Molecular signaling in necrotizing enterocolitis: regulation of intestinal COX-2 expression

Necrotizing enterocolitis (NEC) is the most common surgical emergency in premature infants. The underlying etiology of NEC remains unknown, although bacterial colonization of the gut, formula feeding, and perinatal stress have been implicated as putative risk factors. The disease is characterized by exuberant gut inflammation leading to ischemia and coagulation necrosis of the intestinal epithelium. The molecular and cellular mechanisms responsible for these pathologic changes are poorly understood. It has been shown that various exogenous and endogenous mediators such as lipopolysaccharide, inflammatory cytokines, platelet activating factor, and nitric oxide may play a role in the pathogenesis of NEC. Recent studies in our laboratory and others have established a link between NEC and activation of cyclooxygenase-2, the enzyme that catalyzes the rate-limiting step in the biosynthesis of prostanoids. The challenge is in defining the molecular signaling pathways leading to accumulation of these mediators early in the disease progression, before the onset of tissue necrosis and systemic sepsis. Identification and characterization of these pathways could lead to the development of novel treatment strategies to alleviate the morbidity and mortality associated with NEC.     

An isolation method for assessment of brain mitochondria function in neonatal mice with hypoxic-ischemic brain injury

This work was undertaken to develop a method for the isolation of mitochondria from a single cerebral hemisphere in neonatal mice. Mitochondria from the normal mouse brain hemisphere isolated by the proposed method exhibited a good respiratory control ratio of 6.39 +/- 0.53 during glutamate-malate-induced phosphorylating respiration. Electron microscopy showed intact mitochondria. The applicability of this method was tested on mitochondria isolated from na?ve mice and their littermates subjected to hypoxic-ischemic insult. Hypoxic-ischemic insult prior to reperfusion resulted in a significant (p < 0.01) inhibition of phosphorylating respiration compared to na?ve littermates. This was associated with a profound depletion of the ATP content in the ischemic hemisphere. The expression for Mn superoxide dismutase and cytochrome C (markers for the integrity of the mitochondrial matrix and outer membrane) was determined by Western blot to control for mitochondrial integrity and quantity in the compared samples. Thus, we have developed a method for the isolation of the cerebral mitochondria from a single hemisphere adapted to neonatal mice. This method may serve as a valuable tool to study mitochondrial function in a mouse model of immature brain injury. In addition, the suggested method enables us to examine the mitochondrial functional phenotype in immature mice with a targeted genetic alteration.     

Quantitative structure-activity relationship studies of [(biphenyloxy)propyl]isoxazole derivatives. Inhibitors of human rhinovirus 2 replication

The 50% cytotoxic concentration (CC50) in HeLa cells, the 50% inhibitory concentration (IC50) against human rhinovirus 2 (HRV-2), and the selectivity index (SI = CC50/IC50) of [(biphenyloxy)propyl]isoxazole derivatives were used to develop quantitative structure-activity relationships (QSAR) based on simplex representation of molecular structure. Statistic characteristics for partial least-squares models are quite satisfactory (R2 = 0.838 - 0.918; Q2 = 0.695 - 0.87) for prediction of CC50, IC50, and SI values and permit the virtual screening and molecular design of new compounds with strong anti-HRV-2 activity. The quality of prognosis for designed compounds was additionally estimated by analysis of domain applicability for each QSAR model. A hypothesis to the effect that terminal benzene substituents must have negative electrostatic potential and definite length (approximately 5.5-5.6 A) to possess strong antiviral activity has been suggested. The quality of developed analysis, i.e., high level of antiviral action of three new designed compounds, has been confirmed experimentally.     

Cellular vitamin C increases chromate toxicity via a death program requiring mismatch repair but not p53

