Membrane fatty acid levels as a predictor of treatment response in chronic schizophrenia.

Abnormal fatty acid composition in neural membranes, that is, the balance between essential polyunsaturated fatty acids (EPUFAs) and saturated fatty acids, has been suggested to be related to the psychotic symptoms and cognitive impairment of schizophrenia. This study was conducted to test the hypothesis that the ability of atypical antipsychotic drugs to ameliorate positive symptoms and cognitive function relevant to daily living would be predicted by baseline EPUFAs concentrations in the erythrocyte membrane in subjects with schizophrenia. A total of 24 actively psychotic patients with schizophrenia participated in the study. After blood drawing, they were treated with olanzapine or perospirone. The Scale for the Assessment of Positive Symptoms (SAPS) and the Scale for Assessment of Negative symptoms (SANS), as well as the script tasks, a measure of event schema recognition, were administered at baseline and 3months after the start of treatment. Erythrocyte membrane fatty acid levels were analysed using a gas chromatography system. Scores of SAPS and SANS, as well as script task performance, were improved during treatment with either antipsychotic drug. Regression analysis indicates baseline EPUFAs concentrations were positively and negatively related with percent improvement of positive symptoms and script task performance, respectively. The results of this study suggest composition of phospholipids in the erythrocyte membrane provide a feasible marker to predict treatment response in patients with schizophrenia.

Effect of perospirone on P300 electrophysiological activity and social cognition in schizophrenia: a three-dimensional analysis with sloreta.

The purpose of this study was to determine if perospirone, a second generation antipsychotic drug and partial agonist at serotonin-5-HT(1A) receptors, enhances electrophysiological activity, such as event-related potentials (ERPs), in frontal brain regions, as well as cognitive function in subjects with schizophrenia. P300 current source images were obtained by means of standardized low resolution brain electromagnetic tomography (sLORETA) before and after treatment with perospirone for 6 months. Perospirone significantly increased P300 current source density in the left superior frontal gyrus, and improved positive symptoms and performance on the script tasks, a measure of verbal social cognition, while verbal learning memory tended to be improved. There was a significant correlation between the changes in P300 amplitude on the left frontal lead and those in social cognition. These results suggest the changes in three-dimensional distribution of cortical activity, as demonstrated by sLORETA, may mediate some of the actions of antipsychotic drugs. The distinct cognition-enhancing profile of perospirone in patients with schizophrenia may be related to its actions on 5-HT(1A) receptors.

Letrozole-, anastrozole-, and tamoxifen-responsive genes in MCF-7aro cells: a microarray approach.

Antiestrogens and aromatase inhibitors are important drugs in the treatment of estrogen-dependent breast cancer. To investigate the effects of these drugs on gene expression in breast cancer cells, we treated estrogen receptor-positive MCF-7 cells stably transfected with the aromatase gene (known as MCF-7aro cells) with testosterone, 17 beta-estradiol, two aromatase inhibitors (letrozole and anastrozole), and an antiestrogen (tamoxifen). We found that testosterone or 17 beta-estradiol induced the proliferation of MCF-7aro cells at a rate six times faster than the untreated cells. In addition, the testosterone-induced proliferation of MCF-7aro cells was effectively suppressed by letrozole, anastrozole, or tamoxifen. Microarray analyses on Affymetrix Human Genome U133A GeneChips (Affymetrix, Santa Clara, CA) were carried out using total RNA isolated from the control and treated cells. At the false discovery rate of 0.05 and a minimum fold-change criteria of 1.5, 104 genes were identified that were up-regulated and 109 genes were identified that were down-regulated by both androgen and estrogen. More than 50% of these hormone-regulated genes were counter-regulated by all three inhibitors and >90% were counter-regulated by at least one of the inhibitors. Comparing the effect of each inhibitor on gene expression, we observed that letrozole and anastrozole are more similar in terms of the genes they affect compared with treatment with tamoxifen. To validate the gene expression profiles identified from microarray analyses, the expression patterns of 13 representative genes were examined by Northern analysis. Finally, the genes identified as statistically significant were classified based on their expression patterns and biological function/pathways. The results of this study provide us with a better understanding of the actions of both aromatase inhibitors and antiestrogens at the molecular level. We believe that the results of this study serve as the first step in identifying unique expression patterns following drug treatment, and that this will ultimately be useful in customizing patient treatment strategies for hormone-dependent breast cancer.

Local estrogen formation by nontumoral, cirrhotic, and malignant human liver tissues and cells.

