Biomedical subjects

N Huh

Publications and source records attributed to N Huh.

At least 19 recordsLinked to original sources

PubMed2003

Neuronal interactions are higher in the cortex than thalamus in the somatosensory pathway.

Previous studies have shown significant correlated discharges (noise correlation) and synergistic information coding among adjacent cortical neurons. In order to investigate whether such interactions are present at an earlier stage of sensory processing, we compared noise correlation and synergistic information transmission in the ventral posterolateral nucleus (VPLn) of thalamus and primary somatosensory cortex (SI) of anesthetized rats. A hind paw was stimulated electrically and responses of several neighboring neurons were recorded simultaneously with a tetrode. Analyses indicated that noise correlation in the SI was about four times higher than in the VPLn, and, interestingly, it was significantly reduced following sensory stimulation in both regions. Spike count distributions of individual VPLn units contained higher amounts of information about the delivery of external stimulation compared with those of SI units. When simultaneously recorded units were considered together, transmission of information was more interactive (synergistic or redundant) among SI than VPLn units. On average, information transmission was independent in the VPLn, but synergistic in the SI. The difference in synergistic information coding was largely attributable to different levels of noise correlation and their modulation by external sensory stimulation. These results indicate that neuronal interactions are relatively low at the thalamic level, but much enhanced at the cortical level along the somatosensory pathway. The enhanced neuronal interactions in the cortex may reflect the role of cortex in extracting higher features of sensory stimuli.

Action Potentials↗

PubMed2001

PKCtheta II, a new isoform of protein kinase C specifically expressed in the seminiferous tubules of mouse testis.

Protein kinase C (PKC) theta, a Ca(2+)-independent isoform of PKC, has been known to be expressed in skeletal muscle and T cells. In the present study, we isolated and characterized a smaller transcript expressed in the mouse testis, the cDNA of which is referred hereafter as PKCthetaII and the original PKCtheta as PKCthetaI. The cDNA clone of PKCthetaII has 2184 base pairs and 464 amino acids in the possible open reading frame, consisting of the 5' unique sequence of 20 amino acids and the PKCthetaI sequence of 444 amino acids. Genomic DNA analysis revealed that transcription of PKCthetaII is initiated from the PKCthetaII-specific exon, which is located between exons 7 and 8 of the PKCtheta gene, indicating that alternative splicing is the mechanism by which PKCthetaII is generated. PKCthetaII is expressed exclusively in the testis in an age-dependent manner with sexual maturation. In situ hybridization and reverse transcription-polymerase chain reaction of microdissected tissues clearly demonstrated that PKCthetaII is expressed in the seminiferous tubules of the mouse testis. Consistent with its molecular structure lacking the C1 regulatory domain, PKCthetaII is constitutively active as determined by an in vitro kinase assay, being independent of PKC activators, e.g. phosphatidylserine and phorbol ester. PKCthetaII may play a crucial role in spermatogenesis or some related function of the testis.

Alternative Splicing↗

PubMed2001

Calmin, a protein with calponin homology and transmembrane domains expressed in maturing spermatogenic cells.

A cDNA named calmin of approximately 3.2 kb was isolated by RNA differential display applied to developing mouse skin. Calmin cDNA encodes 1021 amino acids with two calponin homology (CH) domains in tandem on the N-terminal side and a transmembrane domain on the C-terminal side. The region covering the CH domains showed a high level of homology with beta-spectrin, alpha-actinin, and dystrophin. Among the proteins with the tandem CH domains, calmin is unique in having a transmembrane domain. Three alternative splicing sites were identified at the 3'-side of calmin, giving rise to polymorphic protein products with or without the transmembrane domain. The calmin transcript was detected in adult testis, liver, kidney, and large intestine; the expression in testis was far stronger than that in the other tissues. In situ hybridization and immunostaining revealed that calmin was expressed in maturing spermatogenic cells at later stages. Human calmin cDNA was also isolated, and its exon/intron organization was determined.

Actinin↗

PubMed2001

Screening for genes involved in tissue invasion based on placenta formation and cancer cell lines with low and high metastatic potential.

