Nancy I. Hutchinson

Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase

Mice deficient in inducible nitric oxide synthase (iNOS) were generated to test the idea that iNOS defends the host against infectious agents and tumor cells at the risk of contributing to tissue damage and shock. iNOS-/-mice failed to restrain the replication of Listeria monocytogenes in vivo or lymphoma cells in vitro. Bacterial endotoxic lipopolysaccharide (LPS) caused shock and death in anesthetized wild-type mice, but in iNOS-/-mice, the fall in central arterial blood pressure was markedly attenuated and early death averted. However, unanesthetized iNOS-/-mice suffered as much LPS-induced liver damage as wild type, and when primed with Propionobacterium acnes and challenged with LPS, they succumbed at the same rate as wild type. Thus, there exist both iNOS-dependent and iNOS-independent routes to LPS-induced hypotension and death.

Susceptibility of stromelysin 1-deficient mice to collagen-induced arthritis and cartilage destruction

OBJECTIVE It has long been proposed that stromelysin is one of the major degradative matrix metalloproteinases responsible for the loss of cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA). This hypothesis was tested by examining the arthritic paws of stromelysin 1 (SLN1)-deficient mice for loss of cartilage and for generation of neoepitopes that would be indicative of aggrecan cleavage. METHODS The SLN1 gene was inactivated in murine embryonic stem cells, and knockout mice deficient in SLN1 activity were bred onto the B10.RIII background. The incidence and severity of collagen-induced arthritis (CIA) were compared in wild-type and knockout mice. Paws from mice with CIA were examined for loss of cartilage and for proteoglycan staining, as well as for the generation of the neoepitope FVDIPEN341. RESULTS SLN1-deficient mice developed CIA, as did the wild-type N2 mice. Histologic analyses demonstrated no significant differences among the B10.RIII, wild-type, and knockout mice in loss of articular cartilage and proteoglycan staining. No decrease in the FVDIPEN341 epitope was observed in the SLN1-deficient mice. CONCLUSION Disruption of the SLN1 gene neither prevents nor reduces the cartilage destruction associated with CIA. Moreover, SLN1 depletion does not prevent the cleavage of the aggrecan Asn341-Phe342 bond.

Organization and expression of the major genes from the long inverted repeat of the human cytomegalovirus genome

The long inverted repeat (TRL:IRL) of the human cytomegalovirus (CMV) genome is a major transcription unit in productively infected human fibroblasts. Cloned DNA fragments of the CMV IRL and complementary DNA (cDNA) copies of RNAs transcribed from the TRL:IRL were used as probes in RNA filter hybridization experiments to characterize and map the RNAs transcribed from this region of the virus genome. In human fibroblasts, three poly A+ RNAs of 2.7, 2.0, and 1.2 kb were detected during the early (E) and late (L) phases of virus gene expression. Analysis of cloned cDNAs and RNA mapping studies indicate that the TRL:IRL can be divided into three transcriptionally active regions. The most highly transcribed region lies between 0.805 and 0.816 map units and encodes a major abundant poly A+ RNA of 2.7 kb that is expressed at E and L times postinfection (p.i.). The second region spans map coordinates 0.792-0.797 and encodes a 1.2-kb poly A+ RNA that is relatively low in abundance at E times p.i. but achieves nearly the same abundance as the 2.7-kb transcript at L times p.i. The third region encompasses map units 0.796-0.804 and encodes a less abundant poly A+ RNA of 2.0 kb that attains maximum expression at E times. The 1.2- and 2.7-kb RNAs are transcribed in the same direction, while the 2.0-kb RNA is transcribed in the opposite direction. The 2.7-, 2.0-, and 1.2-kb RNAs, as well as 5.7- and 1.8-kb transcripts were detected at immediate early times p.i. when human fibroblasts were treated with cycloheximide, but not in cells treated with anisiomycin.

Expression and Immunoaffinity Purification of Human Inducible Nitric-oxide Synthase INHIBITION STUDIES WITH 2-AMINO-5,6-DIHYDRO-4H-1,3-THIAZINE

Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37°C and contained 1.15 ± 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret λmax at 396 nm with a shoulder at 460 nm and contained 0.28-0.64 mol of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (Ki = 22.6 ± 1.9 nM), reversed the type II difference spectrum induced by imidazole (Kd = 17.7 nM), and altered the CO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.

Production, Purification and Characterization of Canine Prostromelysin

One of the best studied animal models of osteoarthritis is a dog model in which the anterior cruciate ligament of the hind limb stifle joint is transected (Pond-Nuki model). To determine whether stromelysin might play a role in this model, it was necessary to purify the enzyme for production of suitable probes. In the present study, dog synovial fibroblasts were stimulated to express a metalloproteinase that was demonstrated to be canine prostromelysin by Northern blot, protein purification and amino-terminal sequence analyses. Unlike rabbit synoviocytes, passaged dog synoviocytes did not express stromelysin mRNA in response to recombinant human IL-1, but expressed stromelysin mRNA only upon treatment with dog monocyte-conditioned medium (dMCM). The aminophenylmercuric acetate (APMA)-activatable metalloproteinase present in the culture supernatants of stimulated dog synoviocytes was purified using a combination of ion-exchange and dye matrix affinity chromatography. The purified canine metalloproteinase co-migrated on reducing SDS-PAGE with recombinant human prostromelysin-1 as a doublet with apparent molecular masses of 54 and 56 kDa. Similar to APMA-activated human prostromelysin-1, the APMA-activated canine metalloproteinase was inhibited by 1,10-phenanthroline or recombinant human tissue inhibitor of metalloproteinase (TIMP). The amino-terminal sequences of the canine pro- and APMA-activated enzymes were compared with those of human, rabbit and rat stromelysin. The striking homologies among the sequences demonstrated that the purified canine metalloproteinase was indeed canine prostromelysin. A rabbit anti-canine prostromelysin polyclonal antiserum was generated and used to localize the enzyme within cultured dog synoviocytes and articular cartilage stimulated with dMCM. The reagents developed in this study should be useful for examining the expression and distribution of prostromelysin in canine models of osteoarthritis.

Characterization of a major early gene from the human cytomegalovirus long inverted repeat; predicted amino acid sequence of a 30-kDa protein encoded by the 1.2-kb mRNA

The human cytomegalovirus (CMV) long inverted repeat (TRL:IRL) encodes three major early mRNAs. One of these RNAs is a 1.2-kb transcript that maps between 0.792 and 0.797 map units on the human CMV genome. Primer extension experiments, in addition to nucleotide sequence analyses of cloned cDNA transcripts and human CMV IRL DNA fragments, demonstrated that the 1.2-kb mRNA was not spliced. A single major open reading frame of 254 amino acids was identified, encoding a basic polypeptide of approximately 30 kDa. This polypeptide contains 19% Arg, Lys, and His residues, and would have a net positive charge of 31 at neutral pH. Examination of the deduced amino acid sequence revealed several potential phosphorylation sites and a hydrophobic carboxy terminus which resembles a membrane anchor sequence. In vitro translation of human CMV infected cell RNA, hybrid selected with either cloned cDNA or human CMV IRL DNA fragments specific for the 1.2-kb mRNA, resulted in a unique translation product that migrated on SDS-polyacrylamide gels with an apparent molecular mass of 37 kDa. Potential transcriptional regulatory sequences were also identified upstream of the gene encoding the 30-kDa protein.