Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species. In mammalian cells, there exist proteins specifically localize in lipid droplets. However, the protein profile in the droplet remains yet to be clarified. In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques. Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction. The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11). Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins. When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction. In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP. These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction.

1 Antihistamine induced cognitive decline was evaluated using positron emission tomography (PET) measurement of histamine H1 receptor (H1R) occupancy and regional cerebral blood¯ow (rCBF). 2 Cognitive performance in attention-demanding task deteriorated dose-dependently and the e ects were statistically signi®cant after the treatment of 2 mg of d-chlorpheniramine. There was no signi®cant change in subjective sleepiness in the same dose. 3 The regional blockade of H1R was observed mainly in the frontal, temporal and anterior cingulate cortices, and the intravenous administration of d-chlorpheniramine as a therapeutic dose (2 mg) blocked over 60% of H1R in the frontal cortices. 4 The results from activation study using visual discrimination tasks demonstrated that enhanced activity in the right prefrontal and anterior cingulate cortices as well as a decreased activity in the left temporal and frontal cortices and midbrain after the treatment of d-chlorpheniramine. 5 There were no changes in global CBF for the subjects treated with 2 mg d-chlorpheniramine (pre; 44.8+3.3 ml dl 71 min 71 vs post; 44.4+4.7 ml dl 71 min 71 ). 6 The results indicated that the attention system of human brain could be altered by therapeutic doses of H1R antagonists. These ®ndings provide the information as to the potential risk of antihistamines in our daily activities.

We describe a method for simultaneous identification and quantitation of proteins within complex mixtures. The method consists of 18O-labeling, a simple stable isotope-coding that requires merely enzymatic digestion in 18O-water, in combination with a capillary-liquid chromatography electrospray ion-trap mass spectrometer. In a separate experiment using the same sample and a spike test, we demonstrate that the difference ration was calculated accurately using the 18O-labeling method even if the protein was part of a complex mixture. Our data also suggest that the accuracy of the quantitation can be improved by averaging the difference ratios of several peptides. In comparing our method with the isotope-coded affinity tag (ICAT) method, we show that the 18O-labeling method has the advantages of better recovery and fewer isotope effects. Therefore, the 18O-labeling method is a powerful tool for large-scale proteomics applications.

Intracellular amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). Mitochondria were found to be the target both for amyloid precursor protein (APP) that accumulates in the mitochondrial import channels and for Aβ that interacts with several proteins inside mitochondria and leads to mitochondrial dysfunction. Here, we have studied the role of mitochondrial γ-secretase in processing different substrates. We found that a significant proportion of APP is associated with mitochondria in cultured cells and that γ-secretase cleaves the shedded C-terminal part of APP identified as C83 associated with the outer membrane of mitochondria (OMM). Moreover, we have established the topology of the C83 in the OMM and found the APP intracellular domain (AICD) to be located inside mitochondria. Our data show for the first time that APP is a substrate for the mitochondrial γ-secretase and that AICD is produced inside mitochondria. Thus, we provide a mechanistic view of the mitochondria-associated APP metabolism where AICD, P3 peptide and potentially Aβ are produced locally and may contribute to mitochondrial dysfunction in AD.

Sixty‐two patients with schizophrenia and 96 normal controls were investigated for genetic association with restriction fragment length polymorphisms (RFLPs) in the serotonin receptor genes. A positive association between the serotonin 2A receptor gene (HTR2A) and schizophrenia was found, but not between schizophrenia and the serotonin 1A receptor gene. The positive association we report here would suggest that the DNA region with susceptibility to schizophrenia lies in the HTR2A on the long arm of chromosome 13. © 1996 Wiley‐Liss, Inc.

Abstract. The acrosomal process of Limulus sperm is an 80-~m long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin illaments at 13-/~ resolution shows that scruin is organized into tv~ equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organiTation of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Proteasesensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOHterminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an ,,~50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four/~-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin.

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Aims/hypothesis: Metformin is widely used as a hypoglycaemic reagent for type 2 diabetes. While the reduction of hepatic gluconeogenesis is thought to be a key effect, the detailed molecular mechanism of action of metformin remains to be elucidated. To gain insight into this, we performed a global gene expression profiling study. Materials and methods: We performed DNA microarray analysis to study global gene expression in the livers of obese diabetic db/db mice 2 h after a single administration of metformin (400 mg/kg). Results: This analysis identified 14 genes that showed at least a 1.5-fold difference in expression following metformin treatment, including a reduction of glucose-6-phosphatase gene expression. The mRNA levels of glucose-6-phosphatase showed one of the best correlations with blood glucose levels among 12,000 genes. Enzymatic activity of glucose-6-phosphatase was also reduced in metformin-treated liver. Moreover, intensive analysis of the expression profile revealed that metformin effected significant alterations in gene expression across at least ten metabolic pathways, including those involved in glycolysis-gluconeogenesis, fatty acid metabolism and amino acid metabolism. Conclusions/interpretation: These results suggest that reduction of glucose-6-phosphatase activity, as well as suppression of mRNA expression levels of this gene, in liver is of prime importance for controlling blood glucose levels in vivo, at least at early time points after metformin treatment. Our results also suggest that metformin not only affects expression of specific genes, but also alters the expression level of multiple genes linked to the metabolic pathways involved in glucose and lipid metabolism in the liver.