During the formation of placenta, trophoblast cells vigorously invade maternal uterine tissues, sharing many features with the invasion of cancers. We applied RNA differential display to placenta tissues from 8.5 to 17.5 days post-coitus (dpc) ICR mice, and isolated 188 cDNA fragments expressed differentially. Among the 25 known cDNA fragments thus far analyzed, six cDNAs have been reported to be relevant to tumor invasion and/or metastasis. Furthermore, 11 of 20 unknown cDNAs isolated showed differential expression between the pairs of cancer cell lines with low and high metastatic potential, indicating potential usefulness of the present two-step approach.

Animals↗

PubMed2001

Suppression of hair follicle development inhibits induction of sonic hedgehog, patched, and patched-2 in hair germs in mice.

Embryonic induction of hair follicles is a fascinating model of localized morphogenesis from a simple homogeneous epithelial cell sheet. Accumulating evidence indicates that Sonic hedgehog (Shh) signaling plays a central role in hair follicle formation. We quantitated the expression levels of Shh and its receptor genes, Patched (Ptc) and Patched-2 (Ptch2), in two distinct experimental systems in which the development of hair follicles was suppressed. Shh, Ptc, and Ptch2 were induced about six- to tenfold in normal embryonic hair germs in vivo as well as in developing skin tissue maintained in organ culture. This induction was almost completely inhibited both in the developing skin tissue of ICR mice cultured with 30ng/ml epidermal growth factor and in embryos of Tabby mutant mice (a model of hypohidrotic ectodermal dysplasia) at 14.5-15.5 days postcoitus. Expression of Shh, Ptc and Ptch2 was induced in the Tabby embryos at 16.5 days postcoitus, indicating that Shh signaling may be involved in the formation not only of the well-studied guard hair but also of the awl hair. The potential of the two biological systems for studying molecular mechanisms in hair follicle formation, particularly at an early phase including Shh signaling, is discussed.

Animals↗

PubMed2000

Cholesterol sulfate, an activator of protein kinase C mediating squamous cell differentiation: a review.

Activity of protein kinase C (PKC) depends on the interaction with polar head-groups of two membrane lipids, i.e., phosphatidylserine and diacylglycerol. We demonstrated that cholesterol metabolism is directly involved in activation of the eta isoform of protein kinase C (PKCeta), which is predominantly expressed in epithelial tissues in close association with epithelial differentiation. We found that PKCeta was activated by cholesterol sulfate (CS), a metabolite of cholesterol formed during squamous cell differentiation. In the presence of CS, phorbol ester only weakly enhanced the activity of PKCeta. CS also activated PKCeta, PKCdelta and PKCepsilon in a dose-dependent manner, when assayed using purified recombinant materials. However, when partially purified materials were used from overexpressing normal human keratinocytes, only PKCeta was activated by CS among the isoforms examined. All the existing lines of evidence, mainly supplied from our laboratory, suggest that CS is involved in a signal transduction of squamous cell differentiation and thereby modifying squamous cell carcinogenesis.

Cell Differentiation↗

PubMed2000

A carcinoembryonic antigen family cDNA from mouse placenta encoding a protein with a rare domain composition.

The carcinoembryonic antigen (CEA) family comprises many members with pleiotropic functions. Among normal tissues, placenta is characterized by the abundant production of many different kinds of CEA-related proteins, which are apparently important for the maintenance of pregnancy. Using RNA differential display applied to mouse placentae at different gestational days, we have isolated a novel CEA-related cDNA designated as Ceacam11. Ceacam11 cDNA encodes 303 amino acids with a possible signal peptide and two immunoglobulin variable region-like domains. This domain composition is observed only in mouse Cea10/Ceacam10 among the many CEA family members thus far reported. The transcript of Ceacam11 was first detected in the placenta at 12.5 days post-coitus (dpc) and the level increased progressively towards 17.5 dpc. The expression of Ceacam11 appears to be reciprocal to that of Ceacam10, since the Ceacam10 transcripts were detected at 8.5 and 10.5 dpc, but not at 12.5 to 17.5 dpc. In situ hybridization showed that the expression of Ceacam11 was localized to the spongiotrophoblast of the placenta. Except for in the placenta, Ceacam11 transcripts were not detected in any adult tissues examined, including brain, lung, glandular stomach, small intestine, colon, liver, kidney and testis.

Amino Acid Sequence↗

PubMed1999

Adenovirus-mediated gene transfer to keratinocytes--a review.

