Search PubMed⌕ Search

Biomedical subjects

K Shiota

Publications and source records attributed to K Shiota.

At least 37 records · Page 2Linked to original sources

Placentomegaly in cloned mouse concepti caused by expansion of the spongiotrophoblast layer.

Hypertrophic placenta, or placentomegaly, has been reported in cloned cattle and mouse concepti, although their placentation processes are quite different from each other. It is therefore tempting to assume that common mechanisms underlie the impact of somatic cell cloning on development of the trophoblast cell lineage that gives rise to the greater part of fetal placenta. To characterize the nature of placentomegaly in cloned mouse concepti, we histologically examined term cloned mouse placentas and assessed expression of a number of genes. A prominent morphological abnormality commonly found among all cloned mouse placentas examined was expansion of the spongiotrophoblast layer, with an increased number of glycogen cells and enlarged spongiotrophoblast cells. Enlargement of trophoblast giant cells and disorganization of the labyrinth layer were also seen. Despite the morphological abnormalities, in situ hybridization analysis of spatiotemporally regulated placenta-specific genes did not reveal any drastic disturbances. Although repression of some imprinted genes was found in Northern hybridization analysis, it was concluded that this was mostly due to the reduced proportion of the labyrinth layer in the entire placenta, not to impaired transcriptional activity. Interestingly, however, cloned mouse fetuses appeared to be smaller than those of litter size-matched controls, suggesting that cloned mouse fetuses were under a latent negative effect on their growth, probably because the placentas are not fully functional. Thus, a major cause of placentomegaly is expansion of the spongiotrophoblast layer, which consequently disturbs the architecture of the layers in the placenta and partially damages its function.

Animals↗

DNA methylation regulates placental lactogen I gene expression.

Expression of rat placental lactogen I is specific to the placenta and never expressed in other tissues. To obtain insight into the mechanism of tissue-specific gene expression, we investigated the methylation status in 3.4 kb of the 5'-flanking region of the rat placental lactogen I gene. We found that the distal promoter region of the rat placental lactogen I gene had more potent promoter activity than that of the proximal area alone, which contains several possible cis-elements. Although there are only 17 CpGs in the promoter region, in vitro methylation of the reporter constructs caused severe suppression of reporter activity, and CpG sites in the placenta were more hypomethylated than other tissues. Coexpression of methyl-CpG-binding protein with reporter constructs elicited further suppression of the reporter activity, whereas treatment with trichostatin A, an inhibitor of histone deacetylase, reversed the suppression caused by methylation. Furthermore, treatment of rat placental lactogen I nonexpressing BRL cells with 5-aza-2'-deoxycytidine, an inhibitor of DNA methylation, or trichostatin A resulted in the de novo expression of rat placental lactogen I. These results provide evidence that change in DNA methylation is the fundamental mechanism regulating the tissue-specific expression of the rat placental lactogen I gene.

Animals↗

Carboxy-PTIO increases the tetrahydrobiopterin level in mouse brain microvascular endothelial cells.

The aim of the present study was to characterize the increase in tetrahydrobiopterin (BH4), which is a cofactor for nitric oxide synthase (NOS), by carboxy-PTIO, a scavenger of nitric oxide (NO), in vascular endothelial cells. BH4 level was determined by oxidation under acidic conditions as biopterin. Addition of lipopolysaccharide (LPS) to endothelial cells increased mRNA levels of inducible NOS (iNOS) and GTP-cyclohydrolase I (GTPCH), which is a rate-limiting enzyme for BH4 synthesis, and the biopterin level. NOS inhibitors, NO-donors and L-arginine, a substrate of NOS, did not affect the increase in the biopterin level induced by LPS, suggesting that BH4 synthesis is unlikely to be modulated by NO produced by iNOS during LPS treatment. However, carboxy-PTIO increased the biopterin level in the absence and the presence of LPS. Carboxy-PTIO did not affect the expression of GTPCH mRNA level. Moreover, 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTPCH, inhibited only about 30% of the carboxy-PTIO-induced increase in the biopterin level. Whereas, N-acetylserotonin, an inhibitor of sepiapterin reductase, strongly inhibited the increase in biopterin level. Carboxy-PTIO inhibited the accumulation of pterin, a decomposition product of BH4 in endothelial cells. These findings suggest that carboxy-PTIO accumulates BH4 under basal and LPS-treated conditions in vascular endothelial cells due to both inhibition of the decomposition of BH4 to pterin and activation of the salvage pathway of BH4 synthesis via sepiapterin reductase.

