Toshihito Nagata

Quantitative evaluation of extracellular glutamate concentration in postischemic glutamate re-uptake, dependent on brain temperature, in the rat following severe global brain ischemia

Changes in brain temperature are known to modulate the marked neuronal damage caused by an approximately 10-min intra-ischemic period. Numerous studies have suggested that the extracellular glutamate concentration ([Glu](e)) in the intra-ischemic period and the initial postischemia period is strongly implicated in such damage. In this study, the effects of intra-ischemic brain temperature (32, 37, 39 degrees C) on [Glu](e) were investigated utilizing a dialysis electrode combined with ferrocene bovine serum albumin (BSA), which allows oxygen-independent real-time measurement of [Glu](e). This system allowed separate quantitative evaluation of intra-ischemic biphasic glutamate release from the neurotransmitter and metabolic pools, and of postischemic glutamate re-uptake in ischemia-reperfusion models. The biphasic [Glu](e) elevation in the intra-ischemic period did not differ markedly among intra-ischemic brain temperatures ranging from 32 to 39 degrees C. Intra-ischemic normothermia (37 degrees C) and mild hyperthermia (39 degrees C) markedly inhibited [Glu](e) re-uptake during the postischemic period, although the intra-ischemic [Glu](e) elevation did not differ from that during intra-ischemic hypothermia (32 degrees C). It was assumed that normothermia or mild hyperthermia in the intra-ischemic period influences intracellular functional abnormalities other than the intra-ischemic [Glu](e) elevation, thereby inhibiting glutamate re-uptake after reperfusion rather than directly modulating intra-ischemic [Glu](e) dynamics.

Clinical Application of Oligonucleotide Probe Array for Full-Length Gene Sequencing of TP53 in Colon Cancer

Objective:TP53 mutations are the most frequent genetic alterations in colon cancer. We studied whether the recently developed oligonucleotide microarray technique, GeneChip p53 assay, can be applied to sensitive detection of TP53 gene mutations in surgical specimens from colon cancer patients. Methods:TP53 gene mutations in exons 2–11 in 20 colon cancers and the corresponding histopathologically normal mucosa at the surgical margins were assessed by GeneChip p53 assay, and the results were further evaluated by direct sequencing of the involved exon or by mutant-allele-specific amplification (MASA). The expression of TP53 protein was also evaluated immunohistochemically and the result was compared with the gene alteration. Results: The GeneChip p53 assay detected TP53 mutations in 65% of primary cancers; 61% of the mutations were within the evolutionarily conserved regions, and 46% of the mutations were within the zinc-binding domains (regions of loop 2 and loop 3). Direct sequencing confirmed these mutations. Immunohistochemical examination detected TP53 protein overexpression in 47% of primary cancers, but this protein did not accumulate with all types of TP53 mutations. In addition, the GeneChip assay detected a mutation identical to that in the primary tumor in 2 samples from the surgical margins, and MASA confirmed both mutations, implying the presence of occult cancer cells. Conclusion: The GeneChip p53 assay is sufficiently sensitive to detect TP53 mutations in surgical specimens from colon cancers and may be applicable to screening examination in clinical laboratories as a routine procedure.

Transcriptional profiling in hepatoblastomas using high-density oligonucleotide DNA array.

Hepatoblastoma is a common hepatic tumor in children. Although evidence regarding cytogenetic and molecular genetic alterations in hepatoblastomas has been reported, the molecular events affecting the biologic characteristics of this tumor, including alterations of the gene expression profile, are largely unknown. To identify genes differentially expressed between nondiseased liver (NDL) and hepatoblastoma tumor (HBT), we analyzed the gene expression profile in 14 NDL and 16 HBT samples using a high-density oligonucleotide DNA array. Using Mann-Whitney U test followed by the k-nearest neighbor algorithm, we identified 26 genes (predictor genes) that were able to assign unknown samples derived from NDL and HBT to either the NDL group or HBT group with 100% accuracy. Using a cross-validation approach, we confirmed that the k-nearest neighbor algorithm assigned the particular samples derived from NDL and HBT to either the NDL or HBT group with 93.3% (28/30 samples) accuracy. In the 26 predictor genes, we found alteration of the expression of genes regulating cell division (NAP1L1, STMN1, CCNG2, and CDC7L1) and tumor cell growth (IGF2 and IGFBP4) in HBT. Four predictor genes (ETV3, TPR, CD34, and NR1I3) were also found to be mapped to the chromosomal region 1q21 approximately q32, which has been reported to be frequently involved in the development of hepatoblastoma. The findings obtained in this study suggest that alteration of the expression of some genes regulating cell division and tumor cell growth may be characteristics of the gene expression profile in HBT, and that alteration of the expression of the four predictor genes mapped to chromosomal region 1q21 approximately q32 may also contribute to the differences in gene expression profile between NDL and HBT.