The introduction and expression of a foreign gene provide a powerful tool for investigating functions and regulation of a gene of interest; however, keratinocytes have a major drawback in that foreign genes are hardly transfected by conventional methods and stable transformants are most difficult to establish in normal keratinocytes with a limited short life span. To overcome these problems, we used an adenovirus vector, Ax, developed by Saito et al, which yields desired recombinant viruses at an efficiency about 100-fold that of conventional methods, and by which genes are expressed at a high level under the control of a composite CAG promoter. We established Ax vectors carrying various isoforms of protein kinase C (PKC). Using these vectors, we found that the eta and delta isoforms of PKC, but not the alpha and zeta isoforms, mediate terminal differentiation in normal human keratinocytes. These Ax-vectors are also applicable to organ culture of mouse embryos. Advantages and disadvantages of adenovirus vectors and their use for keratinocyte biology are reviewed.

Adenoviridae↗

PubMed1998

Differential expression of Hox genes in multistage carcinogenesis of mouse skin.

We surveyed expression of Hox genes in multiple carcinogenesis of mouse skin by RT-PCR cloning. According to the sequences of the amplified DNA fragments within the homeobox of Hox genes, 25 of the 39 known Hox genes were amplified in the normal dorsal skin of adult mice. In the papilloma and carcinoma, clones of Hox A7 and Hox B7 were preferentially isolated among others. The entire Hox D locus was silent in both papilloma and carcinoma. Our present results suggest that the majority of Hox gene family members are expressed in normal mouse skin, while the repertoire is substantially limited in the papilloma and carcinoma.

Animals↗

PubMed1998

Isolation and characterization of a putative keratin-associated protein gene expressed in embryonic skin of mice.

Embryonic mouse skin undergoes substantial morphologic changes from 13 days post-coitus (dpc) to 16 dpc, i.e., from simple layers of epithelial cells and periderm at 13.5 dpc to almost fully differentiated stratified epithelium with the rudiments of hair follicles at 16.5 dpc. Using RNA differential display, we isolated a gene involved in the development of mouse epidermis. This gene, tentatively designated as 4C32, encodes 197 amino acids containing six direct repeats of 10 amino acids with the CQ motif. The repetitive structure with the CQ motif is seen in most keratin-associated protein families, which are known to be specifically expressed in hair follicles. 4C32 is expressed in the outermost layer of the embryonic epidermis at 15.5 and 16.5 dpc, and abruptly disappears at 17.5 dpc, suggesting that 4C32 is expressed in the periderm. The periderm is a superficial layer of embryonic epidermis, and is known to disappear at 17 dpc in mouse embryos. The 4C32 transcripts were also detected in the developing and matured tongue tissues and in the tail scale, but not at any stage in hair follicles.

Amino Acid Sequence↗

PubMed1997

Specific inhibition of hair follicle formation by epidermal growth factor in an organ culture of developing mouse skin.

Embryonic mouse skin undergoes a drastic morphological change from 13 to 16 gestational days, i.e., formation of rudiments of hair follicles and stratification and cornification of interfollicular epidermis. To investigate underlying molecular mechanisms of the morphogenesis, we established an organ culture system that allows skin tissues isolated from 12.5- or 13.5-days postcoitus embryos to develop in a manner that is histologically and temporally similar to the process in vivo. Expression of differentiation markers of epidermal keratinocytes including cholesterol sulfotransferase and cytokeratin K1 was induced in culture, as it occurs also in vivo. The morphogenic process was observed by time-lapse videomicrography. In this culture system, epidermal growth factor (EGF) and transforming growth factor alpha specifically and completely inhibited the hair follicle formation with marginal effects on interfollicular epidermis. The inhibitory action by EGF was reversible and stage specific, i.e., at an early stage of the development of hair rudiments. Among known ligands to the EGF receptor, Schwannoma-derived growth factor and heparin-binding EGF were expressed in in vivo epidermis during the period of the initial formation of hair follicles. EGF receptor is expressed in epidermis throughout the developing period examined. Using an adenovirus vector, we demonstrated that the lacZ gene was transduced into the epidermal and dermal cell layers without appreciable toxicity. These results indicate that the present culture system provides a unique opportunity to investigate molecular mechanisms of skin morphogenesis including the role of EGF signaling under defined experimental conditions.

Animals↗

PubMed1997

Rat liver 4S-benzo[a]pyrene-binding protein is distinct from glycine N-methyltransferase.