Alcohol Oxidoreductases↗

Ontogenesis of anti-oxidative enzymes in mouse embryos and fetuses: an immunohistochemical study.

The ontogenesis of two anti-oxidative enzymes, thioredoxin (TRX) and glutaredoxin (GRX), was examined immunohistochemically in mouse embryos and fetuses at various developmental stages. They were found to be localized in various tissues, with some tissue specificity and temporal sequence. Both TRX and GRX began to be expressed in many tissues at embryonic (E) day E10 or E11 and tended to increase as the developmental stage advanced. In the heart and neuroepithelium, however, their immunoreactivity was already positive at E8.5. In some fetal organs like the liver, pancreas and kidney, TRX and GRX showed heterogeneous localization, suggesting that their expression may reflect the variable functional states of the cell. These results suggest that TRX and GRX may be associated with tissue differentiation in embryos and fetuses are involved in the acquisition of the capacity of resistance against oxygen radicals.

Animals↗

Pads and flexion creases on the plantar surface of hammertoe mutant mouse (Hm).

The purpose of the present work was to determine the effects of the hereditary malformation of Hammertoe mutant mice (gene symbol Hm) on the surrounding morphological structures and, specifically, on the volar pads, i.e., the sites of the epidermal ridge patterns (dermatoglyphics). The hindlimbs of the wild-type (+/+) Hammertoe mice show no anomalies and their major pad and flexion crease configurations correspond to those of normal mice. The heterozygous (Hm/+) and homozygous (Hm/Hm) mice display a fusion of the interdigital tissues involving all digits with the exception of digit I. In Hm/Hm mice, this webbing extends to the distal phalanx and the markedly flexed digits form a shape resembling a hammer. In Hm/+ mice, the interdigital webbing does not extend as far and the digits show moderate flexion compared to those of Hm/Hm mice. Both Hm/Hm and Hm/+ have a rudimentary extra digit in the postaxial area of the hindlimbs. The ventral volar skin of the flexed digits is incompletely developed. The more posterior digits show the more severe camptodactyly. These aberrant configurations are related to the abnormal occurrence of the programmed cell death (PCD) in the interdigital zones II-IV and the proximal part of the postaxial margin during hindlimb development. They are limited to the pads on the plantar surface of the postaxial area; the preaxial area is not affected. As a result of a severe camptodactyly of digit V, its volar skin is shifted into the distal portion of the hypothenar area. This shifting affects the number, size, and location of the pads, especially of the hypothenar pad, resulting in varying pad configurations, such as a displacement of the distal and proximal components of the hypothenar pad, or a fusion of the two components of the hypothenar pad, leading to a reduced final pad number. These pad modifications are induced by the postaxial plantar surface shifting proximally and are not affected by the presence of an extra rudimentary digit. The pad modifications in Hammertoe mice with webbed digits and postaxial polydactyly resemble closely those of the previously studied mice with genetic preaxial polydactyly.

Animals↗

PAL31, a novel nuclear protein, expressed in the developing brain.

We cloned a cDNA encoding a novel protein (PAL31) predominantly expressed in the fetal rat brain by differential display. PAL31 contains leucine-rich repeat domains, a highly acidic region and a putative nuclear localization signal. PAL31 has 50-70% similarity to SSP29, APRIL, LANP, PHAP I, and PP32. Expression of PAL31 mRNA in the brain was high during the fetal period and decreased after birth. Immunohistochemical studies showed that PAL31 is expressed in the entire embryonic brain, whereas in the adult brain its expression is restricted to the subventricular zone where there are neural progenitor cells. It was also revealed that PAL31 is colocalized with PCNA in the nucleus, indicating that the PAL31 expression is developmentally regulated. Considering the primary structure of PAL31 and its spatiotemporal expression pattern, PAL31 is a novel nuclear protein related to the development of the brain through the proliferation of neuronal cells.

