Heterologous facilitation of G protein-activated K(+) channels by beta-adrenergic stimulation via cAMP-dependent protein kinase

To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.     

Establishment of Myotis myotis Cell Lines - Model for Investigation of Host-Pathogen Interaction in a Natural Host for Emerging Viruses

Bats are found to be the natural reservoirs for many emerging viruses. In most cases, severe clinical signs caused by such virus infections are normally not seen in bats. This indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. Due to the strict protection of European bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. Here, we report about the establishment and functional characterization of Myotis myotis derived cell lines from different tissues: brain (MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol) after immortalization by SV 40 large T antigen. The usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mRNA patterns of immune-relevant genes after poly I:C stimulation. Performed experiments indicated varying susceptibility to lyssavirus infection with MmBr being considerably less susceptible than the other cell lines. Further investigation demonstrated a strong activation of interferon mediated antiviral response in MmBr contributing to its resistance. The pattern recognition receptors: RIG-I and MDA5 were highly up-regulated during rabies virus infection in MmBr, suggesting their involvement in promotion of antiviral responses. The presence of CD14 and CD68 in MmBr suggested MmBr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (CNS). Thus the expression pattern of MmBr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the CNS controlling the lyssavirus infection. Overall, the established cell lines are important tools to analyze antiviral innate immunity in M. myotis against neurotropic virus infections and present a valuable tool for a broad spectrum of future investigations in cellular biology of M. myotis.     

The mammalian homologue of Prp16p is overexpressed in a cell line tolerant to Leflunomide, a new immunoregulatory drug effective against rheumatoid arthritis.

Prp2p, Prp16p, Prp22p, and Prp43p are members of the DEAH-box family of ATP-dependent putative RNA helicases required for pre-mRNA splicing in Saccharomyces cerevisiae. Recently, mammalian homologues of Prp43p and Prp22p have been described, supporting the idea that splicing in yeast and man is phylogenetically conserved. In this study, we show that a murine cell line resistant to the novel immunoregulatory drug Leflunomide (Arava) overexpresses a 135-kDa protein that is a putative DEAH-box RNA helicase. We have cloned the human counterpart of this protein and show that it shares pronounced sequence homology with Prp16p. Apart from its N-terminal domain, which is rich in RS, RD, and RE dipeptides, this human homologue of Prp16p (designated hPrp16p) is 41% identical to Prp16p. Significantly, homology is not only observed within the phylogenetically conserved helicase domain, but also in Prp16p-specific sequences. Immunofluorescence microscopy studies demonstrated that hPrp16p co-localizes with snRNPs in subnuclear structures referred to as speckles. Antibodies specific for hPrp16p inhibited pre-mRNA splicing in vitro prior to the second step. Thus, like its yeast counterpart, hPrp16p also appears to be required for the second catalytic step of splicing. Taken together, our data indicate that the human 135-kDa protein identified here is the structural and functional homologue of the yeast putative RNA helicase, Prp16p.     

Peptidoglycan Structural Dynamics during Germination of Bacillus subtilis 168 Endospores

