Combining one-dimensional stray-field micro-imaging with mechanical field-cycling NMR: a new spectrometer design.

A new spectrometer design combining stray-field micro-imaging with mechanical field-cycling Nuclear Magnetic Resonance (FC-NMR), allowing for one dimensional spatial resolution in the order of 10 µm is described. The field-cycle is implemented by moving the probe in the stray-field of a superconducting gradient magnet. In this way a field range between 10 mT and 6.3 T is covered. The maximum transfer time is less than 5 s. Further, methods to correct for some of the imaging artefacts found in previous studies are implemented. The main objective of this design is a depth- and field-dependent investigation of the defect structure caused by heavy-ion irradiation of ionic crystals.

Spatially resolved nuclear spin relaxation, electron spin relaxation and light absorption in swift heavy ion irradiated LiF crystals.

Spatially resolved (19)F and (7)Li spin-lattice relaxation rates are measured for LiF single crystals after irradiation with two kinds of swift heavy ions ((12)C of 133 MeV and (208)Pb of 1.78 GeV incident energy). Like in earlier studies on (130)Xe and (238)U irradiated LiF crystals, we found a strong enhancement of the nuclear spin-lattice relaxation rate within the ion penetration depth and a slight--but still significant--enhancement beyond. By evaluating the nuclear relaxation rate enhancement within the ion range after irradiation with different projectiles, a universal relationship between the spin-lattice relaxation rate and the dose is deduced. The results of accompanying X-band electron paramagnetic resonance relaxation measurements and optical absorption spectroscopy are included in a physical interpretation of this relationship. Also the reason for the enhanced relaxation rate beyond the ion range is further discussed.

Histological findings on fetal striatal grafts in a Huntington's disease patient early after transplantation.

Cell transplantation is a promising therapeutic approach that has the potential to replace damaged host striatal neurons and, thereby, slow down or even reverse clinical signs and symptoms during the otherwise fatal course of Huntington's disease (HD). Open-labeled clinical trials with fetal neural transplantation for HD have demonstrated long-term clinical benefits for HD patients. Here we report a postmortem analysis of an individual with HD 6 months after cell transplantation and demonstrate that cells derived from grafted fetal striatal tissue had developed into graft-derived neurons expressing dopamine-receptor related phosphoprotein (32 kDa) (DARPP-32), neuronal nuclear antigen (NeuN), calretinin and somatostatin. However, a fully mature phenotype, considered by the expression of developmental markers, is not reached by engrafted neurons and not all types of interneurons are being replaced at 6 months, which is the earliest time point human fetal tissue being implanted in a human brain became available for histological analysis. Host-derived tyrosine hydroxylase (TH) fibers had already heavily innervated the transplants and formed synaptic contacts with graft-derived DARPP-32 positive striatal neurons. In parallel, the transplants contained a considerable number of immature neuroepithelial cells (doublecortin+, Sox2+, Prox-1+, ss3-tubulin+) that exhibited a pronounced migration into the surrounding host striatal tissue and considerable mitotic activity. Graft-derived astrocytes could also be found. Interestingly, the immunological host response in the grafted area showed localized increase of immunocompetent host cells within perivascular spaces without deleterious effects on engrafted cells under continuous triple immunosuppressive medication. Thus this study provides for a better understanding of the developmental processes of grafted human fetal striatal neurons in HD and, in addition, has implications for stem cell-based transplantation approaches in the CNS.

High temperature mechanical field-cycling setup.

A new design of a mechanical field-cycling setup, operating in the wide temperature range up to 1200 K has been implemented. The sample is moved by a stepping motor in the stray field of a superconducting magnet inside a furnace of homogeneous temperature profile. For a field range from 0.75 to 7 T (transfer length 24 cm), the transfer time is less than 100 ms. The temperature profile is homogenized to better than 1% of the absolute set temperature. The main objective of this design is to extend the T1 relaxation dispersion range covered by electronic field-cycling to higher frequencies.

Predominantly neuronal expression of cytochrome P450 isoforms CYP3A11 and CYP3A13 in mouse brain.

Despite the very small amounts of cytochrome P450 enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function, and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450 isoforms is determined by controversial interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the expression of cytochrome P450 3A11 and 3A13 in mouse brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed identical distribution of their proteins and mRNAs throughout the brain especially in neuronal populations, and to some extent in astrocytes. The cerebral expression of these P450 isoforms was confirmed by Western blot and RNAse protection assay analysis. The well-known testosterone-metabolizing capacity and the inducibility of cytochrome P450 3a isoforms by xenobiotics as well as their presence in steroid hormone-sensitive areas and neurons (e.g. hippocampus) clarify the significance of these isoforms for impairment of steroid hormone actions by P450-inducing environmental substances. Therefore, investigation of inducible cerebral P450 isoforms which are able to metabolize xenobiotics as well as steroid hormones might help us to understand neuroendocrine regulation of brain's plasticity.

Expression and localization of the CYP2B subfamily predominantly in neurones of rat brain.

