Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression.

Large scale sequencing of cDNAs provides a complementary approach to structural analysis of the human genome by generating expressed sequence tags (ESTs). We have initiated the large-scale sequencing of a 3'-directed cDNA library from the human liver cell line HepG2, that is a non-biased representation of the mRNA population. 982 random cDNA clones were sequenced yielding more than 270 kilobases. A significant portion of the identified genes encoded secretable proteins and components for protein-synthesis. The abundance of cDNA species varied from 2.2% to less than 0.004%. Fifty two percent of the mRNA were abundant species consisting of 173 genes and the rest were non-abundant, consisting of about 6,600 genes.

Regulation of PLAP-1 expression in periodontal ligament cells.

Periodontal-ligament-associated protein-1 (PLAP-1) is preferentially expressed in the periodontal ligament (PDL) and encodes a novel small leucine-rich repeat proteoglycan protein. PLAP-1 expression was induced during the course of cytodifferentiation of PDL cells into mineralized-tissue-forming cells in vitro, suggesting the possible involvement of PLAP-1 in the mineralization process of PDL cells. In this study, we hypothesized that PLAP-1 expression is regulated by mineralization-related cytokines in PDL cells. PLAP-1 expression was clearly down-regulated when the cytodifferentiation of PDL cells was reversibly inhibited by fibroblast growth factor-2 (FGF-2). In contrast, bone morphogenetic protein-2 (BMP-2) enhanced PLAP-1 expression. Up-regulation of PLAP-1 expression by BMP-2 was confirmed at the protein level when PDL cells were immunostained with anti-PLAP-1 polyclonal antibody. These results revealed the cytokine-mediated regulatory mechanisms of PLAP-1 expression and suggested that PLAP-1 expression may be associated with the process of cytodifferentiation of PDL cells.

Tissue-specific and non-tissue-specific heavy-chain isoforms of myosin in the brain as revealed by monoclonal antibodies.

Four types of monoclonal antibody (BM-1, BM-2, BM-3 and BM-4) each having distinctive tissue specificity were obtained by immunizing mice with purified bovine cerebrum myosin. Both BM-1 and BM-2 reacted most efficiently with cerebrum myosin and less efficiently with myosins from other limited nonmuscle tissues, the tissue specificity of BM-1 being much narrower than that of BM-2. BM-3 reacted more efficiently with several other nonmuscle myosins than with cerebellar or cerebral myosin. BM-4 recognized various nonmuscle and smooth muscle myosins with a nearly equal efficiency. Cerebral myosin as well a cerebellar myosin contained two or more electrophoretic variants of the heavy chains. BM-1 and BM-3 as well as BM-2 and BM-3 were found to recognize selectively these distinct heavy-chain isoforms. The antigenic sites of the three tissue-specific antibodies (BM-1, BM-2 and BM-3) were all localized near the head/tail junction of the myosin molecules, while that of non-tissue-specific antibody BM-4 was near the center of the tail. These and additional results indicate that mammalian brain tissues as well as several other nonmuscle tissues contain multiple heavy-chain isoforms of myosin, the levels of which differed considerably from one tissue to another.

Gene expression profiling of mouse postnatal cerebellar development.

Expression patterns of 1,869 genes were determined using adapter-tagged competitive PCR (ATAC-PCR) at 6 time points during mouse postnatal cerebellar development. The expression patterns were classified into 12 clusters that were further assembled into 3 groups by hierarchical cluster analysis. Among the 1,869 genes, 1,053 known genes were assigned to 90 functional categories. Statistically significant correlation between the clusters or groups of gene expression and the functional categories was ascertained. Genes involved in oncogenesis or protein synthesis were highly expressed during the earlier stages of development. Those responsible for brain functions such as neurotransmitter receptor and synapse components were more active during the later stages of development. Many other genes also showed expression patterns in accordance with literature information. The gene expression patterns and the inferred functions were in good agreement with anatomical as well as physiological observations made during the developmental process.

Eighteen novel human genes regionally mapped on chromosome 11.

