Localization of Glial Cell Line-derived Neurotrophic Factor (GDNF) mRNA in Embryonic Rat by In Situ Hybridization

The localization of glial cell line-derived neurotrophic factor (GDNF) mRNA was studied by in situ hybridization in rat from embryonic (E) day E10 to E15. At E10, GDNF mRNA is found in the urogenital field and the cranial part of the gut. At E11, the most abundant expression of GDNF mRNA is seen in the epithelial cells of the second, third and fourth pharyngeal pouches, the third and fourth pharyngeal arches and pharynx. Also mesenchymal cells of the gut and mesonephric tubules contain GDNF mRNA. At E13, expression is observed in the mesenchymal cell layers of the oesophagus, intestine and stomach, the mesenchymal cells around the condensing cartilages and metanephric kidney mesenchyme. Also, the epithelia of Rathke's pouch and pharynx are intensely labelled. High expression of GDNF mRNA continues at El5 in kidney, gastrointestinal tract and cartilage. At that stage, GDNF mRNA is seen also in whisker pad and skeletal muscles. The distribution of GDNF mRNA in embryonic rat suggests important roles for GDNF in the early differentiation of the kidney tubules, the innervation of the gastrointestinal tract and the differentiation process of the cartilage and muscle. Our results indicate novel functions for GDNF outside the nervous system.     

Effect of quinpirole on striatal dopamine release and locomotor activity in nicotine-treated mice

The effect of chronic oral nicotine treatment which in its intermittent delivery resembles human smoking was studied on the sensitivity of dopamine autoreceptors in mice. On the 50th day of nicotine administration in the drinking water or after 23-25 h withdrawal quinpirole (D2/D3 agonist, 0.01-0.1 mg/kg s.c.) was given, and accumbal and dorsal striatal dopamine outflow, locomotor activity and body temperature were measured. Dorsal striatal extracellular dopamine concentration and locomotor activity were found to be elevated during nicotine administration. Chronic nicotine did not alter the effects of small, autoreceptor preferring doses of quinpirole on accumbal or dorsal striatal dopamine, locomotor activity or body temperature. However, quinpirole's locomotor activity reducing effect was slightly diminished in mice treated repeatedly with nicotine (0.4 mg/kg twice daily for 10 days s.c.). Thus, although repeated nicotine treatment for 5-14 days decreases dopamine autoreceptor sensitivity, after long-term oral nicotine treatment such a decrease is not seen. Thus, the changes occurring in the sensitivity of D2-like dopamine receptors probably play a minor role in regulating the dopaminergic transmission during long-term nicotine administration.     

Multivariate data analysis as a fast tool in evaluation of solid state phenomena

A thorough understanding of solid state properties is of growing importance. It is often necessary to apply multiple techniques offering complementary information to fully understand the solid state behavior of a given compound and the relations between various polymorphic forms. The vast amount of information generated can be overwhelming and the need for more effective data analysis tools is well recognized. The aim of this study was to investigate the use of multivariate data analysis, in particular principal component analysis (PCA), for fast analysis of solid state information. The data sets analyzed covered dehydration phenomena of a set of hydrates followed by variable temperature X-ray powder diffractometry and Raman spectroscopy and the crystallization of amorphous lactose monitored by Raman spectroscopy. Identification of different transitional states upon the dehydration enabled the molecular level interpretation of the structural changes related to the loss of water, as well as interpretation of the phenomena related to the crystallization. The critical temperatures or critical time points were identified easily using the principal component analysis. The variables (diffraction angles or wavenumbers) that changed could be identified by the careful interpretation of the loadings plots. The PCA approach provides an effective tool for fast screening of solid state information.     

Fumonisin B1-induced apoptosis in neuroblastoma, glioblastoma and hypothalamic cell lines

Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium verticilliodes, which commonly infects corn across the world. Fusarium fungi may also be found in moisture-damaged buildings. In this study, we investigated the role of apoptosis in the toxicity of FB(1) in four different cell lines. Activation of caspase-3-like protease, DNA fragmentation and expression of p53 and Bcl-2 family proteins were studied in mouse GT1-7 hypothalamic, rat C6 glioblastoma, human U-118MG glioblastoma, and human SH-SY5Y neuroblastoma cells exposed to 0.1-100microM FB(1) for 0-144h. Caspase-3-like protease activity increased in all cell lines, except SH-SY5Y, at 48-144h, and internucleosomal DNA fragmentation occurred in all of the cell lines, pointing to a role for apoptosis in the toxicity of FB(1). However, the expressions of p53 or pro- or antiapoptotic Bcl-2 family proteins (Bax, Bcl-2, Bcl-X(L) and Mcl-1) were not affected in any of the cell lines even after prolonged exposure to FB(1) at high doses. The results of this study, together with the results of our previous studies, provide evidence that FB(1) is a potential neurotoxin, but that the toxicity of FB(1) varies between different cell lines. The sensitivity of these cell lines towards FB(1) is as follows: U-118MG>GT1-7>C6>SH-SY5Y cells. These results are consistent with the assumption that cells of glial origin may be more sensitive towards FB(1) than cells of neural origin.     