We have investigated the activity and expression of aromatase enzyme in nontumoral, cirrhotic, and malignant human liver tissues and cells using both chromatographic and reverse transcription (RT)-PCR analyses. After 24- and 72-h incubation of tissue minces or hepatic cell lines with either testosterone or androstenedione as androgen precursor, human hepatocellular carcinoma (HCC) tissues and HepG2 hepatoma cells showed elevated aromatase activity, with estrogen formation rates being 20 and >95%, respectively, as opposed to nontumoral hepatic tissues and nonmalignant Chang liver (CL) cells, where no aromatase activity could be detected. Cirrhotic samples exhibited intermediate enzyme activity. Notably, exposure of HepG2 cells to the aromatase inhibitor Letrozole resulted in a striking decrease of estrogen formation, which became virtually absent at a Letrozole dose of 0.4 nM. RT-PCR analysis revealed markedly lower aromatase mRNA in both CL cells and nontumoral liver tissues, as compared with HepG2 cells and HCC samples. Cirrhotic specimens displayed variable transcript levels, in turn comparable with those observed in nontumoral or HCC tissues. Exon-specific RT-PCR showed prominent expression of exon I.3A-containing message and exon I.4-containing message in CL and HepG2 cells, as in nontumoral and HCC tissues, respectively. The present evidence implies that locally elevated estrogen formation in malignant human liver tissues and cells may have a role in the development and/or maintenance of human HCC, eventually leading to develop alternative strategies for treatment of HCC patients using antiaromatase agents.

Growth inhibition of estrogen receptor-positive and aromatase-positive human breast cancer cells in monolayer and spheroid cultures by letrozole, anastrozole, and tamoxifen.

Two third-generation aromatase inhibitors, letrozole and anastrozole, and the antiestrogen tamoxifen, were compared for growth-inhibiting activity in two estrogen receptor (ER)-positive aromatase-overexpressing human breast cancer cell lines, MCF-7aro and T-47Daro. Inhibition of hormone (1 nM testosterone)-stimulated proliferation was evaluated in both monolayer cultures and in three-dimensional spheroid cultures. Letrozole and anastrozole were also compared for effectiveness of aromatase inhibition, and relative affinity for aromatase, under both monolayer and spheroid growth conditions. Letrozole was an effective inhibitor of MCF-7aro monolayer cell proliferation, with an estimated 50% inhibitory concentration (IC50) of 50-100 nM, whereas an IC50 was not reached with anastrozole at any concentration tested (100-500 nM). An IC50 of tamoxifen was 1000 nM. Proliferation of T-47Daro monolayer cells was more sensitive to inhibition by all three agents; as with MCF-7aro cells, letrozole was the most effective inhibitor. MCF-7aro spheroids were slightly less sensitive than monolayer cells proliferation-inhibiting effects of letrozole (IC50 about 200 nM), and there was no significant inhibition with 100-200 nM anastrozole or 200-1000 nM tamoxifen. Letrozole and anastrozole significantly inhibited T-47Daro spheroid cell proliferation, at 15-25 and 50 nM, respectively, consistent with the greater sensitivity of T-47Daro monolayer cells to inhibition of proliferation by these agents. Tamoxifen failed to significantly inhibit T-47Daro spheroid cell proliferation over a 100-500 nM concentration range. Determination of aromatase inhibition in monolayers of both cell lines by a direct-access microsomal assay and an intact-cell assay revealed that letrozole was more active than anastrozole in monolayers of both cell lines and in both assays. In MCF-7aro spheroids following cell lysis, only letrozole significantly inhibited aromatase activity, supporting the conclusion that letrozole binds stronger to aromatase than anastrozole does. Our results demonstrate that MCF-7aro and T-47Daro spheroids could be a suitable model for evaluation of growth-inhibitory effects of agents used in hormonal therapy of breast cancer.

Remodeling of Human Sperm Chromatin Mediated by Nucleoplasmin from Amphibian Eggs: (human sperm/chromatin remodeling/protamine removal/nucleoplasmin).

The molecular events associated with decondensation of human sperm nuclei were analyzed by incubating sperm with egg extracts from an amphibian, Bufo japonicus. Acid-urea-Triton polyacrylamide gel electrophoresis (AUT-PAGE) showed that the nuclear basic proteins of human sperm consist mainly of protamines (HPI, HPII) with minor amounts of nucleosomal histones. On incubation of lysolecithin (LC)- and dithiothreitol (DTT)-treated human sperm with the egg extract, the nuclei lost HPI and HPII within 15 min in association with extensive nuclear decondensation, and the acquirement of a whole set of nucleosomal histones. Incubation of LC-DTT-sperm with nucleoplasmin purified from Bufo eggs also induced nuclear decondensation and loss of protamines within 30 min. Native-PAGE and Western blot analyses of incubation medium indicated tight association of the released protamines to nucleoplasmin, strongly suggesting that protamines are removed from sperm nuclei not enzymatically but by their specific binding to nucleoplasmin. On incubation of LC-DTT-sperm with nucleoplasmin and exogenous nucleosomal core histones, micrococcal nuclease-protected DNA fragments were released, although their unit repeat length was slightly less than that of somatic nucleosomes. Thus remodeling of human sperm during fertilization can be mimicked under defined conditions with nucleoplasmin and exogenous histones.

