Frictional properties of contacting surfaces in the hemelytra-hindwing locking mechanism in the bug<Emphasis Type="Italic"> Coreus marginatus</Emphasis> (Heteroptera,Coreidae)

The structure and function of the hemelytra-to-hindwing locking mechanism of the bug Coreus marginatus were analysed. The system consists of a cuticular protrusion in the ventral side of the hemelytra, which locks the subcostal border of the hindwing in flight. The speed and distance slid by both surfaces against one another during flight were assessed using a combination of high-speed video recordings and a 2D geometrical model. The friction coefficient between sliding surfaces was assessed using a micromanipulator, coupled with force transducers. This was done under three experimental conditions: freshly dissected, air dried and rehydrated ethanol preserved samples. The results showed a high speed of sliding, approximately 0.18 m s^–1, with a relatively low friction coefficient (0.2 ). There was no evident difference in the friction measured under the various treatments, with the exception of the rehydrated condition, which was lower. The surface morphology of the wing locking mechanism, namely outgrowths of one part having rounded edges, and completely flat surface on the counterpart, effectively aids in the reduction of friction at the microscopic level. The structure is effective even dry, and after being preserved in ethanol, suggesting that no cuticle secreted lubrication substance is responsible for its effectiveness. The ultrastructure presumably confers mechanical stability to the system under the high load it is subjected to in flight.     

Automated apparatus for quantitation of social approach behaviors in mice

Mouse models of social dysfunction, designed to investigate the complex genetics of social behaviors, require an objective methodology for scoring social interactions relevant to human disease symptoms. Here we describe an automated, three chambered apparatus designed to monitor social interaction in the mouse. Time spent in each chamber and the number of entries are scored automatically by a system detecting photocell beam breaks. When tested with the automated equipment, juvenile male C57BL/6J mice spent more time in a chamber containing a stranger mouse than in an empty chamber (sociability), similar to results obtained by the observer scored method. In addition, automated scoring detected a preference to spend more time with an unfamiliar stranger than a more familiar conspecific (preference for social novelty), similar to results obtained by the observer scored method. Sniffing directed at the wire cage containing the stranger mouse correlated significantly with time spent in that chamber, indicating that duration in a chamber represents true social approach behavior. Number of entries between chambers did not correlate with duration of time spent in the chambers; entries instead proved a useful control measure of general activity. The most significant social approach behavior took place in the first five minutes of both the sociability and preference for social novelty tests. Application of these methods to C57BL/6J, DBA/2J and FVB/NJ adult males revealed that all three strains displayed tendencies for sociability and preference for social novelty. To evaluate the importance of the strain of the stranger mouse on sociability and preference for social novelty, C57BL/6J subject mice were tested either with A/J strangers or with C57BL/6J strangers. Sociability and preference for social novelty were similar with both stranger strains. The automated equipment provides an accurate and objective approach to measuring social tendencies in mice. Its use may allow higher-throughput scoring of mouse social behaviors in mouse models of social dysfunction.     

Pharmacokinetic investigation of a 14C-labelled beta 3/alpha tetrapeptide in rats

The solid-phase synthesis and an ADME investigation with albino and pigmented male rats of the doubly 14C-labelled beta/alpha-tetrapeptide derivative Ac-beta3 hTyr-(D)Trp-beta3 hLys-beta3 hThr-lactone (3; Fig. 3) are described. After intravenous (i.v.) and peroral (p.o.) administration of the peptide, its concentration in blood and plasma, its tissue distribution, and the metabolism and the excretion of the peptide were analyzed over a period of up to 7 days post dose. The tetrapeptide in its ring opened form, 5, has a bioavailability of ca. 25%; radioactivity is distributed in the animals in an organ-specific way, and the compound appears to pass the blood-brain barrier to a very small extent, if at all (Tables 1-3 and Figs. 2-6). Excretion (37% renal, 44% fecal, including biliary) of the tetrapeptide 4 days after i.v. administration is almost complete, with only 4.3% remaining in the carcass; 4 days after p.o. administration 97% of the dose has been excreted in the feces. Radiochromatograms taken of plasma (0.5 and 24 h after i.v. dosing) and of urine and feces extracts (0-48 h collected) reveal the presence of lactone 3 and/or the corresponding hydroxy acid 5 with essentially no or very minor other peaks, respectively, representing possible metabolites (Tables 4-6, and Fig. 7 and 8). A comparison with a previous ADME investigation of a beta-nonapeptide show that--except for the lack of metabolism--all aspects of exposure, distribution, and elimination are different (structure-specific properties). The investigated tetrapeptide 3 is a potent and highly specific agonist of the somatostatin receptor hsst4, rendering the results described herein promising for diagnostic and therapeutic applications of beta-peptides.     

