Selective decontamination,induced colonization resistance and connected immunological changes in piglets

14-d-old conventional piglets were picked from normal piggery, washed with disinfectants, placed into isolators suitable for germfree work, fed a sterile diet and treated with peroral antibiotics (nalidixic acid, kanamycin, and nystatin). Beginning with day 5 or 7, Enterobacteriaceae were not found in feces. The absence of these bacteria was proved by inoculation of germfree newborn piglets with caecal content. In selectively decontaminated piglets, the white blood cell count in blood had fallen to 6 X 10(9)/L; this decrease was due to an extremely low number of granulocytes (to 0.8 X 10(9)/L). On day 35, IgG-positive cells almost disappeared from the spleen, whereas IgA cells were found in an unusually great amount. Corresponding changes in serum levels were established. The colonization resistance effect in Enterobacteriaceae-deprived piglets was confirmed; settling of introduced various E. coli strains did not occur or was delayed.     

Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.     

A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics,ionic charge and molecular size

High-affinity binding of [³H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose^® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (M_r approx. 25 000) in the front effluent and a smaller more acidic peak (M_r approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (M_r approx 25 000), typically 5–10% of the total binding activity.     

Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density separation in Percoll,and selective substrate adherence

Summary This paper presents a study on the structure and function of Kupffer cells (KC) and liver endothelial cells (LEC) isolated by a simple and rapid technique involving 1) perfusion of the liver with collagenase; 2) cell separation by means of density centrifugation in Percoll; and 3) cell culture, taking advantage of the fact that KC and LEC differ in their preferences for growth substrate. The KC, which attach and spread under serum-free conditions on surfaces of glass or plastic during the first 15 min in culture exhibit a typical macrophage-like morphology including membrane ruffling and a heterogenous content of vacuoles. Moreover, these cells express (a) Fc receptors (FcR) for binding and phagocytosis of erythrocytes covered with immune globulin G (E-IgG), and (b) complement receptors (CR) for binding and serum dependent phagocytosis of erythrocytes covered with either human C3b or mouse inactivated C3b (iC3b). The cells also bind fluid phase fluoresceinated C3b. Approximately 30% of the KC express immune response-associated (Ia)-antigens.The LEC attach and spread on fibronectin coated surfaces, but not on glass or plastic surfaces, during the first two hours in culture with or without serum, and are morphologically distinct from KC. Cultured LEC are well spread out with no membrane ruffling and with numerous large vesicles surrounding the regularly shaped nucleus. These cells bind, but do not ingest E-IgG via the FcR, but no binding of fluid phase C3b or particle fixed C3b or iC3b can be observed. Incubation of LEC with fluorescein amine conjugates of ovalbumin or formaldehyde treated serum albumin, but not with fluoresceinated native serum albumin, results in accumulation of fluorescence specifically localized in the large perinuclear vesicles. Neither KC nor any other cell types tested have the ability to accumulate fluorescence upon incubation with these compounds. Iaantigens are not present on the LEC.Cytochemical demonstration of unspecific esterase, acid phosphatase, and peroxidase reveals different patterns and intensities of staining in KC as compared to LEC.Abbreviations Used KC Kupffer cells - LEC Liver endothelial cells - C Complement - C3b Major fragment of C3 activation - iC3b C3b that has been cleaved by factor I (C3b inactivator), present in serum - meC3b C3b produced by treating purified human C3 with methyl amine - trC3b C3b produced by treating purified human C3 with trypsin - CR Complement receptors for C3b and iC3b - IgG Immune globulin G - IgM Immune globulin M - E Erythrocytes - E-IgG E covered with anti-E IgG - E-IgM E covered with anti-E IgM - E-C3b(h) E-IgM reacted with purified human C1, C4, oxidized C2 and C3 (E-IgMC14^xyC2C3b) - E-iC3b(m) E-IgM incubated with C5 deficient serum from AKR mice - FcR Receptors for the Fc portion of IgG - FITC Fluorescein isothiocyanate - FITC-meC3b FITC conjugated to meC3b - FITC-trC3b FITC conjugated to trC3b - FA Fluorescein amine - FA-OA Ovalbumin conjugated with FA - FA-SA Serum albumin conjugated with FA - FA-FSA Formaldehyde-treated serum albumin conjugated with FA - Ia Immune response-associated AcE Acid unspecific esterase acting on alpha naphtyl acetate - NASDAE Unspecific esterase acting on naphthol AS-D acetate - NASDCAE Unspecific esterase acting on napthol AS-D chloroacetate     