Rat liver 4S-benzo[a]pyrene-binding protein (BAP) was reported to be identical to the subunit (Mr 32,500) of the tetrameric glycine N-methyltransferase (GNMT). We have reevaluated this study. When a liver cytosol was subjected to Sephacryl S-200 gel permeation chromatography, enzyme activity as well as cross-reactivity with anti-GNMT antibody were found only at the elution position of the purified GNMT. Chromatograph of the cytosol pretreated with [3H]benzo[a]pyrene showed two peaks in the void volume and at the position of an approximate Mr of 40,000. The latter, corresponding to the 4 S BAP, did not cross-react with the anti-GNMT antibody. An extract of nuclei in which GNMT was proposed to act as a mediator of cytochrome P4501A1 gene expression contained the tetrameric GNMT but no binding activity. The lung, in which no GNMT mRNA occurred, had the 4 S BAP. Extracts of Escherichia coli and COS-7 cells expressing large amounts of GNMT showed no 4 S BAP. These findings suggest that the 4 S BAP is distinct from the subunit of GNMT.

Aldehyde Dehydrogenase↗

PubMed1997

Mechanistic insights into chemopreventive effects of phenethyl isothiocyanate in N-nitrosobis(2-oxopropyl)amine-treated hamsters.

The influence of phenethyl isothiocyanate (PEITC) on cell kinetics in the target organs of N-nitrosobis(2-oxopropyl)amine (BOP) tumorigenicity and on xenobiotic-metabolizing enzymes was investigated in hamsters. Female 5-week-old Syrian hamsters were given a single s.c. dose of 0, 20 or 50 mg/kg of BOP 2 h after receiving PEITC by gavage at a dose of 0, 100 or 250 mumol/animal (0, 16.3 or 40.8 mg/animal). Six and 22 h after the BOP administration, hamsters were killed and tissues were sampled. Proliferating cell nuclear antigen immunohistochemistry demonstrated significant reduction (P < 0.05-0.001) by PEITC of the labeling indices in the pancreatic acini and ducts, bronchioles, and renal tubules of the BOP-treated animals in a dose-dependent manner. In the lungs, the PEITC pretreatment significantly (P < 0.001) reduced the O6-methyldeoxyguanosine levels as compared to the BOP-alone value. Immunoblot analysis of liver cytochrome P450 isoenzymes showed CYP 2B1 to be mainly involved in the metabolic activation of BOP. PEITC significantly (P < 0.05) inhibited the induction of several isoenzymes, including CYP 2B1, while lowering the hepatic glutathione S-transferase activity as well as glutathione levels, regardless of BOP administration. Our results thus suggest that PEITC exerts its chemopreventive activity against BOP initiation of carcinogenesis in hamsters by decreasing cell turnover and DNA methylation in the target organs, and by influencing hepatic xenobiotic-metabolizing phase I enzymes, although the relationship, if any, of the latter with the former events remains to be investigated.

Animals↗

PubMed1996

DMSO induces apoptosis in SV40-transformed human keratinocytes, but not in normal keratinocytes.

We found that dimethyl-sulfoxide (DMSO) at concentrations of 2.5% induced apoptosis in SV40-immortalized human keratinocytes, while normal keratinocytes were arrested at the boundary of G1/S phase under the same conditions. DMSO-induced apoptosis in SV-40 immortalized keratinocytes was not associated with change in phosphorylated state of the retinoblastoma susceptibility gene. When SV40-immortalized cells were treated with 2.5% DMSO, dissociation of the complex was observed by immunoblotting of SV40 T antigen from immunoprecipitated p53 protein fraction.

Antigens, Viral, Tumor↗

PubMed1996

Sequence and expression patterns of mouse SPR1: Correlation of expression with epithelial function.

