Immunohistochemical localization of inducible nitric oxide synthase on human fetal amnion in intra-amniotic infection.

OBJECTIVES: Amniotic fluid levels of nitric oxide metabolites are significantly elevated in intra-amniotic infection. We hypothesized that fetal amnion is a possible site for the production of nitric oxide. Because inducible nitric oxide synthase is the key enzyme responsible for the generation of nitric oxide in patients with intra-amniotic infection, we used immunohistochemistry to localize it on human fetal amnion. STUDY DESIGN: Human fetal amnions were obtained from patients with and without intra-amniotic infection (n = 5, respectively). Intra-amniotic infection was diagnosed by positive amniotic fluid cultures and placental pathologic features. Human fetal amniotic membranes were processed into tissue blocks and embedded in paraffin. A rabbit polyclonal antibody against human inducible nitric oxide synthase was used as the primary antibody, followed by avidin-biotin immunoperoxidase localization. Normal rabbit serum was used as a negative control and ovarian carcinoma cells were used as the positive control. RESULTS: Anti-inducible nitric oxide synthase labeling of human fetal amniotic membranes in patients with intra-amniotic infection showed positive immunostaining of epithelial cells, specifically in the cytoplasm of the perinuclear area. In contrast, no anti-inducible nitric oxide synthase immunostaining on human fetal amniotic membranes could be identified in patients without intra-amniotic infection. CONCLUSIONS: Our data provide important evidence that inducible nitric oxide synthase can be induced on human fetal amnion in intra-amniotic infection. These findings strongly support our hypothesis that human fetal amnion may be a possible site for the synthesis of nitric oxide after inducible nitric oxide synthase is induced in response to infectious products in intra-amniotic infection.

An MHC interaction site maps to the amino-terminal half of the T cell receptor alpha chain variable domain.

We have used cloned T cell receptor (TCR) genes from closely related CD4 T cell lines to probe the interaction of the TCR with several specific major histocompatibility complex (MHC) class II ligands. Complementarity determining region 3 (CDR3) equivalents of both alpha and beta TCR chains are required for antigen-MHC recognition. Our data provide novel information about the rotational orientation of TCR-MHC contacts in that exchange of the amino terminal portion of the TCR alpha chain containing the putative CDR1 and CDR2 regions results in both gain and loss of MHC class II specificity by the resulting receptor. These two TCRs differ primarily in recognition of polymorphisms in the second hypervariable region of the MHC class II alpha chain. These results document the involvement of CDR1 and/or CDR2 of the TCR alpha chain in MHC recognition and suggest a rotational orientation of this TCR to its MHC ligand.

A specific class of IS10 transposase mutants are blocked for target site interactions and promote formation of an excised transposon fragment.

We report the identification and characterization of a class of IS10 transposase mutants that carry out only some of the steps required for transposition. These mutants were identified among transposition-defective mutants as a specific subclass that retains the wild-type ability to induce SOS functions in the presence of transposon ends. Mutants of this class successfully promote excision of the element from its donor site, but do not promote transfer of the transposon sequences to a target site. SOS induction presumably results from the degradation of the donor site. Uniquely among transposition-defective mutants, SOS+ Tnsp- mutants promote the formation of a new product, the excised transposon fragment (ETF), which consists of the transposon excised from the original donor molecule by double-strand breaks at the transposon ends. SOS+ Tnsp- mutants identified thus far define two patches of amino acids that might correspond to regions of different function. A single additional mutation maps within a region that is highly conserved among IS element transposases. The existence of SOS+ Tnsp- mutants and the structure of the ETF provide strong support for the previously proposed nonreplicative model of Tn10/IS10 transposition.