Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location.

Phase memory relaxation times (T(M) or T(2)) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T(M) and x. T(M) values of seven positions are between 1.6 micros for the most buried residue (L79C) and 4.7 micros for a residue at the protein surface (W245C). In deuteriated buffer, longer T(M) are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T(M) as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.     

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (K_D ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC₅₀ typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.     

Recovery of murine lens epithelial cells from single and fractionated doses of X rays and neutrons

Subpopulations of mouse lens epithelial cells, differing in proliferative status, were irradiated with either X rays or fission spectrum neutrons given singly or in four weekly fractions. After various times, epithelia were mitogenically stimulated by wounding and DNA synthesis responses were determined by incorporation of [3H]thymidine. At 1 h following both X and neutron irradiations, significant suppression of the wound response after single doses and a sparing effect of fractionation were evident in both the mitotically quiescent and the slowly proliferating subpopulations. At 1 week following single or fractionated doses of both radiations, recovery was evident in both subpopulations. By 4 weeks, the quiescent subpopulation showed significant recovery after both single and fractionated doses of X rays or neutrons. In contrast, a marked decreased ability to respond after neutron irradiation and, in addition, a significant enhancement effect of neutron fractionation were observed for the slowly proliferating subpopulation. Per gray, neutrons were about 7.5 times more effective than X rays as a single dose and 25 times more effective in four equal fractions. The shift from an initial sparing to a final enhancing effect of neutron fractionation for the slowly proliferating subpopulation has importance for understanding divergent early and late radiation responses following dose fractionation.     

Effects of noninhibitory alpha-1-antitrypsin on primary human monocyte activation in vitro

A major function of alpha-1-antitrypsin (AAT) is the inhibition of overexpressed serine proteinases during inflammation. However, it is also known that the biological activity of AAT is affected by chemical modifications, including oxidation of the reactive-site methionine, polymerization, and cleavage by unspecific proteases, all of which will result in AAT inactivation and/or degradation. All inactive forms of AAT can be detected in tissues and fluids recovered from inflammatory sites. To test for a possible link between the inflammation-generated, noninhibitory, cleaved form of AAT and cellular processes associated with inflammation, we studied the effects of this form at varying concentrations on human monocytes in culture. We found that cleaved AAT at concentrations ranging between 1 and 10 microM in monocyte cultures over 24 h induces elevation in monocyte chemoattractant protein-1 (MCP-1) and pro-inflammatory cytokines such as TNFalpha and IL-6 and also increases production of interstitial collagenase (MMP-1) and gelatinase B (MMP-9), members of two different classes of matrix metalloproteinase. Moreover, monocytes stimulated with higher doses of cleaved AAT show an increase in cellular oxygen consumption by about 30%, while native AAT under the same experimental conditions inhibits oxygen consumption by about 50%. These results indicate that the cleaved form of AAT may play a role in monocyte recruitment and pro-inflammatory activation during inflammatory processes, and also suggest that changes in structure occurring upon AAT cleavage could alter its functional properties with potential pathological consequences.     

Exclusion of epidermal growth factor and high-resolution physical mapping across the Rieger syndrome locus.

We have evaluated the 4q25-4q26 region where the autosomal dominant disorder Rieger syndrome has been previously mapped by linkage. We first excluded epidermal growth factor as a candidate gene by carrying out SSCP analysis of each of its 24 exons using a panel of seven unrelated individuals with Rieger syndrome. No evidence for etiologic mutations was detected in these individuals, although four polymorphic variants were identified, including three that resulted in amino acid changes. We next made use of two apparently balanced translocations, one familial and one sporadic, to identify a narrow physical localization likely to contain the gene or to be involved in regulation of gene function. Somatic cell hybrids were established from individuals with these balanced translocations, and these hybrids were used as a physical mapping resource for, first, preliminary mapping of the translocation breakpoints using known sequence tagged sites from chromosome 4 and then, after creating YAC and cosmids contigs encompassing the region, for fine mapping of those breakpoints. A cosmid contig spanning these breakpoints was identified and localized the gene to within approximately 150 kb of D4S193 on chromosome 4. The interval between the two independent translocations is approximately 50 kb in length and provides a powerful resource for gene identification.     

The role of the DNA damage response in zebrafish and cellular models of Diamond Blackfan anemia

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis – a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.KEY WORDS: Ribosomal protein deficiency, Rps19, Rpl11, p53, ATR, RNR, Chk1, ATP, AMPK, Exogenous nucleosides     

Variations among clinical isolates of Staphylococcus aureus to induce expression of E-selectin and ICAM-1 in human endothelial cells

Eighteen clinical isolates of Staphylococcus aureus, nine methicillin-sensitive and nine methicillin-resistant, were investigated for their ability to induce expression of E-selectin and ICAM-1 in human endothelial cells. Upregulation of adhesion molecules varied between isolates; 17 isolates induced expression of E-selectin and 13 of ICAM-1. Some isolates induced a significant expression of E-selectin without stimulation of ICAM-1, whereas the opposite was not found. Bacterial viability was required for induction of the adhesion molecules. The kinetics of ICAM-1 expression in S. aureus-infected cells differed from those stimulated with interleukin-1beta (IL-1beta). On the other hand, expression of E-selectin was very similar in S. aureus-infected and IL-1beta-stimulated cells. There was no correlation between ability of S. aureus to induce expression of cell adhesion molecules, methicillin susceptibility, pulse field gel electrophoresis patterns, biochemical characteristics, phage typing and toxin production.