Soybean phytophthora resistance gene Rps8 maps closely to the Rps3 region.

Root and stem rot is one of the major diseases of soybean. It is caused by the oomycete pathogen Phytophthora sojae. A series of resistance genes (Rps) have been providing soybean with reasonable protection against this pathogen. Among these genes, Rps8, which confers resistance to most P. sojae isolates, recently has been mapped. However, the most closely linked molecular marker was mapped at about 10 cM from Rps8. In this investigation, we attempted to develop a high-density genetic map of the Rps8 region and identify closely linked SSR markers for marker-assisted selection of this invaluable gene. Bulk segregant analysis was conducted for the identification of SSR markers that are tightly linked to Rps8. Polymorphic SSR markers selected from the Rps8 region failed to show cosegregation with Phytophthora resistance. Subsequently, bulk segregant analysis of the whole soybean genome and mapping experiments revealed that the Rps8 gene maps closely to the disease resistance gene-rich Rps3 region.

Expression of focal adhesion kinase (p125 FAK) and proline-rich tyrosine kinase 2 (PYK2/CAKb) in cerebral metastases, correlation with VEGF-R-, ecNOS III-labelling and morphometric data.

BACKGROUND: Cerebral metastasis occurs in about 20% of all neurosurgical patients. Cerebral metastases have a typical spherical morphology with a common central necrosis and perifocal oedema. It has been proposed that oedema extension, tumour volumes and infiltrative behaviour are partially mediated by vascular endothelial growth factor (VEGF) and nitric oxide (NO). In several systemic tumour entities NO is suggested as a factor which influences the metastatic potential. VEGF has recently been reported to influence the matrix related migratory activity by interaction with focal adhesion kinase (p125FAK) and proline-rich tyrosine kinase beta (PYK2/CAK beta). Nitric oxide, which is produced in metastases by three different NOS isozymes is capable of antagonizing the binding of FAK to matrix integrins. NO, VEGF and FAK/PYK 2 are therefore considered to be important mediators of the cerebral metastatic incidence, growth, infiltration and oedema extension. The aim of our present study was to investigate the expression of p125FAK and the coexpression with PYK2/CAK beta, VEGF-receptor FLT-1, NOS isozymes NOS I-III, capillary density and the histology in 130 specimens of resected cerebral metastatic tumours. A further analysis was performed to morphometrically evaluate tumour and oedema volumes and to correlate the immunohistochemical data in a subgroup of 40 patients. MATERIALS AND METHODS: Cryosections (N = 130) of metastatic resections were investigated immunohistologically using a 4-step scoring evaluation for the expression of NOS I-III, VEGF-receptor FLT-1, and capillary vessel presence by endothelial Von-Willebrand-Factor (VWF) staining. Tumour and oedema extension was measured in preoperative MRI (N = 40) scans by an image-processing device (Kontron) and the ratios of oedema volumes to total tumour volumes were calculated. The data were analysed statistically (Spearman rank order correlation and Kruskal-Wallis ANOVA) and correlated with the clinical data. RESULTS: FAK immunoexpression was observed in 50% of the specimens (31.2% gradings 2 and 3). We observed a significant coexpression (p = 0.0001) with PYK 2 labelling which occurred frequently in 74% of the specimens (42% gradings 2 and 3). The VEGF receptor FLT-1 could be detected in 70% of them, 24% at higher expression values 2 and 3. The expression of NO synthase was frequently observed. NOS I was detected in 83.6% of the specimens, values 2 and 3 in 40.5%. NOS III, the endothelial isoform, was observed in 39.4% of the specimens (gradings 2 and 3) and inducible NOS II in 29.4% (grading 2 and 3) of them. Coexpressions were statistically significant for FAK and NOS III (Spearman p = 0.008) and FAK and VEGF-R (p = 0.03). The morphometric evaluation resulted in tumour volumes between 2.0 and 83 cm3 (mean 22.5 +/- 19.1 SD) with oedema ratios between 0 and 100% (mean 62.2 +/- 22.5 SD). FAK expression correlated significantly (p = 0.06) with tumour volumes and histology. CONCLUSION: The frequent histotypic occurrence of FAK and PYK2 in metastases could be an important factor in the modulation of metastatic capacity and infiltrative behaviour and might influence the disease course. Judging from its frequent expression PYK2 may generate the more relevant signals. A further aspect is the possible interaction with endothelial NOS III and VEGF receptor, which could be important for the infiltrative behaviour in a latent hypoxic scenery and environment.

Expression of nitric oxide synthase isozymes (NOS I-III) by immunohistochemistry and DNA in situ hybridization. Correlation with macrophage presence, vascular endothelial growth factor (VEGF) and oedema volumetric data in 220 glioblastomas.

