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Biomedical subjects

D Sakai

Publications and source records attributed to D Sakai.

At least 19 recordsLinked to original sources
PubMed2006

Expression of CD90 on keratinocyte stem/progenitor cells.

BACKGROUND: The identification and purification of keratinocyte stem cells (KSCs) that are capable of self-renewal and maintenance of differentiating cell populations could contribute both to our understanding of the biology of these cells, and to significant clinical applications, such as the culturing of keratinocytes for transplantation to severe burn wounds. Here, we report the detection of CD90(+) cells in cultured normal human epidermal keratinocytes and adult skin. OBJECTIVES: To investigate the biological function of CD90(+) and CD90(-) keratinocytes. METHODS: CD90(+) and CD90(-) keratinocytes were purified from adult skin and cultured keratinocytes using fluorescent activated cell sorting, and their biological abilities were analysed using both in vitro and in vivo assays. RESULTS: Flow cytometry (FCM) analysis identified approximately 18% of post-primary neonatal keratinocytes as CD90(+). However, during expansion of the culture, the expression level of CD90 rapidly decreased to about 2.5% at passage 10, while most of the keratinocytes maintained expression of alpha6 integrin. Purified CD90(+) keratinocytes demonstrated a sixfold higher cell growth rate than CD90(-) cells and the ability to form large (over 3 mm in diameter) colonies. We then quantitatively evaluated both populations using a previously described in vivo human epidermal cyst formation assay. Enhanced green fluorescent protein (EGFP)-labelled CD90(+) or CD90(-) keratinocytes were subcutaneously injected into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Six weeks after transplantation, EGFP(+) cell clusters in human epidermal cysts were evaluated using image analysis software. EGFP(+) cell cluster areas in the basal layer, derived from EGFP(+) CD90(+) cells, were eightfold larger than clusters of EGFP(+) CD90(-) cells. Furthermore, immunohistochemical staining and FCM analysis indicated that CD90 was expressed in most of the basal layer of the normal human epidermis. CONCLUSIONS: These results indicated that CD90 is a useful marker for the detection of human KSC-enriched populations in cultured human keratinocytes.

Adult↗
PubMed2005

Analysis of the pilU gene for the prepilin peptidase involved in the biogenesis of type IV pili encoded by plasmid R64.

In many type IV pili, the N-terminal amino acid of the pilin subunit is N-methylated phenylalanine. A prepilin peptidase removes the leader peptide from the precursor and methylates the amino group of the newly formed phenylalanine. PilS, the precursor of the pilin encoded by plasmid R64, is processed by the prepilin peptidase PilU, but the N-terminal amino acid of the mature pilin is a non-methylated tryptophan that is otherwise modified. To study the relationship between the structure and function of PilU, 42 missense pilU mutations were constructed by PCR and site-directed mutagenesis, and the ability of these pilU mutants to complement a pilU null mutant for mating in liquid culture was analyzed. Although practically no conjugation was noted for 21 of the mutants, the remaining 21 supported varying levels of residual plasmid transfer activity. Two mutants with aspartic acid replacements in conserved motifs exhibited no PilU activity, suggesting that the product of the pilU gene is an aspartic acid peptidase, like TcpJ, the prepilin peptidare of Vibrio cholerae. No PilS processing was detected in 21 of the mutants, but the remaining 21 exhibited varying levels of residual PilS processing. A close correlation was noted between residual PilS processing activity and conjugative transfer, suggesting that the pilU gene product possesses prepilin peptidase activity, but is unable to methylate the N-terminal tryptophan. Based on the activity of pilU-phoA and pilU-lacZ fusion genes encoding different segments of PilU, a model for the membrane topology of the protein is also proposed. Furthermore, some amino acid substitutions in the pilU portion of the pilU-phoA and pilU-lacZ fusion genes were found to alter the membrane topology of the product.

Amino Acid Sequence↗
PubMed2001

Atpase activity and multimer formation of Pilq protein are required for thin pilus biogenesis in plasmid R64.