Karyotypic Analyses of Hepatoblastoma: Report of Two Cases and Review of the Literature Suggesting Chromosomal Loci Responsible for the Pathogenesis of this Disease

Two cases of fetal hepatoblastoma with unique karyotypic changes are described. One was a 17-month-old boy with multiple unbalanced chromosomal translocations, resulting in four types of derivative chromosomes involving chromosomal loci at 1q21, 1q32, 2q23, 6q27, 7p22, and 21p12, partial tetrasomy of 1q, partial trisomy of 2q, and partial monosomy of 21p. The clonal karyotype of this tumor was 46,XY,der(2)t(1;2)(q32;q37), der(6)t(1;6)(q12;q27), der(7)t(2;7)(q23;p22), der(21)t(2;21) (q23;p12). In the other case, a 4-year-old girl, karyotypic analyses revealed trisomy 2 and 8, and the clonal karyotype of this case was 48,XX,+2,+8. Review of these cases together with previous reports suggested the significance of chromosomal changes including numerical abnormalities of 1q, 2(or 2q), 20, and 8 (or 8q), and breakage of 1q and 2q in the development of hepatoblastoma. The results presented herein underscore the significance of numerical abnormalities of chromosomal regions 1q and 2q and of chromosome 8 in the development of hepatoblastoma, in addition to abnormalities of 6q27, 7p22, and 21p12-13 as other chromosomal loci that may be responsible for the pathogenesis of this embryonal type of tumor.

Global profiling of influence of intra-ischemic brain temperature on gene expression in rat brain.

Mild to moderate differences in brain temperature are known to greatly affect the outcome of cerebral ischemia. The impact of brain temperature on ischemic disorders has been mainly evaluated through pathological analysis. However, no comprehensive analyses have been conducted at the gene expression level. Using a high-density oligonucleotide microarray, we screened 24000 genes in the hippocampus under hypothermic (32 degrees C), normothermic (37 degrees C), and hyperthermic (39 degrees C) conditions in a rat ischemia-reperfusion model. When the ischemic group at each intra-ischemic brain temperature was compared to a sham-operated control group, genes whose expression levels changed more than three-fold with statistical significance could be detected. In our screening condition, thirty-three genes (some of them novel) were obtained after screening, and extensive functional surveys and literature reviews were subsequently performed. In the hypothermic condition, many neuroprotective factor genes were obtained, whereas cell death- and cell damage-associated genes were detected as the brain temperature increased. At all intra-ischemic brain temperatures, multiple molecular chaperone genes were obtained. The finding that intra-ischemic brain temperature affects the expression level of many genes related to neuroprotection or neurotoxicity coincides with the different pathological outcomes at different brain temperatures, demonstrating the utility of the genetic approach.

Circadian rhythm of melatonin and prostaglandin in modulation of stress-induced gastric mucosal lesions in rats.

We previously demonstrated the circadian variation of water‐immersion restraint stress (WRS)‐induced gastric mucosal lesions in rats.

