The cell death protease Kex1p is essential for hypochlorite-induced apoptosis in yeast

Following microbial pathogen invasion, the human immune system of activated phagocytes generates and releases the potent oxidant hypochlorous acid (HOCl), which contributes to the killing of menacing microorganisms. Though tightly controlled, HOCl generation by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils/monocytes may occur in excess and lead to tissue damage. It is thus of marked importance to delineate the molecular pathways underlying HOCl cytotoxicity in both microbial and human cells. Here, we show that HOCl induces the generation of reactive oxygen species (ROS), apoptotic cell death and the formation of specific HOCl-modified epitopes in the budding yeast Saccharomyces cerevisiae. Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect. The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification. By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes.     

Receptor Binding Sites and Antigenic Epitopes on the Fiber Knob of Human Adenovirus Serotype 3

The adenovirus fiber knob causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s), the interaction of labeled cell membrane proteins to synthetic peptides covering the adenovirus type 3 (Ad3) fiber knob was studied. Peptide P6 (amino acids [aa] 187 to 200), to a lesser extent P14 (aa 281 to 294), and probably P11 (aa 244 to 256) interacted specifically with cell membrane proteins, indicating that these peptides present cell receptor binding sites. Peptides P6, P11, and P14 span the D, G, and I β-strands of the R-sheet, respectively. The other reactive peptides, P2 (aa 142 to 156), P3 (aa 153 to 167), and P16 (aa 300 to 319), probably do not present real receptor binding sites. The binding to these six peptides was inhibited by Ad3 virion and was independent of divalent cations. We have also screened the antigenic epitopes on the knob with recombinant Ad3 fiber, recombinant Ad3 fiber knob, and Ad3 virion-specific antisera by enzyme-linked immunosorbent assay. The main antigenic epitopes were presented by P3, P6, P12 (aa 254 to 269), P14, and especially the C-terminal P16. Peptides P14 and P16 of the Ad3 fiber knob were able to inhibit Ad3 infection of cells.     

Validation of density–elasticity relationships for finite element modeling of human pelvic bone by modal analysis

In total hip arthroplasty and particularly in revision surgery, computer assisted pre-operative prediction of the best possible anchorage strategy for implant fixation would be a great help to the surgeon. Computer simulation relies on validated numerical models. In the current study, three density–elasticity relationships (No. 1–3) from the literature for inhomogeneous material parameter assignment from CT data in automated finite element (FE) modeling of long bones were evaluated for their suitability for FE modeling of human pelvic bone. Numerical modal analysis was conducted on 10 FE models of hemipelvic bone specimens and compared to the gold standard provided by experimental modal analysis results from a previous in-vitro study on the same specimens. Overall, calculated resonance frequencies came out lower than measured values. Magnitude of mean relative deviation of numerical resonance frequencies with regard to measured values is lowest for the density–elasticity relationship No. 3 (−15.9%) and considerably higher for both density–elasticity relationships No. 1 (−41.1%) and No. 2 (−45.0%). Mean MAC values over all specimens amount to 77.8% (No. 1), 78.5% (No. 2), and 83.0% (No. 3). MAC results show, that mode shapes are only slightly influenced by material distribution. Calculated resonance frequencies are generally lower than measured values, which indicates, that numerical models lack stiffness. Even when using the best suited (No. 3) out of three investigated density–elasticity relationships, in FE modeling of pelvic bone a considerable underestimation of model stiffness has to be taken into account.     

Estimation of paracellular conductance of primary rat alveolar epithelial cell monolayers.

Freshly isolated rat type II pneumocytes, when grown on permeable tissue culture-treated polycarbonate filters, form confluent alveolar epithelial cell monolayers (RAECM). Cells in RAECM undergo transdifferentiation, exhibiting over time morphological and phenotypic characteristics of type I pneumocytes in vivo. We recently reported that transforming growth factor-beta(1) (TGF-beta(1)) decreases overall monolayer resistance (R(te)) and stimulates short-circuit current in a dose-dependent manner. In this study, we investigated the effects of TGF-beta(1) (50 pM) or 10% newborn bovine serum (NBS) on modulation of paracellular passive ion conductance and its contribution to total passive ion conductance across RAECM. On days 5-7 in culture, tight-junctional resistance (R(tj), kOmegacm(2)) of RAECM, cultured in minimally defined serum-free medium (MDSF) with or without TGF-beta(1) or NBS, was estimated from the relationship between observed transmonolayer voltage and resistance after addition of gramicidin D to apical potassium isethionate Ringer solution under open-circuit conditions. NaCl Ringer solution bathed the basolateral side throughout the experimental period. Results showed that transmonolayer conductance (1/R(te)) and tight-junctional conductance (1/R(tj)) are 0.59 and 0.14 mS/cm(2) for control monolayers in MDSF, 1.59 and 0.38 mS/cm(2) for monolayers exposed to TGF-beta(1), and 0.38 and 0.18 mS/cm(2) for monolayers grown in the presence of NBS. The contributions to total transepithelial ion conductance by the paracellular pathway are estimated to be 23, 23, and 47% for control, TGF-beta(1)-exposed, and newborn bovine serum (NBS)-treated RAECM, respectively.     

