Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis.

Measles virus infection of the central nervous system in the murine model of experimental measles virus-induced encephalitis is successfully controlled by virus-specific T-helper lymphocytes. T cells from BALB/c mice that are resistant to measles virus encephalitis proliferate well against measles virus in vitro, and bulk cultures recognize viral nucleocapsid and hemagglutinin as well as fusion proteins. The measles virus-specific T cells secrete large amounts of interleukin 2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) but no IL-4, IL-6, or IL-10, and hence the cytokine pattern is consistent with that of subtype 1 T-helper lymphocytes. In contrast, cells obtained from measles virus-infected susceptible C3H mice recognize measles virus proteins only weakly and secrete little IFN-gamma and TNF-alpha. Treatment of infected mice with anti-TNF-alpha antibodies has no effect on survival or virus clearance from the brain. Upon neutralization of IFN-gamma in vivo, the phenotype of measles virus-specific T-helper cells isolatable from BALB/c mice is reversed from subtype 1 to subtype 2-like. Anti-IFN-gamma antibody-treated BALB/c mice are susceptible to measles virus encephalitis, and viral clearance from the central nervous system is impaired. These results indicate that IFN-gamma plays a significant role in the control of measles virus infection of the central nervous system.     

LFchimera protects HeLa cells from invasion by <Emphasis Type="Italic">Yersinia</Emphasis> spp. in vitro

Yersinia pestis is the causative agent of plague. As adequate antibiotic treatment falls short and currently no effective vaccine is available, alternative therapeutic strategies are needed. In order to contribute to solving this problem we investigated the therapeutic potential of the peptide construct LFchimera against the safer-to-handle Y. pestis simulants Yersinia enterocolitica and Yersinia pseudotuberculosis in vitro. LFchimera is a heterodimeric peptide construct mimicking two antimicrobial domains of bovine lactoferrin, i.e. lactoferrampin and lactoferricin. LFchimera has been shown to be a potent antimicrobial peptide against a variety of bacteria in vitro and in vivo. Also Y. enterocolitica and Y. pseudotuberculosis have been shown to be susceptible for LFchimera in vitro. As Yersiniae spp. adhere to and invade host cells upon infection, we here investigated the effects of LFchimera on these processes. It was found that LFchimera has the capacity to inhibit host-cell invasion by Yersiniae spp. in vitro. This effect appeared to be host-cell mediated, not bacteria-mediated. Furthermore it was found that exposure of human HeLa epithelial cells to both LFchimera and the bacterial strains evoked a pro-inflammatory cytokine release from the cells in vitro.     

The effect of different growing conditions on water relations parameters of leaf epidermal cells of Tradescantia virginiana L.

Summary Tradescantia virginiana L. plants were cultivated under contrasting conditions of temperature, humidity, light quality and intensity, and nutrient status in order to investigate the effect of growth conditions on the water relations parameters of the leaf epidermal cells. Turgor pressure (P), volumetric elastic modulus (), half-time of water potential equilibration (T _1/2), hydraulic conductivity (L _p ) were measured with the miniaturized pressure probe in single cells of the upper and lower epidermis of leaves. Turgor differed (range: 0.1 bar to 7.2 bar) between treatments with lowest values under warm and humid conditions and additional supply of fertilizer, and highest values under conditions of low air humidity and low nutrient supply. The volumetric elastic modulus changed by 2 orders of magnitude (range: 3.0 bar to 350 bar, 158 cells), but was only affected by the treatments, in as much as it was dependent on turgor. The turgor dependence of , measured on intact leaves of T. virginiana, was similar to that for cells of the isolated (peeled) lower epidermis, where as a function of turgor was linear over the whole range of turgors. This result has implications for the discussion of pressure/volume curves as measured by the pressure bomb where changes in bulk leaf are frequently discussed as adaptations to certain treatments. The measurements of the hydraulic conductivity indicate that this parameter varies between treatments (range of means: 2.4×10^-6 cm s^-1 bar^-1 to 13.4×10^-6 cm s^-1 bar^-1). There was a negative correlation for L _p in cells of intact leaves as a function of turgor which was altered by the growing conditions. However, a correlation with turgor could not be found for cells from isolated epidermis or cells from a uniform population of plants. The large variation in L _p from cell to cell observed in the present and in previous studies was accounted for in a study of 100 cells from a uniform population of plants by the propagation of measurement errors in calculating L _p . The results suggest that in T. virginiana cellular water relations are changed mainly by the turgor dependence of .     

