Nadine L. N. Halligan

Non-heme iron protein: A potential target of nitric oxide in acute cardiac allograft rejection

We examined iron nitrosylation of non-heme protein and enzymatic activity of the Fe-S cluster protein, aconitase, in acute cardiac allograft rejection. Heterotopic transplantation of donor hearts was performed in histocompatibility matched (isografts: Lewis → Lewis) and mismatched (allografts: Wistar–Furth → Lewis) rats. On postoperative days (POD) 4–6, Western blot analysis and immunohistochemistry revealed inducible nitric-oxide synthase (iNOS) protein in allografts but not isografts. EPR spectroscopy revealed background signals at g = 2.003 (for semiquinone) and g = 2.02 and g = 1.94 (for Fe-S cluster protein) in isografts and normal hearts. In contrast, in allografts on POD4, a new axial signal at g = 2.04 and g = 2.02 appeared that was attributed to the dinitrosyl–iron complex formed by nitrosylation of non-heme protein. Appearance of this signal occurred at or before significant nitrosylation of heme protein. Iron nitrosylation of non-heme protein was coincidental with decreases in the nonnitrosylated Fe-S cluster signal at g = 1.94. Aconitase enzyme activity was decreased to ≈50% of that observed in isograft controls by POD4. Treatment with cyclosporine blocked the (i) elevation of plasma nitrate + nitrite, (ii) up-regulation of iNOS protein, (iii) decrease in Fe-S cluster EPR signal, (iv) formation of dinitrosyl–iron complexes, and (v) loss of aconitase enzyme activity. Formation of dinitrosyl–iron complexes and loss of aconitase activity within allografts also was inhibited by treatment of recipients with a selective iNOS inhibitor, l-N6-(1-iminoethyl)lysine. This report shows targeting of an important non-heme Fe-S cluster protein in acute solid organ transplant rejection.

The influence of genetic variation in surfactant protein B on severe lung injury in African American children

Objective:To determine whether genetic variations in the gene coding for surfactant protein B are associated with lung injury in African American children with community-acquired pneumonia. Design:A prospective cohort genetic association study of lung injury in children with community-acquired pneumonia. Setting:Two major tertiary care childrens hospitals. Subjects:African American children with community-acquired pneumonia (n = 395) either evaluated in the emergency department or admitted to the hospital. Interventions:None. Measurements and Main Results:Three hundred ninety-five African American children (14 days to 18 yrs of age) with community-acquired pneumonia were enrolled. Thirty-seven patients required mechanical ventilation and 26 of these were diagnosed with acute lung injury or acute respiratory distress syndrome. Genotyping was performed on seven linkage disequilibrium-tag single nucleotide polymorphisms in the surfactant protein B gene. Univariate analysis demonstrated two linkage disequilibrium-tag single nucleotide polymorphisms, rs1130866 (also known as SP-B + 1580 C/T) and rs3024793, were associated with the need for mechanical ventilation in African American children (p = .016 and p = .030, respectively). Multivariable analysis indicated that both of these single nucleotide polymorphisms are independently associated with need for mechanical ventilation (p = .040 and p = .012, respectively) as was rs7316 when its interaction with age was considered (p = .015). Multivariable analysis examining acute lung injury demonstrated a significant association of rs7316 with acute lung injury (p = .031). Haplotype analysis was also performed. Two haplotypes, GTGCGCG and ATATAAG, were associated with need for mechanical ventilation using either univariate (p = .041 and p = .043, respectively) or multivariable analysis (odds ratios of 2.62, p = .048, and 3.12, p = .033, respectively). Conclusions:Genetic variations in the gene coding for surfactant protein B are associated with more severe lung injury as indicated by the association of specific single nucleotide polymorphism genotypes and haplotypes with the need for mechanical ventilation in African American children with community-acquired pneumonia.

Genetic variation in MYLK and lung injury in children and adults with community-acquired pneumonia.

Objective: To investigate whether selected single nucleotide polymorphisms in the myosin light chain kinase gene are associated with more severe lung injury in children and adults with community-acquired pneumonia. Previous studies have demonstrated an association between single nucleotide polymorphisms in the myosin light chain kinase gene and increased severity of acute lung injury in adults. Design: Prospective, case-control genetic association study. Setting: Three tertiary childrens hospitals and one adult healthcare system. Patients: A total of 800 pediatric patients and 393 adult patients. Interventions: None. Measurements and Main Results: Genetic variation in the myosin light chain kinase gene was examined. The pediatric cohort was predominantly composed of African American (n = 443) and Caucasian (n = 253) children. A total of 393 patients made up the adult cohort. Within the pediatric cohort, single nucleotide polymorphisms rs16834493, rs820463, and rs9840993 were genotyped in the African American patients, whereas single nucleotide polymorphisms rs960224, rs33264, rs11718105, and rs9289225 were genotyped in the Caucasian patients. One single nucleotide polymorphism (rs820336) was genotyped in both groups. Genotyping in the adult cohort included rs820336, rs860224, rs33264, and rs11718105. Genotyping was performed using the Taqman Assay. Data were analyzed separately for African Americans and Caucasians and for children and adults. No associations were observed between the myosin light chain kinase gene single nucleotide polymorphisms genotyped in children with community-acquired pneumonia and increased severity of lung injury. Similarly, no associations were observed between myosin light chain kinase gene single nucleotide polymorphisms genotyped in adults with community-acquired pneumonia and increased severity of lung injury. Conclusions: No association between the selected single nucleotide polymorphisms in the myosin light chain kinase gene and either the need for positive-pressure ventilation or the development of acute lung injury/acute respiratory distress syndrome was observed in children with community-acquired pneumonia. This suggests that variation in this gene may play less of a role in lung injury in children or adults with community-acquired pneumonia than in adults with sepsis or trauma.