Ascorbate (Asc) plays a key role in reductive activation ofcarcinogenic chromium(VI) in vivo. In addition to much higherrates (t_1/2 = 1 min for 1 mM Asc), its reactions at physiologicalconditions differ from other reducers by low yields of Cr(V)intermediates. Human cells in culture are severely Asc deficient,which results in distorted metabolism and potentially abnormalresponses to Cr(VI). We found that restoration of physiologicalAsc levels in human lung cells (primary IMR90 fibroblasts andepithelial H460 cells) increased clonogenic lethality and apoptosisby Cr(VI). Enhanced cytotoxicity in mass cultures was more evidentafter normalization for lower Cr uptake caused by leakage ofAsc into media. Asc did not change uptake-adjusted yields ofCr–DNA adducts and had no effect on cytotoxicity whendelivered shortly after Cr(VI) exposure. Protein and Ser-15phosphorylation levels of p53 did not show any association withthe presence of Asc and there were no increases in p53-drivenreporter activity in Cr-treated cells. Stable silencing of p53expression by short hairpin RNA (shRNA) had no effect on toxicityof Cr(VI) in both –Asc and +Asc IMR90 and H460 cells.In contrast, shRNA-mediated depletion of essential componentsof MutS or MutL mismatch repair complexes greatly improved survivalof all Cr-treated cells and eliminated Asc-potentiated effectson cell death. Thus, mismatch repair-mediated enhancement ofCr(VI) cytotoxicity by Asc should promote the selection of MSI+/wt-p53phenotype found among chromate-induced human lung cancers. Ourfindings also indicate that Asc plays a dual role in Cr(VI)toxicity: protective outside and potentiating inside the cell. Abbreviations: Asc, ascorbate; DHA, dehydroascorbic acid; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; shRNA, short hairpin RNA Received December 12, 2006; revised January 25, 2007; accepted February 3, 2007.     

“Hearing voices” in schizophrenia: Who’s voices are they?

Paranoid schizophrenia is a subtype within the group of schizophrenia disorders. In the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV), delusions and hallucinations are the first and second symptoms required for the diagnosis of schizophrenia. Empirical data and clinical observations allow us to present the hypothesis that paranoid schizophrenia can be divided into two subgroups: (1) Hallucinatory subgroup, patients with prominent hallucinations and delusions influenced by auditory hallucinations, (2) Delusional subgroup, patients with prominently impaired thought content, in which hallucinations are not significant clinical factors. Furthermore, we believe that auditory hallucinations are not disturbances of perception but rather of thought – or “pseudo-perceptions”.     

Inclusion fluorescent-antibody test as a screening assay for detection of antibodies to Chlamydia pneumoniae

A study was conducted to determine the ability of the inclusion immunofluorescence assay (inclusion IFA) to act as a screening test to detect samples with antibodies to Chlamydia pneumoniae; microimmunofluorescence (MIF) was used as the "gold standard." In addition, the inclusion IFA was compared using HEp-2 cells infected with either C. pneumoniae CM-1 or Chlamydia trachomatis serovar E. A total of 331 serum samples representing a range of MIF titers were evaluated. The sensitivities of the inclusion IFA for detecting samples with C. pneumoniae MIF titers of > or = 16 were 96.9 and 74.8% with C. pneumoniae- and C. trachomatis-infected cells, respectively. For samples with an elevated C. pneumoniae MIF titer of > or = 512, the sensitivities of the C. pneumoniae- and C. trachomatis-based inclusion IFA were 97.0 and 8.8%, respectively. These results suggest that the inclusion IFA is not a genus-specific test, as evidenced by the failure of the C. trachomatis-infected cells to detect a significant number of samples with C. pneumoniae antibodies. Samples that had elevated C. pneumoniae inclusion IFA and MIF titers but that were found negative (titer, <16) by the C. trachomatis inclusion IFA were further tested by an in vitro neutralization assay for functional antibodies that might not have been detected by the serological assays. The in vitro neutralization results corroborated the serological results in that all seven sera tested had a neutralization titer for C. pneumoniae (range, 20 to 225), while all but one failed to have any effect on the infectivity of C. trachomatis serovar E. While the C. pneumoniae inclusion IFA had a high sensitivity for detecting chlamydial antibodies, depending on whether it was used as a screening test for detecting samples with low (> or = 16) or elevated (> or = 512) MIF titers, its specificity ranged from 53.4 to 77.1%. In conclusion, the inclusion IFA with C. pneumoniae-infected cells was best suited as a sensitive screening test for identifying specimens with elevated MIF titers (those associated with a possible acute infection with C. pneumoniae).     

Elevated frequency of ATM gene missense mutations in breast cancer relative to ethnically matched controls

Studies of families of patients with ataxia telangiectasia (A-T) show an increased risk of breast cancer in heterozygous A-T carriers. However, expected increased levels of mutations in the ATM gene among unselected breast cancer patients have not been found to date. Previous methods of mutation detection were biased toward the detection of truncating mutations, and single nucleotide substitutions were likely to have been underreported. In this study, genomic DNA from 43 breast cancer patients and 43 control individuals were scanned for mutations in the entire ATM coding region (exons 4-65) and adjacent intronic splice regions (three megabases total) using detection of virtually all mutation-single-strand conformation polymorphism (SSCP), a modification of SSCP with sufficient redundancy to detect virtually all mutations. Excluding a polymorphism found commonly in cases and controls, there were missense changes in 12 breast cancer patients, one of whom also had a protein truncating mutation, versus six controls (P=0.09). When all structural changes common to the cases and controls were excluded, missense or truncating changes were found in 10 cases compared to two in controls (P=0.013). The background of missense changes in controls is high. There is a trend towards elevation of all structural changes in cases, but the results are not statistically significant. Cohort-specific structural changes are significantly more prevalent in the breast cancer patients. The data are compatible with certain missense mutations in ATM predisposing to breast cancer.     