Plasmid R64 pilQ gene is essential for the formation of thin pilus, a type IV pilus. The pilQ product contains NTP binding motifs and belongs to the PulE-VirB11 family of NTPases. The pilQ gene was overexpressed with an N-terminal His tag, and PilQ protein was purified. Purified His tag PilQ protein displayed ATPase activity with a V(max) of 0.71 nmol/min/mg of protein and a K(m) of 0.26 mm at pH 6.5. By gel filtration chromatography, PilQ protein was eluted at the position corresponding to 460 kDa, suggesting that PilQ protein forms a homooctamer. To analyze the relationship between structure and function of PilQ protein, amino acid substitutions were introduced within several conserved motifs. Among 11 missense mutants, 7 mutants exhibited various levels of reduced DNA transfer frequencies in liquid matings. Four mutant genes (T234I, K238Q, D263N, and H328A) were overexpressed with a His tag. The purified mutant PilQ proteins contained various levels of reduced ATPase activity. Three mutant PilQ proteins formed stable multimers similar to wild-type PilQ, whereas the PilQ D263N multimer was unstable. PilQ D263N monomer exhibited low ATPase activity, while PilQ D263N multimer did not. These results indicate that ATPase activity of the PilQ multimer is essential for R64 thin pilus biogenesis.

Adenosine Triphosphatases↗
PubMed2000

Role for cGATA-5 in transcriptional regulation of the embryonic chicken pepsinogen gene by epithelial-mesenchymal interactions in the developing chicken stomach.

A gene encoding embryonic chicken pepsinogen (ECPg), a zymogen of the digestive enzyme pepsin, is expressed specifically in epithelial cells of glands of embryonic stage proventriculus (glandular stomach) under the influence of mesenchyme. We found four GATA and one Sox binding motifs in 1.1 kb of the 5' flanking region of the ECPg gene which are essential to the organ-specific expression of the gene. The expression of cGATA-5 and cSox2 in the proventriculus from day 6 to day 12 of incubation was therefore analyzed. cGATA-5 was more strongly expressed in glandular epithelial cells than in luminal epithelial cells, while cSox2 gene expression was weaker in glandular epithelial cells. Using heterologous recombination explants we also discovered that the expression of cGATA-5 and cSox2 in epithelial cells was affected by mesenchyme when the latter induced ECPg gene expression in epithelial cells. Introduction of expression constructs into epithelial cells by electroporation demonstrated that cGATA-5 upregulated transcription of a reporter luciferase gene via a cis element in the 5' flanking region of the ECPg gene. The gel mobility shift assay revealed that the cGATA-5 protein specifically binds to the GATA binding sites. cSox2 downregulated the activity of luciferase but it was not through the Sox binding motif. These results suggest that cGATA-5 positively regulates transcription of the ECPg gene and is involved in spatial regulation of the pepsinogen gene during development.

Animals↗
PubMed2000

Reconstitution of damage DNA excision reaction from SV40 minichromosomes with purified nucleotide excision repair proteins.

We previously constructed the cell-free nucleotide excision repair (NER) assay system with UV-irradiated SV40 minichromosomes to analyze the mechanism of NER reaction on chromatin DNA. Here we investigate the factor that acts especially on nucleosomal DNA during the damage excision reaction, and reconstitute the damage excision reaction on SV40 minichromosomes. NER-proficient HeLa whole cell extracts were fractionated, and the amounts of known NER factors involved in the column fractions were determined by immunoblot analyses. The column fractions were quantitatively and systematically replaced by highly purified NER factors. Finally, damage DNA excision reaction on SV40 minichromosomes was reconstituted with six highly purified NER factors, XPA, XPC-HR23B, XPF-ERCC1, XPG, RPA and TFIIH, as those essential for the reaction with naked DNA. Further analysis showed that the damages on chromosomal DNA were excised as the same efficiency as those on naked DNA for short incubation. At longer incubation time, however, the damage excision efficiency on nucleosomal DNA was decreased whereas naked DNA was still vigorously repaired. These observations suggest that although the six purified NER factors have a potential to eliminate the damage DNA from SV40 minichromosomes, the chromatin structure may still have some repressive effects on NER.

DNA Damage↗
PubMed2000

The pilL and pilN genes of IncI1 plasmids R64 and ColIb-P9 encode outer membrane lipoproteins responsible for thin pilus biogenesis.

The predicted amino acid sequences of the pilL and pilN genes, required for the thin pilus formation of IncI1 plasmids R64 and ColIb-P9, contain N-terminal lipoprotein signal peptide motifs. The pilL and pilN products were labeled with [(3)H]palmitic acid as 38- and 57-kDa proteins, respectively, indicating that they are lipoproteins. Both PilL and PilN were localized to the outer membrane.

