Biomedical subjects
Publications and source records attributed to T Sassa.
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The central event in prion disease is thought to be conformational conversion of the cellular isoform of prion protein (PrP(C)) to the insoluble isoform PrP(Sc). We generated polyclonal and monoclonal antibodies by immunizing PrP(C)-null mice with native PrP(C). All seven monoclonal antibodies (mAbs) immunoprecipitated PrP(C), but they immunoprecipitated PrP(Sc) weakly or not at all, thereby indicating preferential reactivities to PrP(C) in solution. Immunoprecipitation using these mAbs revealed a marked loss of PrP(C) in brains at the terminal stage of illness. Histoblot analyses using these polyclonal antibodies in combination of pretreatment of blots dissociated PrP(C) and PrP(Sc) in situ and consistently demonstrated the decrease of PrP(C) at regions where PrP(Sc) accumulated. Interestingly, same mAbs showed immunohistochemical reactivities to abnormal isoforms. One group of mAbs showed reactivity to materials that accumulated in astrocytes, while the other group did so to amorphous plaques in neuropil. Epitope mapping indicated that single mAbs have reactivities to multiple epitopes, thus implying dual specificities. This suggests the importance of octarepeats as a part of PrP(C)-specific conformation. Our observations support the notion that loss of function of PrP(C) may partly underlie the pathogenesis of prion diseases. The conversion of PrP(C) to PrP(Sc) may involve multiple steps at different sites.
Lithium salts have been widely used in the treatment of mood disorders, but the mechanism of action is still not clear. In this work, a methodology for two-dimensional Lithium-7 imaging on clinical systems is presented. The data were acquired using a phosphorus volume head coil that was re-tuned for the Lithium-7 frequency. A spectroscopic sequence was used to acquire the free induction decay (FID) after volume excitation using a hard pulse. The results obtained on the head of patients undergoing lithium treatment (n = 7, 0.6 mEq/l average serum level) demonstrate that images of adequate signal to noise ratio (100:1) can be obtained in acceptable imaging times (55 min) using the proposed methodology. The distribution of 7Li appears uniform in the brains of the patients studied.
Fractionation guided by immunomodulatory activity of the EtOAc extract of the Ascomycete Eupenicillium crustaceum has afforded two new naturally occurring products, 4'-oxomacrophorin D (1) and 4'-oxomacrophorin A (2), as the immunosuppressive components of this fungus [1: 3-hydroxy-3-methylglutaryl (HMG) conjugate of 2]. The structures including the absolute configurations of 1 and 2 have been determined on the basis of chemical correlation of 1 with macrophorin D (3). The absolute configuration of the HMG moiety in 3 has been revised from 3R to 3S.
Cotylenin A, which has a diterpenoid tricarbocyclic skeleton, has been isolated as a plant growth regulator, has been shown to affect several physiological processes of higher plants and have differentiation-inducing activity in several myeloid leukaemia cell lines. We examined the effect of cotylenin A on the differentiation of leukaemic cells that were freshly isolated from acute myeloid leukaemia (AML) patients in primary culture. Cotylenin A significantly stimulated both functional and morphological differentiation of leukaemia cells in 9 out of 12 cases. This differentiation-inducing activity was more potent than those of all-trans retinoic acid and 1alpha,25-dihydroxyvitamin D3 (VD3). Treatment with a combination of cotylenin A and VD3 was more effective than cotylenin A or VD3 alone at inducing the monocytic differentiation of AML cells.
Recent studies of the brain using magnetic resonance imaging (MRI) have suggested progressive structural changes in schizophrenics. However, those studies were conducted over periods of less than 5 years and thus lacked sufficient capacity to determine the course and nature of this process. In this study, MRI scans were obtained in 15 schizophrenics and 12 controls at baseline and after 4- and 10-year follow ups. Volumes of the lateral ventricles were measured. Patients were assessed by the Brief Psychiatric Rating Scale (BPRS) at the same two time points: at baseline and at 10-year follow up. After 10 years, a significant lateral ventricular enlargement was found in patients (mean percentage change: +22.9%) but not in controls (5.1%). Although our results are not in disagreement with the neurodevelopmental hypothesis, they do provide strong evidence that in schizophrenia progressive brain reduction occurs even in its chronic stage.
