Cytotoxic T cell responses in HLA-A2.1 transgenic mice. Recognition of HLA alloantigens and utilization of HLA-A2.1 as a restriction element

Previous studies have indicated that the frequency of murine CTL precursors (CTLp) for human class I molecules is one to two orders of magnitude lower than that for murine class I alloantigens, and that this is due to species-specific structural differences between these molecules. Transgenic mice expressing the human class I MHC Ag HLA-A2.1 were used to examine changes in the frequency of class I HLA-specific precursors after T cell differentiation in an HLA-A2.1 positive environment. The HLA-A2.1 gene product was expressed at levels comparable to those of the endogenous H-2Db molecule in thymus, bone marrow, and spleen. By limiting dilution analysis, it was observed that the frequencies of CTLp in transgenic mice responding to the human alloantigens HLA-B7 or HLA-A2.2 were comparable to or lower than those in normal C57BL/6 mice, regardless of whether the Ag was presented on human or murine cells. Thus, expression of a human class I molecule in these animals did not result in an expansion of the number of CTLp specific for other human class I Ag. In addition, the frequency of HLA-A2.1-restricted, influenza specific CTLp was substantially lower than the frequency of H-2b restricted CTLp, indicating a poor utilization of HLA-A2.1 as a restricting element. Finally, the frequencies of CTLp for HLA-A2.1 expressed on syngeneic murine tumor cells were decreased significantly. Thus, expression of HLA-A2.1 in these animals appeared to induced tolerance to this Ag. Interestingly, however, these mice were not tolerant to the HLA-A2.1 molecule expressed on human cells. This indicates that the HLA-A2.1 associated epitopes expressed on murine and human cells differ and suggests that, under these circumstances, HLA-A2.1 acts as a restricting element for human nominal Ag. These results are discussed in the context of current models of T cell repertoire development.     

Extensive changes in cytokeratin expression patterns in pathologically affected human gingiva

The stratified squamous epithelium of the oral gingiva and the hard palate is characterized by a tissue architecture and a cytoskeletal composition similar to, although not identical with, that of the epidermis and fundamentally different from that of the adjacent non-masticatory oral mucosa. Using immunocytochemistry with antibodies specific for individual cytokeratins, in situ hybridization and Northern blots of RNA with riboprobes specific for individual cytokeratin mRNAs, and gel electrophoresis of cytoskeletal proteins of microdissected biopsy tissue samples, we show changes in the pattern of expression of cytokeratins and their corresponding mRNAs in pathologically altered oral gingiva. Besides a frequently, although not consistently, observed increase in the number of cells producing cytokeratins 4 and 13 (which are normally found as abundant components in the sulcular epithelium and the alveolar mucosa but not in the oral gingiva) and a reduction in the number of cells producing cytokeratins 1, 10 and 11, the most extensive change was noted for cytokeratin 19, a frequent cytokeratin in diverse one-layered and complex epithelia. While in normal oral gingiva cytokeratin 19 is restricted to certain, sparsely scattered cells of --or near--the basal cell layer, probably neuroendocrine (Merkel) cells, in altered tissue of inflamed samples it can appear in larger regions of the basal cell layer(s) and, in apparently more advanced stages, also in a variable number of suprabasal cells. Specifically, our in situ hybridization experiments show that this altered suprabasal cytokeratin 19 expression is more extended at the mRNA than at the protein level, indicating that cytokeratin 19 mRNA synthesis may be a relatively early event during the alteration. These changes in cytokeratin expression under an external pathological influence are discussed in relation to other factors known to contribute to the expression of certain cytokeratins and with respect to changes occurring during dysplasia and malignant transformation of oral epithelia.     

Measurement of ammonia and glutamine in cell culture media by gas sensing electrodes

Summary This paper describes the use of a commercially available off-line gas sensing electrode for determination of ammonia and glutamine in cell culture media. The measurement technique was tested in different media preparations with different serum concentrations. The glutamine decomposition was studied as a function of pH for cell culture medium and the results were compared to those obtained by conventional methods,i.e., HPLC. Finally, glutamine and ammonia metabolism were studied during the cultivation of a hybridoma cell line.     