BACKGROUND: Nitric oxide (NO) is synthesized from arginine by three different isozymes of nitric oxide synthase (NOS I-III). NO has been identified as a powerful metabolite of vascular smooth muscle cell function, cerebral blood circulation and oedema induction. NOS induction by different cytokines has been shown previously in glioblastoma cell cultures and NOS III expression due to astrocytoma grading has been shown in several tumors recently. The aim of the present study was to study the coexpression of NOS I-III, macrophage and capillary presence with VEGF, EGF and their receptors and to investigate a possible mechanism in peritumoral oedema generation. MATERIALS AND METHODS: We have investigated the expression (4-grade values, blinded assay by two observers) of NOS I-III together with those of VEGF, VEGF- R (Flt-1), EGF-R1, von-Willebrand-factor (VWF) and a pan-macrophage marker (Ki-M1P) immunohistochemically in tumor specimens from 220 patients and performed tumor volume morphometry by image analysis in a subgroup of 32 cases to test for any correlation with the peritumoral oedema volumes. Inducible NOS II was further investigated by in situ labelling with a DNA oligonucleotide probe cocktail. RESULTS: All of the specimens revealed some NOS expression, NOS II was expressed in macrophages, microglia and endothelial cells, NOS III and I was localized in glioblastoma cells, NOS III in endothelial cells as well. The highest degrees of expression were observed in 46% (NOS I), 22% (NOS II) and 75% (NOS III) of all specimens. Inducible NOS II in any expression grade was observed in 47.5% of the specimens. Significant correlations were observed for the expression of the macrophage marker Ki-M1P with NOS II (p = 0.024), endothelial NOS III with NOS I (p = 0.0003), VEGF-R1 with NOS II (p = 0.0008) and NOS III (p = 0.011) The oedema volumes could not be correlated significantly with NOS or VEGF-R1 expression values but with those of endothelial staining (p = 0.02). We observed a trend towards higher Ki-M1P expression values together with higher oedema volume extensions. In situ hybridization demonstrated reaction products in endothelial and perivascular regions and sometimes scattered throughout the specimens revealing the labelling of macrophages. CONCLUSIONS: The main source of NO is NOS I and NOS III. The latter is located in endothelial cells and glioblastoma cells. The expression of NOS II in glioblastomas is restricted to infiltrating macrophages. NOS II and III expressions were observed significantly together with that of VEGF-R1. Neither NOS I-III nor VEGF-R expression could be correlated with the extension of the peritumoral oedema.

Oedema extension in cerebral metastasis and correlation with the expression of nitric oxide synthase isozymes (NOS I-III).

BACKGROUND: The development of a peritumoral oedema is a common radiological sign in preoperative CT- and MRI scans of patients with cerebral metastasis. Large tumours can be accompanied by a marginally extended oedema and vice versa. Several cytokines (VEGF) have been identified as mediators of vascular induction and permeability. Transmitters such as nitric oxide (NO) have been identified as specific mediators of vascular dilation and tumour blood flow in primary brain tumours in which different NOS isozymes (NOS I and III) are induced as a result of the latent hypoxic metabolic scenery. Other authors have considered NO as an endothelial stabilising metabolite. Inducible NOS II is expressed by microglia and macrophages invading during tumour growth. At present, no data exist on NO synthesising enzymes in cerebral metastasis. MATERIALS AND PATIENTS: Cryosections (N = 96) of metastatic resections were investigated immunohistologically using a 4-step grading evaluation for the expression of NOS I-III, VEGF-receptor FLT-1, a pan-macrophage marker Ki-M1P, and capillary vessel presence by endothelial Von-Willebrand-Factor staining. The tumour and oedema extension was measured in preoperative MRI scans by an image processing device (Kontron) and calculated for the ratios of oedema volumes to total tumour volumes. The data were analysed statistically (Pearson Chi2 and Kruskal-Wallis analysis of variances) and correlated with the clinical data. Inducible NOS II was further investigated by in situ hybridization with a (4x30 mer) DNA oligoprobe cocktail. RESULTS: Between 1987 and 1996 289 patients in our department suffered from a metastatic disease in the brain or spinal cord. In 96 cases resected tumour material was processed for the immunohistological investigation. The age distribution ranged from 14 to 85 years with a median age of 58 years. The mean duration of symptoms before diagnosis was estimated as 53 days. The expression of NO synthase was frequently observed. NOS I was detected in 83.6%, gradings 2 and 3 in 40.5% of them. NOS III, the endothelial isoform, was observed in 39.4% (gradings 2 and 3), inducible NOS II in 29.4% (grading 2 and 3) of the specimens. The VEGF receptor FLT-1 could be detected in 70% of them, 24% in higher expression 2 and 3. The pan macrophage marker Ki-M1P was observed in 72% of all cases. Fifty seven percent of the specimens exhibited strong labelling with antibodies against VWF. Coexpressions were statistically significant for the VEGF receptor and NOS I-III (p < 0.01), Ki-M1P and NOS I and II (p < 0.05). A negative correlation was detected for the oedema index (oedema volume/total volume) and the labelling data for NOS III (r = -0.44, p = 0.13) and VEGF-R (r = -0.42, p = 0.022). No correlation existed for Ki-M1P, VWF and NOS I. CONCLUSIONS: The objective of the study was to investigate oedema morphometry, expression of NOS I-III and VEGF-R, presence of capillary vessels and macrophages in cerebral metastasis. A further aim was to investigate a putative oedema induction by NO producing isozymes. Nitric oxide synthase expression was statistically significantly correlated with the expression of the VEGF receptor and the presence of macrophages and microglia. There was a negative correlation between oedema extension and the presence of NOS III and VEGF-R. The results seem to indicate a specific oedema modulating role of NO in cerebral metastasis.