Expression sequence tags (EST) obtained by sequencing a randomly primed cDNA library and gene signatures (GS) obtained by sequencing a 3'-directed cDNA library can identify genes that are active in the source cells. Eight ESTs and ten GSs which represent novel human genes, except for one GS, and which have been assigned to human chromosome 11 were used to select cosmids from a chromosome 11-specific cosmid library. These cosmids were regionally mapped using the fluorescence in situ hybridization technique.

Expression of a novel member of the ATP1G1/PLM/MAT8 family, phospholemman-like protein (PLP) gene, in the developmental process of mouse cerebellum.

We have identified a new member of the ATP1G1/PLM/MAT8 family, named phospholemman-like protein (PLP), from a mouse cerebellum cDNA library. The family consists of small transmembrane proteins that modulate the activities of some ion channels. The deduced amino acid sequence of PLP consists of 93 residues that contain the ATP1G1/PLM/MAT8 motif and a single transmembrane domain, and is most similar to the sequence of mouse phospholemman. In situ hybridization analysis showed that the PLP gene is highly expressed in cerebellar granule cells. PLP expression is elevated in the postnatal developing cerebellum. Thus, it may be implicated in the proliferation, differentiation, and axon elongation of granule cells as they mature and migrate to the internal granule layer.

The addition of 5'-coding information to a 3'-directed cDNA library improves analysis of gene expression.

Large-scale sequencing of a 3'-cDNA library permits one to analyse gene expression profiles in various tissues. However, many such sequences lack enough information about the encoded proteins. To overcome this problem, we tested a new library, consisting of a 3'-directed cDNA sequence fused to a to a 5' sequence of about 300 bp. Such 'joint molecules' of about 600 bp were amplified by PCR and directly sequenced. About 40% of these joint molecules included the 5' and 3' terminal portions of the mRNA, and most of the remaining clones contained the middle portion and 3' end of the mRNA. The upstream sequences contained sufficient information with which to search for similarity, ORFs, motifs and hydropathy, thus allowing the mRNAs to be categorized and their functions predicted. The rapid categorization of the cDNAs will help to sort those clones that merit further analysis.

Gene expression in mouse cerebellum during its development.

Genes expressed during the cerebellar development of the mouse were identified in 3'-directed cDNA libraries prepared from the postnatal day 4, day 12, and week 6 cerebellar tissues. Among about 5500 clones selected randomly from each library, there were approximately 3500 distinct species. A total of 7728 species were identified in the three libraries, 1346 of which were known genes in the GenBank, 3041 EST-matching genes, and 3341 new genes. Relative expression levels at the three postnatal stages were quantitated by adapter-tagged competitive PCR for 130 known genes that appeared six times or more in one of the libraries. Genes for ribosomal proteins and some cytoskeletal and nuclear proteins were abundantly expressed at the early stage, coincidently with extensive proliferation of granule cells as the major cerebellar component. Genes related to brain functions, including those for mitochondrial activities and some ion channel systems, were more active at a later stage when the majority of granule cells were engaged in axon extension and synapse formation or the cerebellum had reached maturity. Compared to these stage-specifically expressed genes, genes for transcriptional regulation, signal transduction, protein modification, and basic cellular functions, in general, were not abundantly expressed at any stage of development.

A human cDNA sequence homologue of bovine phosphatidylethanolamine-binding protein.

Sequencing of about 1000 3'-directed cDNA clones from the human HepG2 cell line revealed that about half of them represent transcripts of abundantly or moderately expressed genes, about 70% of which are novel. We identified one of these clones as encoding the human homologue of bovine phosphatidylethanolamine-binding protein.

Expression profile of active genes in human periodontal ligament and isolation of PLAP-1, a novel SLRP family gene.