Amino Acid Motifs↗

Extracellular matrix environment influences chondrogenic pattern formation in limb bud micromass culture: experimental verification of theoretical models.

Various theoretical models have been proposed to explain the periodicity in the pattern of limb chondrogenesis, but experimental comparison of these models have seldom been performed properly. In the present study, micromass culture of limb bud mesenchyme cells was undertaken to test the validity of three theoretical models: the reaction-diffusion model, the cell sorting model, and the mechanochemical model. Computer simulations were undertaken to predict the factors that can affect the coarseness of the chondrogenic pattern. According to the predictions, we performed micromass culture of limb mesenchyme in collagen and agarose gel. Then we carried out time-lapse observation to analyze the cell movement during pattern formation. From computer simulations it was theoretically predicted that changes in the surrounding extracellular matrix should alter the periodicity of the chondrogenic pattern in vitro, and we found that pattern changes actually occurred under different culture conditions. When compared with the culture in a liquid medium, the chondrogenic pattern became less coarse when the cells were cultured in collagen or agarose gel, and the pattern change appeared to be independent of the cell differentiation. Time-lapse observation revealed a decrease in cell motility when the cells were cultured in gel. It was found that both the reaction-diffusion and cell sorting models fit the pattern change produced and that the mechanochemical model is not primarily important in the chondrogenic pattern formation in vitro.

Agar↗

TGFbeta2 acts as an "activator" molecule in reaction-diffusion model and is involved in cell sorting phenomenon in mouse limb micromass culture.

It was previously speculated that TGFbeta acts as an "activator"-molecule in chondrogenic pattern formation in the limb micromass culture system, but its precise role and relationship with the cell sorting phenomenon have not been properly studied. In the present study, we examined whether the TGFbeta2 molecule satisfies the necessary conditions for an "activator"-molecule in the reaction-diffusion model. Firstly, we showed that TGFbeta2 became localized at chondrogenic sites during the establishment of a chondrogenic pattern, and exogenous TGFbeta2 promoted chondrogenesis when added in the culture medium. Secondly, TGFbeta2 protein was shown to promote the production of its own mRNA after 3 hr, indicating that a positive feedback mechanism exists which may be responsible for the emergence of the chondrogenic pattern. We then found that when locally applied with beads, TGFbeta2 suppressed chondrogenesis around the beads, indicating it induces the lateral inhibitory mechanism, which is a key element for the formation of the periodic pattern. We also examined the possible effects of TGFbeta2 on the cell sorting phenomenon and found that TGFbeta2 exerts differential chemotactic activity on proximal and distal mesenchyme cells of the limb bud, and at very early phases of differentiation TGFbeta2 promotes the expression of N-cadherin protein which is known to be involved in pattern formation in this culture system. These findings suggest that TGFbeta2 acts as an "activator"-like molecule in chondrogenic pattern formation in vitro, and is possibly responsible for the cell sorting phenomenon.

Animals↗

A novel method for analysis of the periodicity of chondrogenic patterns in limb bud cell culture: correlation of in vitro pattern formation with theoretical models.

To experimentally examine whether the pattern changes predicted by theoretical models of pattern formation actually occur in a limb bud cell culture system, we developed a practical method to automatically measure the periodicity of chondrogenic patterns in vitro. The method utilizes binary image processing to quantify the total number of peak and valley pixels in a pattern to obtain the average interval between stripes in the chondrogenic pattern, and we named it the peak length method. The reliability of the peak length method was examined by using computer simulation results. The peak length method enabled us quantitatively obtain the average interval between chondrogenic islands, and the values obtained by this method were closely correlated with the average intervals obtained by manual measurement and two-dimensional Fourier transformation. The average intervals obtained by the peak length method were shown to be stable over a wide range of pattern variations that are frequently observed in actual experiments. By applying the peak length method to actual experimental data, we compared the validity of two theoretical models of pattern formation (cell sorting model and reaction-diffusion model) and it was concluded that the peak-length method is a useful tool to quantitatively analyze chondrogenic patterns in limb micromass culture and to relate theoretical predictions and experimental results of pattern formation.

Animals↗

Neural tube closure in humans initiates at multiple sites: evidence from human embryos and implications for the pathogenesis of neural tube defects.