Modulation of the activity of phosphoenolypyruvate carboxylase during potassium-induced swelling of guard-cell protoplasts of Vicia faba L. after light and dark treatments

Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) activity was found to be modulated by light and darkness when measured in the presence of K⁺, which had been added to induce swelling of guard-cell protoplasts (GCPs) from Vicia faba L., whereas no modulation was detected in the absence of K⁺ (PEPcase activity remained constant at 1.5±0.15 pmol PEP metabolized · GCP^–1 ·h^–1; subsequently, pmol GCP^–1 ·h^–1 will be used). The activity of PEPCase increased by 100% (from 1.5 to 3 pmol·protoplast^–1·h^–1) in darkness and by 200% (from 1.7 to 5 pmol·protoplast^–1· h^–1) in light and oscillations in activity of these magnitudes were repeated at intervals of 2 min (dark) and 2.5 min (light) for a period of 10 min during K⁺-induced increase in the volume of GCPs. The oscillations were reflected in changes in malate-pool sizes determined in plastids, mitochondria and the supernatant fraction (consisting of the cytosol and the vacuole). Malate probably functioned as a mitochondrial substrate, thus supplying ATP for K⁺ uptake and the swelling of the protoplasts. On the basis of the present paper and previous results (H. Schnabl and B. Michalke 1988, Life Sci. Adv. Plant Physiol. 7, 203–207) involving adenine nucleotidepool sizes in fractionated GCPs, a model is proposed to explain the cause-effect relationship between K⁺, PEPCase, the cytosolic and mitochondrial malate levels and ATP levels during the K⁺-induced increase of GCP volume.Abbreviations GCP dtguard-cell protoplast - PEP phosphoenol-pyruvate - PEPCase PEP carboxylase The authors thank Professor Hermann Schnabl, University of Stuttgart (FRG), for his assistance in applying the graph theory analysis. This work was supported by Deutsche Forschungsgemeinschaft to H.S.     

A microdeletion of less than 250 kb, including the proximal part of the FMR-1 gene and the fragile-X site, in a male with the clinical phenotype of fragile-X syndrome

A gene designated "FMR-1" has been isolated at the fragile-X locus. One exon of this gene is carried on a 5.1-kb EcoRI fragment that exhibits length variation in fragile-X patients because of amplification of or insertion into a CGG-repeat sequence. This repeat probably represents the fragile site. The EcoRI fragment also includes an HTF island that is hypermethylated in fragile-X patients showing absence of FMR-1 mRNA. In this paper, we present further evidence that the FMR-1 gene is involved in the clinical manifestation of the fragile-X syndrome and also in the expression of the cellular phenotype. A deletion including the HTF island and exons of the FMR-1 gene was detected in a fragile X-negative mentally retarded male who presented the clinical phenotype of the fragile-X syndrome. The deletion involves less than 250 kb of genomic DNA, including DXS548 and at least five exons of the FMR-1 gene. These data support the hypothesis that loss of function of the FMR-1 gene leads to the clinical phenotype of the fragile-X syndrome. In the fragile-X syndrome, there are pathogenetic mechanisms other than amplification of the CGG repeat that do have the same phenotypic consequences.     

Enzymes Involved in Anaerobic Polyethylene Glycol Degradation by Pelobacter venetianus and Bacteroides Strain PG1

In extracts of polyethylene glycol (PEG)-grown cells of the strictly anaerobically fermenting bacterium Pelobacter venetianus, two different enzyme activities were detected, a diol dehydratase and a PEG-degrading enzyme which was characterized as a PEG acetaldehyde lyase. Both enzymes were oxygen sensitive and depended on a reductant, such as titanium citrate or sulfhydryl compounds, for optimal activity. The diol dehydratase was inhibited by various corrinoids (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, and methylcobalamin) by up to 37% at a concentration of 100 μM. Changes in ionic strength and the K⁺ ion concentration had only limited effects on this enzyme activity; glycerol inhibited the enzyme by 95%. The PEG-degrading enzyme activity was stimulated by the same corrinoids by up to 80%, exhibited optimal activity in 0.75 M potassium phosphate buffer or in the presence of 4 M KCI, and was only slightly affected by glycerol. Both enzymes were located in the cytoplasmic space. Also, another PEG-degrading bacterium, Bacteroides strain PG1, contained a PEG acetaldehyde lyase activity analogous to the corresponding enzyme of P. venetianus but no diol dehydratase. Our results confirm that corrinoid-influenced PEG degradation analogous to a diol dehydratase reaction is a common strategy among several different strictly anaerobic PEG-degrading bacteria.     

