Influence of the probiotic Bacillus cereus var. toyoi on the intestinal immunity of piglets.

In a feeding trial, sows and piglets were fed with the probiotic bacterium Bacillus cereus var. toyoi as a feed additive, and the effects on immune cell populations were examined. The development of the gut immune system was determined for piglets at the ages of 14, 28, 35 and 56 days post partum. Tissue samples of the Jejunum and the continuous Peyer's patch were used for enumeration of intraepithelial lymphocyte populations by fluorescence activated flow cytometry and fluorescence microscopy. Both independent methods of investigation led to similar results: the population of intraepithelial CD8+ T cells was significantly enhanced in the probiotic group piglets (p< or =0.05), and the numbers of gammadelta T cells tended to be higher in the intestinal epithelium (p<0.1) at the time of weaning (day 28). Lamina propria lymphocytes were also influenced by the treatment. Application of B. cereus var. toyoi resulted in significantly more CD25+ lymphocytes and gammadelta T cells in the probiotic group post-weaning. The occurrence of pathogenic Escherichia coli serogroups was also less frequent in the feces of piglets from the probiotic group. The finding that the CD8+ T cell population in the intestinal mucosa showed changes on day 28 indicated that the influence of B. cereus var. toyoi supplementation on the intestinal immune system started before weaning, an observation supported by changes in the intestinal microflora observed during the suckling-period. The results suggest that feeding of B. cereus var. toyoi to sows may result in beneficial effects on piglet health status independent of their feed supplementation.

Influence of a probiotic Enterococcus faecium strain on development of the immune system of sows and piglets.

The influence of the probiotic bacterium Enterococcus faecium SF68 on the immune system and the intestinal colonization of pigs were determined in a feeding experiment with sows and piglets. Mucosal immunity of the developing piglets was monitored by isolation and detection of intestinal lymphocyte cell populations from the proximal jejunal epithelium and the continuous Peyers patches by the use of flow cytometry. The levels of intestinal IgA in both groups of piglets were compared, as well as total IgG in the serum of sows and piglets. Feces of the sows and intestinal contents of the piglets were taken for determination of total anaerobe and coliform bacterial counts in both probiotic and control groups. Villus length and depth of the crypts were measured in the jejunum of sacrificed piglets to monitor the development of the intestinal mucosal surface amplification. Total serum IgG of the sows appeared to be unaffected. Piglets of both groups showed similar IgG levels up to 5 weeks after birth with a slight tendency toward lower values in the probiotic group. At an age of 8 weeks the total IgG levels of the probiotic animals were significantly lower (p<0.01). No differences were observed in the populations of CD4+ and CD8+ T cells in the Peyers patches. However, the levels of cytotoxic T cells (CD8+) in the jejunal epithelium of piglets of the probiotic group were significantly reduced. The depth of the jejunal crypts and length of the villi were similar in both groups, suggesting the relative T-cell population differences were not due to alterations in the epithelial cell numbers. The total anaerobe and coliform bacterial populations were not significantly affected by the probiotic treatment, either in sows or in the piglets. However, a remarkable decline in the frequency of beta-haemolytic and O141 serovars of Escherichia coli was observed in the intestinal contents of probiotic piglets, suggesting an explanation for the reduction in cytotoxic T-cell populations.

Cytokine profile in canine immune-mediated polyarthritis and osteoarthritis.

The purpose of this study was to determine the cytokine profile in 21 dogs with canine immune-mediated polyarthritis (IMA) and 15 dogs with osteoarthritis (OA) caused by cranial cruciate ligament rupture (CCLR). The mRNA expression of interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon (IFN)-gamma, transforming growth factor (TGF)-beta, and tumour necrosis factor (TNF)-alpha were analysed in synovial fluid by semi-quantitative RT-PCR, while TNF-alpha protein was determined by L929 cytotoxicity assay. The frequency of lymphocytes was analysed using FACScan. Both disorders reveal a similar cytokine expression pattern, except for significant lower IL-1beta expression in OA. Th1 cytokines IL-2 and IFN-gamma were detected, while IL-4 was nearly absent in IMA and OA. Furthermore, the bioassay demonstrates a significantly higher production of TNF-alpha in synovial fluid of dogs with IMA, compared to dogs with OA (p < 0.05). The frequency of CD4+, CD8+ and MHC class II+ cells was relatively higher in synovial fluids compared to peripheral blood in IMA. These findings reveal that the difference between the cytokine pattern of canine IMA and OA seems to be rather quantitative than qualitative. Both joint disorders show predominance of pro-inflammatory cytokines and absence of TH2 cytokine expression, indicating the potential of IL-4 for a gene therapeutic approach.