The closure of the neural tube (NT) in the human embryo has generally been described as a continuous process that begins at the level of the future cervical region and proceeds both rostrally and caudally. On the other hand, multiple initiation sites of NT closure have been demonstrated in mice and other animals. In humans, based on the study of neural tube defects (NTD) in clinical cases, van Allen et al. (1993) proposed a multisite NT closure model in which five closure sites exist in the NT of human embryos. In the present study, we examined human embryos in which the NT was closing (Congenital Anomaly Research Center, Kyoto University) grossly and histologically, and found that NT closure in human embryos initiates at multiple sites but that the mode of NT closure in humans is different from that in many other animal species. In addition to the future cervical region that is widely accepted as an initiation site of NT closure (Site A), the mesencephalic-rhombencephalic boundary was found to be another initiation site (Site B). The second closure initiating at Site B proceeds bidirectionally and its caudal extension meets the first closure from Site A over the rhombencephalon, and the rostral extension of the second closure meets another closure extending from the rostral end of the neural groove (Site C) over the prosencephalon, where the anterior neuropore closes. The caudal extension of the first closure initiating at Site A was found to proceed all the way down to the caudal end of the neural groove where the posterior neuropore is formed, indicating that in humans, NT closure does not initiate at the caudal end of the neural groove to proceed rostrally. Since there is a considerable species difference in the mode of NT closure, we should be careful when extrapolating the data from other animals to the human. It seems that the type of NTD affects the intrauterine survival of abnormal embryos. Almost all the embryos with total dysraphism appear to die by 5 weeks of gestation, those with an opening over the rhombencephalon by 6.5 weeks, and those with a defect at the frontal and parietal regions survive beyond 7 weeks.

Age Factors↗

Pathomorphologic study on the mechanism of fatty change in small hepatocellular carcinoma of humans.

BACKGROUND/AIMS: Fatty change is frequently observed in small hepatocellular carcinoma (HCC) of the early stage. However, the mechanism of fatty change and its pathomorphological features in small HCC are not yet fully understood. These issues are addressed here. METHODS: Histological examinations were conducted on 260 HCC nodules (< or =3 cm in diameter) which were surgically obtained from 249 patients. According to the distribution pattern, fatty changes were classified into two types: 'diffuse type' when the change was found throughout the cancerous nodule; and 'focal type' when the change was localized in part of the nodule. To study the pathogenesis of fatty change in HCC in relation to angioarchitecture, the number of arterial tumor vessels and intratumoral portal tracts in 104 of the 260 nodules was counted. RESULTS: Fatty change was found in 51 of the 260 nodules (19.6%), the frequency was highest (36.4%) in the nodules whose diameter was 1.1 to approximately 1.5 cm, and the frequency decreased with the increase in tumor diameter. Small well-differentiated HCCs were often associated with a diffuse type fatty change. With the increase in tumor diameter, moderately differentiated cancerous tissues without associated fatty change appeared, and the focal type was found more frequently. According to the angioarchitecture, in HCCs < or =1.5 cm, the number of intratumoral arteries was significantly smaller in HCCs with fatty change (p<0.05), though the number of intratumoral portal tracts was not significantly different compared with HCCs without fatty change. CONCLUSION: These findings suggest that fatty change of small HCC is closely related to the tumor size, the histological grade and insufficient development of the arterial tumor vessels.

Arteries↗

Immunohistochemical localization of thioredoxin and glutaredoxin in mouse embryos and fetuses.

Although oxygen is essential for promoting energy metabolism and for enhancing cell proliferation, early mouse embryos are very sensitive to high oxygen concentration. Because the tissue-specificity and sequential changes of the expression of antioxidative enzymes in rodent embryos have not been investigated systematically, we examined the ontogenesis of thioredoxin (TRX) and glutaredoxin (GRX) in mouse embryos and fetuses by using immunohistochemical methods. These compounds were found to be localized in most tissues examined, with some tissue specificity and temporal sequence. In many tissues, both TRX and GRX began to be expressed at embryonic day 11 (E11) or E13 and appeared to increase later in development, but the heart and great vessels of E8.5 embryos were already positive for their immunoreactivity. The stage at which the antioxidative enzymes begin to be expressed seems to coincide with the stage at which rodent embryos acquire the capacity of aerobic energy metabolism. Although TRX and GRX were co-localized in many tissues and showed similar sequential changes of expression, their expression patterns were different in the fetal cartilage, suggesting that they may play different roles in endochondral ossification. Their immunoreactivity was not homogeneous in the liver and the epithelium of uriniferous tubules, probably because their expression is associated with the proliferating and metabolic activities of the cell, as suggested by previous investigators. These results suggest that TRX and GRX play some tissue-specific roles in mammalian morphogenesis as well as general roles as antioxidant enzymes.