Responses of efferent octopaminergic thoracic unpaired median neurons in the locust to visual and mechanosensory signals

Insect thoracic ganglia contain efferent octopaminergic unpaired median neurons (UM neurons) located in the midline, projecting bilaterally and modulating neuromuscular transmission, muscle contraction kinetics, sensory sensitivity and muscle metabolism. In locusts, these neurons are located dorsally or ventrally (DUM- or VUM-neurons) and divided into functionally different sub-populations activated during different motor tasks. This study addresses the responsiveness of locust thoracic DUM neurons to various sensory stimuli. Two classes of sense organs, cuticular exteroreceptor mechanosensilla (tactile hairs and campaniform sensilla), and photoreceptors (compound eyes and ocelli) elicited excitatory reflex responses. Chordotonal organ joint receptors caused no responses. The tympanal organ (Müller's organ) elicited weak excitatory responses most likely via generally increased network activity due to increased arousal. Vibratory stimuli to the hind leg subgenual organ never elicited responses. Whereas DUM neurons innervating wing muscles are not very responsive to sensory stimulation, those innervating leg and other muscles are very responsive to stimulation of exteroreceptors and hardly responsive to stimulation of proprioceptors. After cutting both cervical connectives all mechanosensory excitation is lost, even for sensory inputs from the abdomen. This suggests that, in contrast to motor neurons, the sensory inputs to octopaminergic efferent neuromodulatory cells are pre-processed in the suboesophageal ganglion.     

Role of lectins in plant--microorganism interactions. IV. Ultrastructural localization of soybean lectin binding sites of Rhizobium japonicum

The binding of purified, ferritin-labeled soybean seed lectin to the cell surfaces of Rhizobium japonicum 31 lb 138 has been examined by whole mount, thin section, and freeze-etch electron microscopy. The ferritin-labeled lectin binds in a biochemically specific manner to the capsular material of this bacterium. The lectin does not bind to the outer membranes of the cells or to flagella. Labeled lectin binds to sites throughout the capsular structure, although the density of labeling is somewhat greater on the outer surface of the capsule. Some cells appear to be partially encapsulated. Preservation of the capsular material proved difficult, and methods for retaining most of the capsular material were developed.     

Genetic and structural analysis of a virulence determinant in polyomavirus VP1.

The LID strain of polyomavirus differs from other laboratory strains in causing a rapidly lethal infection of newborn C3H/Bi mice. This virulent behavior of LID was attenuated by dilution, yet at sublethal doses LID was able to induce tumors at a high frequency, like its parent virus PTA. By constructing and assaying LID-PTA recombinant viruses and by DNA sequencing, the determinant of virulence in LID was mapped to the major viral capsid protein, VP1. The VP1s of LID and PTA differed at two positions: at 185, LID has phenylalanine and PTA has tyrosine, and at 296, LID has alanine and PTA has valine. Results obtained with viruses constructed by site-directed mutagenesis showed that alanine at position 296 is sufficient to confer a fully virulent phenotype regardless of which amino acid is at position 185. However, with valine at position 296, an effect of phenylalanine at position 185 is apparent, as this virus possesses an intermediate level of virulence. A crystal structure of polyomavirus complexed with 3'-sialyl lactose previously indicated van der Waals contacts between the side chain of valine 296 and the sialic acid ring (T. Stehle, Y. Yan, T. L. Benjamin, and S. C. Harrison, Nature [London] 369:160-163, 1994). When this interaction was modeled with alanine, these contacts were greatly reduced. Direct confirmation that the substitutions in VP1 affected receptor binding was obtained by studying virus hemagglutination behavior. The ensemble of results are discussed in terms of the idea that a lower affinity of the virus for its receptor can result in more rapid spread and increased pathogenicity.