Some synchorological aspects of basiphilous pine forests in Fennoscandia

Basiphilous pine forests and related birch forests are herb-and grass-rich forests on calcareous substrate. These forests are complex communities with floristic/ecological elements from different vegetation types occurring in a subtle micromosaic. These elements are e.g. species from acidophilous conifer forests, thermophilous forest-rim communities, calcareous shallow-soil and steppe communities, eutrophic wet meadows and fens, and in northern Fennoscandia also species from alpine Dryas heaths. Four associations are recognized in Fennoscandia: Convallario-Pinetum, Melico-Piceetum pinetosum, Peucedano-Pinetum and Epipacto atrorubentis-Betuletum. The main association is the Convallario-Pinetum, a widespread community in Fennoscandia and Estonia with a considerable floristic variation between the different regions. Examples of the floristic variation along west-east profiles and south-north profiles in Fennoscandia are presented. The basiphilous pine forest complex can be divided into a number of ecological types along the moisture and nutritional gradients. A further subdivision into geographical types (races) is presented.Nomenclature follows Lid (1974) for vascular plants, Nyholm (1954–1969) for musci and Dahl & Krog (1973) for lichens.     

Differences in reactivity of confluent and nonconfluent cultures of human endothelial cells toward thrombin-stimulated platelets or heparinized salt solution

Summary This study examined whether nonconfluent endothelial cell cultures reacted differently than confluent ones toward thrombin-stimulated platelets or a heparinized salt solution. The adherence to the endothelial cell cultures of⁵¹Cr-labeled human platelets stimulated at different thrombin concentrations was studied. There was significantly higher adherence of stimulated platelets to nonconfluent cultures compared with confluent ones. This was confirmed by scanning electron microscopy, which also revealed a tendency for the platelets to adhere at the cell periphery. Electron microscopy also showed that thrombin-stimulated platelets induced endothelial cell contraction. Part of the peripheral endothelial cell surface toward the bottom of the culture dish was inverted, facing the lumen of the dish. This phenomenon was particularly seen in nonconfluent cultures. When⁵¹Cr-labeled endothelial cultures were incubated with a mildly injurious fluid as heparinized sodium acetate and 20% serum, at 20° C for 30 min, the nonconfluent cultures showed significantly more cell detachment and release of⁵¹Cr than the confluent ones. We conclude that under the conditions of the present experiments there are differences in the reactivity of confluent and nonconfluent endothelial cell cultures. These differences probably reflect biological dissimilarities. In experiments where properties of cultured endothelium are studied, care should be taken that the degree of confluency is standardized.     

Properties of action potentials carried by divalent cations in identified leech neurons

Properties of divalent cation potentials carried by either Sr2+ or Ca2+ ions in Na+-free, TEA-Ringer solution were characterized in identified neurons of two species of leeches (Macrobdella and Haementeria). In Macrobdella, the overshoot of the potentials varied logarithmically with [Sr2+]0 (28.5 mV per 10-fold change). The overshoot, Vmax, and duration of the potentials increased with increasing divalent cation concentration and saturated at about 20 to 30 mM [Sr2+]0. The Vmax, amplitude, and duration of the potentials were reversibly blocked by Co2+ and Mn2+. The block by Mn2+ could be well-fitted by a reverse Langmuir-curve with an apparent KI of 100 micromolar. The local anesthetic procaine also reversibly inhibited the Vmax and duration of the potentials. The inhibition was greater at alkaline pH suggesting that procaine blocks the calcium channel from inside the membrane. The identified leech neurons examined in Macrobdella varied considerably in their ability to sustain somatic divalent cation potentials. Stimulation of T cells and most motoneurons produced no or only weak potentials, whereas stimulation of Retzius, N, Nut, and AP cells evoked overshooting potentials of several seconds' duration. Stimulation of the ALG cell of Haementeria in normal Ringer solution evoked a slowly-rising, purely Ca2+-dependent potential of approximately 100 ms duration. This response was TTX-resistant, unaffected by complete removal of Na+ from the Ringer solution, and abolished by 1 mM Mn2+. The overshoot varied logarithmically with a slope of 28 mV/decade change in [Ca2+]0.     

Molecular cloning and sequence analysis of cDNA for human transferrin

A cDNA clone for human transferrin was identified from a human liver cDNA library by pre-screening with different ss-cDNA probes against length-fractionated liver mRNAs, positive hybridization-selection and nucleotide sequence analysis. The insert was of 1 kb, encoding human transferrin from aminoacid 403 through the COOH terminus, with a 3' non coding region of 166 nucleotides. This insert hybridized with a single major mRNA species of about 2.4 kb and several genomic DNA restriction fragments. Hybridization of the Southern blots with different parts of the transferrin insert and at different stringences suggest that the various bands observed correspond to splice sites inside one gene rather than to hybridization to several related genes. Finally, a single or a low number of transferrin gene copies seem to exist in the human genome.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).