Despite the very small amounts of cytochrome P450 (P450, CYP) enzymes expressed in different areas and cell populations of the brain as compared with the liver, there is significant evidence for their specific involvement in brain development, function and plasticity. Nevertheless, the current discussion about occurrence and importance of cerebral cytochrome P450s is determined by inconsistent interpretations of their function in general and with respect to single isoforms. Continuing a series of publications about brain P450 isoforms, we now present evidence for the constitutive expression of CYP2B1 and CYP2B2 mRNAs in rat brain. Immunocytochemical and non-radioactive in situ hybridization studies revealed the same expression pattern throughout the brain predominantly in neuronal populations, but to some extent in astrocytes of corpus callosum and olfactory bulb. The well known testosterone-metabolizing capacity and the presence of CYP2B isoforms shown in steroid hormone-sensitive areas and neurones (e.g. hippocampus) clarify the significance of isoforms like CYP2B1 and CYP2B2 for impairment of steroid hormone actions by P450 inducing environmental substances. We argue that cerebral P450 isoforms which are induced by xenobiotics and are able to metabolize these as well as endogenous substrates help us to understand fundamental aspects of brain's functioning.

Identification, induction and localization of cytochrome P450s of the 3A-subfamily in mouse brain.

Several cytochrome P450 subfamilies are inducible by specific exogenous compounds like the antiepileptic drug phenytoin. Some of these P450 enzymes are involved in the metabolism of gonadal hormones also contributing to neuronal differentiation. CYP3A enzymes have the capacity to catalyze the hydroxylation of testosterone and a wide variety of therapeutic agents, but little is known about the expression and potential function of this subfamily in mouse brain. Here, we report the identification of mouse CYP3A isoforms, their induction and localization in mouse brain. Western blot analysis with anti-CYP3A1 antibodies revealed the phenytoin-inducible expression of CYP3A in brain microsomes, and also a constitutive expression of members of this subfamily in brain mitochondria. Using RT-PCR with a consensus primer pair for known mouse liver CYP3A-isoforms we could demonstrate the expression of CYP3A11 and 3A13 mRNA in mouse brain. Finally, using double immunofluorescence labeling we analyzed the histoanatomical distribution of CYP3A throughout the brain with confocal laser scanning microscopy. We found strong immunoreactivity in neurons of hippocampus and hypothalamic areas which are sensitive to steroid hormones. CYP3A immunoreactivity was apparent also in neurons of the cerebellum, the thalamus and the olfactory bulb. Non-neuronal expression of CYP3A could be found in some astrocyte populations and in vascular as well as ventricular border lines. The presence of CYP3A predominantly in neurons but also in cells contributing to the blood-brain and blood-liquor barrier suggests important roles of this subfamily in mediation of steroid hormone action in mouse brain as well as in preventing the brain from potentially cytotoxic compounds.

Expression of transgenic carcinoembryonic antigen (CEA) in tumor-prone mice: an animal model for CEA-directed tumor immunotherapy.

Carcinoembryonic antigen (CEA) is a tumor marker for the most common forms of adenocarcinomas. We have previously described C57BL/6 mice transgenic for the complete human CEA gene. Compared with humans, these mice reveal a conserved spatiotemporal CEA expression pattern. To establish animal models for CEA-targeted tumor immunotherapy, we have crossed CEA transgenic mice with mice that are genetically predisposed to tumor development. These immunocompetent animals should allow optimization of immunotherapy strategies for maximal destruction of tumor tissues with minimal damage to CEA-expressing normal tissues. To develop a breast tumor model, CEA transgenic mice were cross-bred with mice transgenic for the rat neu protooncogene controlled by the mouse mammary tumor virus long terminal repeat. Female offspring developed poorly differentiated breast tumors, none of which, however, expressed CEA. As a model for colorectal tumors, mice bearing a mutation in the Apc gene (Min mice) and the CEA transgene developed multiple intestinal adenomas with strong CEA expression in all tumor cells. CEA expression had no significant effect on tumor growth. Occasional, well-differentiated breast adenocarcinomas in female offspring expressed CEA focally in tumor cells lining pseudolumina. Cross-breeding Apc(Min/+) mice with neu transgenic mice did not reveal a synergistic effect on the kinetics of breast tumor formation. Finally, CEA transgenic mice crossbred with mice transgenic for the SV40 large T antigen regulated by the surfactant protein-C promoter, developed multiple lung adenocarcinomas that revealed a mosaic CEA expression pattern.

Down-regulation of carcinoembryonic antigen family member 2 expression is an early event in colorectal tumorigenesis.

Carcinoembryonic antigen gene family member 2 (CGM2), a member of the carcinoembryonic antigen (CEA) family, is expressed in normal colon and rectum but is down-regulated in colorectal adenocarcinomas. In situ hybridization studies demonstrate that CGM2 expression is limited to epithelial cells in the upper third of the crypts. Two other CEA family members, biliary glycoprotein (BGP) and nonspecific cross-reacting antigen (NCA), are similarly expressed, whereas CEA transcripts were found down to the base of the crypts but were less predominant in the upper region. Only low CGM2 and BGP mRNA levels were seen in colorectal tumors. CEA mRNA was expressed at an equivalent level in normal epithelia and in tumor cells, whereas NCA transcript levels were upregulated in tumor cells. Monoclonal antibodies that recognize the CGM2 protein reveal its presence on the apical membranes of epithelial cells in the upper third of the crypts but its absence from colorectal tumors, which do express the CEA and NCA-50/90 proteins. The newly cloned CGM2 3'-untranslated region was used to probe RNAs from adenomas, colorectal tumors at different stages of progression, and liver metastases of colorectal adenocarcinomas. This showed that CGM2 is already down-regulated in adenomas when compared to normal mucosae. The CGM2 expression pattern along with its sequence homology to BGP suggests a similar tumor suppressor function for CGM2.