Comparative approaches to avian song system function: insights into auditory and motor processing.

Many fundamental advances in our understanding of basic neural function have been made using bird song learning and performance as a model system. These advances have included a greater understanding of higher-order neural processing, developmental and hormonal influences on behavior, and the realization that neurogenesis plays an important role in normal adult brain function. The great diversity of passerine birds and song-related behaviors they exhibit suggest that oscine songbirds are ideally suited for comparative studies. While the comparative approach has been used successfully in the past to study song-related phenomena at anatomical and behavioral levels, it has been underutilized in addressing questions at the neurophysiological level. Most neurophysiological studies of songbird auditory and motor processing have been performed in one species, the zebra finch (Taeniopygia guttata). We present and compare neurophysiological studies we have performed in zebra finches and song sparrows (Melospiza melodia), species that differ markedly in their singing behavior and song repertoire characteristics. Interspecific similarities, and striking differences, in song neural processing are apparent. While preliminary, these data suggest that comparative neurophysiological studies of species carefully chosen for their vocal repertoire and singing behavior will contribute significantly to our understanding of vertebrate sensory and motor neural processing.

Biomarkers of joint tissue metabolism in canine osteoarthritic and arthritic joint disorders.

OBJECTIVE: To explore the levels of matrix metalloprotease-3 (MMP-3), tissue inhibitor of metalloproteases-1 (TIMP-1), 5D4 keratan sulfate, and two 3B3 chondroitin-sulfate epitopes in several canine osteoarthritic and inflammatory arthropathies. METHODS: Blood and synovial fluid were obtained from 103 dogs with rupture of the anterior cruciate ligament (ACLR), osteochondritis dissecans (OCD), fragmented coronoid process (FPC), patella luxation (PL), hip dysplasia (HD) or infectious arthritis. Dogs with non-musculosceletal disorders were used as controls. The biomarkers were measured by immunoassays. RESULTS: Median levels of synovial MMP-3, TIMP-1 and molar ratios of MMP/TIMP-1 were significantly higher in the arthritis than in the control group. The release of 5D4 keratan sulfate epitope and serum 3B3 neoepitope was reduced in arthritis patients. Increases in synovial TIMP-1 in OA were less pronounced and the molar ratio of MMP-3/TIMP-1 remained far below 1.0, demonstrating a surplus of the protease inhibitor. In osteoarthritic patients median levels of synovial 5D4 keratan sulfate were up-regulated after ACLR and PL and were inversely correlated with increasing duration of lameness. Serum TIMP-1 levels were significantly reduced in the joint disorder group when compared with the control group. CONCLUSION: Our observations present the TIMP-1 serum level as a potential marker for the detection of degenerative changes in cartilage and also indicate that in canine OA, the MMP-3 mediated matrix destruction is not of major importance. However MMP-3 seems to be a sensitive marker for the local inflammation in canine arthritis.

Molecular cloning, expression and characterization of the Canis familiaris interleukin-4.

Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. The rapid amplification of cDNA ends (RACE) was used to clone the canine IL-4 gene. It was expressed in mammalian cells and Escherichia coli. Monoclonal antibodies were raised to rcIL-4 for use in an enzyme-linked immunosorbent assay (ELISA). This will facilitate studies on the role of cIL-4 in inflammatory diseases, particularly rheumatoid arthritis.

Enzymatic depalmitoylation of viral glycoproteins with acyl-protein thioesterase 1 in vitro.

Many glycoproteins of enveloped viruses as well as cellular proteins are covalently modified with fatty acids. Palmitoylation is often reversible, but the enzymology of this hydrophobic protein modification is not understood. Recently a cytosolic enzyme designated acyl-protein thioesterase 1 (APT1) was purified, which depalmitoylates several cellular proteins. Since hitherto no transmembrane proteins have been tested as substrates for APT1 we have investigated whether palmitoylated viral membrane glycoproteins can be deacylated by use of this enzyme. Recombinant APT1 was purified from Escherichia coli, and depalmitoylation of [3H]palmitate-labeled glycoproteins present in virus particles was measured by SDS-PAGE, fluorography, and scanning densitometry. We find that APT1 causes rapid and almost complete cleavage of fatty acids from the G-protein of vesicular stomatitis virus, hemagglutinin proteins of influenza A and C virus, and E2 of Semliki Forest virus (SFV). In contrast, E1 of SFV is largely resistant against APT1 activity. This substrate specificity of APT1 was also observed using microsomes prepared from SFV-infected cells. Our data emphasize the potential of APT1 as a tool for functional analysis of protein-bound fatty acids.