Natural CD8<Superscript>+</Superscript> T-cell responses against MHC class I epitopes of the HER-2/<Emphasis Type="Italic">neu</Emphasis> oncoprotein in patients with epithelial tumors

HER-2/neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/neu-positive (⁺) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2–binding nona-peptide 369-377 (HER-2(9₃₆₉)). In the present study, we examined patients with HER-2/neu⁺ breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/neu–derived and HLA-A2–restricted "cytotoxic" peptides and to a novel one spanning amino acids 777–785 also with HLA-A2–binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an interferon (IFN-) ELISpot assay detecting T cells at the single cell level secreting IFN-. CTLp were defined as peptide-specific precursors per 10⁶ peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/neu-negative (^-) tumors and healthy individuals. Of the HER-2/neu⁺ patients examined, 31% had increased CTLp to HER-2(9₉₅₂), 19% to HER-2(9₆₆₅), 16% to HER-2(9₆₈₉), and 12.5% HER-2(9₄₃₅), whereas only 2 of 32 patients (6%) responded to HER-2(9₇₇₇). The CTLp recognizing HER-2(9₉₅₂) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu^- patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN- production, preexisting CTL immunity to all five HER-2/neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9₄₃₅), HER-2(9₉₅₂), HER-2(9₆₈₉), HER-2(9₆₆₅), and HER-2(9₇₇₇) is present in patients with HER-2/neu⁺ tumors of distinct histology, (2) HER-2(9₇₇₇) is a naturally processed peptide expressed on the surface of HER-2/neu⁺ tumors, as are the other four peptides, and (3) HER-2/neu⁺ prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/neu-based immunotherapy.     

CC chemokine receptor 2 expression in donor cells serves an essential role in graft-versus-host-disease

The complete repertoire of cellular and molecular determinants that influence graft-vs-host disease (GVHD) is not known. Using a well-established murine model of GVHD (B6-->bm12 mice), we sought to elucidate the role of the donor non-T cell compartment and molecular determinants therein in the pathogenesis of GVHD. In this model the acute GVHD-inducing effects of purified B6 wild-type (wt) CD4(+) T cells was inhibited by wt non-T cells in a dose-dependent manner. Paradoxically, unlike the chronic GVHD phenotype observed in bm12 mice transplanted with B6wt unfractionated splenocytes, bm12 recipients of B6ccr2-null unfractionated splenocytes developed acute GVHD and died of IFN-gamma-mediated bone marrow aplasia. This switch from chronic to acute GVHD was associated with increased target organ infiltration of activated CD4(+) T cells as well as enhanced expression of Th1/Th2 cytokines, chemokines, and the antiapoptotic factor bfl1. In vitro, ccr2(-/-) CD4(+) T cells in unfractionated splenocytes underwent significantly less activation-induced cell death than B6wt CD4(+) T cells, providing another potential mechanistic basis along with enhanced expression of bfl1 for the increased numbers of activated T cells in target organs of B6ccr2(-/-) splenocyte-->bm12 mice. Collectively, these findings have important clinical implications, as they implicate the donor non-T cell compartment as a critical regulator of GVHD and suggest that ccr2 expression in this cellular compartment may be an important molecular determinant of activation-induced cell death and GVHD pathogenesis.     

Neurovirulence in mice of H5N1 influenza virus genotypes isolated from Hong Kong poultry in 2001

We studied the pathogenicity of five different genotypes (A to E) of highly pathogenic avian H5N1 viruses, which contained HA genes similar to those of the H5N1 virus A/goose/Guangdong/1/96 and five different combinations of "internal" genes, in a mouse model. Highly pathogenic, neurotropic variants of genotypes A, C, D, and E were isolated from the brain after a single intranasal passage in mice. Genotype B virus was isolated from lungs only. The mouse brain variants had amino acid changes in all gene products except PB1, NP, and NS1 proteins but no common sets of mutations. We conclude that the original H5N1/01 isolates of genotypes A, C, D, and E were heterogeneous and that highly pathogenic neurotropic variants can be rapidly selected in mice.     

