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S Kitazawa

Publications and source records attributed to S Kitazawa.

At least 19 recordsLinked to original sources
PubMed1999

Transcriptional regulation of rat cyclin D1 gene by CpG methylation status in promoter region.

Cyclin D1, a G(1)/S cell cycle-regulating oncogene, is known to be transcriptionally regulated by numerous growth factors. We cloned and characterized the rat cyclin D1 gene 5'-flanking region and, by species- and subspecies-matched transient transfection studies, found that a basic promoter structure with a cAMP response element and two continuous Sp1-binding sites was crucial for the steady-state expression of the cyclin D1 gene. Furthermore, the methylation status especially around two continuous Sp1-binding sites was found to be an important epigenetical mechanism determining the steady-state expression level in rat leukemic cell lines K4D, K4DT, and K4D16. Whether or not epigenetic control of the cyclin D1 gene existed among normal rat tissues was further examined by high sensitivity mapping of the methylated cytosine. In normal rat tissues, the methylated cytosines at non-CpG loci within two continuous Sp1-binding sites were observed in uterine stromal cells of the basal layer and found to be demethylated in the functioning layer, possibly by a passive demethylation mechanism through cell division. Since in the passive demethylation process Sp1-binding sites remain methylated in a part of the cell population, methylated cytosines at Sp1-binding sites may be essential for keeping a number of the stromal cells in the basal layer live against estrogen-induced proliferation that leads to either apoptosis or compaction.

Animals↗
PubMed1999

Promoter structure of mouse RANKL/TRANCE/OPGL/ODF gene.

Receptor activator of NF-kappa B ligand (RANKL)/tumor necrosis factor-related activation induced cytokine (TRANCE)/osteoprotegerin ligand (OPGL)/osteoclast differentiation factor (ODF) is a membrane-bound signal transducer responsible for differentiation and maintenance of osteoclasts. To elucidate the mechanism regulating RANKL/TRANCE/OPGL/ODF gene expression, we cloned the 5'-flanking basic promoter region of the mouse RANKL/TRANCE/OPGL/ODF gene and characterized it by transient transfection studies and genomic Southern blot analysis. Inverted TATA- and CAAT-boxes and a putative Cbfa1/Osf2/AML3 binding domain constituted the basic promoter structure. The repeated half-sites for the vitamin D3 (VitD3) and glucocorticoid receptors were located at -935 and -640, respectively. Transient transfection studies revealed that short-term treatment with 1alpha,25(OH)2 VitD3 or dexamethasone increased luciferase activity up to 204% and 178%, respectively; on the other hand, treatment with dibutyryl cyclic AMP did not affect the promoter activity. Since the expression of Cbfa1/Osf2/AML3 is also regulated by VitD3, 1alpha,25(OH)2 VitD3 might affect RANKL/TRANCE/OPGL/ODF gene expression both directly and indirectly. CpG methylation was observed dominantly in mouse stromal cells, ST2, of a later passage which ceased to support in vitro osteoclastogenesis, suggesting that the methylation status of the CpG loci in the RANKL/TRANCE/OPGL/ODF gene promoter may be one of the influential cis-regulating factors.

Animals↗
PubMed1999

Involvement of mutations in the DPC4 promoter in endometrial carcinoma development.

To define the target of chromosome 18q loss of heterozygosity, which is prevalent in endometrial carcinomas, we made a deletion map from 64 tumors. Loss of heterozygosity on 18q was found in 20 tumors. Among these, 14 tumors carried deletions at the 18q21.1 region, where the DPC4 gene is located. DPC4 transcription was disturbed in all six of the tumors with deletions at 18q21.1 examined, which sharply contrasted with the positive transcription in 12 tumors that retained heterozygosity at the 18q21.1 region. However, in the 14 tumors with the 18q21.1 deletions, the remaining allele had the wild-type sequence of the DPC4 coding region instead of somatic mutations in the DPC4 coding region. We found a one- and two-base substitutions in the DPC4 promoter in two of the six tumors that showed disturbed DPC4 transcription. Chloramphenicol acetyltransferase assays clearly demonstrated that the mutant promoters had the potential to suppress or silence DPC4 transcription, implicating the DPC4 gene in endometrial carcinoma.

