The properties of peptidyl diazoethanes and chloroethanes as protease inactivators

Earlier work has demonstrated the irreversible inactivation of serine and cysteine proteinases by peptides with a C-terminal chloromethyl ketone group. With a C-terminal diazomethyl ketone, on the other hand, peptides become reagents specific for cysteine proteinases. We have now synthesized and examined the properties of reagents with an additional methyl side chain near the reactive grouping with the goal of diminishing side reactions in a cellular environment. Derivatives of neutral amino acids as well as of lysine and arginine have been prepared. The chloroethyl ketones are about 60% less reactive to chemical nucleophiles than the chloromethyl ketones. However, the susceptibilities of the proteases examined varied remarkably. Cathepsins B and L of the papain family of cysteine proteinases were much less susceptible (about 2 orders of magnitude less) to both peptidyl diazoethyl and chloroethyl ketones. In marked contrast, clostripain, a cysteine proteinase of a separate family was decisively more susceptible to chloroethyl ketones. The serine proteinases showed a drop in susceptibility to the chloroethyl ketones generally, and this was similar to the drop in chemical reactivity in proceeding from the chloromethyl to the chloroethyl ketone.     

Evaluation of inhibitor constants and alkylation rates for a series of thrombin affinity labels.

The kinetics for the inactivation of thrombin (EC 3.4.21.5) by a series of peptides containing C-terminal arginyl chloromethane in the presence of substrate were determined. The inhibitor effectiveness was analysed so as to allow for both the evaluation of the affinity with which the enzyme binds the inhibitor before irreversible modification and also the rate of covalent-bond formation between enzyme and inhibitor. The results obtained show that the observed large range in inhibitor effectiveness can be accounted for almost entirely by marked differences in affinity, with only small variations in rates of formation of covalent complex.     

A Chemokine-Like Viral Protein Enhances Alpha Interferon Production by Plasmacytoid Dendritic Cells but Delays CD8+ T Cell Activation and Impairs Viral Clearance

Murine cytomegalovirus encodes numerous proteins that act on a variety of pathways to modulate the innate and adaptive immune responses. Here, we demonstrate that a chemokine-like protein encoded by murine cytomegalovirus activates the early innate immune response and delays adaptive immunity, thereby impairing viral clearance. The protein, m131/129 (also known as MCK-2), is not required to establish infection in the spleen; however, a mutant virus lacking m131/129 was cleared more rapidly from this organ. In the absence of m131/129 expression, there was enhanced activation of dendritic cells (DC), and virus-specific CD8⁺ T cells were recruited into the immune response earlier. Viral mutants lacking m131/129 elicited weaker production of alpha interferon (IFN-α) at 40 h postinfection, indicating that this protein exerts its effects during early rounds of viral replication in the spleen. Furthermore, while wild-type and mutant viruses activated plasmacytoid dendritic cells (pDC) equally at this time, as measured by the upregulation of costimulatory molecules, the presence of m131/129 stimulated more pDC to secrete IFN-α, accounting for the stronger IFN-α response than from the wild-type virus. These data provide evidence for a novel immunomodulatory function of a viral chemokine and expose the multifunctionality of immune evasion proteins. In addition, these results broaden our understanding of the interplay between innate and adaptive immunity.     

Reliability of two-point discrimination thresholds using a 4-2-1 stepping algorithm

Purpose: A 4-2-1 stepping algorithm reliably captures light touch thresholds but has not been used to assess two-point discrimination (TPD) thresholds. Therefore, the purpose of this investigation was to determine the intra- and inter-rater reliability of a 4-2-1 stepping algorithm at determining TPD thresholds.

Materials and methods: Fifteen healthy, physically active young adults were assessed twice over a 1-week period using digital calipers and a 4-2-1 stepping algorithm. TPD thresholds were assessed by an expert and a novice examiner at each time point. Reliability was assessed on the plantar surface of the foot at the head of the first and base of the fifth metatarsal.

Results: Three intra-rater intraclass correlation coefficient (ICC) values exceeded 0.75 and were interpreted as good. The inter-rater reliability was good with ICC values ranging from 0.76 to 0.93 at both sites during both test sessions.

Conclusions: The 4-2-1 stepping algorithm demonstrates good intra- and inter-tester reliability at determining TPD thresholds on the plantar surface of the foot at the head of the first and base of the fifth metatarsal in young healthy adults.     

Allergic airways disease develops after an increase in allergen capture and processing in the airway mucosa

Airway mucosal dendritic cells (AMDC) and other airway APCs continuously sample inhaled Ags and regulate the nature of any resulting T cell-mediated immune response. Although immunity develops to harmful pathogens, tolerance arises to nonpathogenic Ags in healthy individuals. This homeostasis is thought to be disrupted in allergic respiratory disorders such as allergic asthma, such that a potentially damaging Th2-biased, CD4(+) T cell-mediated inflammatory response develops against intrinsically nonpathogenic allergens. Using a mouse model of experimental allergic airways disease (EAAD), we have investigated the functional changes occurring in AMDC and other airway APC populations during disease onset. Onset of EAAD was characterized by early and transient activation of airway CD4(+) T cells coinciding with up-regulation of CD40 expression exclusively on CD11b(-) AMDC. Concurrent enhanced allergen uptake and processing occurred within all airway APC populations, including B cells, macrophages, and both CD11b(+) and CD11b(-) AMDC subsets. Immune serum transfer into naive animals recapitulated the enhanced allergen uptake observed in airway APC populations and mediated activation of naive allergen-specific, airway CD4(+) T cells following inhaled allergen challenge. These data suggest that the onset of EAAD is initiated by enhanced allergen capture and processing by a number of airway APC populations and that allergen-specific Igs play a role in the conversion of normally quiescent AMDC subsets into those capable of inducing airway CD4(+) T cell activation.     

