3D-SSP analysis for amyloid brain PET imaging using 18F-florbetapir in patients with Alzheimer's dementia and mild cognitive impairment.

INTRODUCTION: The aim of this study is to use 3D-SSP and a population-comparable normal database to investigate the associations between amyloid deposition detected by 18Fflorbetapir PET and neurocognitive performance of participants with mild cognitive impairment (MCI) and Alzheimer's disease (AD). MATERIALS AND METHODS: 18F-florbetapir PET and 18F-FDG PET imaging was prospectively performed on 78 subjects (20 cognitively healthy controls [HC], 27 MCI patients, and 31 AD patients) within 6 weeks of their neurocognitive assessments. The PET datasets from 19 HCs were used to create an NBD. The 3D-SSP analysis and Z-score mapping of 18F-florbetapir accumulations in the brain were further staged based on their accumulation patterns. Global and regional standard uptake value ratios (SUVRs) of 18Fflorbetapir were calculated using the cerebellar cortex as the normalised region. The relationships between the 18Fflorbetapir PET results, the clinical diagnoses and Thai Mini- Mental State Examination (TMSE) scores were determined. RESULTS: There was high agreement between the visual assessment results and the semiquantitative analysis (κ = 0.793 and 0.845). The stages of amyloid deposition were consistent with neurocognitive status across participants. Significantly higher SUVRs were found in AD than MCI and HC. Visual assessment and stage were not significantly correlated with TMSE scores. A significant negative correlation between the SUVRs and TMSE scores was partially demonstrated in MCI and AD, but not HC. CONCLUSIONS: 3D-SSP analysis of 18F-florbetapir PET provides special patterns and intensity of beta amyloid accumulation semi-quantitatively that are associated with the diagnosis and neurocognitive performances in MCI and AD patients.

Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase.

Autosomal recessive juvenile parkinsonism (AR-JP), one of the most common familial forms of Parkinson disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. We previously cloned PARK2, mutations of which cause AR-JP (ref. 2), but the function of the gene product, parkin, remains unknown. We report here that parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7, and that mutant parkins from AR-JP patients show loss of the ubiquitin-protein ligase activity. Our findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. Our findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis.

Positional cloning of the APECED gene.

Autoimmune polyglandular syndrome type I (APS 1, also called APECED) is an autosomal-recessive disorder that maps to human chromosome 21q22.3 between markers D21S49 and D21S171 by linkage studies. We have isolated a novel gene from this region, AIRE (autoimmune regulator), which encodes a protein containing motifs suggestive of a transcription factor including two zinc-finger (PHD-finger) motifs, a proline-rich region and three LXXLL motifs. Two mutations, a C-->T substitution that changes the Arg 257 (CGA) to a stop codon (TGA) and an A-->G substitution that changes the Lys 83 (AAG) to a Glu codon (GAG), were found in this novel gene in Swiss and Finnish APECED patients. The Arg257stop (R257X) is the predominant mutation in Finnish APECED patients, accounting for 10/12 alleles studied. These results indicate that this gene is responsible for the pathogenesis of APECED. The identification of the gene defective in APECED should facilitate the genetic diagnosis and potential treatment of the disease and further enhance our general understanding of the mechanisms underlying autoimmune diseases.

Insertion of beta-satellite repeats identifies a transmembrane protease causing both congenital and childhood onset autosomal recessive deafness.

Approximately 50% of childhood deafness is caused by mutations in specific genes. Autosomal recessive loci account for approximately 80% of nonsyndromic genetic deafness. Here we report the identification of a new transmembrane serine protease (TMPRSS3; also known as ECHOS1) expressed in many tissues, including fetal cochlea, which is mutated in the families used to describe both the DFNB10 and DFNB8 loci. An 8-bp deletion and insertion of 18 monomeric (approximately 68-bp) beta-satellite repeat units, normally present in tandem arrays of up to several hundred kilobases on the short arms of acrocentric chromosomes, causes congenital deafness (DFNB10). A mutation in a splice-acceptor site, resulting in a 4-bp insertion in the mRNA and a frameshift, was detected in childhood onset deafness (DFNB8). This is the first description of beta-satellite insertion into an active gene resulting in a pathogenic state, and the first description of a protease involved in hearing loss.

Identification of 11 novel mutations in USH2A among Japanese patients with Usher syndrome type 2.

