The tumor necrosis factor-alpha converting enzyme (TACE): a unique metalloproteinase with highly defined substrate selectivity

TNF alpha converting enzyme (TACE) processes precursor TNF alpha between Ala76 and Val77, yielding a correctly processed bioactive 17 kDa protein. Genetic evidence indicates that TACE may also be involved in the shedding of other ectodomains. Here we show that native and recombinant forms of TACE efficiently processed a synthetic substrate corresponding to the TNF alpha cleavage site only. For all other substrates, conversion occurred only at high enzyme concentrations and prolonged reaction times. Often, cleavage under those conditions was accompanied by nonspecific reactions. We also compared TNF alpha cleavage by TACE to cleavage by those members of the matrix metalloproteinase (MMP) family previously implied in TNF alpha release. The specificity constants for TNF alpha cleavage by the MMPs were approximately 100-1000-fold slower relative to TACE. MMP 7 also processed precursor TNF alpha at the correct cleavage site but did so with a 30-fold lower specificity constant relative to TACE. In contrast, MMP 1 processed precursor TNF alpha between Ala74 and Gln75, in addition to between Ala76 and Val77, while MMP 9 cleaved this natural substrate solely between Ala74 and Gln75. Additionally, the MMP substrate Dnp-PChaGC(Me)HK(NMA)-NH(2) was not cleaved at all by TACE, while collagenase (MMP 1), gelatinase (MMP 9), stromelysin 1 (MMP 3), and matrilysin (MMP 7) all processed this substrate efficiently. All of these results indicate that TACE is unique in terms of its specificity requirements for substrate cleavage.     

Selective interleukin-12 synthesis defect in 12/15-lipoxygenase-deficient macrophages associated with reduced atherosclerosis in a mouse model of familial hypercholesterolemia

Targeted gene disruption or overexpression of 12/15-lipoxygenase in mice on the genetic background of apolipoprotein E or low density lipoprotein-receptor (LDL-R) deficiency has implicated 12/15-lipoxygenase in atherogenesis. The data support indirectly a role for 12/15-lipoxygenase in the oxidative modification of low density lipoprotein. In this study we set out to explore other potential mechanisms for 12/15-lipoxygenase in atherosclerosis using apolipoprotein B mRNA editing catalytic polypeptide-1/LDL-R double-deficient mice, a model highly related to the human condition of familial hypercholesterolemia. 12/15-Lipoxygenase deficiency in this strain led to approximately 50% decrease in aortic lesions in male and female mice at 8 months on a chow diet in the absence of cholesterol differences. While studying 12/15-lipoxygenase-deficient macrophages in culture, we discovered a remarkable selective defect (75-90% decrease) in interleukin-12 production but not in tumor necrosis factor-alpha or nitric oxide release, in response to lipopolysaccharide in the presence or absence of interferon-gamma priming. The lipopolysaccharide/interferon-gamma response was associated with a 33-50% decrease in nuclear interferon consensus sequence-binding protein, which is consistent with interferon consensus sequence-binding protein containing protein complex-dependent regulation of the interleukin-12 p40 gene. The decrease in interleukin-12 production was recapitulated in vivo in mouse aortas of the triple knockout group and was reflected in a marked decrease in interferon-gamma expression. The data provide support for a novel mechanism linking the 12/15-lipoxygenase pathway to a known immunomodulatory Th1 cytokine in atherogenesis.     

The structuring role of free-floating versus submerged plants in a subtropical shallow lake

In shallow temperate lakes many ecological processes depend on submerged macrophytes. In subtropical and tropical lakes, free-floating macrophytes may be equally or more important. We tested the hypothesis that different macrophyte growth forms would be linked with different bottom-up and top-down mechanisms in out-competing phytoplankton. We compared experimentally the effects of submerged and free-floating plants on water chemistry, phytoplankton biomass, zooplankton and fish community structure in a shallow hypertrophic lake (Lake Rodó, 34°55S 56°10W, Uruguay). Except for the retention of suspended solids, we found no other significant bottom-up process connected with either Eichhornia crassipes or Potamogeton pectinatus. Free-floating plants had a lower abundance of medium-sized zooplankton than any other microhabitat and submerged plants were apparently preferred by microcrustaceans. Fish showed a differential habitat use according to species, size-class and feeding habits. Dominant omnivore-planktivores, particularly the smallest size classes, preferred submerged plants. In contrast, omnivore-piscivores were significantly associated with free-floating plants. The density of omnivorous-planktivorous fish, by size class, significantly explained the distribution of medium-sized zooplankton, the high number of size 0 fish being the main factor. The abiotic environment and the structure of the zooplankton community explained little of the fish distribution pattern. Our results suggest that bottom-up effects of free-floating plants are weak when cover is low or intermediate. Top-down effects are complex, as effects on zooplankton and fish communities seem contradictory. The low piscivores:planktivores ratio in all microhabitats suggests, however, that cascading effects on phytoplankton through free-floating plant impacts on piscivorous fish are unlikely to be strong.     

