Comparative gene expression profiling by oligonucleotide fingerprinting.

The use of hybridisation of synthetic oligonucleotides to cDNAs under high stringency to characterise gene sequences has been demonstrated by a number of groups. We have used two cDNA libraries of 9 and 12 day mouse embryos (24 133 and 34 783 clones respectively) in a pilot study to characterise expressed genes by hybridisation with 110 hybridisation probes. We have identified 33 369 clusters of cDNA clones, that ranged in representation from 1 to 487 copies (0.7%). 737 were assigned to known rodent genes, and a further 13 845 showed significant homologies. A total of 404 clusters were identified as significantly differentially represented (P < 0.01) between the two cDNA libraries. This study demonstrates the utility of the fingerprinting approach for the generation of comparative gene expression profiles through the analysis of cDNAs derived from different biological materials.

Expression of cytochrome P45011B1 mRNA in the brain of normal and hypertensive transgenic rats.

Cytochrome P45011B1 (11 beta-hydroxylase) was detected in the brain of male rats by in situ hybridization methods. Normal Sprague-Dawley rats were compared to the transgenic strain TGR(mRen2)27, characterized by the expression of the murine Ren-2d renin gene and the development of severe hypertension. Specific riboprobes were generated by in the vitro transcription of a 152 base-pair long cDNA template 35S-labeled riboprobes were hybridized to cryostat sections from adrenal glands and from two different levels of the brain using standard protocols and varying washing conditions. After exposure of the radiolabeled sections to X-ray film, the signals were quantified and compared. Following autoradiography and counterstaining, cytochrome P45011B1 mRNA was clearly localized in the zona fasciculata/reticularis of the adrenal cortex and in distinct layers of the cerebral cortex. High signal densities were obtained in the layers II-IV of the neocortex and in the layer II of the piriform cortex, although the concentrations of cytochrome P45011B1 mRNA were remarkably lower in the central nervous system as compared to adrenal glands. As revealed by the semi-quantitative analysis, there was a slight increase in adrenal 11 beta-hydroxylase mRNA in the transgenic rats, whereas the brain seems to express nearly the same amount of this enzyme in both strains. The cytochrome P45011B1 mRNA expression in distinct cells, probably nerve cells, and especially in regions with high densities of glucocorticoid receptors points to a possible function of brain derived corticosterone in receptor activation.

Basic fibroblast growth factor expression and tenascin C immunoreactivity after partial unilateral hemitransection of the rat brain.

Basic fibroblast growth factor (bFGF) gene expression as well as its immunoreactivity were studied after partial unilateral hemitransection of the rat brain during a time course of 24 h, 72 h, 7 and 14 days. The mechanical injury resulted in a global increase of bFGF gene expression at the 24-h time interval. This global increase was seen at the ipsilateral site at the level of the lesion as well as rostral to the lesion in the ipsilateral hemisphere. The upregulation in bFGF gene expression was in most of the areas investigated due to an upregulation in glial cells as seen by means of nonradioactive in situ hybridization compared with immunocytochemistry for glial fibrillary acidic protein (GFAP). Basic FGF immunoreactivity (IR) was increased around the lesion in glial cell nuclei 7 days after the injury. This increase was also detected in GFAP positive glial cells surrounding small vessels in the lesioned area. Moreover, in the present paper we demonstrate increased tenascin immunoreactivity in the lesioned area 7 days after injury. The tenascin IR was increased at the edges of the lesion as well as in vessel like structures. The tenascin IR was partially codistributed with GFAP IR in the lesioned area. The lesion was also characterized by an increase in vimentin IR as well as in laminin IR. It is suggested that the observed changes in the expression of bFGF, matrix proteins (laminin, tenascin) and intermediate filaments (vimentin) are involved in (a) tissue repair, (b) protection of neuronal cells from excitotoxic influences and (c) formation of new vessels in the lesioned area.