5-Hydroxytryptamine 4(a) receptor expressed in Sf9 cells is palmitoylated in an agonist-dependent manner.

The mouse 5-hydroxytryptamine 4(a) receptor [5-HT(4(a))] was expressed with a baculovirus system in insect cells and analysed for acylation. [(3)H]Palmitic acid was effectively incorporated into 5-HT(4(a)) and label was sensitive to the treatment with reducing agents indicating a thioester-type bond. Analysis of protein-bound fatty acids revealed that 5-HT(4(a)) contains predominantly palmitic acid. Treatment of infected Sf9 (Spodoptera frugiperda) cells with BIMU8 [(endo-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dehydro-2-oxo-3-(prop-2-yl)-1H-benzimid-azole-1-carboxamide], a 5-HT(4) receptor-selective agonist, generated a dose-dependent increase in [(3)H]palmitate incorporation into 5-HT(4(a)) with an EC(50) of approx. 10 nM. The change in receptor labelling after stimulation with agonist was receptor-specific and did not result from general metabolic effects. We also used both pulse labelling and pulse-chase labelling to address the dynamics of 5-HT(4(a)) palmitoylation. Incorporation studies revealed that the rate of palmitate incorporation was increased approx. 3-fold after stimulation with agonist. Results of pulse-chase experiments show that activation with BIMU8 promoted the release of radiolabel from 5-HT(4(a)), thereby reducing the levels of receptor-bound palmitate to approximately one-half. Taken together, our results demonstrate that palmitoylation of 5-HT(4(a)) is a reversible process and that stimulation of 5-HT(4(a)) with agonist increases the turnover rate for receptor-bound palmitate. This provides evidence for a regulated cycling of receptor-bound palmitate and suggests a functional role for palmitoylation/depalmitoylation in 5-hydroxytryptamine-mediated signalling.

Lipid composition of virosomes modulates their fusion efficiency with cryopreserved bull sperm cells.

Virosomes derived from different fusogenic enveloped viruses have been generated for potential application in gene targeting to sperm cells. Comparative characterization of reconstitution products revealed that virosomes derived from influenza viruses are superior to those generated from Sendai viruses, with respect to the fusion rates with cryopreserved bull sperm cells and to sperm cell vitality after fusion. Modulation of the lipid composition during virosome reconstitution affects fusion sites on target sperms and allows optimization of the fusion rate and sperm cell vitality. A fluorescence-based microscopic fusion assay combined with a vital cell stain revealed that about 90% of sperm cells fused with influenza virosomes containing exogenous cholesterol, sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. About 85% of the fused sperm cells remained vital. Such optimized influenza-derived virosomes provide the basis for ongoing experiments, which aim at eventually generating biologically active transgenic sperms.

Slow synaptic inhibition mediated by metabotropic glutamate receptor activation of GIRK channels.

Glutamate is the predominant excitatory neurotransmitter in the vertebrate CNS. Ionotropic glutamate receptors mediate fast excitatory actions whereas metabotropic glutamate receptors (mGluRs) mediate a variety of slower effects. For example, mGluRs can mediate presynaptic inhibition, postsynaptic excitation, or, more rarely, postsynaptic inhibition. We previously described an unusually slow form of postsynaptic inhibition in one class of projection neuron in the song-control nucleus HVc of the songbird forebrain. These neurons, which participate in a circuit that is essential for vocal learning, exhibit an inhibitory postsynaptic potential (IPSP) that lasts several seconds. Only a portion of this slow IPSP is mediated by GABA(B) receptors. Since these cells are strongly hyperpolarized by agonists of mGluRs, we used intracellular recording from brain slices to investigate the mechanism of this hyperpolarization and to determine whether mGluRs contribute to the slow synaptic inhibition. We report that mGluRs hyperpolarize these HVc neurons by activating G protein-coupled, inwardly-rectifying potassium (GIRK) channels. MGluR antagonists blocked this response and the slow synaptic inhibition. Thus, glutamate can combine with GABA to mediate slow synaptic inhibition by activating GIRK channels in the CNS.

Combining phage display and screening of cDNA expression libraries: a new approach for identifying the target antigen of an scFv preselected by phage display.

