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Age-related decrease of testosterone levels in blood and brain is believed to be associated with neurodegenerative diseases such as Alzheimer's disease. However, the effect of testosterone on brain function is not well understood. Therefore, we investigated the impact of testosterone deprivation on mitochondrial gene expression in the brain of male gonadectomized (GDX) rats. We found that peripheral castration led to testosterone deficiency in the brain and caused a significant reduction in protein and mRNA expression of genes encoded by mitochondrial DNA, namely NADPH dehydrogenase subunit 1, subunit 4, cytochrome b, and cytochrome c oxidase subunit 1 and subunit 3 in the hippocampus. In addition, gene expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), which is a master regulator of mitochondrial biogenesis, and its downstream transcriptional factors, nuclear respiratory factors 1 and 2 and mitochondrial transcription factors A and B2, were also decreased in the hippocampus of GDX rats. These reductions in the expression of mitochondrial gene and transcriptional coactivators and factors were recovered by androgen replacement. These findings indicate that androgen plays an important role in mitochondrial gene expression in the hippocampus.

Various clustering methods are used in cluster analyses, with each clustering method demonstrating unique advantages. Therefore, it is important to make the best use of the advantages each method provides. We have recognized that it is necessary in the evaluation of X-ray images to classify observers quantitatively according to visual characteristics (grouping of observers) and have clustered observers using the UPGMA method, which is one of the clustering methods. We found that the observers were clustered into two different groups, one with radiologist-like characteristics and the other with medical physicist-like characteristics. Furthermore, we suggested that the group with radiologist-like characteristics was suitable for QC of X-ray images. However, it is doubtful whether the UPGMA method is most suitable for the grouping of observers. In this work we clustered observers using various clustering methods and examined the most suitable method for the evaluation of X-ray images. The results showed that the ward method was least suitable for the grouping of observers, and they were distinctly grouped into two different categories by using a further method.

Exposure of PC12 cells to C(2)-ceramide results in dose- dependent apoptosis. Here, we investigate the involvement of death-associated protein (DAP) kinase, initially identified as a positive mediator of the interferon-gamma-induced apoptosis of HeLa cells, in the C(2)-ceramide-induced apoptosis of PC12 cells. DAP kinase is endogenously expressed in these cells. On exposure of PC12 cells to 30 microm C(2)-ceramide, both the total (assayed in the presence of Ca(2+)/calmodulin) and Ca(2+)/calmodulin-independent (assayed in the presence of EGTA) DAP kinase activities were transiently increased 5.0- and 12.2-fold, respectively, at 10 min, and then decreased to 1.7- and 3.4-fold at 90 min. After 10 min exposure to 30 microm C(2)-ceramide, the Ca(2+)/calmodulin independent activity/ total activity ratio increased from 0.22 to 0.60. These effects were dependent on the C(2)-ceramide concentration. C(8)-ceramide, another active ceramide analog, also induced apoptosis and activated DAP kinase, while C(2)-dihydroceramide, an inactive ceramide analog, failed to induce apoptosis and increase DAP kinase activity. Furthermore, transfection studies revealed that overexpression of wild-type DAP kinase enhanced the sensitivity to C(2)- and C(8)-ceramide, while a catalytically inactive DAP kinase mutant and a construct containing the death domain and C-terminal tail of DAP kinase, which act in a dominant-negative manner, rescued cells from C(2)-, and C(8)-ceramide-induced apoptosis. These findings demonstrate that DAP kinase is an important component of the apoptotic machinery involved in ceramide-induced apoptosis, and that the intrinsic DAP kinase activity is critical for ceramide-induced apoptosis.

Death-associated protein kinase (DAP-kinase) is Ca2+/calmodulin-dependent serine/threonine kinase that contains ankyrin repeats and the death domain. It has been isolated as a positive mediator of interferon-γ-induced apoptotic cell death of HeLa cells. In order to reveal the physiological role of DAP-kinase, the tissue distribution and developmental changes in mRNA expression of DAP-kinase were investigated by Northern blot and in situ hybridization analyses. DAP-kinase mRNA was predominantly expressed in brain and lung. In brain, DAP-kinase mRNA had already appeared at embryonic day 13 (E13) and was, thereafter, detected throughout the entire embryonic period. High levels of expression were detected in proliferative and postmitotic regions within cerebral cortex, hippocampus, and cerebellar Purkinje cells. These findings suggest that DAP-kinase may play an important role in neurogenesis where a physiological type of cell death takes place. The overall expression of DAP-kinase mRNA in the brain gradually declined at postnatal stages, and the expression became restricted to hippocampus, in which different expression patterns were observed among rostral, central, and caudal coronal sections, suggesting that DAP-kinase may be implicated in some neuronal functions. Furthermore, it was found that the expression of DAP-kinase mRNA was increased prior to a certain cell death induced by transient forebrain ischemia, indicating a possible relationship between DAP-kinase and neuronal cell death. J. Neurosci. Res. 58:674–683, 1999. © 1999 Wiley-Liss, Inc.