Out-of-field dose equivalents delivered by proton therapy of prostate cancer

Measurements were performed to assess the dose equivalent outside a primary proton treatment field, using a silicon-on-insulator (SOI) microdosimeter. The SOI microdosimeter was placed on the surface of an anthropomorphic phantom and dose equivalents were determined as a function of lateral distance from a typical passively scattered and modulated prostate treatment field. Measurements were also completed within a polystyrene plate phantom as a function of depth for a distance of 5 cm from the field edge, as function of lateral distance from field edge at two different depths, and as a function of distance from the distal edge on the central beam axis. The dose equivalent at the surface of the anthropomorphic phantom decreases from 3.9 to 0.18 mSv/Gy when the lateral distance from the proton field edge increases from 2.5 to 60 cm. Measurements along the proton depth dose distribution at a constant distance of 5 cm from the primary field edge indicate a decrease in dose equivalent as a function of depth, with a 38% decrease relative to the surface dose at a depth of 5 cm in polystyrene. Measurements completed as a function of lateral distance from the primary field at two separate depths within polystyrene illustrate a convergence of the dose equivalent at approximately 20 cm from the primary field edge. Past the distal edge of the spread-out Bragg peak dose equivalents decrease exponentially for increasing distance, with an initial value of 1.6 mSv/Gy at 0.6 cm from the distal edge. Silicon microdosimetry measurements were also compared with published results obtained utilizing different measurement techniques. This study demonstrates the applicability of SOI microdosimetry in determining the dose equivalent outside proton treatment fields, and provides valuable information on the dose equivalent both at the surface and at depth experienced by prostate cancer patients treated with protons.     

Impact of sex and ozone exposure on the course of pneumonia in wild type and SP-A (-/-) mice

Female mice exhibited higher survival rate than males after pneumonia, with a reversal of this pattern following ozone exposure. Surfactant protein A (SP-A) plays an important role in innate immunity and SP-A (-/-) mice were more susceptible to pneumonia than wild type mice. Here, we investigated underlying mechanisms of the differential susceptibility of mice to pneumonia. Wild type and SP-A (-/-) C57BL/6J male and female mice were exposed to ozone or filtered air (FA) and then infected intratracheally with Klebsiella pneumoniae. Blood, spleen, and lung were analyzed for bacterial counts, lung and spleen weights, and sex hormone and cortisol levels were measured in plasma within two days post-infection. We found: 1) in the absence of ozone-induced oxidative stress, males had higher level of bacterial dissemination compared to females; ozone exposure decreased pulmonary clearance in both sexes and ozone-exposed females were more affected than males; 2) ozone exposure increased lung weight, but decreased spleen weight in both sexes, and in both cases ozone-exposed females were affected the most; 3) plasma cortisol levels in infected mice changed: ozone-exposed>FA-exposed, females>males, and infected>non-infected; 4) no major sex hormone differences were observed in the studied conditions; 5) differences between wild type and SP-A (-/-) mice were observed in some of the studied conditions. We concluded that reduced pulmonary clearance, compromised spleen response to infection, and increased cortisol levels in ozone-exposed females, and the higher level of lung bacterial dissemination in FA-exposed males, contribute to the previously observed survival outcomes.     

Thirteen novel mutations in the NR0B1 (DAX1) gene as cause of adrenal hypoplasia congenita

X-linked adrenal hypoplasia congenita (AHC) is a rare developmental disorder associated with primary adrenal insufficiency and combined primary and secondary male hypogonadism. It is caused by deletions or mutations of the NR0B1 (DAX1) gene encoding DAX1, an atypical orphan member of the nuclear receptor superfamily. The continuous molecular genetic analysis of male patients with primary adrenal insufficiency revealed 13 novel mutations within the coding region of the NR0B1 gene which are predicted to inactivate the DAX1 function. These were three nonsense mutations (c.312C>A, p.Cys104X, c.670C>T, p.Gln224X; and c.873G>A, p.Trp291X), five duplications (c.269_270dup, c.421_422dup, c.895_896dup, c.989dup, c.999_1000dup), and five deletions (c.483del, c.745_746del, c.734_740del, c.1092del, and c.1346del). All of the mutations resulted in a premature stop codon destroying the ligand binding domain of the predictive DAX1 protein.