Horses as Sentinels for the Circulation of Flaviviruses in Eastern-Central Germany.

Since 2018, autochthonous West Nile virus (WNV) infections have been regularly reported in eastern-central Germany. While clinically apparent infections in humans and horses are not frequent, seroprevalence studies in horses may allow the tracing of WNV and related flaviviruses transmission, such as tick-borne encephalitis virus (TBEV) and Usutu virus (USUV), and consequently help to estimate the risk of human infections. Hence, the aim of our study was to follow the seropositive ratio against these three viruses in horses in Saxony, Saxony Anhalt, and Brandenburg and to describe their geographic distribution for the year 2021. In early 2022, i.e., before the virus transmission season, sera from 1232 unvaccinated horses were tested using a competitive pan-flavivirus ELISA (cELISA). In order to estimate the true seropositive ratio of infection with WNV, TBEV, and USUV for 2021, positive and equivocal results were confirmed by a virus neutralization test (VNT). In addition, possible risk factors for seropositivity using questionnaires were analyzed using logistic regression based on questionnaires similar to our previous study from 2020. In total, 125 horse sera reacted positive in the cELISA. Based on the VNT, 40 sera showed neutralizing antibodies against WNV, 69 against TBEV, and 5 against USUV. Three sera showed antibodies against more than one virus, and eight were negative based on the VNT. The overall seropositive ratio was 3.3% (95% CI: 2.38-4.40) for WNV, 5.6% (95% CI: 4.44-7.04) for TBEV, and 0.4% (95% CI: 0.14-0.98) for USUV infections. While age and number of horses on the holding were factors predicting TBEV seropositivity, no risk factors were discovered for WNV seropositivity. We conclude that horses are useful sentinels to determine the flavivirus circulation in eastern-central Germany, as long as they are not vaccinated against WNV.

Disruption of gallbladder smooth muscle function is an early feature in the development of cholesterol gallstone disease.

UNLABELLED: BACKGROUND; Decreased gallbladder smooth muscle (GBSM) contractility is a hallmark of cholesterol gallstone disease, but the interrelationship between lithogenicity, biliary stasis, and inflammation are poorly understood. We studied a mouse model of gallstone disease to evaluate the development of GBSM dysfunction relative to changes in bile composition and the onset of sterile cholecystitis. METHODS: BALB/cJ mice were fed a lithogenic diet for up to 8 weeks, and tension generated by gallbladder muscle strips was measured. Smooth muscle Ca(2+) transients were imaged in intact gallbladder. KEY RESULTS: Lipid composition of bile was altered lithogenically as early as 1 week, with increased hydrophobicity and cholesterol saturation indexes; however, inflammation was not detectable until the fourth week. Agonist-induced contractility was reduced from weeks 2 through 8. GBSM normally exhibits rhythmic synchronized Ca(2+) flashes, and their frequency is increased by carbachol (3 µm). After 1 week, lithogenic diet-fed mice exhibited disrupted Ca(2+) flash activity, manifesting as clustered flashes, asynchronous flashes, or prolonged quiescent periods. These changes could lead to a depletion of intracellular Ca(2+) stores, which are required for agonist-induced contraction, and diminished basal tone of the organ. Responsiveness of Ca(2+) transients to carbachol was reduced in mice on the lithogenic diet, particularly after 4-8 weeks, concomitant with appearance of mucosal inflammatory changes. CONCLUSIONS & INFERENCES: These observations demonstrate that GBSM dysfunction is an early event in the progression of cholesterol gallstone disease and that it precedes mucosal inflammation.

Spread monomolecular films of monohydroxy bile acids and their salts: influence of hydroxyl position, bulk pH, and association with phosphatidylcholine.

Undissociated dihydroxy bile acids, alone or with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), lie with their long axes parallel to aqueous-lipid interfaces [Fahey, D. A., Carey, M. C., and Donovan, J. M. (1995) Biochemistry 34, 10886-10897]. To test the generality of this orientation, we used an automated Langmuir-Pockels surface balance to examine pressure-molecular area isotherms and dipole moments of insoluble monohydroxy bile acids and their salts, which are sparingly soluble because of their presumed high Krafft points. We studied lithocholic acid (LCA) (the natural 3alpha-OH isomer), glycolithocholic acid (GLCA) (its glycine conjugate), and the semisynthetic isomers, 7alpha-OH- and 12alpha-OH-cholanoic acids with and without POPC, at pH values ranging from 2 to 12. Monolayer collapse pressures increased sigmoidally with ionization, giving apparent pK values of 7.0-8.5 and implying a stronger affinity of the bile salt anions for the interface. At monolayer collapse, the molecular area of LCA was approximately 85 A(2) independent of pH, consistent with the steroid nucleus lying flat. In contrast, the interfacial area of 7-OH-cholanoic acid decreased from approximately 80 A(2) at pH 2 to approximately 40 A(2) above pH 9, consistent with a more vertical orientation and approximating 12-OH-cholanoic acid, which exhibited a molecular area of approximately 45 A(2) at all pH values. All monohydroxy bile acids condensed POPC monolayers more effectively at low than at high (ionized) pH. We conclude that the 3-OH group is crucial for anchoring bile acids and their salts to the aqueous interface, with all monohydroxy species condensing phospholipid membranes regardless of ionization state.

Immobilized artificial membrane chromatography: a rapid and accurate HPLC method for predicting bile salt-membrane interactions.

