IntroductionDegenerative joint diseases including osteoarthritis (OA) are common, particularly in the elderly. Early signs of OA include progressive loss from articular cartilage of the proteoglycan aggrecan, reflected by a loss of safranin O staining, excessive damage to type II collagen, and general degeneration and fibrillation of the cartilage surface, resulting ultimately in a loss of articular cartilage (1).One of the primary targets of this disease is type II collagen, the major structural collagen found in articular cartilage in healthy individuals. There is ordinarily a strict balance between the production of type II collagen and degradation of this protein by catabolic enzymes during normal remodeling of cartilage (1). Pathological conditions such as OA are characterized by a loss of this balance with increased proteolysis (1-5) and upregulation of the synthesis of type II procollagen (5) and aggrecan (6).Matrix metalloproteinases (MMPs) comprise a family of zinc-dependent enzymes that degrade extracellular matrix components. MMPs are synthesized in articulating joints by synovial cells and chondrocytes. In mature articular cartilage, chondrocytes maintain the cartilage-specific matrix phenotype. Elevated expression of MMPs is associated with cartilage degradation (1). MMP-13, also known as human collagenase-3, is thought to play an important role in type II collagen degradation in articular cartilage and especially in OA (4, 7-9). Type II collagen is the preferred substrate for MMP-13 (4, 7, 10). Expression and contents of MMP-1 (collagenase-1) and 11,12), expression of MMP-8 (collagenase-2), and collagenase activity (4,8) are upregulated in human OA cartilage.Spontaneous development of focal sites degeneration has been described in aging guinea pigs (13). Sublines of the inbred STR/ORT strain of mice also develop spontaneous OA with aging (14). Mice exhibit upregulated expression of MMP-13 and collagenase activity is upregulated in focal lesions (15). In guinea pigs, MMP-1 and MMP-13 are also upregulated in OA lesions associated with increased collagenase activity (16). It has been suggested that increased collagenase-3 (MMP-13) activity plays a pivotal role in the pathogenesis of osteoarthritis (OA). We have used tetracycline-regulated transcription in conjunction with a cartilage-specific promoter to target a constitutively active human MMP-13 to the hyaline cartilages and joints of transgenic mice. Postnatal expression of this transgene resulted in pathological changes in articular cartilage of the mouse joints similar to those observed in human OA. These included characteristic erosion of the articular cartilage associated with loss of proteoglycan and excessive cleavage of type II collagen by collagenase, as well as synovial hyperplasia. These results demonstrate that excessive MMP-13 activity can result in articular cartilage degradation and joint pathology of the kind observed in OA, suggesting that excessive activity of this proteinase can lead to this disease.

Three mouse strains in the C3H lineage--C3H/HeJ, C3HeB/FeJ, and C3H/HeNCr1BR--were tested for their ability to be protected against infection with Salmonella typhimurium by a panel of nonviable vaccines and by passive transfer of hyperimmune serum. These strains differ in their innate susceptibilities to infection with S. typhimurium, but all are histocompatible. The same vaccines showed a widely different ability to protect different mouse strains. Ability to protect was not closely related to the capacity of the mice to make either agglutinating or anti-O antibody (as shown by ELISA) in response to a particular vaccine. Passive transfer of antibody was shown to protect inherently resistant mice but not inherently susceptible strains. These observations suggest that reported discrepancies in vaccine efficacy among laboratories may be attributable to differences in the mouse strains used and raise the question as to what might be an appropriate mouse model for human infections with Salmonella species.

Oxidoreductases of the thioredoxin superfamily possess the C-X-X-C motif. The redox potentials vary over a wide range for these proteins. A crucial determinant of the redox potential has been attributed to the variation of the X-X dipeptide. Here, we substitute Lys for Gly at the first X of Escherichia coli thioredoxin to investigate how a positive charge would affect the redox potential. The substitution does not affect the protein's redox potential. The equilibrium constant obtained from pairwise reaction between the mutant and wild-type proteins equals 1.1, indicating that the replacement does not significantly affect the thiol-disulfide redox equilibrium. However, the catalytic efficiency of thioredoxin reductase on the G33K mutant decreases approximately 2.8 times compared to that of the wild type. The mutation mainly affects K(m), with little effect on k(cat). The mutation also inhibits thioredoxin's ability to reduce insulin disulfide by approximately one-half. Whether the mutant protein supports the growth of phages T3/7 and f1 was tested. The efficiency of plating (EOP) of T3/7 on the mutant strain decreases 5 times at 37 degrees C and 3 x 10(4) times at 42 degrees C relative to that of the wild-type strain, suggesting that interaction between phage gene 5 protein and thioredoxin is hindered. The mutation also reduces the EOP of phage f1 by 8-fold at 37 degrees C and 1.5-fold at 42 degrees C. The global structure of the mutant protein does not change when studied by CD and fluorescence spectra. Therefore, G33K does not significantly affect the overall structure or redox potential of thioredoxin, but primarily interferes with its interaction with other proteins. Together with the G33D mutation, the overall results show that a charged residue at the first X has a greater influence on the molecular interaction of the protein than the redox potential.