Roles for asparagine endopeptidase in class II MHC-restricted antigen processing

The adaptive immune response depends on the creation of suitable peptides from foreign antigens for display on MHC molecules to T lymphocytes. Similarly, MHC-restricted display of peptides derived from self proteins results in the elimination of many potentially autoreactive T cells. Different proteolytic systems are used to generate the peptides that are displayed as T cell epitopes on class I compared with class II MHC molecules. In the case of class II MHC molecules, the proteases that reside within the endosome/lysosome system of antigen-presenting cells are responsible; surprisingly, however, there are relatively few data on which enzymes are involved. Recently we have asked whether proteolysis is required simply in a generic sense, or whether the action of particular enzymes is needed to generate specific class II MHC-associated T cell epitopes. Using the recently identified mammalian asparagine endopeptidase as an example, we review recent evidence that individual enzymes can make clear and non-redundant contributions to MHC-restricted peptide display.     

PKA accelerates rate of force development in murine skinned myocardium expressing alpha- or beta-tropomyosin

In myocardium, protein kinase A (PKA) is known to phosphorylate troponin I (TnI) and myosin-binding protein-C (MyBP-C). Here, we used skinned myocardial preparations from nontransgenic (NTG) mouse hearts expressing 100% alpha-tropomyosin (alpha-Tm) to examine the effects of phosphorylated TnI and MyBP-C on Ca2+ sensitivity of force and the rate constant of force redevelopment (k(tr)). Experiments were also done using transgenic (TG) myocardium expressing approximately 60% beta-Tm to test the idea that the alpha-Tm isoform is required to observe the mechanical effects of PKA phosphorylation. Compared with NTG myocardium, TG myocardium exhibited greater Ca2+ sensitivity of force and developed submaximal forces at faster rates. Treatment with PKA reduced Ca2+ sensitivity of force in NTG and TG myocardium, had no effect on maximum k(tr) in either NTG or TG myocardium, and increased the rates of submaximal force development in both kinds of myocardium. These results show that PKA-mediated phosphorylation of myofibrillar proteins significantly alters the static and dynamic mechanical properties of myocardium, and these effects occur regardless of the type of Tm expressed.     

Luminous vibriosis in rock lobster Jasus verreauxi (Decapoda: Palinuridae) phyllosoma larvae associated with infection by Vibrio harveyi

Studies were conducted to determine the cause of outbreaks of luminous vibriosis in phyllosoma larvae of the packhorse rock lobster Jasus verreauxi reared in an experimental culture facility. On 2 separate occasions mortalities of up to 75% over a period of 4 wk were observed in 4th to 5th and 8th to 10th instar phyllosomas at water temperatures of 20 and 23 degrees C, respectively. Affected larvae became opaque, exhibited small red spots throughout the body and pereiopods, and were faintly luminous when viewed in the dark. Histopathology showed that the gut and hepatopancreas tubules of moribund phyllosomas contained massive bacterial plaques. The hepatopancreas tubules of moribund larvae were atrophic and some contained necrotic cells sloughed into the lumen. Dense, pure cultures of a bacterium identified as Vibrio harveyi were isolated from moribund larvae. The disease syndrome was reproduced by in vivo challenge and V. harveyi was successfully reisolated from diseased larvae after apparently healthy larvae were exposed by immersion to baths of more than 10(4) V. harveyi ml(-1) at 24 degrees C. Injured larvae were more susceptible to infection than were healthy larvae. Survival of larvae experimentally and naturally exposed to V. harveyi was improved when antibiotics were administered via bath exposures.     

Induction of AIDS virus-specific CTL activity in fresh, unstimulated peripheral blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen

The observed role of CTL in the containment of AIDS virus replication suggests that an effective HIV vaccine will be required to generate strong CTL responses. Because epitope-based vaccines offer several potential advantages for inducing strong, multispecific CTL responses, we tested the ability of an epitope-based DNA prime/modified vaccinia virus Ankara (MVA) boost vaccine to induce CTL responses against a single SIVgag CTL epitope. As assessed using both 51Cr release assays and tetramer staining of in vitro stimulated PBMC, DNA vaccinations administered to the skin with the gene gun induced and progressively increased p11C, C-->M (CTPYDINQM)-specific CD8+ T lymphocyte responses in six of six Mamu-A*01+ rhesus macaques. Tetramer staining of fresh, unstimulated PBMC from two of the DNA-vaccinated animals indicated that as much as 0.4% of all CD3+/CD8alpha+ T lymphocytes were specific for the SIVgag CTL epitope. Administration of MVA expressing the SIVgag CTL epitope further boosted these responses, such that 0.8-20.0% of CD3+/CD8alpha+ T lymphocytes in fresh, unstimulated PBMC were now Ag specific. Enzyme-linked immunospot assays confirmed this high frequency of Ag-specific cells, and intracellular IFN-gamma staining demonstrated that the majority of these cells produced IFN-gamma after peptide stimulation. Moreover, direct ex vivo SIV-specific cytotoxic activity could be detected in PBMC from five of the six DNA/MVA-vaccinated animals, indicating that this epitope-based DNA prime/MVA boost regimen represents a potent method for inducing high levels of functionally active, Ag-specific CD8+ T lymphocytes in non-human primates.