Hidehiko Sugino

Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer

When neural cells were collected from the entire cerebral cortex of developing mouse fetuses (15.5–17.5 days postcoitum) and their nuclei were transferred into enucleated oocytes, 5.5% of the reconstructed oocytes developed into normal offspring. This success rate was the highest among all previous mouse cloning experiments that used somatic cells. Forty-four percent of live embryos at 10.5 days postcoitum were morphologically normal when premature and early-postmitotic neural cells from the ventricular side of the cortex were used. In contrast, the majority (95%) of embryos were morphologically abnormal (including structural abnormalities in the neural tube) when postmitotic-differentiated neurons from the pial side of the cortex were used for cloning. Whereas 4.3% of embryos cloned with ventricular-side cells developed into healthy offspring, only 0.5% of those cloned with differentiated neurons in the pial side did so. These facts seem to suggest that the nuclei of neural cells in advanced stages of differentiation had lost their developmental totipotency. The underlying mechanism for this developmental limitation could be somatic DNA rearrangements in differentiating neural cells.

Underexpression of mitochondrial-DNA encoded ATP synthesis-related genes and DNA repair genes in systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by various systemic symptoms and multiple organ damage. We clarify biological and functional abnormalities in SLE by comparing the gene expression profiles of SLE patients with those of healthy individuals.MethodsGene expression profiles from the peripheral blood of 21 SLE patients and 45 healthy individuals were obtained using a DNA microarray. Gene ontology analysis and network pathway analysis were performed on the genes differentially expressed between SLE and healthy individuals.ResultsA total of 2,329 upregulated genes and 1,884 downregulated genes were differentially expressed. Gene ontology analysis revealed that the upregulated genes were classified as response to biotic stimulus genes, which mainly includes genes related to immune response. Abnormalities in other categories such as cell motility and regulation of apoptosis were also revealed. Downregulated genes were mainly sorted into two gene categories, sensory perception and response to radiation/light. The sensory perception genes included ATPase/ATPase domain-containing genes, myosin-related genes, and two excision repair cross-complementing genes, which are involved in DNA repair. Other genes in this group - including three crystallin genes, genes encoding the receptor protein for melanocyte-stimulating hormone, and six mitochondrial-DNA encoded genes, which are involved in ATP synthesis - were also categorized as response to radiation genes. Using network pathway analysis, IL-6, transforming growth factor beta 1, TNF, and hepatocyte nuclear factor 4α were found to play central roles in the networks of sensory perception-related molecules.ConclusionsFunctional abnormalities in ATP synthesis and DNA repair are implicated in peripheral blood cells from SLE patients.

Interactions among type I and type II interferon, tumor necrosis factor, and β-estradiol in the regulation of immune response-related gene expressions in systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by various clinical manifestations. Several cytokines interact and play pathological roles in SLE, although the etiopathology is still obscure. In the present study we investigated the network of immune response-related molecules expressed in the peripheral blood of SLE patients, and the effects of cytokine interactions on the regulation of these molecules.MethodsGene expression profiles of peripheral blood from SLE patients and from healthy women were analyzed using DNA microarray analysis. Differentially expressed genes classified into the immune response category were selected and analyzed using bioinformatics tools. Since interactions among TNF, IFNγ, β-estradiol (E2), and IFNα may regulate the expression of interferon-inducible (IFI) genes, stimulating and co-stimulating experiments were carried out on peripheral blood mononuclear cells followed by analysis using quantitative RT-PCR.ResultsThirty-eight downregulated genes and 68 upregulated genes were identified in the functional category of immune response. Overexpressed IFI genes were confirmed in SLE patient peripheral bloods. Using network-based analysis on these genes, several networks including cytokines – such as TNF and IFNγ – and E2 were constructed. TNF-regulated genes were dominant in these networks, but in vitro TNF stimulation on peripheral blood mononuclear cells showed no differences in the above gene expressions between SLE and healthy individuals. Co-stimulating with IFNα and one of TNF, IFNγ, or E2 revealed that TNF has repressive effects while IFNγ essentially has synergistic effects on IFI gene expressions in vitro. E2 showed variable effects on IFI gene expressions among three individuals.ConclusionsTNF may repress the abnormal regulation by IFNα in SLE while IFNγ may have a synergistic effect. Interactions between IFNα and one of TNF, IFNγ, or E2 appear to be involved in the pathogenesis of SLE.

Cytokine networks in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease more prominent in women and characterized by multiple organ damage. Imbalance in cytokine production and cytokine levels correlates with SLE progression, making the understanding of SLE cytokine networks very important for SLE treatment strategy and drug development. In this article, we review cytokine networks that may be involved in the pathogenesis of SLE by briefly describing abnormal cytokine production and serum cytokine levels in SLE patients. We also focus on the pathological roles of cytokines and their interactions in immunoregulatory networks and suggest how their disturbances may implicate in pathological conditions in SLE. Finally, we further discuss the influence of estrogen on these cytokine networks.

Somatic mutations of synaptic cadherin(CNR family)transcripts in the nervous system

Cadherin‐related neuronal receptor (CNR) family genes have been identified in the nervous system by screening molecules bound to Fyn‐tyrosine kinase. The CNR family is comprised of diverse synaptic cadherins. The genomic organization of the CNR genes, composed of variable and constant regions, is similar to that of the immunoglobulin gene cluster. The nervous system is characterized by the acquisition of diverse function. This feature is similar to the immune system. In the immune system, the generation and selection of immunoglobulin gene mutants is the underlying basis for acquired immunity. We therefore examined somatic regulation of the CNR family genes in the nervous system to determine whether a similar mechanism controls nervous system development.