Immunization with avirulent Salmonella typhimurium strain SL3235, a smooth, aroA derivative, was shown to induce high levels of resistance to challenge with virulent S. typhimurium in innately hypersusceptible C3H/HeJ mice and inherently resistant C3H/HeNCrlBR mice. Strain SL3235 is one of a class of avirulent aroA-derivatives made from various strains and species of SalmoneUla that are being considered as vaccine candidates for cattle and humans. This paper supports their efficacy and potential utility in this regard. In C3H/HeJ mice, immunity against over 1,000 50% lethal doses of virulent S. typhimurium was evident as early as 3 days after immunization and persisted for at least 7 months. Further, the vaccine was effective over a broad * Corresponding author. immunity against challenge with virulent S. typhimurium strains in both mouse strains. In addition, short-term crossprotection against Listeria monocytogenes is evident. The period of nonspecific resistance is accompanied by marked splenomegaly. Immune macrophage-rich fractions, but not immune T cells, were able to transfer protection into naive C3H/HeJ recipients. MATERIALS AND METHODS Mice. Female C3H/HeJ and C3H/HeNCrlBR mice, 8 weeks old, were purchased from Jackson Laboratories, Bar Harbor, Maine, and Charles River Laboratories, Wilmington, Mass., respectively. They were housed in plastic cages with Absorb-Dri for bedding. Purina Mouse Chow and fresh water were available ad libitum. Bacterial strains. S. typhimurium strain SL3235, aroA-(nonreverting), was obtained from Bruce Stocker, Depart

The adamalysins are a family of proteins in the metzincin superfamily of metalloproteases, which also includes the matrix metalloproteinases. There are two subfamilies of adamalysins: the snake venom metalloproteases (SVMPs) and the ADAMs (proteins containing a disintegrin and metalloprotease domain). At least 23 ADAMs have been identified to date. The ADAMs are expressed by a wide variety of cell types, and are involved in functions as diverse as sperm-egg binding, myotube formation, neurogenesis, and proteolytic processing of cell surface proteins. An overview of the ADAM family and their functions will be presented. TACE is a unique member of the ADAM family that cleaves membrane-bound TNF-alpha to generate soluble TNF-alpha. Mice lacking proteolytically active TACE have been generated and characterized. The TACE knock-out results in perinatal lethality. Cells from the TACE-deficient mice release 80-90% less soluble TNF-alpha than do wild-type cells. Irradiated mice that are reconstituted with TACE knock-out hematopoeitic stem cells have markedly reduced levels of serum TNF-alpha following LPS challenge, compared to irradiated mice reconstituted with wild-type cells, suggesting that TACE is the major TNF-alpha converting enzyme in vivo. TACE-deficient cells are compromised in the generation of other soluble proteins that are produced as the result of cleavage of a membrane precursor form, suggesting that TACE is involved in multiple shedding events.

To date, few animal models of chronic obstructive pulmonary disease (COPD) exist that are ideal for the evaluation of pathophysiology, as they typically require many months of cigarette smoke exposure in inhalation facilities. Here we show that pulmonary inflammation and some of the inflammatory hallmarks of COPD can be induced in mice by cigarette smoke-conditioned media (CS) administered by the intranasal route. Balb/c mice were challenged with CS for up to 40 days. At the end of smoke treatment, mice were sacrificed and bronchoalveolar lavage (BAL) fluid collected. Total cell counts and cell differentials were performed. Enzyme-linked immunosorbent assays (ELISAs) for KC and tumor necrosis factor alpha (TNF-alpha) were performed on BAL fluid. Lungs and nasal cavities were examined histologically. Intranasal CS treatment significantly increased BAL neutrophils, lymphocytes, KC, TNF-alpha, and mucin. Changes in pulmonary reactivity to methacholine were also observed in mice challenged with CS for 40 days. The model described above demonstrates that within 1 to 8 weeks of intranasal instillation of CS, mice develop pulmonary inflammation and cellular lung changes that are characteristic of human COPD and therefore may be a good short-term in vivo model that can be utilized to monitor intervention strategies targeted for COPD.

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The matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases that play a key role in both physiological and pathological tissue degradation. In our preceding paper, we have reported on a series of novel and orally active N-hydroxy-alpha-phenylsulfonylacetamide derivatives. However, these compounds had two drawbacks (moderate selectivity and chirality issues). To circumvent these two problems, a series of novel and orally active N-substituted 4-benzenesulfonylpiperidine-4-carboxylic acid hydroxyamide derivatives have been synthesized. The present paper deals with the synthesis and SAR of these compounds. Among the several compounds synthesized, derivative 55 turned out to be a potent, selective, and an orally active MMP inhibitor in the clinically relevant advanced rabbit osteoarthritis model. Detailed pharmacokinetics and metabolism data are described.