A final event in the terminal differentiation of stratified squamous epithelia is the formation of a cornified cell envelope, which is a complex of several proteins cross-linked together by transglutaminases. One set of proteins is the family of small proline rich (SPR) proteins. In human foreskin epidermal cell envelopes, SPRs serve as cross-bridging proteins among the more abundant loricrin. In order to study further their evolution and expression, we have isolated and sequenced cDNAs encoding two mouse SPR1 proteins, SPR1a and SPR1b Comparative sequence analysis showed the preservation of the overall structure of mammalian SPR1 proteins with highly conserved termini and a central peptide domain repeated 13 (SPE1a) or seven (SPR1b) times. Tissues obtained from mouse fetal, newborn, and adult skin were tested by Northern blot analyses, in situ hybridization and immunohistochemistry using an antibody raised to a synthetic peptide corresponding to the C terminus of the SPR1a protein. Skin expression was first detected in fetal periderm in anagen hair follicles of newborn and older mice, and in the thickened epidermis of the lip and footpad, but no signal was detected in interfollicular trunk epidermis. High levels of SPR1a expression were found in epithelia from the forestomach and penis, and in benign squamous papillomas. Other epithelia expressing SPR1a include the tongue, esophagus, and vagina. Whenever detected, SPR1a positive staining was present in the spinous and granular layers. In the forestomach and papillomas, the periphery of cells in the cornified layer was also stained. Our results suggest that SPR1a participates widely in the construction of cell envelopes in cornifying epithelia characterized by either increased thickness or a requirement for extreme flexibility. Based on its likely function as a cross-bridging protein in cell envelopes, we conclude that the mechanical attributes of cell envelopes may be determined in part by the SPR1 content, in accordance with the specific function of the epithelium.

Animals↗

PubMed1995

Growth enhancement of normal human keratinocytes by the antisense oligonucleotide of retinoblastoma susceptibility gene.

We have found that the growth of normal human keratinocytes, grown in serum-free medium, was significantly stimulated by the antisense oligonucleotide of retinoblastoma susceptibility gene (Rb). Normal human keratinocytes were exposed to phosphorothionate oligonucleotides which were complementary to translation initiation codon of Rb gene. The growth of keratinocytes was enhanced by the antisense, but not the sense, oligonucleotide of Rb gene in a dose-dependent manner from 1 to 10 microM. The Rb antisense oligonucleotide, however, did not result in any appreciable change in transcription of the gene when examined by reverse-transcription polymerase chain reaction (RT-PCR) analysis or in the protein expression and the phosphorylation pattern when examined by immunoprecipitation and Western blotting.

Base Sequence↗

PubMed1995

Detection of O6-methylguanine, O4-methylthymine and O4-ethylthymine in human liver and peripheral blood leukocyte DNA.

O6-Methylguanine, O4-methylthymine and O4-ethylthymine were determined by a recently developed, highly sensitive and specific method (PREPI, pre-fractionation, 32P-post-labeling and immunoprecipitation) in human liver DNA obtained from 15 autopsy specimens and in 15 peripheral blood leukocyte DNA samples obtained from healthy volunteers. All the cases had no obvious history of recent occupational or therapeutic exposure to alkylating agents. In the human liver DNA O6-methylguanine was detected in 13 cases at levels of 1.1-6.7 adducts/10(7) guanine; two cases were below the detection limit of approximately 1.1 x 10(-8). O4-Methylthymine and O4-ethylthymine were detected in all the liver samples at levels of 0.1-14 adducts and 0.5-140 adducts/10(7) thymine respectively. The mean value of the ratio of O6-methylguanine to O4-methylthymine was about 6. Among the three DNA adducts measured there was no significant correlation between any two combinations of adducts. In peripheral leukocytes of healthy volunteers O6-methylguanine was detected at 0.7-4.6 adducts/10(8) guanine, this being approximately 3-6% of that in livers. Neither O4-methylthymine nor O4-ethylthymine was above the detection limits. These results suggest that humans are exposed to exogenous and/or endogenous methylating and ethylating agents in daily life.

Adolescent↗

PubMed1994

Growth inhibition of SV40-transformed human keratinocytes by TGF-beta s is not linked to dephosphorylation of the Rb gene product.

We found that transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 2 inhibited the growth of normal human keratinocytes and their SV40-transformed counterpart in a dose dependent manner. Both normal and SV-40 transformed keratinocytes accumulated in G1 when treated with TGF-beta s. The hyperphosphorylated form of Rb gene product (RB) was reduced by TGF-beta s in normal keratinocytes. In contrast, phosphorylation of RB was not essentially affected in SV-40 transformed cells. This uncoupling of growth kinetics and phosphorylation states of RB in SV-40 transformed keratinocytes suggests that RB is not the primary target of action for TGF-beta s and that additional factors or pathways may be involved in mechanisms of the growth inhibition.

Blotting, Western↗