FKBP12 binding to RyR1 modulates excitation-contraction coupling in mouse skeletal myotubes

The skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel or ryanodine receptor (RyR1) binds four molecules of FKBP12, and the interaction of FKBP12 with RyR1 regulates both unitary and coupled gating of the channel. We have characterized the physiologic effects of previously identified mutations in RyR1 that disrupt FKBP12 binding (V2461G and V2461I) on excitation-contraction (EC) coupling and intracellular Ca2+ homeostasis following their expression in skeletal myotubes derived from RyR1-knockout (dyspedic) mice. Wild-type RyR1-, V246I-, and V2461G-expressing myotubes exhibited similar resting Ca2+ levels and maximal responses to caffeine (10 mm) and cyclopiazonic acid (30 microm). However, maximal voltage-gated Ca2+ release in V2461G-expressing myotubes was reduced by approximately 50% compared with that attributable to wild-type RyR1 (deltaF/Fmax = 1.6 +/- 0.2 and 3.1 +/- 0.4, respectively). Dyspedic myotubes expressing the V2461I mutant protein, that binds FKBP12.6 but not FKBP12, exhibited a comparable reduction in voltage-gated SR Ca2+ release (deltaF/Fmax = 1.0 +/- 0.1). However, voltage-gated Ca2+ release in V2461I-expressing myotubes was restored to a normal level (deltaF/Fmax = 2.9 +/- 0.6) following co-expression of FKBP12.6. None of the mutations that disrupted FKBP binding to RyR1 significantly affected RyR1-mediated enhancement of L-type Ca2+ channel activity (retrograde coupling). These data demonstrate that FKBP12 binding to RyR1 enhances the gain of skeletal muscle EC coupling.     

The control of seizure-like activity in the rat hippocampal slice

The sudden and transient hypersynchrony of neuronal firing that characterizes epileptic seizures can be considered as the transitory stabilization of metastable states present within the dynamical repertoire of a neuronal network. Using an in vitro model of recurrent spontaneous seizures in the rat horizontal hippocampal slice preparation, we present an approach to characterize the dynamics of the transition to seizure, and to use this information to control the activity and avoid the occurrence of seizure-like events. The transition from the interictal activity (between seizures) to the seizure-like event is aborted by brief (20-50 s) low-frequency (0.5 Hz) periodic forcing perturbations, applied via an extracellular stimulating electrode to the mossy fibers, the axons of the dentate neurons that synapse onto the CA3 pyramidal cells. This perturbation results in the stabilization of an interictal-like low-frequency firing pattern in the hippocampal slice. The results derived from this work shed light on the dynamics of the transition to seizure and will further the development of algorithms that can be used in automated devices to stop seizure occurrence.     

Terminal sialylation is altered in airway cells with impaired CFTR-mediated chloride transport

Reduced terminal sialylation at the surface of airway epithelial cells from patients with cystic fibrosis may predispose them to bacterial infection. To determine whether a lack of chloride transport or misprocessing of mutant cystic fibrosis transmembrane conductance regulator (CFTR) is critical for the alterations in glycosylation, we studied a normal human tracheal epithelial cell line (9/HTEo(-)) transfected with the regulatory (R) domain of CFTR, which blocks CFTR-mediated chloride transport; DeltaF508 CFTR, which is misprocessed, wild-type CFTR; or empty vector. Reduced cAMP-stimulated chloride transport is seen in the R domain and DeltaF508 transfectants. These two cell lines had consistent, significantly reduced binding of elderberry bark lectin, which recognizes terminal sialic acid in the alpha-2,6 configuration. Binding of other lectins, including Maakia amurensis lectin, which recognizes sialic acid in the alpha-2,3 configuration, was comparable in all cell lines. Because the cell surface change occurred in R domain-transfected cells, which continue to express wild-type CFTR, it cannot be related entirely to misprocessed or overexpressed CFTR. It is associated most closely with reduced CFTR activity.