Expression of CD 73 (ecto-5'-nucleotidase) in 165 glioblastomas by immunohistochemistry and electronmicroscopic histochemistry.

BACKGROUND: CD 73 (5'-nucleotidase) is an ectoenzyme, which is expressed on normal and neoplastic glial plasma membranes. The enzyme binds to intracellular filamentous actin and the extracellular matrix proteins laminin and fibronectin. CD 73 is a signalling pathway metabolite in the immune response of lymphocytes. The ectoenzyme catalyzes the conversion of purine and pyrimidine ribo- and deoxyribo-nucleoside monophosphates (AMP, GMP, IMP) and leads to elevation of the corresponding nucleosides (adenosine) in the extracellular space and might therefore modulate neuronal signalling and vascular perfusion. CD 73 has also been called a cellular motility factor. There is an increasing amount of evidence for the modulatory role of PKC-mediated CD 73 activity in ischemia, regeneration and repair, glioma cell proliferation and a possible invasion promoting feature of the ectoenzyme. The aim of the present study was to investigate the expression patterns of CD 73 together with the labelling of PKC and EGFR. The latter is known as a marker for primary glioblastomas. PATIENTS AND METHODS: We investigated the expression of CD 73 in 165 glioblastoma specimens together with the expression patterns of PKC and EGFR by immunocytochemistry on cryosections with a 4-step grading evaluation by two independent observers. CD 73 was further investigated morphologically by electron-microscopic histochemistry in cell cultures of glioblastoma specimens. RESULTS: With these methods it was possible to demonstrate a dense labelling pattern of glioblastoma specimens with anti-CD 73. 95.7% of the glioblastomas were identified with staining products, 63% with labelling grades 2 and 3. The dense staining of the endoplasmatic reticulum, vesicles, caveolar structures and glial membranes was demonstrated by electron-microscopic histochemistry. Some free enzymatic activity was located bound to the ECM components. We observed a significant coexpressions of CD 73 with PKC (p = 0.001) and CD 73 with EGFR (p = 0.022), which is a prospective marker for a high rate of early recurrency. CONCLUSIONS: The CD 73 activity was densely distributed on the membranes of glioblastoma cells in vivo and in cell cultures. The electron-microscopic histochemical studies could demonstrate enzymatic activity at the cell membranes and in vesicular structures and caveolae. Free staining deposits located on ECM components may result in a migration- and infiltration-promoting activity. The CD 73 expression could be correlated with the expression grades of PKC and EGFR. The latter has been identified as a prognostic factor which is expressed mainly on primary glioblastomas. PKC is a known tumour metabolite in several proliferation promoting pathways of EGF receptor signalling.

Postmortem delay and temperature conditions affect the in situ end-labeling (ISEL) assay in brain tissue of mice.

Apoptotic cell changes occurring under certain developmental, physiological, and pathological conditions have been of increasing interest during recent years. Due to occasional difficulties in detecting apoptosis in routinely stained sections, various methods have been developed to facilitate tissue examination. Fragmentation of DNA during the process of apoptosis is a prerequisite for detection in the in situ end-labeling (ISEL) procedure. It is yet unclear whether other mechanisms of cell change that induce DNA fragmentation such as necrosis and postmortem autolysis also show positive staining with the ISEL technique. To investigate whether the ISEL assay visualizes autolytic DNA changes along with apoptotic DNA fragmentation, we tested the technique on brain tissue of mice after different time intervals (0, 6, 12, 24, 48, 72 h) of postmortem delay (PMD) and at 2 different temperatures of postmortem storage (4 degrees C and room temperature (RT)). Our semiquantitative results show that up to 24 h of PMD no prominent difference in labeling is observable at both temperatures. After 48 and 72 h of PMD at RT clusters of labeled cells begin to appear. Clusters of stained cells should therefore not be considered as apoptosis when using the ISEL assay.