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 2 (USH2) is the most common type of USH and is frequently caused by mutations in USH2A, which accounts for 74-90% of USH2 cases. This is the first study reporting the results of scanning for USH2A mutations in Japanese patients with USH2. In 8 of 10 unrelated patients, we identified 14 different mutations. Of these mutations, 11 were novel. Although the mutation spectrum that we identified differed from that for Caucasians, the incidence of mutations in USH2A was 80% for all patients tested, which is consistent with previous findings. Further, c.8559-2A>G was identified in four patients and accounted for 26.7% of mutated alleles; it is thus a frequent mutation in Japanese patients. Hence, mutation screening for c.8559-2A>G in USH2A may prove very effective for the early diagnosis of USH2.

Two independent mechanisms for escaping epidermal growth factor-mediated growth inhibition in epidermal growth factor receptor-hyperproducing human tumor cells.

Human squamous cell carcinoma cell lines often possess increased levels of epidermal growth factor (EGF) receptor. The growth of these EGF receptor-hyperproducing cells is usually inhibited by EGF. To investigate the mechanism of EGF-mediated inhibition of cell growth, variants displaying alternate responses to EGF were isolated from two squamous cell carcinoma lines, NA and Ca9-22; these cell lines possess high numbers of the EGF receptor and an amplified EGF receptor (EGFR) gene. The variants were isolated from NA cells after several cycles of EGF treatment and they have acquired EGF-dependent growth. Scatchard plot analysis revealed a decreased level of EGF receptor in these ER variants as compared with parental NA cells. Southern blot analysis and RNA dot blot analysis demonstrated that the ER variants had lost the amplified EGFR gene. One variant isolated from Ca9-22 cells, CER-1, grew without being affected by EGF. CER-1 cells had higher numbers of EGF receptor than parental Ca9-22 but similar EGFR gene copy number. Flow cytometric analysis indicated an increase in ploidy and cell volume which may give rise to the increase in receptor number per cell. The EGF receptors on both Ca9-22 and CER-1 cells were autophosphorylated upon EGF exposure in a similar manner suggesting no obvious alteration in receptor tyrosine kinase. However, very efficient down-regulation of the EGF receptor occurred in CER-1 cells. These data suggest two independent mechanisms by which EGF receptor-hyperproducing cells escape EGF-mediated growth inhibition: one mechanism is common and involves the loss of the amplified EGFR genes, and another is novel and involves the efficient down-regulation of the cell-surface receptor.

Changes in cerebral metabolic activity in men undergoing androgen deprivation therapy for non-metastatic prostate cancer.

OBJECTIVE: Androgen deprivation therapy (ADT) is a common treatment option for men with biochemical relapse from prostate cancer. ADT is associated with changes in mood, cognition, and quality of life, and most recently with increased risk for Alzheimer's disease (AD). This study examined changes in brain metabolism using positron emission tomography (PET) in men undergoing intermittent ADT. METHODS: Nine men with prostate cancer and a rising PSA (biochemical recurrence) without evidence of metastases were treated with intermittent ADT consisting of 9 months of complete androgen blockade achieved with combined leuprolide acetate and flutamide. Patients underwent resting [Fuorine-18] fluorodeoxyglucose PET (18F-FDGPET) at baseline (before treatment) and again after 9 months of ADT. RESULTS: Whole-brain mapping analysis after 9 months of androgen deprivation compared to pretreatment baseline revealed decreased regional cerebral glucose metabolism in the cerebellum, posterior cingulate, and medial thalamus bilaterally. Associations of brain metabolism with measurements of cognition and mood while on androgen deprivation revealed positive correlations between the posterior cingulate, left inferior parietal lobule (BA40), and left mid temporal gyrus (BA39) and spatial reasoning and a negative correlation between left inferior parietal lobule and verbal memory. Several mood indices were negatively correlated with hypothalamus and brainstem. CONCLUSION: These findings suggest that complete androgen deprivation may result in changes in regional brain metabolism associated with variation in mood, verbal memory, and spatial performance. Brain regions that were impacted from ADT are similar and overlap with brain regions with metabolic decline found in early AD and diabetes, suggesting possible common mechanisms.

In vivo manganese MR imaging of calcium influx in spontaneous rat pituitary adenoma.

