No abstract

S U M M A R Y Group II phospholipase A2 (PLA2) is an acute-phase protein and an important component of the host defense against bacteria. In this study we investigated the distribution of PLA2 protein by immunohistochemistry and the distribution of mRNA of PLA2 by Northern blotting and in situ hybridization in rat tissues. PLA2 protein was localized in the Paneth cells of the intestinal mucosa, chondrocytes and the matrix of cartilage, and megakaryocytes in the spleen. By Northern blotting, mRNA of PLA2 was found in the gastrointestinal tract, lung, heart, and spleen. By in situ hybridization, PLA2 mRNA was localized in the Paneth cells of the small intestinal mucosa but in no other cell types. Our results show specific distribution of PLA2 in a limited number of cell types in rat tissues. The reagents developed in this study (the anti-rat PLA2 antibody and probes for Northern blotting and in situ hybridization of mRNA of rat PLA2) will provide useful tools for future studies concerning the role of PLA2 in various experimental disease models.

We present a novel method of statistical analysis for the comparison of electrophoretic data. The method is based on the squared Euclidian distance of normalized signal data vectors of electrophoretic lanes. The differences in the electrophoretic patterns are evaluated by a statistical test based on Hubert's statistics which measures the significance of the signal grouping. We demonstrate the validity and applicability of the method in a large data set derived from automated fluorescent mRNA differential display analysis of the expression of acute-phase proteins during experimental Escherichia coli infection in mice. The current testing method is capable of finding theoretically similar natural groupings to be similar in a statistically significant way whereas theoretically dissimilar or random groupings can be recognized to be artifactual. We also show how the calculated pairwise signal distances can be utilized in methodological problem solving. These analytical methods can be applied to the study of other related problems of similarity analysis of electrophoretic patterns, and also provide useful tools for the development of automated recognition of differentially expressed mRNAs.

We present a modification of mRNA differential display in which increased throughput results from the use of an automated fluorescent sequencer. The sequence analysis is performed directly on purified fragments without further cloning. The amplified fragments carry a T7 RNA polymerase promoter sequence tag for in vitro transcription of riboprobes for nonradioactive in situ hybridization. We compared changes in gene expression in the liver and colon of group II phospholipase A2 transgenic and group II phospholipase A2 deficient mice during the course of experimental Escherichia coli infection. Fluorescent mRNA differential display comprising a 7 x 24 set of primers was used to study a total of 31,257 amplified cDNA fragments. Sequence analysis of the displayed fragments associated with infection identified classical acute-phase proteins in the liver and host defense proteins in the colon. The displayed mRNAs associated to transgenicity were the transgene itself, i.e., human group II phospholipase A2, and glutathione-S-transferase in the liver. In the colon, the displayed mRNAs associated with transgenicity were the pancreatitis-associated protein and mucin. The results show that fluorescent mRNA differential display is a reliable method to identify differences in the expression of the genes of acute-phase proteins.

We analyzed changes in gene expression in human colonic carcinoma by fluorescent mRNA differential display. RNA isolated from two samples of normal colon and four cases of colonic adenocarcinoma were amplified with a 15 x 32 set of primers resulting in 2880 cDNAs analyzed with an automated sequencer. Electrophoretic patterns implying constitutive gene expression as well as upregulated and downregulated expression in carcinomas were identified. Forty such cDNA fragments were purified by a novel fluorescent polyacrylamide gel electrophoresis (PAGE)-based method and identified by cyclic sequencing. Most genes showing differential expression were upregulated in colonic carcinoma. Upregulated genes included those for various ribosomal and mitochondrial proteins, heat shock proteins, nucleolar RNA-helicase and phosphoserine aminotransferase. Downregulated genes included histone H3.3. In conclusion, genes associated with vital cellular functions such as transcription, protein synthesis and mitochondrial metabolism were upregulated in colonic carcinoma. Fluorescent mRNA differential display can be applied to the identification of novel cancer-related genes for diagnostic, prognostic and therapeutic purposes.

We present a novel method for the automated detection of fragments showing dissimilar expression in mRNA differential display. The analysis is based on aligning the numerical electrophoretic lane data in respect of a given distance function defined on a set of fragments, or signal peaks in general. We presume that significant dissimilarities between peaks result in extreme score values computed for aligned peak pairs. Whereas in sequence comparison, an overall sequence similarity score is conventionally used, the current method defines a special dissimilarity score for searching the peak pairs showing the largest relative differences between the lanes. The output of the analysis is a highly reduced list of peak pairs, along with a set of associated features extracted from the lanes. Only the peaks of this list need to be visually confirmed instead of the vast amount of peaks in the original electrophoretic results. The results obtained by the algorithm correlate well with results of visual evaluation of the same electropherograms. The current algorithm may be applied to the study of complex expression patterns in multiple lanes and, in general, to automated recognition of variously defined patterns of quantitative electrophoretic data.