Amino Acid Sequence↗
PubMed2000

Ileal carcinoid tumor complicating carcinoid heart disease and secondary retroperitoneal fibrosis.

A 70-year-old man with metastatic liver tumors showed carcinoid syndrome with clinical symptoms of facial flushing, palpitation, dyspnea, and an itching sensation. Regurgitation of the tricuspid and aortic valves was observed by echocardiography. An elevated serum level of serotonin and a high urine excretion of serotonin metabolites were confirmed. Autopsy confirmed a serotonin-immunoreactive 1 cm ileal carcinoid tumor with metastasis to the liver, bone and peritoneum. The tumor cells were argyrophilic but not argentaffin, and showed erythrophagocytosis in the primary lesion. All the four heart valves and bilateral atrial endocardium showed fibromyxoid thickening, indicating the association of carcinoid heart disease. Desmoplastic reaction with deposition of sulfated acid mucopolysaccharides was also observed in the retroperitoneal space (secondary retroperitoneal fibrosis). Stenosis of the bilateral ureters and inferior mesenteric artery provoked hydronephrosis and lethal ischemic necrosis of the left-sided colon, respectively. Rarity of functioning ileal carcinoid tumor and pathogenesis of systemic fibroplasia are discussed.

Aged↗
PubMed1995

Osteoclast molecular phenotyping by random cDNA sequencing.

The osteoclast is a cell type that is highly specialized for its bone resorption function. In order to decipher the numerous biochemical functions of osteoclasts, a description of the gene expression profile of osteoclasts would be beneficial. We have sought to identify genes that are highly expressed in osteoclasts by partially sequencing 194 randomly chosen cDNA clones from a representative rabbit osteoclast cDNA library. Comparison to nucleic acid and protein sequence databases indicates that 135 of these cDNAs are identical to or homologous to known mammalian genes. Reverse transcription-polymerase chain reaction (RT-PCR) assays with microisolated osteoclasts were used to verify the osteoclast expression of some of these genes. Fifty-nine cDNAs, including two abundantly expressed species, have no significant similarity to the sequence databases and likely represent novel genes. The most abundant of the osteoclast expressed genes encode cofilin and the vacuolar H(+)-ATPase 16 kd subunit. Each were represented at a frequency of 4.1% of the clones in the library (95% confidence interval = 2.4-6.6%). The high expression of these gene products is consistent with the high motility of osteoclasts and their very active hydrogen ion secretion. Other abundantly expressed sequences include beta-actin (95% C.I. = 2.0-6.0%), creatine kinase B (95% C.I. = 1.2-4.9%), c-fms and ribosomal protein L18 (95% C.I. = 0.8-4.3%), and cathepsin-OC2, cyclophilin, delta-aminolevulinate synthetase, 16S mitochondrial rRNA, and two novel gene sequences (95% C.I. = 0.5-3.6%).

5-Aminolevulinate Synthetase↗
PubMed1993

The mouse Eb meiotic recombination hotspot contains a tissue-specific transcriptional enhancer.

A meiotic recombination hotspot exists within the second intron of the mouse major histocompatibility complex (MHC) gene, Eb. In the present study, a small fragment from the intron which contains two potential transcriptional regulatory elements was cloned into an expression vector and its effect on transcription was tested. This fragment was found to contain tissue-specific transcriptional enhancer activity. An octamer-like sequence and a B motif may contribute to this enhancer activity. Similar regulatory sequences with the same orientation and distance from one another are found in another mouse MHC recombination hotspot.

Animals↗
PubMed1984

Estrogen receptor transformation to a high-affinity state without subunit-subunit interactions.