BACKGROUND AND PURPOSE: Rapid uptake of the calcium analog manganese (Mn2+) into spontaneous pituitary adenoma during MR imaging of aged rats generated the hypothesis that neuroendocrine tumors may have a corresponding increase in calcium influx required to trigger hormonal release. A goal of this study was to investigate the potential for clinical evaluation of pituitary adenoma by MR imaging combined with administration of Mn2+ (Mn-MR imaging). MATERIALS AND METHODS: Mn-MR imaging was used to characterize the dynamic calcium influx in normal aged rat pituitary gland as well as spontaneous pituitary adenoma. To confirm the validity of Mn2+ as a calcium analog, we inhibited Mn2+ uptake into the olfactory bulb and pituitary gland of normal rats by using the calcium channel blocker verapamil. Rats with adenomas received fluorodeoxyglucose-positron-emission tomography (FDG-PET) scanning for characterization of tumor metabolism. Mn2+ influx was characterized in cultured pituitary adenoma cells. RESULTS: Volume of interest analysis of the normal aged pituitary gland versus adenoma indicated faster and increased calcium influx in adenoma at 1, 3, 11, and 48 hours. Mn2+ uptake into the olfactory bulb and pituitary gland of normal rats was inhibited by calcium channel blockers and showed dose-dependent inhibition on dynamic MR imaging. FDG-PET indicated correlation between tumor energy metabolism and Mn2+ influx as well as tumor size. CONCLUSION: These results indicate that adenomas have increased activity-dependent calcium influx compared with normal aged pituitary glands, suggesting a potential for exploitation in the clinical work-up of pituitary and other neuroendocrine tumors by developing Mn-MR imaging for humans.

The KMDB/MutationView: a mutation database for human disease genes.

The KMDB/MutationView is a graphical database of mutations in human disease-causing genes and its current version consists of nine category-based sub-databases including diseases of eye, heart, ear, brain, cancer, syndrome, autoimmunity, muscle and blood. The KMDB/MutationView stores mutation data of 97 genes involved in 87 different disease and is accessible through http://mutview.dmb.med. keio.ac.jp.

Effects of long-term hormone therapy on cholinergic synaptic concentrations in healthy postmenopausal women.

Experimental evidence suggests that gonadal steroids regulate brain neurochemical systems associated with cognitive function, such as the cholinergic system. This study examines the effect of long-term postmenopausal hormone therapy on the brain concentrations of cholinergic synaptic terminals in women using single photon emission computed tomography and the radiotracer [(123)I]iodobenzovesamicol ([(123)I]IBVM). [(123)I]IBVM labels the vesicular acetylcholine transporter (VAChT) located in the presynaptic terminals of these neurons. Sixteen healthy women treated with hormone therapy since the menopause and 12 women not treated with hormones were studied. There were no significant differences in regional IBVM binding indexes between the 2 groups. The length of hormone replacement therapy correlated positively with VAChT binding indexes in multiple cortical areas (P < 0.05): frontal cortex (Spearman rank correlation: rho = 0.79), parietal cortex (rho = 0.62), temporal cortex (rho = 0.80), anterior cingulate (rho = 0.71), and posterior cingulate (rho = 0.63), but not in the basal ganglia (rho = 0.35; P = 0.2). An earlier onset of menopause in hormone-treated women was associated with higher VAChT indexes in the anterior cingulate (rho = -0.56; P = 0.02) and posterior cingulate (rho = -0.63; P = 0.01). The opposite was found in the posterior cingulate of women not treated with hormones (rho = 0.58; P = 0.04). Women treated with estrogen alone also showed higher VAChT indexes than women treated with estrogen and progestin in the posterior cingulate cortex (by Mann-Whitney U test: z = 2.42; P = 0.015). Although an overall effect of postmenopausal hormone therapy was not found, associations between an index of cortical cholinergic terminal concentrations and the length of hormonal replacement suggest that hormone therapy may influence the survival or plasticity of these cells in postmenopausal women. The data also suggest possible differential effects of estrogen and estrogen with progestin treatments in brain areas critical for cognitive processing.

Brain activation in PTSD in response to trauma-related stimuli.