Animals↗

A novel secretory protein produced by rat spongiotrophoblast.

The placenta secretes various factors in stage- and cell-specific manners. We have identified a cDNA encoding a novel protein with 124 amino acids, which was named spongiotrophoblast specific protein (SSP). SSP is highly homologous to mouse 4311, showing 81% and 59% similarity at the nucleotide and amino acid levels, respectively. Northern blot analysis showed that SSP mRNA was first detected on Day 14 of pregnancy, peaked on Day 16, and remained elevated until term. In situ hybridization analysis showed that SSP mRNA was specifically expressed in spongiotrophoblast cells of Day 20 placenta but not in Day 12 placenta. No expression was detected from the differentiated or undifferentiated rat choriocarcinoma Rcho-1 cell line. Native SSP was detected as a 19-kDa molecule by Western blotting in cell extracts prepared from the junctional zone. SSP was predicted to be a secretory protein, because 1) a hydropathy test revealed that SSP contained an N-terminal hydrophobic region and 2) native SSP was also detected in the cultured media of junctional zone explants. To further investigate a potential signal peptide of this protein, sets of recombinant SSP were generated using a COS7 transfection system. The N-terminal amino acid sequence of secreted recombinant SSP confirmed that the N-terminal 17 amino acids had been cleaved to produce a secretory protein. Thus, we have identified and cloned a novel secretory protein, SSP, which is specifically expressed by rat spongiotrophoblast cells during the latter half of pregnancy.

Amino Acid Sequence↗

Suckling dysfunction caused by defects in the olfactory system in genetic arhinencephaly mice.

Mouse newborns find their mother's nipples and suckle milk by themselves. It has been argued which sense organ they use when locating their mother's nipples to suckle milk. Olfactory or tactile sensory systems are primary candidates. In the present study, we investigated the trigeminal-whisker sensory and olfactory systems in genetic arhinencephaly mouse embryos (Pdn/Pdn). Pdn/Pdn newborns do not suckle milk and die within 1 day after birth. Dysfunction of nipple-searching behavior was clear in Pdn/Pdn newborns. Pdn/Pdn newborns had a complete developmental failure in the olfactory nerve projection to the central nervous system and no olfactory bulb architecture. The trigeminal-whisker system was intact in this strain. From the results of these experiments, it was suggested that the olfactory system is essential for nipple-searching behavior and suckling milk and that the trigeminal-whisker system is not able to substitute for the lack of olfactory input in mouse newborns.

Animals↗

Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm.

From early stages of development, Sox2-class transcription factors (Sox1, Sox2 and Sox3) are expressed in neural tissues and sensory epithelia. In this report, we show that Sox2 function is required for neural differentiation of early Xenopus ectoderm. Microinjection of dominant-negative forms of Sox2 (dnSox2) mRNA inhibits neural differentiation of animal caps caused by attenuation of BMP signals. Expression of dnSox2 in developing embryos suppresses expression of N-CAM and regional neural markers. We have analyzed temporal requirement of Sox2-mediated signaling by using an inducible dnSox2 construct fused to the ligand-binding domain of the glucocorticoid receptor. Attenuation of Sox2 function both from the late blastula stage and from the late gastrula stage onwards causes an inhibition of neural differentiation in animal caps and in whole embryos. Additionally, dnSox2-injected cells that fail to differentiate into neural tissues are not able to adopt epidermal cell fate. These data suggest that Sox2-class genes are essential for early neuroectoderm cells to consolidate their neural identity during secondary steps of neural differentiation.

Animals↗