PET examination of [11C]NNC 687 and [11C]NNC 756 as new radioligands for the D1-dopamine receptor

The benzazepines NNC 687 and NNC 756 have in animal studies been described as selective D₁-dopamine receptor antagonists. Both compounds have been labeled with¹¹C for examination by positron emission tomography (PET). In the present study central receptor binding was studied in monkeys and healthy men. After IV injection of both radioligands in Cynomolgus monkeys radioactivity accumulated markedly in the striatum, a region with a high density of D₁-dopamine receptors. This striatal uptake was displaced by high doses of the selective D₁-antagonist SCH 23390 (2 mg/kg) but not by the 5HT₂-antagonist ketanserin (1.5 mg/kg) or the selective D₂-antagonist raclopride (3 mg/kg). The cortical uptake after injection of [¹¹C]NNC 687 was not reduced in displacement experiments with ketanserin. The cortical uptake of [¹¹C]NNC 756 was reduced in displacement and protection experiments with ketanserin by 24–28% (1.5 mg/kg), whereas no reduction could be demonstrated on striatal uptake. In healthy males both compounds accumulated markedly in the striatum. For [¹¹C]NNC 687 the ratio of radioactivity in the putamen to cerebellum was about 1.5. For [¹¹C]NNC 756 the ratio was about 5. This ratio of 5 for [¹¹C]NNC 756 is the highest obtained so far for PET radioligands for the D₁-dopamine receptor.     

The ubiquitin ligase Rnf6 regulates local LIM kinase 1 levels in axonal growth cones

LIM kinase 1 (LIMK1) controls important cellular functions such as morphogenesis, cell motility, tumor cell metastasis, development of neuronal projections, and growth cone actin dynamics. We have investigated the role of the RING finger protein Rnf6 during neuronal development and detected high Rnf6 protein levels in developing axonal projections of motor and DRG neurons during mouse embryogenesis as well as cultured hippocampal neurons. RNAi-mediated knock-down experiments in primary hippocampal neurons identified Rnf6 as a regulator of axon outgrowth. Consistent with a role in axonal growth, we found that Rnf6 binds to, polyubiquitinates, and targets LIMK1 for proteasomal degradation in growth cones of primary hippocampal neurons. Rnf6 is functionally linked to LIMK1 during the development of axons, as the changes in axon outgrowth induced by up- or down-regulation of Rnf6 levels can be restored by modulation of LIMK1 expression. Thus, these results assign a specific role for Rnf6 in the control of cellular LIMK1 concentrations and indicate a new function for the ubiquitin/proteasome system in regulating local growth cone actin dynamics.     

Evidence that tissue mast cells derive from mononuclear phagocytes

A new method has been developed that allows in vitro differentiation of rat mast cells from precursor cells in the absence of feeder layers. In the present investigation, the precursor cells are further characterized as to their nature, state of activation and distribution in different organs of the rat. The data support our previously published evidence, based on morphology and enzyme-cytochemical reaction patterns that the precursor cells are mononuclear phagocytes. Peritoneal macrophages elicited by mineral oil, thioglycollate or proteose-peptone have lost the ability to differentiate into mast cells. Single cell suspensions of spleen and bone marrow contain mast cell precursors, while cells from mesenteric lymph nodes and thymus or purified lymphocytes never yield mast cells in this culture system.     

Activin controls skin morphogenesis and wound repair predominantly via stromal cells and in a concentration-dependent manner via keratinocytes

The transforming growth factor-beta family member activin is a potent regulator of skin morphogenesis and repair. Transgenic mice overexpressing activin in keratinocytes display epidermal hyper-thickening and dermal fibrosis in normal skin and enhanced granulation tissue formation after wounding. Mice overexpressing the secreted activin antagonist follistatin, however, have the opposite wound-healing phenotype. To determine whether activin affects skin morphogenesis and repair via activation of keratinocytes and/or stromal cells, we generated transgenic mice expressing a dominant-negative activin receptor IB mutant (dnActRIB) in keratinocytes. The architecture of adult skin was unaltered in these mice, but delays were observed in postnatal pelage hair follicle morphogenesis and in the first catagen-telogen transformation of hair follicles. Although dnActRIB-transgenic mice showed slightly delayed wound re-epithelialization after skin injury, the strong inhibition of granulation tissue formation seen in follistatin-transgenic mice was not observed. Therefore, although endogenous activin appeared to affect skin morphogenesis and repair predominantly via stromal cells, overexpressed activin strongly affected the epidermis. The epidermal phenotype of activin-overexpressing mice was partially rescued by breeding these animals with dnActRIB-transgenic mice. These results demonstrate that activin affects both stromal cells and keratinocytes in normal and wounded skin and that the effect on keratinocytes is dose-dependent in vivo.     