A potential method for identifying new tumor-specific antibody structures as well as tumor-associated antigens is by selecting scFv phage libraries on tumor cells. This phage display technique involves multiple rounds of phage binding to target cells, washing to remove non-specific phage and elution to retrieve specific binding phage. Although the binding properties of an isolated tumor-specific scFv can be evaluated by ELISA, FACS and immunohistochemistry, it still remains a challenge to define the corresponding antigen. Here, we provide evidence that the target antigen of a given scFv displayed on phages can be detected in an immobilized lambda phage cDNA expression library containing thousands of irrelevant clones. The library contained CD30-negative breast-cancer specific cDNA as well as human CD30 receptor cDNA. The interaction of anti-CD30 scFv phages and their target antigen after blotting onto nitrocellulose filters was documented under defined conditions. Screening of different ratios between CD30 receptor and breast cancer specific clones (1:1 and 1:200) revealed that the CD30 antigen could be detected by anti-CD30 scFv phages using at least 5x10(12) plaque forming units of filamentous phages per blot. These investigations demonstrate that it is possible to detect the target antigen of a preselected scFv displayed on filamentous phages in lambda phage cDNA expression libraries.

Acylation of Galpha(13) is important for its interaction with thrombin receptor, transforming activity and actin stress fiber formation.

Palmitoylation of alpha-subunits in heterotrimeric G proteins has become a research object of growing attention. Following our recent report on the acylation of the mono-palmitoylated Galpha(12) [Ponimaskin et al., FEBS Lett. 429 (1998) 370-374], we report here on the identification of three palmitoylation sites in the second member of the G(12) family, Galpha(13), and on the biological significance of fatty acids on the particular sites. Using mutants of alpha(13) in which the potentially palmitoylated cysteine residues (Cys) were replaced by serine residues, we find that Cys-14, Cys-18 and Cys-37 all serve as palmitoylation sites, and that the mutants lacking fatty acids are functionally defective. The following biological functions of Galpha(13) were found to be inhibited: coupling to the PAR1 thrombin receptor, cell transformation and actin stress fiber formation. Results from established assays for the above functions with a series of mutants, including derivatives of the constitutively active mutant Galpha(13)Q226L, revealed a graded inhibitory response on the above mentioned parameters. As a rule, it appears that palmitoylation of the N-proximal sites (e.g. Cys-14 and Cys-18) contributes more effectively to biological function than of the acylation site located more internally (Cys-37). However, the mutant with Cys-37 replaced by serine is more severely inhibited in stress fiber formation (80%) than in cell transformation (50%), pointing to the possibility of a differential involvement of the three palmitoylation sites in Galpha(13).

Modification of the cytoplasmic domain of influenza virus hemagglutinin affects enlargement of the fusion pore.

The fusion activity of chimeras of influenza virus hemagglutinin (HA) (from A/fpv/Rostock/34; subtype H7) with the transmembrane domain (TM) and/or cytoplasmic tail (CT) either from the nonviral, nonfusogenic T-cell surface protein CD4 or from the fusogenic Sendai virus F-protein was studied. Wild-type or chimeric HA was expressed in CV-1 cells by the transient T7-RNA-polymerase vaccinia virus expression system. Subsequently, the fusion activity of the expression products was monitored with red blood cells or ghosts as target cells. To assess the different steps of fusion, target cells were labeled with the fluorescent membrane label octadecyl rhodamine B-chloride (R18) (membrane fusion) and with the cytoplasmic fluorophores calcein (molecular weight [MW], 623; formation of small aqueous fusion pore) and tetramethylrhodamine-dextran (MW, 10,000; enlargement of fusion pore). All chimeric HA/F-proteins, as well as the chimera with the TM of CD4 and the CT of HA, were able to mediate the different steps of fusion very similarly to wild-type HA. Quite differently, chimeric proteins with the CT of CD4 were strongly impaired in mediating pore enlargement. However, membrane fusion and formation of small pores were similar to those of wild-type HA, indicating that the conformational change of the ectodomain and earlier fusion steps were not inhibited. Various properties of the CT which may affect pore enlargement are considered. We surmise that the hydrophobicity of the sequence adjacent to the transmembrane domain is important for pore dilation.

Sendai virosomes revisited: reconstitution with exogenous lipids leads to potent vehicles for gene transfer.

A reliable new procedure is described for the reconstitution of Sendai viral envelopes suitable for gene transfer. Both fusion and hemagglutinin-neuraminidase glycoproteins were extracted from purified Sendai virus and reconstituted together with DNA in the presence of cholesterol:sphingomyelin:phosphatidylcholine:phosphatidylethanolamin e (Chol:SM:PC:PE) in a molar ratio of 3.5:3.5:2:1. Before reconstitution, the DNA to be transferred was condensed by pretreatment with polylysine. Exogenous lipid addition and the DNA-condensation step were essential for maximal size as well as for fusogenic activity of the resulting virosomes, the analysis of which revealed (1) the absence of any genomic material originating from Sendai virus, (2) the presence of fusogenic spikes in a functional orientation, (3) the encapsulation of reporter genes, and (4) high-transfer activity for plasmids carrying the green fluorescent protein (GFP) gene and double-stranded nucleotides into different mammalian cells. Transfer rates were up to 10-fold higher than those obtained with different cationic lipids. Gene delivery by means of our lipid-enriched Sendai virosomes extends the known gene transfer strategies, including those based on Sendai virus previously published.