BACKGROUND: Repetitive recall of traumatic memories and chronic intermittent hyperarousal are characteristic of posttraumatic stress disorder (PTSD). Hyperarousal and memory dysfunction implicates "limbic" brain regions, including the amygdaloid complex, hippocampal formation, and limbic cortex, such as the orbitofrontal and anterior cingulate areas. To investigate the neurobiologic role of these brain regions in PTSD, we measured regional cerebral blood flow in PTSD with single photon emission computerized tomography (SPECT) during a symptom provocation paradigm. METHODS: Fourteen Vietnam veterans with PTSD, 11 combat control subjects, and 14 normal control subjects were studied with [99mTc]HMPAO in two sessions 48 hours apart: one session after exposure to white noise and the other following exposure to combat sounds. Skin conductance, heart rate, and subjective experience were recorded at the time of the studies. RESULTS: Activation for all three groups occurred in the anterior cingulate/middle prefrontal gyrus. Activation in the region of the left amygdala/nucleus accumbens was found in PTSD patients only. Deactivation was found in all three groups in the left retrosplenial region. CONCLUSIONS: These findings implicate regions of the "limbic" brain, which may mediate the response to aversive stimuli in healthy individuals and in patients suffering from PTSD.

Regional cerebral blood flow effects of nicotine in overnight abstinent smokers.

BACKGROUND: Most people agree that dependence to tobacco is mediated by the effects of nicotine on the central nervous system, albeit the neural pathways involved are not clearly delineated. We investigated the effect of nasal nicotine spray on regional cerebral blood flow (rCBF) in a sample of habitual smokers, with H2 15O and positron emission tomography (PET). METHODS: Eighteen volunteer smokers were studied after 12 hours of smoking deprivation. Regional cerebral blood flow measures were obtained with PET and 50 mCi H2 15O in six consecutive scans. Nicotine spray and a placebo spray were administered in a single-blind design, preceded and followed by baseline studies. Images were coregistered and anatomically standardized. Square (9-mm side) regions of interest were placed in 10 preselected brain regions, bilaterally. The effects of the experimental condition and gender were tested with two-way repeated-measures analysis of variance in each of the regions studied. RESULTS: Nicotine reduced rCBF in the left anterior temporal cortex and in the right amygdala. Increases were noted in the right anterior thalamus. CONCLUSIONS: In habitual smokers after overnight abstinence, nicotine induced differing effects on regional blood flow relative to whole brain blood flow. Increases were observed in the thalamus, a region rich in nicotinic receptors, and reductions in limbic and paralimbic (amygdala, anterior temporal cortex) regions.

Stereotaxic summation analysis of human cerebral benzodiazepine binding maps.

Summation analysis strategies are recognized throughout diverse scientific fields as powerful means of differentially enhancing experimental signals over random fluctuations (noise). Such techniques, applied to emission tomographic cerebral blood flow scans, reveal subtle alterations in neuronal activity during specific behavioral states. In the present work, we extend the principles of intersubject image summation analysis to the evaluation of emission tomographic ligand-binding studies. A general methodology is presented that may be applied to a wide variety of binding site determinations. The procedure consists of anatomic standardization of individual brains to a common stereotaxic orientation, followed by statistical analyses of group versus group or individual versus group differences. We develop and evaluate performance of our technique with the use of positron emission tomographic [11C]flumazenil scans from normal volunteers, depicting the regional cerebral distribution of benzodiazepine binding sites.

Localization of 16 exons to a 450-kb region involved in the autoimmune polyglandular disease type I (APECED) on human chromosome 21q22.3.

As a step toward identifying the pathogenic genes for autoimmune polyglandular disease type I (APECED) and other disorders mapped to the PFKL locus on chromosome 21q22.3, we have constructed a cosmid/BAC (bacterial artificial chromosome) contig of 450 kb covering markers D21S1460-D21S25-PFKL-D21S154 and performed exon trapping. We isolated 22 distinct exons including 6 exons derived from two known genes (PFKL and EHOC-1). Among 16 novel exons, 2 exons matched with human expressed sequence tags (EST) and 7 exons showed homology at predicted amino acid sequence level with proteins from other species. These 16 exons were mapped back to the cosmid contigs, 12 of which were confirmed for their expression by polymerase chain reaction (PCR) screening of human cDNA libraries of various tissues. These exon sequences and a transcript map will aid for isolation of corresponding genes which will be identified as candidate genes involved in the pathogenesis of disorders mapped to the 21q22.3 region.

Cloning of the cDNA encoding human xanthine dehydrogenase (oxidase): structural analysis of the protein and chromosomal location of the gene.