Periodontal ligament (PDL) is one of the most important tissues in maintaining the homeostasis of tooth and tooth-supporting tissue, periodontium. In this study, we investigated the expression profile of active genes in the human PDL obtained by collecting sequences with a 3'-directed cDNA library, which faithfully represents the composition of the mRNA population. We succeeded in obtaining a total of 1752 cDNA sequences by sequencing randomly selected clones and identified a total of 1318 different species as gene signatures (GS) by their sequence identity, 344 of which were known genes in the GenBank, and 974 of which were new genes. The resulting expression profile showed that collagen type I and type III were the most abundant genes and that osteogenesis-related proteins, such as SPARC/osteonectin and osteoblast specific factor 2, were highly expressed. By comparing the expression profile of PDL with 44 profiles similarly obtained with unrelated human cell/tissue, nine novel genes, which are probably expressed specifically in PDL, were discovered. Among them, we cloned a full-length cDNA of GS5096, which is frequently expressed in freshly-isolated periodontal tissue. We found that it encodes a novel protein, which is a new member of the class I small leucine-rich repeat proteoglycan family, and designated it PLAP-1 (periodontal ligament associated protein-1). PLAP-1 mRNA expression was confirmed in in vitro-maintained PDL cells and was enhanced during the course of the cytodifferentiation of the PDL cells into mineralized tissue-forming cells such as osteoblasts and cementoblasts. These findings suggest the involvement of PLAP-1 in the mineralized matrix formation in PDL tissues.

A novel mutation of the KAL1 gene in monozygotic twins with Kallmann syndrome.

OBJECTIVE: Kallmann syndrome is defined by the association of hypogonadotropic hypogonadism and anosmia. The KAL1 gene is responsible for the X-linked form of Kallmann syndrome. In this study we describe monozygotic twins with Kallmann syndrome due to the same mutation in the KAL1 gene. DESIGN: We studied male monozygotic twins with Kallmann syndrome. METHODS: We analyzed the KAL1 gene using the PCR-direct sequencing method. The twins' mother was examined for the identified mutation. RESULTS: We identified a 14 bp deletion from codon 419 in exon 9 (Pro419del14) in both KAL1 genes of the twins. This was a novel mutation in the KAL1 gene and was responsible for Kallmann syndrome. As Pro419del14 was not detected in the mother of the twins, Pro419del14 was a germline mutation originating from them. These monozygotic twins showed different LH and FSH responses to LH-RH stimulation and different phenotypes such as complications, physiques and psychiatric characters. CONCLUSIONS: We report an identical KAL1 gene mutation in the monozygotic twins with Kallmann syndrome. As these monozygotic twins showed different phenotypes in some respects, we suggest that factors other than mutations in the KAL1gene affect the symptomatic features of Kallmann syndrome.

PCR-array gene expression profiling of hepatocellular carcinoma.

Many trials using DNA microarrays have been reported for various human malignancies, but an efficient molecular diagnostic system has yet to be established. Here, we adopted a high throughput quantitative PCR-array system based on adaptor-tagged competitive PCR (ATAC-PCR), as a novel technique for gene expression profiling of hepatocellular carcinoma (HCC). This PCR-array contained 3,072 genes derived from three different cDNA libraries, including 298 additional known genes suspected to be involved in hepatocarcinogenesis. Using this PCR-array with 20 pairs of liver tissues (20 HCC, 20 surrounding nontumor liver), we identified a total of 117 genes differing in expression levels in the two liver tissues. Hierarchical clustering analysis and principal component analysis with these genes revealed distinct gene expression patterns in the HBV-positive group and the HCV-positive groups. Among 117 genes, only 7 (GPAA1, TMEM9, FACL4, ADFP, MAWBP, PACE4, FOS) were common to both groups. In conclusion, this PCR-array analysis with an appropriate set of genes is considered useful for gene expression profiling of HCC, and we identified some genes which may play a common key role in hepatocarcinogenesis.

A study on how to increase the sudden infant death syndrome (SIDS) autopsy rate.

To clarify the real cause of death of sudden infant death syndrome (SIDS), it is the most urgent and important subject to increase the autopsy rate of SIDS. So, we make following three proposals. (1) SIDS must be reported to the police. (2) Autopsy of SIDS must be performed in the area where a medical examiner's system is established. (3) Medical examiner's system or similar system must be established in all big cities. (4) It is desirable to perform autopsy in the area having no medical examiner's system by judicial autopsy or pathological autopsy by pathologists having diploma in postmortem medical examination.

Comparative evaluation of diagnostic guidelines for sudden infant death syndrome (SIDS) in Japan.