The drimane sesquiterpenes, (+)-albicanol (2) and (+)-albicanyl acetate (3), were synthesized from an optically active bicyclic diol [(+)-1] that had been obtained via the recently developed optical resolution of a general synthetic intermediate for drimane sesquiterpenes. The crucial step in the previous syntheses was markedly improved by the modified Wittig methylenation of a silyloxy ketone (7). The high overall yield (77% in 4 or 5 steps from (+)-1) by this total synthesis makes it possible to synthesize the other biologically active drimane sesquiterpenes.
The mass production of pure gibberellin A1 (GA1) by shake-culturing Phaeosphaeria sp. L487 was investigated. Its GA1 production was markedly influenced by natural nitrogen sources and NH4NO3. When the fungus was cultured in an 8% glucose-1.5% oatmeal-0.1% NH4NO3-0.5% KH2PO4-0.1% MgSO4 x 7H2O medium for 3 weeks, the amount of GA1 in the culture filtrate was up to ca. 200 microg/ml: the addition of safflower oil to the culture medium two weeks after inoculation prolonged the GA1-production period to produce 300 microg/ml. Further preparation of [U-13C]GA, as a tool for the analysis of a complex of GA1 and its binding protein was attempted by using the fungus. The fungal culture in a [U-13C]glucose-oatmeal medium gave 6 mg of crystalline 13C-enriched GA1. Its 13C-enrichment of ca. 75% and 1J(CC) values were determined by NMR spectrometry.
We report here kinetic analysis and identification of the two cyclase domains in a bifunctional diterpene cyclase, Phaeosphaeria ent-kaurene synthase (FCPS/KS). Kinetics of a recombinant FCPS/KS protein indicated that the affinity for copalyl diphosphate is higher than that for geranylgeranyl diphosphate (GGDP). ent-Kaurene production from GGDP by FCPS/KS was enhanced by the addition of a plant ent-kaurene synthase (KS) but not by plant CDP synthase (CPS), suggesting that the rate of ent-kaurene production of FCPS/KS may be limited by the KS activity. Site-directed mutagenesis of aspartate-rich motifs in FCPS/KS indicated that the (318)DVDD motif near the N terminus and the (656)DEFFE motif near the C terminus may be part of the active site for the CPS and KS reactions, respectively. The other aspartate-rich (132)DDVLD motif near the N terminus is thought to be involved in both reactions. Functional analysis of the N- and C-terminal truncated mutants revealed that a N-terminal 59-kDa polypeptide catalyzed the CPS reaction and a C-terminal 66-kDa polypeptide showed KS activity. A 101-kDa polypeptide lacking the first 43 amino acids of the N terminus reduced KS activity severely without CPS activity. These results indicate that there are two separate interacting domains in the 106-kDa polypeptide of FCPS/KS.
