Polar invasion and translocation of Neisseria meningitidis and Streptococcus suis in a novel human model of the blood-cerebrospinal fluid barrier

Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.     

Die Problematik der Nomenklaitur bei denTrichogramma-Arten

Ohne Zusammenfassung     

The influence of limiting and non-limiting growth conditions on glucose and maltose metabolism in Lactococcus lactis ssp. lactis strains

Three strains of Lactococcus lactis ssp. lactis, a dairy strain 65.1, a type strain ATCC 19435, and a mutant AS 211, were grown on glucose and on maltose under chemostat conditions. When the culture was shifted from glucose-limiting to non-limiting conditions, the product shifted from mixed acids to lactate. Mixed acids were obtained in all maltose cultures; however, an enhanced lactate formation was observed in 19435 and AS 211. An inorganic-phosphate (P_i)-dependent maltose phosphorylase activity was found to be responsible for the initial conversion of maltose. The activation of maltose phosphorylase by Pi was strain-specific. When growth was on maltose under non-limiting conditions, a correlation was found between high initial maltose phosphorylase and -phosphoglucomutase activities and lactate production. No such correlation was observed in maltose-limited cells. In glucose-grown cells under non-limiting conditions, homo-fermentative lactate formation coincided with high concentrations of fructose 1,6-bisphosphate (Fru1,6P ₂) and pyruvate (Pyr) and low concentrations of phosphoenolpyruvate (PPyr). Under limiting conditions, mixed acid formation coincided with low concentrations of Fru1,6P ₂ and Pyr and high concentrations of PPyr. In maltose-grown cells there was no correlation between intracellular intermediary metabolite concentrations and product formation. Therefore, in addition to intracellular intermediary metabolite concentrations, the product formation on maltose is suggested to be regulated by the transport and initial phosphorylating steps.     

Study of fluxes at low concentrations of l-tri-iodothyronine with rat liver cells and their plasma-membrane vesicles. Evidence for the accumulation of the hormone against a gradient

1. Influx and efflux of l-tri-[(125)I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23 degrees C and in the concentration range that included the concentration of free l-tri-iodothyronine in rat plasma (3-5pm) influx into cells was saturable; an apparent K(t) value of 8.6+/-1.6pm was obtained. 3. At 5pm-l-tri-[(125)I]iodothyronine in the external medium the ratios of the concentrations inside to outside in cells and plasma-membrane vesicles were 38:1 and 366:1 respectively after 7s of incubation. At equilibrium (60s at 23 degrees C) uptake of l-tri-[(125)I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a K(d) value of 6.1+/-1.3pm. 4. Efflux of l-tri-[(125)I]iodothyronine from cells equilibrated with the hormone (5-123pm) was constant up to 21 s; the amount that flowed out was 17.7+/-3.8% when cells were equilibrated with 5pm-hormone. When plasma-membrane vesicles were equilibrated with l-tri-[(125)I]iodothyronine (556-1226pm) 66.8+/-5.8% flowed out after 21 s. 5. From a consideration of the data on efflux from cells and binding of l-tri-[(125)I]iodothyronine to the liver homogenate, as studied by the charcoal-adsorption and equilibrium-dialysis methods, it appears that 18-22% of the hormone exists in the free form in the cell. 6. Vinblastine and colchicine diminished the uptake of l-tri-[(125)I]iodothyronine by cells but not by plasma-membrane vesicles; binding to the cytosol fraction was not affected. Phenylbutazone, 6-n-propyl-2-thiouracil, methimazole and corticosterone diminished the uptake by cells, plasma-membrane vesicles and binding to the cytosol fraction to different extents. 7. These results suggest that at low concentrations of l-tri-[(125)I]iodothyronine rat liver cells and their plasma-membrane vesicles accumulated the hormone against an apparent gradient by a membrane-mediated process. Contribution of cytoplasmic proteins to uptake by plasma-membrane vesicles was negligible. The amount of l-tri-[(125)I]iodothyronine required to achieve half-maximal uptake agrees with that occurring in the free form in the blood, conferring physiological importance to the transporting system in the plasma membrane of the liver cell.     

Cloning,Expression, and Characterization of the Squid Na+–Ca2+ Exchanger (NCX-SQ1)

We have cloned the squid neuronal Na⁺–Ca²⁺ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na⁺–Ca²⁺ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca²⁺ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na⁺-dependent ⁴⁵Ca²⁺ uptake; the uptake was inhibited by injection of Ca²⁺ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na⁺-dependent inactivation, secondary activation by cytoplasmic Ca²⁺, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATPγS, in the presence of F⁻ (0.2 mM) and vanadate (50 μM), and both effects reversed on application of a phosphatidylinositol-4′,5′-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATPγS. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4′,5′-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5–10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na⁺ and Ca²⁺ transport reactions. Outward current transients associated with Na⁺ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca²⁺ extrusion were much larger. For NCX-SQ1, charge movements of Ca²⁺ transport could be defined in voltage jump experiments with a low cytoplasmic Ca²⁺ (2 μM) in the presence of high extracellular Ca²⁺ (4 mM). The rates of charge movements showed “U”-shaped dependence on voltage, and the slopes of both charge–voltage and rate–voltage relations (1,600 s⁻¹ at 0 mV) indicated an apparent valency of −0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.     

Investigations of the seed protein content of several pea genotypes grown in two different years

Summary Seventeen X-ray and neutron induced mutants of the commercial variety Dippes gelbe Victoria were analyzed with regard to their seed protein percentage. The interaction of genotypic and year effects in 1975 (normal weather conditions) and 1976 (extremely hot and dry) was also taken into consideration. To avoid undiscoverable environmental bias, the plants were grown in a nonstandard three-dimensional layout. Biometric analysis was done by using the theory of the general linear model with a formula-processing computer program. In the first year, significant genetically caused differences were found in the material. The bifurcated mutant 157A was especially of considerable interest because an improved protein content was combined with relative good yield. In the second year, no significant differences between the mutants were revealed, but all genotypes showed a similar good protein value of about 27%.     

An Extended Threshold Model for Analyzing Ordered Categorical Data

This paper presents an extended threshold model for analyzing ordered categorical data. The model admits interactions between the position of the thresholds and the levels of the effective factors. These interactions are described according to the approach of Milliken and Graybill (1970). Especially important for practical application is the special assumption that there is a linear relation between interactions and thresholds, and that the slopes of the concerning regression lines may be different for samples. This means that the latent variables are distributed according to the same type of distributions, but may have different expectations and variances. Underlying this submodel, the estimation of parameters and the testing of hypotheses according to the maximum likelihood method is described. The procedure is illustrated by a numerical example, and an outline is given about a cluster analysis using model parameters.     

Genomic Relationships between Enterococcus faecium Strains from Different Sources and with Different Antibiotic Resistance Profiles Evaluated by Restriction Endonuclease Analysis of Total Chromosomal DNA Using EcoRI and PvuII

Forty-seven Enterococcus faecium strains from different sources were evaluated by restriction endonuclease analysis (REA) of total chromosomal DNA. Strains from chicken, pork, and humans were clearly divided into separate clusters, whereas strains from different countries, strains with different antibiotic resistance profiles, or clinical and healthy-subject strains were not.