To predict bile salt-membrane interactions physiologically, we used an immobilized artificial membrane HPLC column that contains dimyristoyl-phosphatidylcholine molecules covalently linked to silica microspheres. Using a 90% aqueous (10% acetonitrile) mobile phase, 22 species of bile salts and 4 species of fusidates were eluted. Glycine conjugates displayed higher affinity for the column at pH 5.5, eluting later than their taurine-conjugated congeners, but this order was reversed at pH 6.5 and 7.4 as glycine conjugates became fully ionized. Capacity factors decreased logarithmically as functions of increasing temperature, permitting determinations of interaction enthalpies, which ranged from -2.86 to -7.67 kcal/mol. A standard curve was developed from which the enthalpy for an uncommon bile salt could be inferred from its capacity factor at room temperature. Bile salt interaction enthalpies were substantially better correlated than hydrophobic indices by octadecylsilane-HPLC (D. M. Heuman, J. Lipid Res. 1989. 30: 719-730) with equilibrium binding to small unilamellar vesicles and literature values reflecting bile salt-membrane interactions (e.g., biliary phosphatidylcholine secretion), but not with bile salt functions that do not require phospholipid (e.g., micellar cholesterol solubility). This new application should prove valuable for evaluating membrane-active physical-chemical properties as well as therapeutic potential of novel bile salts, particularly when they are available in quantities too small for study by conventional techniques.

Calcium affinity for biliary lipid aggregates in model biles: complementary importance of bile salts and lecithin.

BACKGROUND/AIMS: Despite putative roles of calcium in biliary physiology and gallstone formation, quantitative aspects of calcium binding to bile salt (BS) monomers, simple micelles, mixed micelles, and vesicles, which constitute the lipid aggregates in bile, remain unexplored. METHODS: Calcium activity was measured using the calcium electrode in pathophysiologically relevant model biles composed of either individual BS species or a physiological mixture of glycine and taurine conjugates, as functions of lecithin and cholesterol contents and total lipid concentration. RESULTS: Calcium binding increased with increasing BS concentrations and lecithin contents and varied with species (dihydroxy > trihydroxy BS) and with conjugation (unconjugated > glycine conjugates > taurine conjugates). Although lecithin/cholesterol vesicles did not bind detectable calcium, when taurocholate was incorporated into membrane bilayers, calcium binding was substantially greater than with equimolar BS alone. Added cholesterol did not alter calcium binding, despite cholesterol saturation of biliary lipid aggregates and induction of liquid crystalline and solid crystalline-phase transitions. CONCLUSIONS: In model biles, most calcium is bound to mixed micelles, with minor contributions by BS monomers, simple micelles, and vesicles. It is proposed that BS-induced binding of calcium to vesicles and mixed micelles may be important in nucleation of cholesterol and bilirubinates from native bile.

In vitro evidence that phospholipid secretion into bile may be coordinated intracellularly by the combined actions of bile salts and the specific phosphatidylcholine transfer protein of liver.

Using model systems, we explored a potential function of hepatic phosphatidylcholine transfer protein to extract biliary-type phosphatidylcholines from intracellular membranes (e.g., smooth endoplasmic reticulum) and deliver them to canalicular plasma membranes where biliary secretion occurs. We measured transfer rates of parinaroyl phosphatidylcholine, a naturally fluorescent phospholipid, from small unilamellar vesicles composed of sn-1 palmitoyl, sn-2 parinaroyl phosphatidylcholine, and egg yolk phosphatidylcholine (molar ratio 75:25) wherein the fluorophore is self-quenched to small unilamellar vesicles composed of phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and cholesterol (molar ratios 22:22:10:8:38) representing model microsomal and canalicular plasma membranes, respectively. Following addition of phosphatidylcholine transfer protein (purified from bovine liver), fluorescence intensity increased exponentially indicating net phosphatidylcholine transfer from donor to acceptor vesicles. Submicellar concentrations of a wide hydrophobicity range of common and uncommon taurine and glycine conjugated bile salts species (anionic steroid detergent-like molecules), sodium taurofusidate (a conjugated fungal bile salt analog), and sodium dodecyl sulfate and octylglucoside, anionic and nonionic straight chain detergents, respectively, markedly stimulated phosphatidylcholine transfer protein activity. This 40-115-fold effect was most pronounced for the common bile salts and correlated positively with bile salt hydrophobicity. Thermodynamic analysis of net transfer revealed that the rate-limiting step was extraction of phosphatidylcholine molecules from donor vesicles and that bile salts facilitated their capture by enhancing both phosphatidylcholine transfer protein binding as well as perturbing phospholipid packing in vesicle bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Subunit dynamics in Escherichia coli preprotein translocase.

SecY, SecE, and band 1 copurify as the SecY/E integral membrane domain of Escherichia coli preprotein translocase. To measure the in vivo association of these polypeptides and assay possible exchange, plasmid-borne secY and secE genes were placed under control of the ara regulon and fused to DNA encoding the influenza hemagglutinin epitope. Cells were incubated with [35S]methionine, grown for a "chase" period, and then induced with arabinose to express epitope-tagged, nonradioactive SecY and SecE. Both the wild-type and epitope-tagged polypeptides assembled into functional, heterotrimeric SecY/E complex. However, immunoprecipitation with antibody to the epitope tag did not cross-precipitate radiolabeled SecY or SecE. Thus, these subunits normally associate stably in vivo.