3-Carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF), a candidate for uremic toxin, was measured in human hair for examining a possible utility as indicator of renal dysfunction. The serum concentration of CMPF was much higher (32.3 +/- 2.7 microg/ml, n = 17; mean +/- SEM) in uremic patients aged 40-55 years receiving hemodialysis treatment than in healthy younger subjects (3.61 +/- 0.19 microg/ml, n = 22), aged 18-23 years. However, the hair concentration of CMPF tended to be lower in the patients (6.8 +/- 1.7 ng/10 mg hair) than in the healthy younger subjects (15.8 +/- 4.5 ng/10 mg) and was significantly lower than that in the healthy age-matched subjects (22.4 +/- 5.3 ng/10 mg, n = 12), aged 40-47 years. Since CMPF was measurable in the sweat (4.4 +/- 3.7 ng/mg) collected from six out of seven healthy subjects examined, it was suggested that the contribution of sweat to the measurement of CMPF in hair was considerable. The fact that the uremic patients undergoing hemodialysis therapy had less sweat than healthy subjects may explain the lower concentration of CMPF in the patients' hair. The pathophysiological roles of CMPF in the body were attempted to be explored by using excised guinea pig organs, and human platelets and neutrophils. CMPF showed no remarkable effects in the concentration range of < or =10(-4) M except for only slight suppression of spontaneous contracture of guinea pig tenia coli at 10(-4) M. As far as the organs and tissues examined in the present study are concerned, the biological activity of CMPF itself, if any, may be very weak. Precaution should be taken against the delivery of a substance through sweat to hair when a small amount of substance is attempted to be measured in hair by employing a sensitive analytical method.
Using positron emission tomography (PET) and [11C]N-methylspiperone (NMSP), we examined 5-HT2 receptors in the cortex of schizophrenic patients in whom we previously observed decreased prefrontal D1 receptor binding. The subjects were 10 neuroleptic-naive schizophrenic patients, 7 schizophrenic patients who were drug-free but had previously been treated with neuroleptics, and 12 normal controls. A non-significant trend towards decreased prefrontal [11C]NMSP binding was observed in the neuroleptic-treated patients, suggesting a possible effect of previous neuroleptic treatment on the alteration in cortical 5-HT2 function. However, the neuroleptic-naive patients showed no noticeable difference in cortical [11C]NMSP binding compared to controls. Our results do not rule out the role of 5-HT2 function as a crucial site of therapeutic activity of schizophrenia, but they do suggest that cortical 5-HT2 receptors might not be primarily involved in the pathophysiology of schizophrenia.
KKIAMRE is a serine/threonine protein kinase whose transcripts increase in the deep cerebellar nuclei of the rabbit after eyeblink conditioning, a model of associative learning and memory. We here characterized the expression, isoforms, and promoters of murine KKIAMRE gene. The expression of KKIAMRE was detected, by in situ hybridization and immunohistochemistry, in neurons in various brain regions including deep cerebellar nuclei. The gene spans approximately 40 kb and consists of 15 exons. Analysis of cDNA clones revealed multiple variants, having diversity in the putative carboxy-terminal regulatory domain, generated by alternative splicing and intraexonal termination. Furthermore, they had alternative 5' noncoding sequences. Primer extension, RNase protection, and transient expression assays revealed that two alternative promoters linked to distinct noncoding exons direct the expression of KKIAMRE. The gene was mapped on chromosomes 5 and 4 in mouse and human, respectively.
We report here the nucleotide sequence of a full-length cDNA encoding ent-kaurene synthase that was isolated by a reverse-transcription polymerase chain reaction from Gibberella fujikuroi (Gcps/ks). This cDNA encodes 952 amino acid residues with a relative molecular mass of 107 kDa. The sequence similarity between Gcps/ks and ent-kaurene synthase of the gibberellin A1-producing fungus, Phaeosphaeria sp. L487, is very high, suggesting that Gcps/ks is also a bifunctional diterpene cyclase. Its recombinant protein expressed in Escherichia coli converted geranylgeranyl diphosphate to copalyl diphosphate and ent-kaurene.
The Caenorhabditis elegans unc-13, unc-18, and unc-64 genes are required for normal synaptic transmission. The UNC-18 protein binds to the unc-64 gene product C. elegans syntaxin (Ce syntaxin). However, it is not clear how this protein complex is regulated. We show that UNC-13 transiently interacts with the UNC-18-Ce syntaxin complex, resulting in rapid displacement of UNC-18 from the complex. Genetic and biochemical evidence is presented that UNC-13 contributes to the modulation of the interaction between UNC-18 and Ce syntaxin.