Localization of thioredoxin in the rat brain and functional implications.

The immunoreactivity for thioredoxin, which catalyzes protein disulfide reductions, has previously been shown to exist in nerve cells and their axons. Here we demonstrate the localization of thioredoxin mRNA as revealed by in situ hybridization in the rat brain. The gene is expressed in nerve cells of a variety of brain regions, for example, the cerebral cortex, the piriform cortex, the medial preoptic area, the CA3/CA4 region of the hippocampal formation, the dentate gyrus, the paraventricular nucleus of the hypothalamus, the arcuate nucleus, the substantia nigra pars compacta, the locus coeruleus, the ependyma of the 4th ventricle, and the epithelial cells of the choroid plexus. This distribution implicates an important function in nerve cell metabolism, especially in regions with high energy demands and indicates a role of the choroid plexus in nerve cell protection from environmental influences. It was found that after mechanical injury induced by partial unilateral hemitransection the thioredoxin mRNA expression is upregulated in the lesioned area and spreads to the cortical hemispheres at the lesioned level. This induction suggests a function of thioredoxin in the regeneration machinery of the brain following mechanical injury and oxidative stress.

Zone-specific localization of cytochrome P45011B1 in human adrenal tissue by PCR-derived riboprobes.

Cytochrome P45011B1 (11 beta-hydroxylase) was detected in the human adrenal cortex and in human adenomas by in situ hybridization methods. Specific riboprobes were generated by in vitro transcription of 11 beta-hydroxylase--specific synthetic oligonucleotides with attached T7 and SP6 polymerase promotors. [35S]- and digoxigenin-labeled riboprobes were hybridized to sections of an aldosterone-producing adenoma (APA), the non-tumour portion of the corresponding adrenal gland, and two adenomas not related to hyperaldosteronism using standard protocols and varying washing conditions. After exposure of the radiolabeled sections to X-ray film, the signals were quantified and compared by statistical tests. Following autoradiography or immunohistochemical detection of the digoxigenin cytochrome P45011B1 mRNA was clearly localized in the zona fasciculata/reticularis of non-tumour portion of an human adrenal with an APA. Zona glomerulosa, medulla and connective tissue were free of label. As revealed by the semi-quantitative analysis, 11 beta-hydroxylase mRNA signals in the APA were significantly lower than those in the attached non-tumour portion and the other two adenomas. The results confirm known observations on the occurrence of cytochrome P45011B1 in the adrenal cortex of other species, but show, contrary to several immunohistochemical studies, that the enzyme is obviously not expressed in the zona glomerulosa.

Human adrenal CYP11B1: localization by in situ-hybridization and functional expression in cell cultures.

CYP11B1 was detected in the human adrenal cortex and in human adenomas by in situ-hybridization methods. Specific riboprobes were generated and hybridized to sections of an Aldosterone Producing Adenoma (APA), the non-tumour portion of the corresponding adrenal gland and two adenomas not related to hyperaldosteronism. P45011B1 mRNA was clearly localized in the zona fasciculata/reticularis. Semi-quantitative analysis has been performed and seems to be applicable for a further classification of adrenal tumours. Stable expression of CYP11B1 cDNA was performed in V79 cells. The interference of different substances (metyrapone, spironolactone and different imidazole derivatives) with CYP11B1 activity was studied using this cell line. The cell line revealed to be suitable for analysis of the active site of CYP11B1 as well as for analysis of side effects of drugs on steroidogenesis.

[Effectiveness of various forms of therapy in chronic cardiovascular diseases--a vector analysis of the transition probabilities of degrees of severity]. / Effektivität differenter Therapieformen bei chronischen Herz-Kreislauf-Krankheiten--eine Vektorenanalyse der Ubergangswahrscheinlichkeiten von Schweregraden.