Synthesis and characterization of tetrakis(μ-hydroxo)tetrakis(2,2′-dipicolylamine)tetranickel perchlorate, a nickel-hydroxy cubane complex

A novel tetranuclear complex, [Ni(μ₃-OH)(DPA)]₄(ClO₄)₄ (where DPA = 2,2′-dipicolylamine) has been synthesized, with characterization including electronic and infrared spectroscopy, elemental analysis, mass spectrometry, crystal structure analysis, and variable-temperature and variable-field magnetic susceptibility measurements. The complex features a 4Ni-4OH cubane-type cluster, displaying both ferromagnetic and antiferromagnetic intracluster interactions in a 2J model (J₁ = −3.4 cm⁻¹, J₂ = 4.7 cm⁻¹, D = 2.0 cm⁻¹). Each nickel atom sits in a pseudooctahedral environment, with one DPA molecule facially coordinated and the remaining three coordination sites occupied by the bridging hydroxide anions that make up the cubane core.     

Steric and counterion effects on the structure of dipicolylamine nickel complexes

Four nickel complexes each containing an R-2,2′-dipicolylamine ligand species (RDPA; R = benzyl, isopropyl, or tert-butyl) were synthesized and structurally characterized. In the absence of an interfering coordinating counterion, BzDPA and iPrDPA form 1:2 nickel:ligand complexes, with two facial ligands completing an pseudooctahedral nickel(II) coordination environment. In contrast, the sterically hindered tBuDPA ligand instead forms 1:1 metal:ligand complexes, even in the absence of associating counterions. Two novel tBuDPA nickel complexes with different counterions are described: nickel(II) chloride gives rise to an unusual 2Ni-3Cl dimer complex, while nickel(II) nitrate affords a 1:1 nickel:ligand complex which crystallizes with both fac and mer conformations in the same unit cell.     

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy

Accumulation of depolarized mitochondria within beta-cells has been associated with oxidative damage and development of diabetes. To determine the source and fate of depolarized mitochondria, individual mitochondria were photolabeled and tracked through fusion and fission. Mitochondria were found to go through frequent cycles of fusion and fission in a 'kiss and run' pattern. Fission events often generated uneven daughter units: one daughter exhibited increased membrane potential (delta psi(m)) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of non-fusing mitochondria that were found to have reduced delta psi(m) and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1(K38A) or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion. Pulse chase and arrest of autophagy at the pre-proteolysis stage reveal that before autophagy mitochondria lose delta psi(m) and OPA1, and that overexpression of OPA1 decreases mitochondrial autophagy. Together, these findings suggest that fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy.     

Restricted aeroallergen access to airway mucosal dendritic cells in vivo limits allergen-specific CD4+ T cell proliferation during the induction of inhalation tolerance

Chronic innocuous aeroallergen exposure attenuates CD4(+) T cell-mediated airways hyperresponsiveness in mice; however, the mechanism(s) remain unclear. We examined the role of airway mucosal dendritic cell (AMDC) subsets in this process using a multi-OVA aerosol-induced tolerance model in sensitized BALB/c mice. Aeroallergen capture by both CD11b(lo) and CD11b(hi) AMDC and the delivery of OVA to airway draining lymph nodes by CD8α(-) migratory dendritic cells (DC) were decreased in vivo (but not in vitro) when compared with sensitized but nontolerant mice. This was functionally significant, because in vivo proliferation of OVA-specific CD4(+) T cells was suppressed in airway draining lymph nodes of tolerized mice and could be restored by intranasal transfer of OVA-pulsed and activated exogenous DC, indicating a deficiency in Ag presentation by endogenous DC arriving from the airway mucosa. Bone marrow-derived DC Ag-presenting function was suppressed in multi-OVA tolerized mice, and allergen availability to airway APC populations was limited after multi-OVA exposure, as indicated by reduced OVA and dextran uptake by airway interstitial macrophages, with diffusion rather than localization of OVA across the airway mucosal surface. These data indicate that inhalation tolerance limits aeroallergen capture by AMDC subsets through a mechanism of bone marrow suppression of DC precursor function coupled with reduced Ag availability in vivo at the airway mucosa, resulting in limited Ag delivery to lymph nodes and hypoproliferation of allergen-specific CD4(+) T cells.     

A Natural Genetic Variant of Granzyme B Confers Lethality to a Common Viral Infection

Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmB^W) common in wild mouse. While retaining ‘Asp-ase’ activity, GzmB^W has substrate preferences that differ considerably from GzmB^P, which is common to all inbred strains. In vitro, GzmB^W preferentially cleaves recombinant Bid, whereas GzmB^P activates pro-caspases directly. Recombinant GzmB^W and GzmB^P induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmB^W were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmB^W-expressing CD8 T cells isolated from infected mice were unable to kill MCMV-infected targets in vitro. Our results suggest that known virally-encoded inhibitors of the intrinsic (mitochondrial) apoptotic pathway account for the increased susceptibility of GzmB^W mice to MCMV. We conclude that different natural variants of GzmB have a profound impact on the immune response to a common and authentic viral pathogen.