Interhemispheric coordination of premotor neural activity during singing in adult zebra finches.

The song system, a neural network that mediates the learning and production of song by oscine songbirds, is investigated extensively as a model system for understanding the neural basis of complex skill learning. Part of the complexity of birdsong arises from the coordinated recruitment of multiple groups of muscles on both sides of the body. Although the song system is bilaterally organized, little is known about how premotor activities on the two sides are coordinated during singing. We investigated this by unilaterally recording neural activity in the forebrain song nucleus HVc (also known as the high vocal center) during singing and by forcing the premotor activities in the two hemispheres out of synchrony by perturbing neural activity in the contralateral HVc with electrical stimulation. Perturbing the activity in one HVc at any time during a song led to a short-latency readjustment of activity in the contralateral HVc. This readjustment consisted of a true resetting of the temporal pattern of activity in the contralateral HVc rather than merely a transient activity suppression overlaid on an unaltered pattern of premotor activity. These results strongly suggest that the output of song premotor areas in the forebrain is continuously monitored and that an active mechanism exists for resynchronizing the outputs from the two hemispheres whenever their gross temporal patterns differ significantly. The possible anatomical substrates for these coordinating mechanisms and their potential roles in song learning are discussed.

Palmitoylation of rhodopsin with S-protein acyltransferase: enzyme catalyzed reaction versus autocatalytic acylation.

Protein palmitoylation in vitro was studied using bovine rhodopsin as the substrate and a partially purified acylating enzymatic activity (PAT) from placental membranes. PAT incorporates fatty acid into rhodopsin with higher efficiency (10 times higher initial rate), as compared to autoacylation. The activity is sensitive to heat and trypsin, indicating a protein-mediated enzymatic process and requires the native conformation of rhodopsin. The presence of deacylated, free cysteine residues in dark-adapted rhodopsin increases palmitoylation via PAT. The sites for non-enzymatic and enzymatic palmitoylation could not be distinguished by peptide mapping. The reversible palmitoylation described here will provide a tool for the study of the role of palmitoylation in photoreceptor function.

Domain-structure of cytoplasmic border region is main determinant for palmitoylation of influenza virus hemagglutinin (H7).

We have shown previously that the length of cytoplasmic tails influences the selection of lipid substrates for palmitoylation of influenza viral hemagglutinin esterase fusion (HEF) and hemagglutinin (HA) glycoproteins [Veit et al. (1996) Biochem. J. 318, 163-172; Reverey et al. (1996) J. Biol. Chem. 271, 23607-23610]. Using a series of new chimeric mutant proteins derived from acylated influenza virus HA (subtype H7) and from nonacylated Sendai virus fusion protein (F, strain Z), we report here that palmitoylation levels depend on the type of transmembrane or cytoplasmic domain, or both, present in the expression products and that cysteine residues placed close to the cytoplasmic membrane border are not sufficient for acylation. By inserting stretches of the HA transmembrane domain into a nonacylated mutant of Sendai F (FCys), we induce palmitoylation after expression in CV.1 cells, and the level of fatty acid transfer increases with the length of the HA-derived insert. A five-amino-acid shift of the HA transmembrane domain severely augments fatty acid transfer. Our data suggest that the influenza virus HA contains complex conformational signals for palmitoylation that are mainly located within the transmembrane domain but also involve the C-tail region, whereas the extracellular (luminal) domain has only marginal influence on palmitoylation.

A cysteine-11 to serine mutant of G alpha12 impairs activation through the thrombin receptor.

We have recently reported that G alpha12 is acylated with palmitic acid [Veit et al., FEBS Lett. 339 (1994) 160-164]. Here we identify cysteine 11 as the sole palmitoylation site and assess the function of G alpha12 palmitoylation after expression of wild type and acylation-deficient mutant in insect cells. Our experimental approach yielded the following results. (1) Palmitoylation of G alpha12 has no influence on the subunit interactions. (2) Palmitoylation promotes membrane binding of G alpha12 when this protein is expressed alone. Membrane attachment of the heterotrimer occurs independent of the presence of fatty acids in G alpha12. (3) Assays for agonist-stimulated binding of [35S]GTPgammaS after expression of the human thrombin receptor (PAR1) along with G alpha12 and the betagamma subunits revealed a 70% inhibition with the palmitoyl-deficient mutant.