The effect of age and previous damage of Scots pine (Pinus sylvestris) foliage on the larval growth and survival of four Diprionid species was studied in the laboratory and field. One and two‐year‐old needles supported the fastest larval growth and highest pupal weights of Neodiprion sertifer. On current‐year foliage the larval growth was strongly retarded. Damage‐induced reactions in the mature foliage of Scots pine were studied by rearing larvae on needles originating from trees which were partly or totally defoliated during the early season or previous summer. The early season species N. sertifer had a shorter larval period and lower mortality when reared on needles of trees partly defoliated in the early season. A similar trend was observed with the late season species Gilpinia virens. Two late‐season species Diprion pini and Microdiprion pallipes had no difference in larval growth between control and experimental groups, but the mortality of M. pallipes was higher in the experimental group fed on needles of trees defoliated in the early summer. Also, larvae of N. sertifer were growing better on trees which were partly or totally defoliated during the mass outbreak in the previous summer. These results indicate that no damage‐induced defence against sawfly larvae exists in the mature foliage of Scots pine. This lack of induced resistance could explain why outbreaks of Diprionid species can last several years in the same area. The difference in the anti‐herbivore tactics of deciduous and evergreen trees are also briefly discussed. Zusammenfassung Zur Wirkung vorangegangener Schäden auf die Nahrungsqualität von Kiefernnadeln für Diprioniden (Hym., Tenthredinoidea) Es wurde die Wirkung von Alter und Vorschädigung der Nadeln von Pinus sylvestris auf das Larvenwachstum und überleben von vier Diprioniden‐Arten im Freiland und Labor untersucht. Ein‐ und zweijährige Nadeln ergaben das rascheste Larvenwachstum und das größte Puppengewicht von Neodiprion sertifer. Bei Ernährung mit diesjährigen Nadeln wurde das Larvenwachsturn stark verzögert. Weiterhin wurden auf Vorschäden beruhende Wirkungen älterer Kiefernnadeln auf Blattwespenlarven untersucht. Die Frühsaison‐Art N. sertifer zeigte eine kürzere Larvenentwicklung und geringere Mortalität, wenn sie mit Nadeln von teilweise entnadelten Kiefern aufgezogen wurde. Der gleiche Trend wurde bei der Spätsaison‐Art Gilpinia virens beobachtet. Dagegen wiesen die Spätsaison‐Arten Diprion pini und Microdiprion pallipes keine Unterschiede im Larvenwachstum zwischen den Nadelherkünften auf, jedoch war die Mortalität von M. pallipes an Nadeln vorgeschädigter Bäume höher als an anderen. Die Larven von N. sertifer wuchsen auf Bäumen mit Vorschädigung vom vergangenen Sommer besser auf. Aus diesen Ergebnissen geht hervor, daß vollentwickelte Kiefernnadeln keinen schadensinduzierten Abwehrmechanismus gegen Blattwespenlarven besitzen. Dieser Mangel einer induzierten Resistenz könnte erklären, warum Vermehrungen von Diprioniden mehrere Jahre lang in einem Gebiet anhalten können....

Gene expression analysis by electrophoretic methods is currently limited by the labor-intensive visual evaluation of the electrophoretic signal profiles. For this purpose, we present a flexible approach to computer-assisted comparison of quantitative electrophoretic patterns between multiple expression signals. Gaussian curves are first fitted to the complex peak mixtures, and the resulting approximate signals are then aligned and compared on a peak-by-peak basis with respect to specific patterns defined by the investigator. The rationale of the method is to produce a compressed list of exceptional expression patterns quantified by a set of associated numeric features. A score value is attached to each pattern in such a way that large values identify the most potential findings to be focused on in visual analysis instead of the vast amount of original electrophoretic results. The validity of the method is demonstrated by analyzing a large set of electrophoretic data from mRNA differential display experiments monitoring changes in gene expression patterns in human colonic carcinoma. The automated identification of variously defined gene expression patterns agrees well with the visual evaluation of the same electropherograms. The general comparison approach may also be found useful with other gene expression profiling instruments.