A new non-equilibrium enzyme linked immunosorbent assay for a glycogen-derived urinary tetrasaccharide

The tetrasaccharide, Glc1-6Glc1-4Glc1-4Glc, denoted (Glc)₄, is a limit dextrin formed by amylolytic degradation of glycogen. In order to evaluate the possible clinical importance of (Glc)₄ excretion as an indicator of certain physiological and pathological conditions, we have developed a new rapid and inexpensive immunoassay using a monoclonal antibody raised against (Glc)₄ glycosidically-linked to a carrier protein. As the antibody is highly specific, it can be used to measure native (Glc)₄ directly, without the chemical reduction step required in previous methods. A new type of non-equilibrium ELISA inhibition test was developed based on the capacity of free (Glc)₄ to decrease initial rates of antibody binding to (Glc)₄-coated microtiter wells. The method is highly reproducible and is as sensitive and accurate as the gas chromatography method or radioimmunoassay used previously.Abbreviations (Glc)₄ Glc1-6Glc1-4Glc1-4Glc - KLH keyhole limpet hemacyanin - BSA bovine serum albumin - PEG polyethylene glycol     

T-antigen expression by polyoma mutants with modified RNA splicing

Polyoma virus mutants were constructed that could not express all the three T-antigens. The mutagenesis was directed to the two 5' splice sites utilized in the maturation of early RNA. The mutant bc1051 had a base change at the splice site of large T-antigen mRNA, and the mutants dl1061 and dl1062 had deletions at the corresponding splice point of small and middle T-antigen mRNA. The site was removed in mutant dl1061 and altered by fusion to upstream sequences in mutant dl1062. Analysis of viral RNA showed that dl1061 and dl1062 formed only large T-antigen mRNA, whereas bc1051 did not produce this RNA-species. However, the biological properties of dl1062 suggested that it also produced mRNA directing the synthesis of a small T-antigen-related polypeptide, at least in low amounts. Only mutant bc1051 could induce transformation of rat cells. In mouse 3T3 cells dl1062 multiplied to a limited extent, while bc1051 and dl1061 failed to produce virus. However, dl1061 DNA was synthesized at a low rate which could be increased to normal levels by co-transfection with mutant bc1051. This result suggests that polyoma small and middle T-antigen have a previously unrecognized function in the early phase of the infection process, or in viral DNA-synthesis.     

High efficiency polyoma DNA transfection of chloroquine treated cells.

Chloroquine treatment of rodent cells during the first hours of polyoma DNA transfection increase the fraction of cells expressing viral functions. The effect has been observed after DNA absorption using both the DEAE-dextran and calcium phosphate coprecipitation methods. Exposure to chloroquine increased the proportion of transfected mouse cells to approximately 40%. From a culture of one million such cells, microgram quantities of newly synthesized viral DNA could be isolated. Similarly, the transformation frequency of rat cells following polyoma DNA transfection was approximately 6-fold increased by chloroquine treatment. The effect of the compound was even more pronounced in transfections with linear forms of polyoma DNA, suggesting that chloroquine inhibits degradation of DNA absorbed by the cells.     

Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils

Cytosolic free calcium ([Ca2+]i) and fusion of secondary granules with the phagosomal membrane (phagosome-lysosome fusion, P-L fusion) were assessed in single adherent human neutrophils during phagocytosis of C3bi-opsonized yeast particles. Neutrophils were loaded with the fluorescent dye fura2/AM and [Ca2+]i was assessed by dual excitation microfluorimetry. Discharge of lactoferrin, a secondary granule marker into the phagosome was verified by immunostaining using standard epifluorescence, confocal laser scanning and electron microscopy. In Ca2(+)-containing medium, upon contact with a yeast particle, a rapid rise in [Ca2+]i was observed, followed by one or more Ca2+ peaks (maximal value 1,586 nM and median duration 145 s): P-L fusion was detected in 80% of the cells after 5-10 min. In Ca2(+)-free medium the amplitude, frequency and duration of the [Ca2+]i transients were decreased (maximal value 368 nM, mostly one single Ca2+ peak and median duration 75 s): P-L fusion was decreased to 52%. Increasing the cytosolic Ca2+ buffering capacity by loading the cells with MAPT/AM led to a dose-dependent inhibition both of [Ca2+]i elevations and P-L fusion. Under conditions where basal [Ca2+]i was reduced to less than 20 nM and intracellular Ca2+ stores were depleted, P-L fusion was drastically inhibited while the cells ingested yeast particles normally. P-L fusion could be restored in Ca2(+)-buffered cells containing ingested particles by elevating [Ca2+]i with the Ca2(+)-ionophore ionomycin. The present findings directly indicate that although the ingestion step of phagocytosis is a Ca2(+)-independent event, [Ca2+]i transients triggered upon contact with opsonized particles are necessary to control the subsequent fusion of secondary granules with the phagosomal membrane.     

Modulation of the junctional integrity by low or high concentrations of cytochalasin B and dihydrocytochalasin B in associated with distinct changes in F-actin and ZO-1

In a study of Necturus gallbladder epithelium Benzel et al. (Benzel et al., 1980) found that low (0.2–1.2 M) and higher concentrations (1.5 M and more) of cytochalasin B (CB) caused an increase and decrease in the transepithelial electrical resistance (TER), respectively. Moreover, there were slight changes in the height and complexicity of tight junction (TJ) strands, as visualized by freeze-fracture and freeze-etching. To elucidate the mechanisms of these findings, we first demonstrated that the effect is also present in monolayers of Madin-Darby Canine Kidney strain I (MDCK-I) cells. Thus, a low concentration (0.1 ng/ml) cytochalasin B (CB) strengthened the permeability barrier, as evidenced quantitatively by increases in TER on transepithelial electrical measurements. Furthermore, indirect immunofluorescence and confocal microscopy demonstrated that this effect was paralleled with an accumulation of F-actin and the tight junction marker protein, ZO-1, at the level of TJ. Equimolar concentrations of dihydrocytochalasin B (dhCB), on the other hand, did not lead to a tightening of the epithelium. Confirming previous studies, there was a general decrease in epithelial resistance after treatment with high concentrations (1 g/ml) of CB and dhCB, which was accompanied by distinct changes in the F-actin network and distribution of ZO-1. We speculate that the divergent effects of CB and dhCB on the F-actin and ZO-1 organization might be due to specific effects on the transport of monosaccharides across the plasma membrane, or that CB and dhCB in distinct ways involve the turnover of phosphatidylinositols in the membrane, thereby modulating junctional permeability and F-actin structure.     

Secretion of a heat-stable fungalβ-glucanase from transgenic,suspension-cultured barley cells

Transgenic plant cell cultures have a potential for production and secretion of important proteins and peptides. To assess the possibilities of using a stable barley suspension culture for secretion of heterologous proteins in active form, we expressed the cDNA of the thermostable-glucanase (EGI) ofTrichoderma reesei in barley suspension cells. The cDNA coding for EGI and its signal sequence was placed under the control of the CaMV 35S promoter and the construction was transferred to the cells by particle bombardment. Stably transformed lines were obtained by selecting for a cotransformed antibiotic resistance marker. The expression of EGI cDNA led to accumulation of EGI in the culture medium, as shown by analysis with EGI-specific antibodies. Enzymatic assays confirmed that the EGI secreted by the suspension cells retained its activity and thermostable character. Furthermore, it was shown that the enzyme produced by the transgenic suspension culture could be used for degradation of soluble-glucans during mashing.     

Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells.