Immunocytochemical mapping of IgE/IgG binding sites within the birch pollen grain using serum from a patient undergoing hyposensitization therapy. Comparison with biochemically determined data

Serum from an atopic patient undergoing a hyposensitization therapy to birch pollen allergens was used to carry out immunocytochemical mapping of specific IgE/IgG binding sites within ultrathin sections of birch pollen grains. There was a distinct rise in the density of specific IgG labelling in the course of therapy, whereas the density of IgE labelling remained fairly constant. However, the patterns of IgG and IgE binding in the pollen grain did not completely coincide since there was only IgE binding to certain pollen structures such as the apertural region. If the widely accepted concept of specific antibodies as 'blocking antibodies' is taken as a basis, the success of therapy must be questioned in this case because no IgG antibodies were formed to some of the allergens localized in the pollen grain and relevant to this patient. Very probably this result must be attributed to an incomplete pollen extract used in hyposensitization therapy. The results of the biochemical measurements (RAST, ELISA) agreed well with the immunocytochemical observations.     

Predictive control of muscle responses to arm perturbations in cerebellar patients

OBJECTIVES: To examine changes in predictive control of early antagonist responses to limb perturbations in patients with defined lesions of the cerebellum. METHODS: Eight cerebellar patients and eight sex and age matched control subjects participated. Subjects held a handle that was rotated around the elbow joint. They were instructed to hold the forearm at 90 degrees flexion against a mechanical perturbation. Extensor torque (5 Nm) was applied for 140 ms (pulse), or for 1400 ms (step) through an external motor. Motor responses were tested under two different conditions of anticipatory information. In the expected condition, subjects anticipated and received a pulse. Under the unexpected condition, subjects expected steps, but received unexpected pulses. Biceps and triceps EMG as well as angular kinematics were compared between expected and unexpected pulse perturbations to quantify possible effects of prediction. RESULTS: In all healthy subjects, the degree of overshoot in the return flexion movement was significantly less in expected pulse perturbations compared with unexpected trials. The degree of amplitude reduction was significantly smaller in the patient group than in the control group (22.8% v 40.0%). During the expected trials, latency of peak triceps activity was on average 20% shorter in the control group, but 4% larger in the cerebellar patients. CONCLUSIONS: In the expected condition, controls achieved a significant reduction in angular amplitude by generating triceps activity earlier, whereas the ability to use prediction for adjusting early antagonist responses after limb perturbation was impaired in cerebellar patients.     

Reproducibly emitting reference materials for volatile and semi-volatile organic compounds—using finite element modeling for emission predictions

Recent research into emissions of (semi-)volatile organic compounds [(S)VOC] from solid materials has focused on the development of suitable reference materials for quality assurance/quality control of emission test chamber measurements, which fulfill requirements such as homogenous and reproducible (S)VOC release. The approach of this study was to find a method for preparation of a material with predictable (S)VOC emission rates. A VOC (styrene) and an SVOC (2,6-diisopropylnaphthalene, DIPN), loaded into either vacuum grease or a 1:1 mixture of paraffin/squalane, have been tested. For the prediction of the emission rates, a model using the finite element method (FEM) was created to simulate the (S)VOC emission profiles. Theoretical and experimental results obtained in a Micro-Chamber/Thermal Extractor (μ-CTE?) and in 24 L emission test chamber measurements were in good agreement. Further properties were investigated concerning the material applicability, such as shelf life and inter-laboratory comparability. The maximum relative standard deviation in the inter-laboratory study was found to be 20%.