Abnormal networks of immune response-related molecules in bone marrow cells from patients with rheumatoid arthritis as revealed by DNA microarray analysis.

IntroductionRheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic synovitis that progresses to destruction of cartilage and bone. Bone marrow (BM) cells have been shown to contribute to this pathogenesis. In this study, we compared differentially expressed molecules in BM cells from RA and osteoarthritis (OA) patients and analyzed abnormal regulatory networks to identify the role of BM cells in RA.MethodsGene expression profiles (GEPs) in BM-derived mononuclear cells from 9 RA and 10 OA patients were obtained by DNA microarray. Up- and down-regulated genes were identified by comparing the GEPs from the two patient groups. Bioinformatics was performed by Expression Analysis Systemic Explorer (EASE) 2.0 based on gene ontology, followed by network pathway analysis with Ingenuity Pathways Analysis (IPA) 7.5.ResultsThe BM mononuclear cells showed 764 up-regulated and 1,910 down-regulated genes in RA patients relative to the OA group. EASE revealed that the gene category response to external stimulus, which included the gene category immune response, was overrepresented by the up-regulated genes. So too were the gene categories signal transduction and phosphate metabolism. Down-regulated genes were dominantly classified in three gene categories: cell proliferation, which included mitotic cell cycle, DNA replication and chromosome cycle, and DNA metabolism. Most genes in these categories overlapped with each other. IPA analysis showed that the up-regulated genes in immune response were highly relevant to the antigen presentation pathway and to interferon signaling. The major histocompatibility complex (MHC) class I molecules, human leukocyte antigen (HLA)-E, HLA-F, and HLA-G, tapasin (TAP) and TAP binding protein, both of which are involved in peptide antigen binding and presentation via MHC class I molecules, are depicted in the immune response molecule networks. Interferon gamma and interleukin 8 were overexpressed and found to play central roles in these networks.ConclusionsAbnormal regulatory networks in the immune response and cell cycle categories were identified in BM mononuclear cells from RA patients, indicating that the BM is pathologically involved in RA.

Comparative genomic analysis of the mouse and rat amylase multigene family

The rat and mouse amylase gene families were characterized using sequence data from the UCSC genome assembly. We found that the rat genome contains one amylase‐1 and two amylase‐2 genes, lying close to one another on the same chromosome. Detailed analysis revealed at least six additional amylase pseudogenes in the rat genome in the region adjacent to the amylase‐2 genes. In contrast, the mouse has one amylase‐1 gene and five amylase‐2 genes; the latter are tandemly and systematically arranged on the same chromosome and were generated by segmental duplication. Detailed analysis revealed that the mouse has two amylase pseudogenes, located 5′ to the five amylase‐2 segments. Thus, the amylase genes of mouse and rat tend to be amplified; the sequences of some of them are fixed while others have become pseudogenes during evolution. This is the second report of amylase genomic organization in mammals and the first in the rodents.

DNA microarray analysis of rheumatoid arthritis susceptibility genes identified by genome-wide association studies.

normalized using the global ratio median. Only gene expression data that were collected from at least 80% of samples from each group were selected for further analysis. Th e unpaired Mann–Whitney test was used to determine statistically signifi cant diff erences in the mRNA expression levels between the RA and healthy groups. Statistical signifi cance was set at P <0.05. Th e results of our DNA microarray analysis showed that the expressions of four out of the six RA susceptibility genes were signifi cantly higher in RA patients than in healthy individuals (1.0 x 10 –16 to 2.32 x 10 –5 )

ICAM-3, a ligand for DC-SIGN, was duplicated from ICAM-1 in mammalian evolution, but was lost in the rodent genome.

ICAM‐3 is a DC‐SIGN ligand that is constitutively expressed on resting leukocytes, and is thus an important molecule for the first immune response. But, ICAM‐3 has not been isolated form rodents. Thus, we compare the ICAM gene clusters in human, dog, mouse, and rat. ICAM‐1, ‐4, ‐5 and ‐3 are located close to one another on the same chromosome and show genomic synteny in human and dog. Almost the same ICAM gene clusters were found in rodent genome, but only the ICAM‐3 was not present. A phylogenetic tree plotting the cDNAs of human, dog, mouse, rat, and bovine suggested that ICAM‐3 was made from a duplication of ICAM‐1. Thus, ICAM‐3 arose from ICAM‐1 in the mammalian evolution, but was lost in the rodents genome. Our study suggests the different immune response in the rodents in comparison with other mammals.

Banding profiles of LTR of human endogenous retrovirus HERV-A in 24 chromosomes in somatic cell hybrids

The human genome carries multiple copies of sequences related to endogenous retroviral genomes. We investigated the distribution of one of these sequences, HERV-A, in 24 human chromosomes by Southern analyses using DNAs from flow-sorted chromosomes or rodent cells carrying a single human chromosome. The results showed that HERV-A is distributed among all human chromosomes and that each chromosome has a specific Southern blot profile. The chromosome-specific pattern did not show significant polymorphism, except in a few cases, when the same chromosome obtained from different individuals was compared. These chromosome-specific Southern hybridization profiles may be useful for chromosome karyotyping. This would allow the integrity of human chromosomes in human-rodent somatic cell hybrids to be monitored without using conventional cytogenetic methods.