Peptidoglycan structural dynamics during endospore germination of Bacillus subtilis 168 have been examined by muropeptide analysis. The first germination-associated peptidoglycan structural changes are detected within 3 min after the addition of the specific germinant l-alanine. We detected in the spore-associated material new muropeptides which, although they have slightly longer retention times by reversed-phase (RP)-high-pressure liquid chromatography (HPLC) than related ones in dormant spores, show the same amino acid composition and molecular mass. Two-dimensional nuclear magnetic resonance (NMR) analysis shows that the chemical changes to the muropeptides on germination are minor and are probably limited to stereochemical inversion. These new muropeptides account for almost 26% of the total muropeptides in spore-associated material after 2 h of germination. The exudate of germinated spores of B. subtilis 168 contains novel muropeptides in addition to those present in spore-associated material. Exudate-specific muropeptides have longer retention times, have no reducing termini, and exhibit a molecular mass 20 Da lower than those of related reduced muropeptides. These new products are anhydro-muropeptides which are generated by a lytic transglycosylase, the first to be identified in a gram-positive bacterium. There is also evidence for the activity of a glucosaminidase during the germination process. Quantification of muropeptides in spore-associated material indicates that there is a heterogeneous distribution of muropeptides in spore peptidoglycan. The spore-specific residue, muramic δ-lactam, is proposed to be a major substrate specificity determinant of germination-specific lytic enzymes, allowing cortex hydrolysis without any effect on the primordial cell wall.The extreme heat resistance of dormant bacterial endospores has made them an important problem in the production of safe foodstuffs (3). The spore cell wall peptidoglycan is considered to play a major role in the maintenance of heat resistance and dormancy (6). Bacillus subtilis spore peptidoglycan is composed of two layers. A thin, inner layer called the primordial cell wall retains the basic vegetative cell peptidoglycan structure. The primordial cell wall represents 2 to 4% of the total endospore peptidoglycan, is not digested during germination, and serves as the initial cell wall during outgrowth (2, 5, 25, 29). The outer thick layer of peptidoglycan, known as the cortex, is characterized by several unique spore-specific features. Approximately 50% of the muramic acid residues in the glycan strands are present in the δ-lactam form (2, 24). Muramic acid side chains are composed of 26 and 23% of tetrapeptide and single l-alanine, respectively (2).Despite their extreme dormancy and thermostability, bacterial endospores retain an alert sensory mechanism enabling them to respond within minutes to the presence of specific germinants. Spores of B. subtilis respond to at least two different types of germinative stimuli: (i) l-alanine and (ii) a combination of l-asparagine, glucose, fructose, and KCl (AGFK) (34). The germination response is initiated by the interaction of a receptor protein with specific germinants which triggers the loss of spore-specific properties and the transformation of a dormant resistant bacterial spore into a metabolically active vegetative cell. The germination process is characterized by sequential, interrelated biochemical events. The specific hydrolysis of peptidoglycan in the spore cortex layer is an essential event in germination (2, 25). Its degradation removes the physical constraints of the cortex and allows core expansion and outgrowth (9, 25). As a consequence of cortex hydrolysis, peptidoglycan fragments can be detected in the germination exudate (13, 33).A number of bacterial spore germination-specific cortex-lytic enzymes (GSLEs) have been reported to be involved in cortex hydrolysis (9, 18–20). A gene homologous to that encoding the GSLE from Bacillus cereus has been identified and inactivated in B. subtilis, and the resulting mutant germinates more slowly than the wild type (22). Recently a germination-specific muramidase isolated from a germination extract of Clostridium perfringens S40 has been purified and characterized (4).GSLEs have a high substrate specificity, requiring intact spore cortex for activity (9, 23). The muramidase from C. perfringens S40, however, hydrolyzes cortical fragments but has a strict requirement for the presence of the muramic δ-lactam residues (4). Thus, the GSLEs are highly specialized and may exist as proforms which are specifically activated during germination (9).Very little is known about the mechanism by which the cortex is hydrolyzed during germination and the autolytic enzymes involved. Muropeptide analysis provides a method for fine chemical structural determination of spore cortex (2, 24, 25). In this paper, we report the use of muropeptide analysis to determine the peptidoglycan structural dynamics which occur during spore germination of B. subtilis 168 and the evidence for a number of different enzyme activities.     