It is a well-recognized fact among professionals that the diagnosis of sudden infant death syndrome (SIDS) involves difficult elements; a SIDS diagnosis is not uniform throughout Japan; and such a diagnosis is not made based on any internationally recognized definition. Faced with this situation, guidelines have been prepared and proposals have been made to standardize and improve the accuracy of SIDS diagnoses, viz. the following three can be cited: "guideline for diagnosis of SIDS" prepared by a Study Group of the Ministry of Health and Welfare; "case studies of SIDS" and a "guideline for its diagnosis" prepared by the Case Study Committee of Japan SIDS Research Society; and a "proposal on the principles of medico-legal pathology concerning SIDS", included in the research report supported by a Grant-in-Aid for Scientific Research from the Ministry of Education. In the current study, a comparison was made focusing on the discrepancies among these three documents. The major discrepancies among these three are: (1) handling of the patient's age (by months or years) in the diagnosis of SIDS; (2) dealing with those cases for which no autopsy has been conducted; (3) attitudes concerning whether sleeping in a prone posture is a cause for asphyxia and (4) opinions concerning the aspiration of vomited milk. It is anticipated that these discrepancies will invite confusion and affect judgments and recognition of SIDS-related cases that will be brought to court. It is essential that those involved with these three documents have an opportunity at the earliest time to discuss the matter and come to a uniform understanding.

Screening and determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry.

Benzodiazepines are one of the most widely prescribed drugs for the treatment of a wide spectrum of clinical disorders. They are used as anticonvulsants, anxiolytics, hypnotics or muscle relaxants with different duration of action. In this paper, a simple and sensitive method for the determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry (GC/MS) is described. The drugs spiked in whole blood were extracted with an Oasis HLB solid-phase extraction cartridge (Waters), which contains a copolymer designed to have a hydrophilic-lipophilic balance. GC/MS analysis was performed using a Shimadzu QP-5000 equipped with a BPX5 capillary column (15 mx0.32 mm I.D., film thickness 0.25 microm, SGE). Nineteen benzodiazepines and two thienodiazepines were well separated from each other on their SIM chromatograms and also on the TIC with the exception of oxazolam to cloxazolam separation. The blank extract from whole blood gave no peaks that interfered with all benzodiazepines and thienodiazepines on the chromatogram. The calibration curves for selected benzodiazepines with fludiazepam as an internal standard showed excellent linearity over the concentration range 5-500 ng/ml blood with a correlation coefficients of >0.995. The detection limits ranged from 0.2 to 20 ng/ml blood. The method is simple and sensitive for the determination of benzodiazepines in whole blood and seems to be useful in the practice of forensic science.

Research for improving the autopsy rate for infant death--medical economic assessment of the forensic autopsy system in Japan.

The rate at which autopsies are performed in Japan for cases of infant death is not adequate for diagnosing sudden infant death syndrome (SIDS). In Japan, it will be necessary to increase the autopsy rate at the time of infant deaths in order to improve the certainty of diagnosing SIDS and improving the accuracy of determining the cause of death with respect to infant death. The objective of this research is to provide basic documentation required for administrative implementation of this objective. In Japan, the Medical Examiner System and its related Approved Autopsy System are not deployed nationwide. The estimated budget in the case of deploying the Medical Examiner System nationwide for the purpose of improving the infant death autopsy rate is in excess of 5 trillion yen, and that in the case of deploying the Approved Autopsy System nationwide is estimated at roughly 130 million US dollars. However, since the rate of autopsies performed for SIDS has not changed following the implementation of approved autopsies, the efficacy of the Approved Autopsy System has come to be viewed questionably. In addition, it is also necessary to enact legislation that mandates the conducting of autopsies for all cases of infant death as is done in Scandinavia. The required cost in the case of performing autopsies for all cases of abnormal infant death is estimated at 200,000-700,000 US dollars and is considered to be within a range that could be implemented through local government regulations. In addition, the cost per body of an autopsy performed at the State Crime Laboratory in the State of Arkansas in the US in 1999 was about 6000 US dollars. In contrast, the same cost at the Tokyo Medical Examiner Office is much less at only about 4000 US dollars.