Testicular xenografts: a novel approach to study cytotoxic damage in juvenile primate testis

The underlying primary damage to the testis caused by chemotherapeutic regimens during childhood is largely unknown. Xenografting of monkey testes was successfully applied in maturation of juvenile testis to the point of complete spermatogenesis. This allows us to manipulate developing primate testis without direct treatment of patients. This new model is validated establishing the effects of cytotoxic treatment in the immature primate testis. Male castrated nude mice received eight s.c. grafts of juvenile monkey testicular tissue and, 28 weeks later, were injected with busulfan (38 mg/kg, i.p.) or vehicle. Graft numbers, size, and histology were examined. Grafts showed pubertal induction of spermatogenesis to the level of pachytene spermatocytes at point of busulfan treatment and further progressed to the level of round spermatids in control samples at 4 weeks. Busulfan treatment caused a statistically significant decrease in the number of seminiferous tubules containing germ cells. Type B spermatogonia and more advanced stages of spermatogenesis were depleted. A statistically significant decrease to pretreatment level was observed in the number of type A pale and centrally located spermatogonia. Busulfan did not affect type A dark spermatogonia. Occasionally, elongating spermatids were detected in busulfan-treated grafts. Observations show that busulfan selectively destroys differentiating spermatogonia whereas some of the spermatocytes present at the moment of cytotoxic insult are able to continue differentiation. Data indicate that xenografting of testicular monkey tissue is a valid approach to detect the busulfan-induced germ cell damage and serves as a powerful experimental tool to study cytotoxic effects in developing primate testis.     

Hemochromatosis gene mutations among Finnish male breast and prostate cancer patients

Hereditary hemochromatosis (HH), the most common genetic disease in northern Europeans, is an autosomal recessive disorder of iron metabolism. The association between hepatocellular carcinoma and HFE homozygosity is well documented, but recently HFE hetero- and homozygosity has also been linked to nonhepatocellular malignancies, including female breast cancer. We hypothesized that C282Y and H63D mutations in the HFE gene could contribute to male breast cancer (MBC) and prostate cancer (PC) susceptibility at the population level in Finland. We screened the 2 major HFE mutations, H63D and C282Y, from 116 MBC cases diagnosed in Finland between 1967 and 1996, 843 consecutive unselected PC cases diagnosed at the Pirkanmaa Hospital District between 1999 and 2001 and 480 anonymous blood donor controls by minisequencing. Our results indicate that the frequencies of the HFE mutations do not significantly differ between MBC and PC patients and the population-based controls. No significantly altered risks for MBC or PC among carriers of the 2 variants were observed. However, HFE mutations were seen twice as often among carriers of a common BRCA2 mutation 9346(-2)A-->G compared with the rest of the MBC cases, indicating that HFE may be an MBC risk modifier gene among BRCA2 mutation carriers. In conclusion, our results indicate a minor role for the HFE mutations C282Y and H63D in the causation of MBC and PC, but carriers of both BRCA2 9346(-2)A-->G and an HFE mutation may be at an increased risk.     

Association of MBL2 polymorphism with asthma after bronchiolitis in infancy

Background: Mannose‐binding lectin (MBL) is a component of innate immunity and has been linked with the pathogenesis of asthma. The aim of the present study was to evaluate the association of MBL genotypes with preschool asthma and allergy in children with bronchiolitis in early infancy. Methods: In all, 205 infants were hospitalized for bronchiolitis at <6 months of age. Asthma and allergy were studied from a total of 166 children at 6.4 years (mean). A total of 141 (85%) frozen whole blood samples were available for MBL genotyping and MBL2 gene mutations were determined on pyrosequencing for detection of three single‐nucleotide polymorphisms. Results: Ninety‐five children (67.4%) had the wild‐type MBL genotype A/A and 46 had A/O or O/O genotypes. Asthma was present in 16 children (11.3%) at 5–7 years of age. Nine children (19.6%) with non‐AA genotype had asthma (vs 7.4% of those with genotype AA, P= 0.03). The result remained significant after adjustment for age, gender and atopy. There were no significant associations between MBL genotypes and asthma at any age before the study. Atopic dermatitis, allergic rhinitis or paternal and/or maternal asthma had no significant associations with MBL genotypes. Conclusions: The variant non‐A/A MBL genotype is associated with asthma after bronchiolitis in infancy, but not earlier than at 5–7 years of age.