Energy requirements for unfolding and membrane translocation of precursor proteins during import into mitochondria

ATP is involved in conferring transport competence to numerous mitochondrial precursor proteins in the cytosol. Unfolded precursor proteins were found not to require ATP for import into mitochondria, suggesting a role of ATP in the unfolding of precursors. Here we report the unexpected finding that a hybrid protein containing the tightly folded passenger protein dihydrofolate reductase becomes unfolded and specifically translocated across the mitochondrial membranes independently of added ATP. Moreover, interaction of the precursor with the mitochondrial receptor components does not require ATP. The results suggest that ATP is not involved in the actual process of unfolding during membrane translocation of precursors. ATP rather appears to be necessary for preventing the formation of improper structures of precursors in the cytosol and for folding of imported polypeptides on (and release from) chaperone-like molecules in the mitochondrial matrix.     

Unsaturated 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholines (unsaturated platelet-activating factor): aggregation of human platelets after incubation with indomethacin, creatinephosphate/creatinephosphokinase, xylocain and hirudine, and serotonin release after incubation with indomethacin

Unsaturated platelet-activating factor (PAF) aggregates thrombocytes of healthy female volunteers and releases within 1 min up to 30.95% of the platelet serotonin. Indomethacin does not inhibit the aggregation but reduces the release of serotonin induced by unsaturated PAF in citrated platelet-rich plasma (PRP). Creatinephosphate combined with creatinephosphokinase (CP/CPK) inhibits the second phase, whereas xylocain inhibits the first and second phase of aggregation induced by unsaturated PAF. Hirudine shows no influence on the aggregation induced by unsaturated PAF.     

Targeting of proteins involved in sterol biosynthesis to lipid particles of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, three enzymes of the sterol biosynthetic pathway, namely Erg1p, Erg6p and Erg7p, are located in lipid particles. Whereas Erg1p (squalene epoxidase) is also present in the endoplasmic reticulum (ER) to a significant amount, only traces of Erg6p (sterol C-24 methyltransferase) and Erg7p (lanosterol synthase) are found in the ER. We have chosen these three Erg-proteins as typical representatives of lipid particle proteins to study targeting to their destination. Lipid particle proteins do not contain obvious targeting motifs, but the only common structural feature is the presence of one or two hydrophobic domains near the C-termini. We constructed truncated versions of Erg1p, Erg6p and Erg7p to test the role of these hydrophobic domains in subcellular distribution. Our results demonstrate that lack of the hydrophobic domains prevents at least in part the association of the proteins with lipid particles and causes their retention to the ER. This result strongly supports the view that ER and lipid particles are related organelles.     

i-SNAREs: inhibitory SNAREs that fine-tune the specificity of membrane fusion

A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.     

Kiebitze (Vanellus vanellus) kopulieren w?hrend der Brutabl?sung

Zusammenfassung Die Bebrütung von vier Gelegen des Kiebitzes wurde ca. 32 h an 11 Bruttagen verfolgt. Die bisher selten beobachtete Brutablösung mit Kopulation konnte dabei relativ häufig (in 9 von 13 Brutablösungen) registriert werden. Dabei traten 3 unterschiedliche Verhaltensabläufe auf. Durch die Kopulation bei der Brutablösung werden möglicherweise und näher aneinander gebunden und beteiligen sich dann gemeinsam an der Brut- und Nestbewachung. Dadurch könnten sich die Chancen auf eine erfolgreiche Brut erhöhen. Sieht man dagegen die Kopulationen während der Brutphase als Strategie zur Verringerung der Fremdfertilisation bzw. zur Stimulierung des Follikelwachstums an, könnten in kürzeren Abständen Gelegeverluste ausgeglichen werden.

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Lapwings (Vanellus vanellus) copulate during breeding relief

The incubation of four clutches of the lapwing has been observed for thirty hours during eleven days of breeding. Up to now breeding reliefs with copulation have rarely been registered. During this study it happened rather frequently (nine of thirteen breeding reliefs), showing three different types of behaviour. Probably copulation during breeding reliefs serves to strengthen pair-bonds in order to ensure the care of brood and clutches. Thus chances for a successful incubation are increased. If, instead, copulations during the time of breeding were seen as a strategy to avoid fertilisation by strangers or to stimulate follicle growth, loss of clutches could be balanced more easily.