning in vivo.

G(o) protein-dependent survival of primary accessory olfactory neurons.

Extensive G protein-coupled receptor families in both the main and accessory olfactory systems have been implicated in axonal targeting, sensory function, and cell survival. Although sensory function seems to be mediated by G proteins, axonal guidance and cell survival may be G protein-independent processes. In the accessory olfactory system, the G(o)-containing neurons in the basal vomeronasal organ (VNO) project to the posterior accessory olfactory bulb (AOB), whereas more apically located VNO neurons contain G(i2) and project to the anterior AOB. Herein, we investigate the organization of the accessory olfactory system in mice with a targeted deletion in the G(o)alpha gene. The accessory olfactory system seems normal at birth; however, postnatally, the number of G(o)-receptor-containing VNO neurons decreases by half, and apoptotic neurons are detected. The axons of VNO neurons remain restricted to the posterior AOB. The posterior AOB is reduced in size but contains a synaptophysin-positive layer with the normal number of glomeruli. The posterior AOB has reduced mitral cell c-Fos immunoreactivity, consistent with decreased sensory activation of G(o) protein-coupled VNO receptor neurons. Thus, in the accessory olfactory system, receptor-coupled G proteins are required for cell survival.

Granular cell dermatofibroma. A benign tumor that can simulate malignancy.

We report on an unusual dermatofibroma with granular cells. The dermatofibroma contained mitotic figures raising a differential diagnosis that included malignant granular cell tumor. Granular cell dermatofibroma is an uncommon variant, but one that clinicians and surgeons should become aware of to ensure accurate diagnosis.

New polymorphism on platelet glycoprotein IIIa gene recognized by endonuclease Msp I: implications for PlA typing by allele-specific restriction analysis.

BACKGROUND: Five human platelet alloantigen systems have been shown to result from single base pair substitutions in encoding regions of platelet glycoprotein genes IIIa, Ib, IIb, and Ia. For each of the diallelic systems, at least one restriction enzyme is known to cut only one of the two haplotypes. In the PlA system, restriction endonucleases Nci I and Msp I both recognize the PlA2 allele. STUDY DESIGN AND METHODS: A causal observation of an unexpected Msp I restriction pattern of a PlA2/PlA2 individual was made. Samples from 261 blood donors were then typed for antigens of the PlA system by restriction fragment length polymorphism analysis using the Nci I and Msp I restriction enzymes. RESULTS: Applying both enzymes, concordant restriction patterns were found in 258 of 261 blood donors. Three donors had a base pair mutation on the PlA2 allele, which creates an additional restriction site for Msp I 20 base pairs downstream from the PlA polymorphic site. Nucleotide sequence analysis revealed a CT217-->CG217G base exchange resulting in a Leu40-->Arg40 polymorphism of glycoprotein IIIa. CONCLUSION: Presuming that the mutation is not a singular phenomenon and also occurs with the PlA1 haplotype, it could lead to false interpretations of restriction analysis with Msp I. To exclude that possibility, Nci I is preferred for restriction fragment length polymorphism typing in the PlA system.

A point mutation leads to an unpaired cysteine residue and a molecular weight polymorphism of a functional platelet beta 3 integrin subunit. The Sra alloantigen system of GPIIIa.

Recently, we described a low frequency platelet alloantigen on human platelet membrane glycoprotein (GP) IIIa, termed Sra, that was involved in neonatal alloimmune thrombocytopenia. To identify the molecular nature of the Sra alloantigen, we analyzed the nucleotide sequence of polymerase chain reaction-amplified GPIIIa mRNA, and found a C2004-->T substitution in seven Sra positive individuals which results in an Arg636-->Cys polymorphism within the cysteine-rich region of GPIIIa. Analysis of allele-specific recombinant forms of GPIIIa that differed only at amino acid residue 636 showed that anti-Sra alloantibodies reacted with the Cys636, but not the Arg636, recombinant form of GPIIIa. Interestingly, under reducing conditions, the Cys636 form of GPIIIa migrated with a slightly increased apparent molecular weight compared with the Arg636 form. Following treatment with Endoglycosidase H, both allelic forms of GPIIIa exhibited the same mobility, however the Sra epitope was lost. Sra positive platelets express the same number of GPIIb-IIIa complexes on their surface as wild-type Sra negative platelets, and also aggregate normally in response to a variety of platelet agonists. Based upon these results, we conclude that 1) GPIIIa residue 636 specifically controls the formation and expression of the Sra alloantigenic determinant, and 2) an unpaired cysteine residue alters the N-linked glycosylation pattern of the extracellular domain of GPIIIa, but affects neither the degree of surface expression nor the adhesive function of the GPIIb-IIIa complex.

Creutzfeldt-Jakob disease probably acquired from a cadaveric dura mater graft. Case report.

A case of Creutzfeldt-Jakob disease (CJD) is reported in a 28-year-old woman who had received a cadaveric dural graft 19 months earlier after resection of a cholesteatoma. The circumstances of the case point to the graft as the most likely source of the disease. Cadaveric dura should be added to the list of materials that may transmit CJD, and it must be very carefully screened if it is used at all for grafting. Autologous tissue should be considered whenever possible.

Recurrent cutaneous vasculitis in cystic fibrosis.

We treated a patient with cystic fibrosis who experienced recurrent episodes of palpable purpura and arthralgias associated with exacerbations of her pulmonary disease. Skin biopsy demonstrated the classic findings of leukocytoclastic vasculitis, and C3 deposits were detected in the dermal blood vessels. Chronic bacterial infection and antibiotics are possible sources of antigen involved in immune complex formation in patients with chronic lung diseases.

Ectodermal defects and chronic mucocutaneous candidiasis in idiopathic hypoparathyroidism.

Idiopathic hypoparathyroidism is a rare disorder produced by parathyroid hormone deficiency of unknown cause. It is often associated with other endocrine abnormalities. Patients with idiopathic hypoparathyroidism frequently develop ectodermal disease, including dry, rough skin: coarse, brittle hair; and lusterless, distally split nails. All of these complaints are relatively common in a dermatologic practice. Chronic mucocutaneous candidiasis is also a manifestation of idiopathic hypoparathyroidism. A patient is presented with a prominent ectodermal dysplasia and a chronic mucocutaneous candidiasis that were due to the underlying idiopathic hypoparathyroidism. A brief review of idiopathic hypoparathyroidism is included, as well as the implications of this diagnosis in terms of differential diagnosis, associated endocrine disorders, and therapy.

Chronic perianal herpes simplex in immunocompromised hosts.

Four immunosuppressed patients are described with chronic ulcerative herpes simplex virus infection in the sacral and perianal area. Three of these patients were evaluated for decubitus ulcers. Prompt diagnosis was possible when the characteristic morphologic features were recognized and when viral culture and Tzanck smear specimens were obtained. Previously reported cases are reviewed as well. Chronic mucocutaneous herpes simplex infections are complications of immunocompromised hosts and should be recognized early if appropriate therapy is to be initiated.

Avascular necrosis of bone in dyskeratosis congenita.

A case of dyskeratosis congenita, a rare X-linked disorder, is presented. The patient had the classic triad of cutaneous findings: reticulated hyperpigmentation, nail dystrophy, and leukoplakia. In addition, a previously unreported finding developed, avascular necrosis of bone without systemic steroid therapy. Recognition of this disorder is important because pancytopenia and malignancy develop in up to 50 percent of patients.