Hepatocyte nuclear factor-6 (HNF-6) is a liver-enriched transcription factor that contains a single cut domain and a novel type of homeodomain. Here we have studied the developmental expression pattern of HNF-6 in the mouse.In situhybridization experiments showed that HNF-6 mRNA is detected in the liver at embryonic day (E) 9, at the onset of liver differentiation. HNF-6 mRNA disappeared transiently from the liver between E12.5 and E15. In transfection experiments HNF-6 stimulated the expression of HNF-4 and of HNF-3β, two transcription factors known to be involved in liver development and differentiation. HNF-6 was detected in the pancreas from E10.5 onward, where it was restricted to the exocrine cells. HNF-6 was also detected in the developing nervous system. Both the brain and the spinal cord started to express HNF-6 at E9–9.5 in postmitotic neuroblasts. Later on, HNF-6 was restricted to brain nuclei, to the retina, to the ventral horn of the spinal cord, and to dorsal root ganglia. Our observations that HNF-6 contributes to the control of the expression of transcription factors and is expressed at early stages of liver, pancreas, and neuronal differentiation suggest that HNF-6 regulates several developmental programs.

The catalytic domain of death-associated protein kinase (DAPK) has been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method with PEG 8000 and magnesium acetate as precipitants. Complexes with the inhibitor staurosporine and its analogue BDB402 were also crystallized in the presence of PEG 400 and PEG 8000, respectively. Diffraction data were collected to 2.4 A for the native catalytic domain, to 2.9 A for the staurosporine complex and to 2.7 A for the BDB402 complex. All three crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 77.992, b = 109.909, c = 50.063 A for the catalytic domain, a = 78.911, b = 113.162, c = 50.658 A for the staurosporine complex and a = 77.337, b = 108.869, c = 50.186 A for the BDB402 complex. In both complexes the inhibitor molecule was clearly assigned in the difference Fourier map calculated on the basis of the phases obtained from the structure of the catalytic domain.

It is important that the evaluation of X-ray images include the observer's visual characteristics. However, evaluations of X-ray images that include these characteristics are not performed because of the difficulty of quantitatively elucidating visual characteristics. In this study, we classified observers into groups (clusters) by the same criteria of visual decision, using cluster analysis (unweighted Pair-Group method using arithmetic averages), and evaluated X-ray images on the basis of this separation. Clinical application is also discussed. It was found that observer clustering caused a decrease in between-observer variation. Observers were grouped into two different categories: one with the characteristics of radiologists and the other with the characteristics of medical physicists. Our results indicated that the group with the characteristics of radiologists was suitable for the quality control (QC) of X-ray images.

Exposure of PC12 cells to C(2)-ceramide results in dose- dependent apoptosis. Here, we investigate the involvement of death-associated protein (DAP) kinase, initially identified as a positive mediator of the interferon-gamma-induced apoptosis of HeLa cells, in the C(2)-ceramide-induced apoptosis of PC12 cells. DAP kinase is endogenously expressed in these cells. On exposure of PC12 cells to 30 microm C(2)-ceramide, both the total (assayed in the presence of Ca(2+)/calmodulin) and Ca(2+)/calmodulin-independent (assayed in the presence of EGTA) DAP kinase activities were transiently increased 5.0- and 12.2-fold, respectively, at 10 min, and then decreased to 1.7- and 3.4-fold at 90 min. After 10 min exposure to 30 microm C(2)-ceramide, the Ca(2+)/calmodulin independent activity/ total activity ratio increased from 0.22 to 0.60. These effects were dependent on the C(2)-ceramide concentration. C(8)-ceramide, another active ceramide analog, also induced apoptosis and activated DAP kinase, while C(2)-dihydroceramide, an inactive ceramide analog, failed to induce apoptosis and increase DAP kinase activity. Furthermore, transfection studies revealed that overexpression of wild-type DAP kinase enhanced the sensitivity to C(2)- and C(8)-ceramide, while a catalytically inactive DAP kinase mutant and a construct containing the death domain and C-terminal tail of DAP kinase, which act in a dominant-negative manner, rescued cells from C(2)-, and C(8)-ceramide-induced apoptosis. These findings demonstrate that DAP kinase is an important component of the apoptotic machinery involved in ceramide-induced apoptosis, and that the intrinsic DAP kinase activity is critical for ceramide-induced apoptosis.

Lithiierung des optisch aktiven (R)-1-Ferrocenyl-2-propanamins (I) (optische Reinheit 80%) und anschliesende Umsetzung mit Chlorodiphenylphosphin fuhren zu den diastereomeren Phosphinen (II), die dunnschichtchromatographisch trennbar sind.

A pair of diastereoisomeric phosphines, (R)-N,N-dimethyl-1-[2-(diphenylphosphino) ferrocenyl]-2-propanamines, have been prepared and used as ligands for nickel-catalyzed asymmetric cross-coupling of 1-phenylethylmagnesium chloride with vinyl bromide to give 3-phenyl-1-butene of 14–15% enantiomeric excess.