Cloning of the murine cDNA encoding VDJP, a protein homologous to the large subunit of replication factor C and bacterial DNA ligases

A putative full-length 1.7-kb cDNA, encoding a murine protein that specifically binds to the nonamer portion of the V(D)J recombinational signal sequence (RSS) element, has been cloned. By its sequence analysis, this cDNA is identical to a portion of the 4.5-kb murine replication factor C large-subunit-encoding cDNA. By Northern blot analysis, the 1.7-kb mRNA species is observed in murine immature B cells but not in non-lymphoid cells and tissues, while the 4.5-kb replication factor C-encoding cDNA is expressed in all cell types. The deduced VDJP amino-acid sequence includes a region of homology with bacterial DNA ligases at the C terminus of each of the proteins. VDJP has been synthesized as a fusion protein in bacteria, and the purified protein has been previously shown to mediate the joining of DNA fragments in a V(D)J RSS-dependent fashion (Guilliams et al., Biochem. Biophys. Res. Commun. 202 (1994) 1134-1141).

Nitric oxide formation in acutely rejecting cardiac allografts correlates with GTP cyclohydrolase I activity

Inducible nitric oxide synthase (iNOS) is a prominent component of the complex array of mediators in acute graft rejection. While NO production is determined by iNOS expression, BH4 (tetrahydrobiopterin), a cofactor of iNOS synthesized by GTP cyclohydrolase I, has been considered critical in sustaining NO production. In the present study, we examined time-dependent changes in iNOS and GTP cyclohydrolase I in rat cardiac allografts. The increase in iNOS protein and mRNA in allografts was similar at POD4 (post-operative day 4) and POD6. However, the peak increase in intragraft NO level at POD4 was not sustained at POD6. This disparity could not be explained by any decrease in iNOS enzyme activity measured ex vivo with optimal amounts of substrate and cofactors. Lower iNOS activity could be explained by changes in total biopterin levels in allografts at POD4 that was decreased to baseline at POD6. Changes in biopterin production correlated with lower GTP cyclohydrolase I protein levels but not by any change in GTP cyclohydrolase I mRNA. Functionally, allografts displayed bradycardia and distended diastolic and systolic dimensions at POD6 but not at POD4. Likewise, histological rejection scores were increased at POD4 but with a secondary increased stage at POD6. It is hypothesized that the dissimilar amounts of NO at early and later stages of rejection is due to uncoupling of iNOS arising from disproportionate synthesis of BH4. These findings provide insight into a potential pathway regulating NO bioactivity in graft rejection. Such knowledge may potentially assist in the design of newer strategies to prevent acute graft rejection.

Association of polymorphisms in genes of factors involved in regulation of splicing of cystic fibrosis transmembrane conductance regulator mRNA with acute respiratory distress syndrome in children with pneumonia.

BackgroundPrevious work has demonstrated a strong association between lung injury in African American children with pneumonia and a polymorphic (TG)mTn region in cystic fibrosis transmembrane conductance (CFTR) involved in the generation of a nonfunctional CFTR protein lacking exon 9. A number of splicing factors that regulate the inclusion/exclusion of exon 9 have been identified. The objective of this study was to determine whether genetic variants in these splicing factors were associated with acute respiratory distress syndrome (ARDS) in children with pneumonia.MethodsThis is a prospective cohort genetic association study of lung injury in African American and non-Hispanic Caucasian children with community-acquired pneumonia evaluated in the emergency department or admitted to the hospital. Linkage-disequilibrium-tag single nucleotide polymorphisms (LD-tag SNPs) in genes of the following splicing factors (followed by gene name) involved in exon 9 skipping PTB1 (PTBP1), SRp40 (SFRS1), SR2/ASF (SFRS5), TDP-43 (TARDBP), TIA-1 (TIA1), and U2AF65 (U2AF2) were genotyped. SNPs in the gene of the splicing factor CELF2 (CELF2) were selected by conservation score. Multivariable analysis was used to examine association between genotypes and ARDS.ResultsThe African American cohort (n = 474) had 29 children with ARDS and the non-Hispanic Caucasian cohort (n = 304) had 32 children with ARDS. In the African American group multivariable analysis indicated that three variants in CELF2, rs7068124 (p = 0.004), rs3814634 (p = 0.032) and rs10905928 (p = 0.044), and two in TIA1, rs2592178 (p = 0.005) and rs13402990 (p = 0.018) were independently associated with ARDS. In the non-Hispanic Caucasian group, a single variant in CELF2, rs2277212 (p = 0.014), was associated with increased risk of developing ARDS.ConclusionsThe data indicate that SNPs in CELF2 may be associated with the risk of developing ARDS in both African American and non-Hispanic Caucasian children with pneumonia and suggest that the potential role of the splicing factor CELF2 in ARDS should be explored further.