Anticholinergic drug is often used to treat extrapyramidal symptoms. We measured muscarinic cholinergic receptor (mAchR) occupancy by the oral administration of biperiden in eight healthy subjects using positron emission tomography (PET) and [11C]N-methyl-4-piperidylbenzilate (NMPB). After the baseline scan each subject underwent one or two post-dose PET scans. mAchR occupancy was 10-45% in the frontal cortex three hours after the oral administration of 4 mg of biperiden. The occupancy correlated with the plasma concentration of biperiden in a curvilinear manner.
BACKGROUND/AIMS: Blood concentration levels of alpha-fetoprotein like the Lens culinaris agglutinin-reactive fraction (AFP-L3) are a useful marker for predicting the long-term prognosis of hepatocellular carcinoma. This study investigated the relationship between serum AFP-L3 and various imaging modalities. METHODS: Sixty-three patients with small hepatocellular carcinomas < or = 2 cm in diameter were studied. Serum AFP-L3 concentrations were measured by lectin-affinity electrophoresis coupled with antibody-affinity blotting and expressed as % AFP-L3 (the percent of AFP-L3 as total AFP). A clinical "cutoff level" of 10% was used in this study to indicate the presence of hepatocellular carcinoma. Selective hepatic intraarterial digital subtraction angiography (DSA), ultrasonographic angiography with carbon dioxide microbubbles (USAG), and computed tomography during arterial portography (CTAP) were performed to evaluate the hemodynamics of hepatic nodules. RESULTS: Fourteen (22.2%) of the 63 patients were positive for % AFP-L3. The % AFP-L3 levels (n=45, 4.4%) of patients with hypervascular tumors were significantly higher than those (n=15, 0.0%) of patients with isovascular or hypovascular tumors as determined by USAG (p=0.0061). The % AFP-L3 levels (n=53, 4.4%) of patients with a negative portal blood supply were significantly higher than the % AFP-L3 levels (n=7, 0.0%) of patients with a positive portal blood supply as determined by CTAP (p=0.0140). The % AFP-L3 levels of patients with tumors with a long doubling time (DT) were significantly lower than for patients with tumors with a short DT (p=0.0176). CONCLUSION: AFP-L3 is a positive indicator which may be more specific for small advanced hepatocellular carcinoma.
OBJECTIVE: To evaluate the diagnostic efficacy of simultaneous measurements of high-sensitivity des-gamma-carboxy prothrombin (H-DCP) and Lens culinaris agglutinin A-reactive alpha-fetoprotein (AFP-L3) in small hepatocellular carcinoma (HCC). METHODS: Sixty-one patients with small HCCs < or = 2 cm in diameter and 134 controls (chronic hepatitis: 59 cases; cirrhosis: 75 cases) were examined. H-DCP was measured by electrochemiluminescence immunoassay (cut-off 40 mAU/ml (milli-arbitrary units/ml) and AFP-L3% (percentage of AFP-L3/total AFP) by lectin-affinity electrophoresis coupled with the antibody-affinity blotting method (cut-off 10%). Fifty-six patients were histologically diagnosed and the remaining five patients were diagnosed clinically. RESULTS: Of 61 patients, 27 (44.3%) were positive for H-DCP and 14 (23.0%) were positive for AFP-L3. There was no correlation between H-DCP and AFP-L3%. Nineteen patients (31.1%) had positive H-DCP alone. Six patients (9.8%) had positive AFP-L3 alone, and in eight patients (13.1%) both markers were positive. In combination assay, 33 of 61 patients (54.1%) were positive for either marker; specificity and accuracy were 97.8% and 84.1%, respectively. There was a tendency for the AFP-L3% to be elevated in patients with moderately or poorly differentiated HCC (P= 0.0564) and multiple HCC nodules (P= 0.0316), while the H-DCP showed no elevation related to the tumour type. CONCLUSION: The detection rate of small HCC was improved by combination assay with H-DCP and AFP-L3%. Our results indicate that the markers are complementary and useful for the diagnosis and evaluation of small HCC when measured simultaneously.