The primary structure of human xanthine dehydrogenase (hXDH) was determined by cloning and sequence analysis of the cDNAs encoding the enzyme. The nucleotide (nt) sequence has an open reading frame of 3999 nt encoding a protein of 1333 amino acids (aa) with a calculated M(r) of 146,604. The deduced aa sequence of hXDH is homologous to the previously reported rat XDH (rXDH) and Drosophila melanogaster XDH sequences with identities of 90.2 and 52.0%, respectively. The aa residues involved in both the reversible and the irreversible conversion from the dehydrogenase type to the oxidase type of rXDH are completely conserved between the rat and the human enzymes. This implies that the molecular mechanisms of the conversion of hXDH from dehydrogenase to oxidase are common to those of the well-characterized rXDH. Five sequence variations were detected in the isolated cDNA clones. Spot blot hybridization using flow-sorted human chromosome revealed that the hXDH-encoding gene (hXDH) was located on chromosome 2.

Structure, expression and chromosomal localization of the gene encoding human 2',3'-cyclic-nucleotide 3'-phosphodiesterase.

Four human genomic DNA clones for 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) were isolated by screening a human genomic library with human CNP cDNA clones as probes. Restriction mapping and sequence analysis revealed that the human CNP gene is about 8.5 kb long and composed of four exons interrupted by three introns. There are two transcription start points and in human brain, two forms of CNP mRNA are produced from a single gene by alternative splicing, similar to mouse. A homology search of the 5'-flanking regions of exon 0 and exon 1 in the human CNP gene indicated the presence of oligodendroglia-specific elements and myelin basic protein transcription element (MBTE) motif, in addition to TATA-box-like sequences. Spot blot hybridization of flow-sorted human chromosomes with the 3'-noncoding region of the human CNP cDNA showed the localization of CNP to chromosome 17.

Molecular mechanisms of human single-minded 2 (SIM2) gene expression: identification of a promoter site in the SIM2 genomic sequence.

We previously postulated that the single-minded 2 (SIM2) gene identified on the human chromosome 21q22.2 is a good candidate gene for the pathogenesis of mental retardation in Down syndrome because its mouse homolog exhibits preferential expression in the mouse diencephalon during early embryogenesis. We analyzed the genomic sequence of the entire SIM2 gene which consists of 11 exons and spans over 50 kb. As a step toward understanding the molecular mechanisms of SIM2 gene expression, we have analyzed the human SIM2 gene expression in nine established human cell lines. Three transcripts of 3.6, 4.4, and 6.0 kb were detected in the glioblastoma cell line, T98G, neuroblastoma cell line, TGW, and transformed embryonic kidney cell line, 293. The RACE analysis using SIM2-expressing human cell line T98G provided evidence for the transcription start site at approximately 1.2 kb upstream of the translation initiation site. The transfection assay using various deletion constructs with reporter gene suggested the presence of a presumptive promoter region. Transient transfection assay in T98G cell line revealed a significant promoter activity located in the 60 bp sequence between nt -1385 and -1325 upstream region of the translation initiation site. This 60 bp sequence contains cis-elements for c-myb, E47 and E2F transcription factors. Moreover, the gel retardation assay using oligo-DNA of various cis-element sequences indicated the presence of protein factor(s) which bind to the cis-element for c-myb. These results suggested that binding of a protein transcription factor(s) such as c-myb or that alike regulates transcription of the SIM2 gene by binding to a small upstream region.

Characterization and chromosomal mapping of a novel human gene, ANKHZN.

Ankhzn (ankyrin repeats hooked to a zinc finger motif) was originally isolated by means of the gene trap method, as a novel cytoplasmic protein on mouse embryonic stem cells. The Ankhzn protein is ubiquitously expressed in a spatiotemporal-specific manner and is located on endosomes. In the present study, we have cloned human ANKHZN cDNA by PCR using candidate EST clones exhibiting a high homology to mouse Ankhzn cDNA. The human ANKHZN cDNA encoded a 1166aa protein exhibiting 84.9% identity to the mouse one. The size of the transcript was found to be about 7kb on a Northern blot analysis, and ANKHZN mRNA was found to be ubiquitously expressed in human tissues on RT-PCR analysis. Western blot analysis showed that a 130kDa protein was detected at various levels in human tissues and also present in both membrane and soluble fractions obtained on subcellular fractionation. Human ANKHZN is a single copy gene consisting of predicted 25 exons in the human genome, and has been mapped to human chromosome 17p13 by radiation hybrid panel and fluorescence in-situ hybridization.

Human BAC library: construction and rapid screening.

We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.