Preprotein signature for full susceptibility to the co‐translational translocation inhibitor cyclotriazadisulfonamide

Cyclotriazadisulfonamide (CADA) inhibits the co‐translational translocation of human CD4 (huCD4) into the endoplasmic reticulum lumen in a signal peptide (SP)‐dependent way. We propose that CADA binds the nascent huCD4 SP in a folded conformation within the translocon resembling a normally transitory state during translocation. Here, we used alanine scanning on the huCD4 SP to identify the signature for full susceptibility to CADA. In accordance with our previous work, we demonstrate that residues in the vicinity of the hydrophobic h‐region are critical for sensitivity to CADA. In particular, exchanging Gln‐15, Val‐17 or Pro‐20 in the huCD4 SP for Ala resulted in a resistant phenotype. Together with positively charged residues at the N‐terminal portion of the mature protein, these residues mediate full susceptibility to the co‐translational translocation inhibitory activity of CADA towards huCD4. In addition, sensitivity to CADA is inversely related to hydrophobicity in the huCD4 SP. In vitro translocation experiments confirmed that the general hydrophobicity of the h‐domain and positive charges in the mature protein are key elements that affect both the translocation efficiency of huCD4 and the sensitivity towards CADA. Besides these two general SP parameters that determine the functionality of the signal sequence, unique amino acid pairs (L14/Q15 and L19/P20) in the SP hydrophobic core add specificity to the sensitivity signature for a co‐translational translocation inhibitor.     

Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis

LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265–284 and lactoferricin17–30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.     

The Transfer Functions of Cardiac Tissue during Stochastic Pacing

The restitution properties of cardiac action potential duration (APD) and conduction velocity (CV) are important factors in arrhythmogenesis. They determine alternans, wavebreak, and the patterns of reentrant arrhythmias. We developed a novel approach to characterize restitution using transfer functions. Transfer functions relate an input and an output quantity in terms of gain and phase shift in the complex frequency domain. We derived an analytical expression for the transfer function of interbeat intervals (IBIs) during conduction from one site (input) to another site downstream (output). Transfer functions can be efficiently obtained using a stochastic pacing protocol. Using simulations of conduction and extracellular mapping of strands of neonatal rat ventricular myocytes, we show that transfer functions permit the quantification of APD and CV restitution slopes when it is difficult to measure APD directly. We find that the normally positive CV restitution slope attenuates IBI variations. In contrast, a negative CV restitution slope (induced by decreasing extracellular [K⁺]) amplifies IBI variations with a maximum at the frequency of alternans. Hence, it potentiates alternans and renders conduction unstable, even in the absence of APD restitution. Thus, stochastic pacing and transfer function analysis represent a powerful strategy to evaluate restitution and the stability of conduction.     

Desmocollin 3-mediated Binding Is Crucial for Keratinocyte Cohesion and Is Impaired in Pemphigus

Desmocollin (Dsc) 1–3 and desmoglein (Dsg) 1–4, transmembrane proteins of the cadherin family, form the adhesive core of desmosomes. Here we provide evidence that Dsc3 homo- and heterophilic trans-interaction is crucial for epidermal integrity. Single molecule atomic force microscopy (AFM) revealed homophilic trans-interaction of Dsc3. Dsc3 displayed heterophilic interaction with Dsg1 but not with Dsg3. A monoclonal antibody targeted against the extracellular domain reduced homophilic and heterophilic binding as measured by AFM, caused intraepidermal blistering in a model of human skin, and a loss of intercellular adhesion in cultured keratinocytes. Because autoantibodies against Dsg1 are associated with skin blistering in pemphigus, we characterized the role of Dsc3 binding for pemphigus pathogenesis. In contrast to AFM experiments, laser tweezer trapping revealed that pemphigus autoantibodies reduced binding of Dsc3-coated beads to the keratinocyte cell surface. These data indicate that loss of heterophilic Dsc3/Dsg1 binding may contribute to pemphigus skin blistering.Desmogleins (Dsg)² and desmocollins (Dsc) are members of the Ca²⁺-dependent cadherin family of adhesion molecules that extend with their outer domains into the extracellular core of desmosomes. Desmosomal cadherins include four Dsg (Dsg1–4) and three Dsc3 isoforms (Dsc1–3) (1, 2). Desmosomal cadherins share a common domain organization with five N-terminally located extracellular subdomains (EC1–5). The membrane-distal EC1 domain is thought to contain the adhesive interface necessary for trans-interaction as could be concluded from structural analysis and blocking studies using peptides and antibodies (3–5). By establishing trans- and cis-interacting adhesive complexes, desmosomal cadherins participate in providing mechanical strength to stratified epithelia (6). In human epidermis Dsg1 and Dsc1 expression decreases from the outermost granular layer toward deeper layers, whereas Dsg3 and Dsc3 are primarily found in the basal layer and display an inverse expression gradient (7, 8). In contrast to classical cadherins present in adherens junctions that primarily undergo homophilic trans-interaction, desmosomal cadherins are generally believed to mediate both homo- and heterophilic binding (9). Recently, an important role of Dsc3 for integrity of murine epidermis was demonstrated in animals with conditional epidermal Dsc3 deficiency that suffered from severe intraepidermal blister formation (10) comparable with the phenotype of the autoimmune bullous skin disease pemphigus vulgaris (PV) (11). PV is associated with antibodies (Abs) against Dsg3, in part combined with Abs targeting Dsg1, whereas Dsg1 Abs alone are associated with pemphigus foliaceus (PF). However, PV and PF sera usually do not contain autoantibodies targeting Dsc3 (12). In view of the apparently important role of Dsc3 in epidermal adhesion, we addressed whether Dsg1 and Dsg3 might heterophilically interact with Dsc3 and whether Abs in pemphigus might interfere with such type of interaction.