We have tested the ability of monomeric rat estrogen receptor (generated by dissociating the native receptor aggregate with KCl and binding to hydroxylapatite) to undergo heat-induced transformation to a state with altered chromatographic properties and estradiol dissociation kinetics. Participation of nonadsorbed cytosolic components in the transformation process was eliminated by washing the adsorbed receptor prior to incubation at 28-30 degrees C for 30 min. We found that monomeric estradiol-receptor complexes can be converted to a form which coelutes with transformed receptor during hydroxylapatite chromatography and which has a slow estradiol dissociation rate characteristic of transformed receptor. Monomeric receptor does not, however, display cooperative ligand-binding behavior, strongly suggesting adsorbed monomers cannot dimerize or interact with other adsorbed cytosol proteins. Maintenance of the monomer form was further verified by the sedimentation of eluted receptor solely as a 4S species. Although conversion of monomers to the slow-dissociating state was incomplete (congruent to 80%) on hydroxylapatite, eluted receptor showed only a slow dissociation rate component. The data are consistent with a model in which receptor monomers are in equilibrium between low-affinity and high-affinity conformations. Dimerization of the high-affinity receptor may shift the equilibrium to greatly favor the high-affinity (transformed) state.

Animals↗
PubMed1984

Remodeling the estrogen receptor model.

The estrogen receptor model has revised to make it compatible with new data on subcellular localization of the receptor and physical characteristics of solubilized versus immobilized receptors. Our current model suggests that receptors, with or without bound estrogen, are present in the nuclear fraction. Furthermore, the receptor behaves as if it were immobilized or bound to some nuclear constituent at all times. Thus, the association of the estrogen-free receptor (unbound receptor) to a target site in the nucleus is considered to be the critical event in defining the nature of the response to estrogenic hormones.

Animals↗
PubMed1984

Primary cultures of estrogen-responsive cells from rat uteri: induction of progesterone receptors and a secreted protein.

Uterine cells from immature rats can be grown in culture, are estrogen responsive, and contain estrogen receptors. Progesterone receptor is induced within 1 day of 17 beta-estradiol treatment, with maximal response occurring after 2 days of treatment (300-500% of the control value). Induction of progesterone receptor occurred at physiological 17 beta-estradiol concentrations, with half-maximal response at about 5 X 10(-11) M. 17 beta-Estradiol induced the synthesis of a secreted protein (mol wt, 130,000) in a dose-dependent fashion. This 130-K protein was also induced by 16 alpha-estradiol (1-10 nM), but not by progesterone (10 nM), testosterone (1 nM), or dexamethasone (1 nM). Examination of the estrogen-binding properties of the cultured cells shows a saturable binding site (Kd approximately 10(-10) M) which can be translocated to the nucleus. Estrogen receptors were maintained at in vivo levels as uterine cells proliferated throughout 10 days of culture. This was in contrast to estrogen receptor levels in Fischer 344 rat pituitary cell cultures, which dropped off drastically on a DNA basis as cells proliferated. These studies indicate that estrogen receptor-containing rat uterine cells proliferate and are responsive in primary cell cultures.

Animals↗
PubMed1984

Reversible denaturation of the estrogen receptor and estimation of polypeptide chain molecular weight.

The estrogen receptor protein loses its ability to bind to estrogens upon denaturation with sodium dodecyl sulfate and 2-mercaptoethanol. Binding activity is recovered at 60-80% efficiency upon removal of the denaturants, equilibration with 6 M guanidine hydrochloride, and dilution into buffer containing estrogen. Renatured receptor is similar to native receptor in affinity for 17 beta-estradiol and ability to bind DNA. Detection of receptor activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of uterine or pituitary cytosol or of uterine nuclear extracts reveals a single unique polypeptide species of 65,000 mol wt.

Animals↗
PubMed1983

Estrogen receptor development in the absence of growth hormone and prolactin: studies in dwarf mice.

The concentration of high-affinity estradiol binding sites in the uteri and pituitaries of normal and two dwarf strains of mice was studied. The two dwarf strains are characterized by their total absence of growth hormone or prolactin synthesis and low or absent TSH production. The uteri of the dwarf mice are small but the receptor concentration per unit of DNA was similar to the normals. Nuclear and cytoplasmic distribution studies and saturation analysis detected no differences in receptor characteristics between dwarf and normal mice. These results demonstrate that prolactin, growth hormone and TSH do not control the developmental increase in uterine estrogen receptor that normally occurs in the young mouse. The concentration of estrogen receptor per unit of DNA in the pituitaries of the dwarfs was only 20-30% of normal animals of the same strain. This suggests that estrogen receptor in the mouse pituitary is associated to a great extent with lactotrophs and/or somatotrophs which are absent in the dwarfs.

Animals↗