Screening for control genes in rat global cerebral ischemia using high-density oligonucleotide array

From conventional relative gene expression analyses (Northern blotting, in situ hybridization, and RT‐PCR), it has been reported that the expression of control genes, such as glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and β‐actin, used as references may be affected by ischemia. Therefore, we extended searching and evaluation at the mRNA level of transcripts whose expression levels were not changed by cerebral ischemia, using a high‐density oligonucleotide array and statistical analysis in a rat global cerebral ischemia and reperfusion model. We added a hyperthermic factor and localization factor to ischemia and identified transcripts with a stable expression level under conditions even more disadvantageous than ischemia only. Screening of more than 8,000 transcripts with the Rat Genome U34A array yielded 28 transcripts, which we listed and classified according to their expression level. Widely used control genes, GAPDH and β‐actin, were not included, although cyclophilin A was included. In addition, we conducted a functional classification based on gene ontology. Under the functional classification of the 28 transcripts, many genes tended to be associated with metabolism. In conclusion, use of several transcripts is recommended, such as those we identified, as references in the analysis of gene expression in pathological models of ischemia.

Minimal effect of brain temperature changes on glutamate release in rat following severe global brain ischemia : a dialysis electrode study

USING a dialysis electrode, we recently developed an oxygen-independent system for real-time measurement of the glutamate concentration in the extracellular space ([Glu]e) during ischemia. This system allows separate evaluation of intra-ischemic biphase [Glu]e elevation, i. e. release from synaptic vesicles (1st phase), reversed uptake of glutamate from metabolic pools in neuronal cells (2nd phase), and post-ischemic glutamate re-uptake in ischemia—reperfusion models. Using the system, we attempted to clarify the relationship between biphase glutamate release and brain temperature in a model of acute global ischemia produced by transecting both carotid arteries. Our results showed that, in contrast to mild hyperthermia, hypothermia did not inhibit the 1st phase of [Glu]e release, and changes in intra-ischemic brain temperature had a minimal effect on the 2nd phase of [Glu]e elevation during severe acute ischemia. These findings, together with our previous data, indicate that brain temperature change in the intra-ischemic period plays an important role in disturbance of the glutamate re-uptake system during ischemia. NeuroReport 9: 3863–3868

Microarray Analysis of Gene Expression Changes in Aging in Mouse Submandibular Gland

Little is known about the effect of salivary gland function during aging based on gene expression. Recently emerged DNA array technology provides a sensitive, quantitative, rapid approach to the monitoring of the global pattern of gene expression. In this study, we used high-density oligonucleotide arrays to monitor the changes of gene expression levels in the submandibular gland (SMG) by comparing adult mice with elderly adult mice. Of the 1328 genes screened, 160 genes (12.0%) showed more than two-fold changes; 154 (96.3%) of these genes, associated with transcription regulation, transport, signal transduction, and enzymes in the elderly mice, exhibited decreased expression levels. The remaining 6 genes (3.7%) in the elderly mice showed increased expression levels. In mouse SMG, analysis of these data suggests that aging may lead the gene expression to decrease than increase. Thus, DNA array technology can be a powerful tool for the identification of age-associated candidate genes for further analysis in aging.

Hemophagocytic syndrome and hepatosplenic gammadelta T-cell lymphoma with isochromosome 7q and 8 trisomy.

The authors describe a 15-year-old boy with hepatosplenic gammadelta T-cell lymphoma associated with hemophagocytic syndrome (HPS) along with isochromosome 7q and trisomy 8. He presented with prolonged fever, mild anemia, thrombocytopenia, and hepatosplenomegaly. Physical examination, radiography, and ultrasound tomography revealed no lymphoadenopathy. He had elevated levels of serum ferritin, interferon-gamma, soluble interleukin-2 receptor, and interleukin-6. Bone marrow aspirate showed hypercellularity with 50% lymphoblasts and erythrophagocytosis of macrophage. A cytogenetic study of bone marrow revealed an abnormal karyotype, 47,XY,I(7q),+8, in 5/30 cells. Clonal rearrangement of the genes for T-cell receptor gamma and delta chains was elucidated by polymerase chain reaction. He achieved a complete remission after intensive chemotherapy and underwent splenectomy 18 months after diagnosis. Although the patient was clinically in remission, minimal residual disease (MRD) was detected in the removed spleen by polymerase chain reaction. This might mean that this type of lymphoma is refractory, as reported previously, and might indicate that marrow ablative therapy is needed to achieve a cure. The present case illustrates the usefulness of MRD analysis, and MRD studies in this group of disorders may be helpful in the decision of whether to continue a more aggressive therapeutic approach.