The present findings result from a check of probationers representative for approx. 280,000 inhabitants of defined territories, who had been designated as suspects of heart and vessel disease on grounds of X-ray-morphological criterions (classification by Richter). Those about 3,000 suspects, subdivided into 3 comparable patient groups A, B and C, underwent different regimes of treatment of outpatient medical practice after standardized and noninvasive diagnostics in a follow-up during 5 years and had been valued by means of a problem-specific grading. The comparative analysis about the distribution of severe degrees concerning hypertension and coronary heart disease after the conclusion of the intervention showed significant differences concerning the results of treatment to the credit of the patient group A (treated by specialists) contrary to the patient group B (treated by family doctors) and patient group C (principle of announcing the patients themselves). Also the patient group B showed significantly better results of therapy compared with the patient group C. In addition to the concluding rating the estimation of yearly transition of severe degrees gave an insight into the therapeutical decision of the person who looks after as well as the different distribution of severe degrees of special heart diseases in dependence on the starting severe degree in the special period of intervention conditioned on the therapy. The results gain exceptional importance for practice on the grounds of methodics of the study-automatable classification of dv-thorax-X-ray pictures, problem-specific grading of noninvasive, simple parameters, variants of therapy in dependence on the graduated system of medical care.

[Assessment of prognosis with Markov chains in chronic cardiovascular diseases in relation to various therapies]. / Prognoseabschätzung mit Markoff-Ketten bei chronischen Herz-Kreislauf-Krankheiten in Abhängigkeit von Therapievarianten.

On the basis of 5-years intervention results concerning chronic heart and vessel diseases of a check, which is representative for approx. 280.000 inhabitants of defined territories (EBMO-Cor Berlin), the prognose for the time of 5 and 10 years were estimated. It refers to ambulantories with the diagnosis hypertension and coronary heart disease, who were treated in a different way. The fundamental idea was to extract a prognostic index to make possible a choice of the treatment, adequate to the specific disease and its severe degrees, free of chance. The basic requirement of the application of the "Markoff-model" was the evidence of homogeneity concerning transition of severe degrees, used as reference, which was proved by means of the 2-k-chi 2-Felder-test. By using a starting vector of 1000 patients in each case inquiries about the distribution to the severe degrees had been made. The so achieved results illustrate a therapy-dependent susceptibility of these (morphologically defined) population suffering from a heart disease and allow a forecast, estimated also for a longer period, about the extent of the transition of a respecting and with the possibilities of the outpatient practice defined severe degrees of a heart and circulation disease and with that about chance and risk of a patient. By means of the epidemiological reference of the study the result gains special importance for outpatient practice.

[Problem-specific grading in disease course assessment and calculation of the likelihood of transition of chronic cardiovascular diseases]. / Problemspezifisches Grading zur Verlaufsbeurteilung sowie zur Berechnung von Ubergangswahrscheinlichkeiten chronischer Herz-Kreislauf-Krankheiten.

For the course rating of cardiovascular caused drop in efficiency a problem-specific grading is introduced, the characterization of which is composed of simple, non-invasive parameters. The grading is specified for 4 clinical diagnostic classes (hypertension, coronary heart diseases, pulmonary heart, "other heart and vessel diseases") and takes into consideration the frequent coincidence or connection of several diagnoses of heart and vessel diseases with each other. The choice of parameter and connection have to fulfil the demand, which is essential for the course rating of heart and vessel diseases, like practicability proportionate with the frequency of heart and vessel diseases, adequate registration of changes in severe degrees, statement about the patient and not about several parameters. Furthermore it has to make it possible to calculate provisional probabilities under variable conditions as well as to make possible a valuation of prognosis. For that purpose the construction of suitable matrixes is shown and an integral projection is represented, which is used for the interpretation of courses dependent on severe degrees. The conception of grading has the function to modify necessary parameters for inclusion in the hierarchy according to medical starting position (for instance primary mode of registration). The model which is applicable to level of population makes possible an electronic data - processing - based evaluation including algorithmic classification.