Aldosterone regulates paracellular pathway resistance in rabbit distal colon

Summary Regulation of the paracellular pathway in rabbit distal colon by the hormone aldosterone was investigated in vitro in Ussing chambers by means of transepithelial and microelectrode techniques. To evaluate the cellular and paracellular resistances an equivalent circuit analysis was used. For the analysis the apical membrane resistance was altered using the antibiotic nystatin. Under control conditions two groups of epithelia were found, each clearly dependent on the light: dark regime. Low-transporting epithelia (LT) were observed in the morning and high-transporting epithelia (HT) in the afternoon. Na⁺ transport was about 3-fold higher in HT than in LT epithelia. Incubating epithelia of both groups with 0.1 mol·1^-1 aldosterone on the serosal side nearly doubled in LT epithelia the short circuit current and transepithelial voltage but the transepithelial resistance was not influenced. Maximal values were reached after 4–5 h of aldosterone treatment. In HT epithelia due to the effect of aldosterone all three transepithelial parameters remained constant over time. Evaluation of the paracellular resistance revealed a significant increase after aldosterone stimulation in both epithelial groups. This increase suggests that tight junctions might have been regulated by aldosterone. The hormonal effect on electrolyte transport was also dependent on the physiological state of the rabbit colon. Since net Na⁺ absorption in distal colon is, in addition to transcellular absorption capacity, also dependent on the permeability of the paracellular pathway, the regulation of tight junctions by aldosterone may be a potent mechanism for improving Na⁺ absorption during hormone-stimulated ion transport.Abbreviations V _t transepithelial potential difference (mV) - R _t transepithelial resistance (·cm²) - G _t transepithelial conductance (mS·cm^-2) - I_sc calculated short circuit current (A·cm^-2) - V _a apical membrane potential difference (mV) - V _bl basolateral membrane potential difference (mV) - voltage divider ratio - R _a apical membrane resistance (·cm²) - R _bl basolateral membrane resistance (·cm²) - R _c cellular resistance ( of apical and basolateral resistance) (·cm²) - R _p resistance of the paracellular pathway (·cm²) - G _a apical membrane conductance (mS·cm^-2) - G _bl basolateral membrane conductance (mS·cm^-2) - G _p paracellular conductance (mS·cm^-2) - G _t transepithelial conductance (mS·cm^-2) - HT _contr high transporting control epithelia - LT _contr low transporting control epithelia - HT _aldo aldosterone incubated high transporting epithelia - LT _aldo aldosterone incubated low transporting epithelia     

Colony orientation and successful defence behaviour in the conifer aphid, Schizolachnus pineti

We studied colony structure and body orientation behaviour of Schizolachnus pineti, a specialised aphid feeding on needles of Scots pine. Aphids feeding at the margin of a colony always face towards the centre of the colony, bounding the colony with their legs. This behaviour increased the defence success aginst the specialised parasitoid (Pauesia unilachni) and the specialised predator (Scymnus nigrinus). Both enemies had less success when attacking S. pineti from the abdominal site when compared to the front. We assume that the defence orientation displayed by marginally feeding aphids has an adaptive nature and is particularly effective when feeding in a row on linear plant structures such as needles.     

Aphid honeydew and its effect on the phyllosphere microflora of Picea abies (L.) Karst

Aphids of the genus Cinara, feeding on Norway spruce, excrete copious amounts of honeydew, a carbon-rich waste product, which accumulates locally on needles and twigs. We investigated the role of honeydew as a potential source of energy which might promote the growth of micro-organisms in the phyllosphere of conifer trees. To approach this question, we followed the population dynamics of Cinara spp. in a natural forest stand over two seasons. We also studied the amounts of honeydew produced by individual aphids and identified potential parameters which might influence honeydew production. Finally, we determined the growth of micro-organisms on infested and uninfested needles of Norway spruce during the growing season. Confined to Picea abies, the investigated Cinara species only became abundant in midsummer, when needles and shoots were expanding. The populations showed only a single peak in abundance, the timing and magnitude of which may vary from year to year due to weather conditions, changes in plant quality in a yearly cycle or the impact of natural enemies. The amount of honeydew produced by individual aphids was dependent on the developmental stage of the aphid, the nutritional supply of its host plant and on the developmental state of the Norway spruce (e.g. bud burst, end of shoot extension). The presence of honeydew significantly increased the growth of bacteria, yeast and filamentous fungi on the surface of needles and there was a pronounced seasonal trend, with the highest abundance in midsummer correlating with the period of peak aphid abundance. Taken together, these findings indicate that aphids have an influence on microbial ecology in the phyllosphere of trees. The implication of our study, from interactions at the population level to effects and potential consequences for C and N fluxes at the level of forest ecosystems, is discussed.