Transcriptional regulation of aromatase expression in human breast tissue.

Aromatase (CYP19) is the estrogen synthetase that converts androgen to estrogen. The expression of aromatase in breast cancer cells and surrounding stromal cells is up regulated compared to non-cancerous cells. In situ estrogen synthesis is thought to stimulate breast cancer growth in both an autocrine and a paracrine manner. A complex mechanism is involved in the control of human aromatase expression, in that seven promoters have been identified and found to be utilized in a tissue-selective manner. Increased aromatase expression in breast tumors is, in part, attributed to changes in the transcriptional control of aromatase expression. While promoter I.4 is the main promoter that controls aromatase expression in non-cancer breast tissue, promoters II and I.3 are the dominant promoters that drive aromatase expression in breast cancer tissue. During the last several years, our laboratory performed a series of studies to examine the transcription regulatory mechanism of aromatase expression in breast cancer cells. We functionally characterized promoters II and I.3, and carried out DNase 1 footprinting analysis that identified two regulatory elements, S1 and CREaro. Using the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library, we found that four orphan/nuclear receptors, ERR alpha-1, EAR-2, COUP-TFI and RAR gamma, bind to the S1 element, and that CREB1, Snail (SnaH) and Slug proteins bind to the CREaro element. Studies from this and other laboratories have revealed that in cancer tissue versus normal tissue, several positive regulatory proteins (e.g. ERR alpha-1 and CREB1) are present at higher levels and several negative regulatory proteins (e.g. EAR-2, COUP-TFI, RAR gamma, Snail and Slug proteins) are present at lower levels. This may explain why the activity of promoters II and I.3 is up regulated in cancer tissue. An understanding of the molecular mechanisms of aromatase expression between non-cancerous and cancerous breast tissue, at the transcriptional level, may help in the design of a therapy based on the suppression of aromatase expression in breast cancer tissue.

LORETA analysis of three-dimensional distribution of δ band activity in schizophrenia: relation to negative symptoms.

We sought to determine if altered electroencephalography (EEG) activities, such as delta band activity, in specific brain regions are associated with psychotic symptoms. Data were obtained from 17 neuroleptic-naive patients with schizophrenia and age- and sex-matched 17 healthy control subjects. Low Resolution Brain Electromagnetic Tomography (LORETA) was used to generate current source density images of delta, theta, alpha, and beta activities. Localization of the difference in EEG activity between the two groups was assessed by voxel-by-voxel non-paired t-test of the LORETA images. Spearman's correlation coefficient was obtained to relate LORETA values of EEG current density in brain regions showing a significant between-group difference and psychopathology scores. Delta band activity, represented by LORETA current density, was greater for patients in the following areas; the left inferior temporal gyrus, right middle frontal gyrus, right superior frontal gyrus, right inferior frontal gyrus, and right parahippocampal gyrus. LORETA values for delta band activity in the above five brain regions were negatively correlated with negative, but not positive symptoms. The results of this study suggest the role for electrophysiological changes in some of the brain regions, e.g. prefrontal cortex, in the manifestation of negative symptoms.

Serotonin-1A receptor gene polymorphism and the ability of antipsychotic drugs to improve attention in schizophrenia.

INTRODUCTION: The purpose of this study was to determine if the functional single nucleotide polymorphisms of rs6259 C(-1019)G in the promoter region, which regulates serotonin 5-HT(1A) receptor transcription, affects the ability of antipsychotic drugs to improve attention in patients with schizophrenia. METHODS: Subjects were neuroleptic-free and meeting DSM-IV-TR criteria for schizophrenia. Psychopathology and attention were evaluated with the Scale for the Assessment of Positive Symptoms (SAPS) and the Scale for the Assessment of Negative Symptoms (SANS) at baseline and 3 months after treatment with atypical antipsychotic drugs (AAPDs). DNA was extracted from peripheral blood following standard procedures. Genotyping was performed with HS-Taq assay (LaboPass). RESULTS: Data were available from 30 subjects (male/female=19/11), in which 17 had the CC genotype, three had the GG genotype, and 10 were heterozygous. The 3-month treatment with AAPDs was associated with significant improvements in positive and negative symptoms, but not attention as measured by SANS-Attention subscale in the entire subject group. There were no significant differences in the degree of improvements of SAPS and SANS scores between the CC genotype group and the (C/G plus G/G) combined group. On the other hand, improvement of attention was significantly greater for the former group compared to the latter group (P<0.016), suggesting a detrimental influence of the G-allele. CONCLUSION: These results provide additional support to the role of 5-HT(1A) receptors in some of the cognitive disturbances of schizophrenia. Further studies with a larger number of subjects are warranted.