Detection of toluene in an adipoceratous body.

A 24-year-old male was found dead in a car left in a river for about 3 months. The cadaver was almost adipoceratous and autopsy findings revealed that there were neither remarkable injuries nor lethal diseases. Toluene, ethanol, 1-propanol, 2-propanol, 1-butanol, dimethyl sulfide, dimethyl disulfide, isovaleraldehyde and n-butyl n-butyrate were detected in the specimens collected at the autopsy by head space gas chromatography/mass spectrometry (GC/MS). The toluene concentrations (micrograms/g) were 31.0 in brain, 10.6 in liver, 5.4 in kidney, 15.0 in skeletal muscle and 187.1 in adipose tissue. The presence of diatom in lung, liver and kidney suggested that death was caused by drowning. So far as we know, this is the first report of detection of toluene in an adipoceratous body.

An autopsy case of acute selenium (selenious acid) poisoning and selenium levels in human tissues.

A case of fatal suicidal ingestion of "Super Blue (Gun Blue)" (gun-blueing) is presented. Post-mortem examination of the patient revealed pulmonary edema with pleural effusion and congestion of the kidney. Necrosis of proximal tubules was found in the kidney by histological examination. "Super Blue" contains 4% selenious acid and 2.5% cupric sulfate in HCl. Levels of selenium and copper in tissues of the toxic case and normal individuals were determined. The selenium levels of tissues of the patient were 9-90-fold higher than that of normal subjects, whereas concentrations of copper were about 2-fold compared to that of control levels. The highest levels of selenium in the tissues of the patient were found in the lung, kidney and stomach contents.

Direct effects of methamphetamine on hypertrophy and microtubules in cultured adult rat ventricular myocytes.

Morphological alterations occasionally found in the myocardium of methamphetamine (MAP) abusers include hypertrophy, atrophy, disarrangement of myofibrils and fibrosis. These cardiac alterations have been thought to be due to an indirect action of MAP via catecholamines released by MAP administration. However, the direct effect of MAP on cardiomyocytes is not clear. In previous studies, we showed that cell size of isolated adult rat ventricular cardiomyocytes (ARCs) exposed to MAP was larger than that of untreated cells in culture supplemented with 10% fetal calf serum (FCS). In this study, to determine further the direct effect of MAP on cardiomyocytes, cultured ARCs were exposed to 0.05, 0.1 and 0.5 mM MAP for 7 days in culture medium without FCS following 6-day normal culture in medium containing FCS. Myocyte size was measured and microtubular (MT) structures which were associated with functional disorder of hearts were immunohistochemically observed using confocal microscopy. The size in treated ARCs significantly increased time- and dose-dependently as compared with untreated cells, but it decreased 7 days after exposure to 0.5 mM MAP. The increases in cell size, however, were lower than that in serum-supplemented cultures. MT structures in intact ARCs appeared as a filamentous network throughout the cytoplasm and around the nucleus. MAP exposure for 3 days promoted MT assembly, but in 7-day treated cells, MT and actin structures were injured. These results suggested that MAP directly induced cellular hypertrophy and might lead to cardiac functional disorder.

Event-related brain potentials as indicators of visual recognition and detection of criminals by their use.

An event-related potential (ERP) was recorded, using photographs as stimuli, in 12 subjects for attended, 9 subjects for non-attended conditions and 14 subjects for a simulated criminal investigation. An ERP was detected only when a subject recognized a familiar image (target) mixed with other, unfamiliar images (non-target), regardless of whether he was asked to attend to or neglect the target image. ERPs in the subject who watched each picture but tried to ignore the relevant picture (non-attended) were more activated at the parietal region than at the central region, in contrast with ERPs in the subjects who paid attention to each picture without trying to ignore the relevant picture (attended). In the simulated criminal investigation, only a simulated thief, but not a simulated innocent subject elicited ERP only after the picture of a criminal site or thing was intermingled with pictures bearing no relationship to the crime. These findings indicate that the ERP using photographs as stimuli is useful as an objective indicator of crime-relevance.