Pathogenic bacteria of the species Yersinia, including Yersinia pestis, block phagocytosis by macrophages. This process involves the YopE protein, which induces disruption of the host cell actin microfilament structure. Here, we show that the contact between the pathogen and the mammalian cell induces expression and then polarized transfer of YopE into the eukaryotic cell. While the bacteria remain at the surface of the target cell, the YopE cytotoxin is transferred through the host cell plasma membrane and YopE is only recovered within the cytosol of the target cell. The results suggest that the pathogen senses cell structures and focuses the transfer of YopE to occur solely at the interaction zone between the bacterium and the eukaryotic cell. The regulation of this process is shown to involve surface-located YopN sensor protein of the bacterium.     

Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T₀), and four of the 90 T₀ spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T₁). The four transgenic shoots of Toivo (the T₀ plant) produced 25 plantlets as T₁ progeny. Altogether fifteen of these T₁ plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T₂ progeny.     

Effects of ammonium sulphate application on the rhizosphere,fine-root and needle chemistry in aPicea abies (L.) Karst. stand

Rhizosphere, fine-root and needle chemistry were investigated in a 28 year old Norway spruce stand in SW Sweden. The uptake and allocation pattern of plant nutrients and aluminium in control plots (C) and plots repeatedly treated with ammonium sulphate (NS) were compared. Treatments started in 1988. Current year needles, one-year-old needles and cylindrical core samples of the LFH-layer and the mineral soil layers were sampled in 1988, 1989 and 1990. Compared to the control plots, pH decreased significantly in the rhizosphere soil in the NS plots in 1989 and 1990 while the SO₄-S concentration increased significantly. Aluminium concentration in the rhizosphere soil was generally higher in the NS plots in all soil layers, except at 0–10 cm depths, both in 1989 and 1990. Calcium, Mg and K concentrations also increased after treatment with ammonium sulphate. Ammonium ions may have replaced these elements in the soil organic matter. The NS treatment significantly reduced Mg concentrations in fine roots in all layers in 1990. A similar trend was found in the needles. Ca concentrations in fine roots were significantly lower in the NS plots in the LFH layer in 1990 and the same pattern was found in the current needles. The N and S concentrations of both fine roots and needles were significantly higher in the NS plots. It was suggested that NS treatment resulted in displacement of Mg, Ca and K from exchange sites in the LFH layer leading to leaching of these cations to the mineral soil. Further application of ammonium sulphate may damage the fine roots and consequently adversely affect the water and nutrient uptake of root systems.     

The Effect of Transport Stress on Plasma Levels of Catecholamines,Cortisol, Corticosteroid-Binding Globulin,Blood Cell Count,and Lymphocyte Proliferation in Pigs

The effect of transport stress on the plasma levels of catecholamines, Cortisol, and corticosteroid-binding globulin were studied in 6 gilts. To assess the effect on immune status, white blood cells were also counted and the cell-mediated immunity was estimated. The adrenaline level increased significantly during transport, from a basal mean level of 0.03 ng/ml to a plateau level of 0.11 to 0.12 ng/ml. The noradrenaline level fluctuated, but not constantly, during transport. The mean plasma Cortisol level before loading was approximately 40 nmol/1 and rose immediately after the start of transport to 70 nmol/1 (p< 0.05) and to 87 nmol/1 (p< 0.01) within 10 and 30 min, respectively. After unloading the Cortisol level rapidly decreased and a minimum level was seen 4 h after the transport, whereafter the diurnal rhythm was resumed. The plasma corticosteroid-binding globulin level increased nonsignificantly during the day of transport, from 25 nmol/1 to a level of 34 nmol/1, and it continued to increase until a plateau level was reached on the second day after transportation. The total white blood cell number increased significantly (from 13.7 to 15.5 × 109 cells/1), the number of lymphocytes decreased significantly (from 8.4 to 7.0 × 109 cells/1), and the number of polymorphonuclear neutrophils increased significantly (from 4.3 to 7.2 × 109 cells/1) during transport. No significant variation in the proliferation response was seen in the whole blood cell cultures. The main results were the significant signs of simultaneous activity of both the adrenal cortex and the adrenal medulla during transport.