Alleles↗
PubMed1999

In situ hybridization with polymerase chain reaction-derived single-stranded DNA probe and S1 nuclease.

A rapid and simplified protocol for in situ hybridization (ISH) with polymerase chain reaction (PCR)-derived single-stranded DNA probes and S1 nuclease revealed transcripts of bone matrix proteins on decalcified skeletal bone specimens. Mouse bone tissue was fixed with 4% paraformaldehyde, decalcified with 20% EDTA, and embedded in paraffin. Each pair of primers for reverse transcriptase -PCR was designed to amplify a 280-bp DNA fragment from the coding region of the mature protein of mouse osteonectin (ON) and a 320-bp fragment from the coding region of mouse osteopontin (OP). Initial PCR products were eluted, purified, and reamplified by unidirectional PCR in the presence of the digoxigenin (DIG)-labeled dUTP. ISH was carried out by proteinase K treatment, hybridization, and washing. The unhybridized single-stranded DNA probe was selectively removed by S1 nuclease treatment. Hybridized probes were visualized with the alkaline phosphatase-conjugated anti-DIG antibody. The transcripts of ON and OP were clearly detected on the thin sections of the decalcified bone. Because this protocol does not require cloning or in vitro transcription, reliable and stable ISH can be done in an ordinary laboratory equipped with a thermal cycler.

Animals↗
PubMed1999

Expression of platelet-derived growth factor proteins and their receptor alpha and beta mRNAs during fracture healing in the normal mouse.

Platelet-derived growth factor (PDGF), abundant in bone tissue, has been reported to stimulate mesenchymal cell proliferation and migration. To elucidate the functional roles of PDGF during fracture healing, we investigated the expression of PDGF-A and -B chain proteins and receptor alpha and beta mRNAs in fractured mouse tibiae. Twelve-week-old male BALB/c mice were operated on to make a closed fracture on the proximal tibia. On days 2, 4, 7, 10, 14, 21, and 28 after the operation, the fractured tibiae were excised, fixed with 4% paraformaldehyde, decalcified with 20% EDTA, and embedded in paraffin to prepare 7-microm sections. Immunohistochemistry using polyclonal antibodies against human PDGF-A and -B chains was carried out by the avidin-biotin-peroxidase method. For in situ hybridization, we used digoxigenin-labeled single-stranded DNA probes specific for mouse PDGF receptors alpha and beta generated by unidirectional polymerase chain reaction. In the inflammatory phase on days 2-4 after the fracture, mesenchymal cells gathering at the fracture site expressed the PDGF-B chain and beta receptor mRNA. At the stage of cartilaginous callus formation on day 7, the immunoreactivity for PDGF-A and -B chains on proliferating and hypertrophic chondrocytes and the signals of alpha and beta receptor mRNAs on proliferating chondrocytes became manifest. At the stage of bony callus and bone remodeling on days 14-21, the predominant expression of the PDGF-B chain and beta receptor was observed on both osteoclasts and osteoblasts. On day 28, signals for PDGF ligand proteins and receptor mRNAs diminished. The coincidental localization of PDGF ligands and their receptors implies a paracrine and autocrine mechanism. Our data suggested that PDGF contributed in part to the promotion of the chondrogenic and osteogenic changes of mesenchymal cells from the early to the midphase of fracture healing; the functions mediated by the beta receptor, including cell migration, might be prerequisites to the recruitment of mesenchymal cells in the initial step and to the interaction between osteoclasts and osteoblasts in the bone remodeling phase.

Animals↗
PubMed1999

Estimated nutritional intake based on the reference growth curves for extremely low birthweight infants.

BACKGROUND: Although the optimal goal of extremely low birthweight (ELBW) infants has been debated, it can be argued that the best nutritional strategy for ELBW infants is one which achieves better growth while preventing neurological sequelae due to nutritional inadequacy, even if it does not mimic fetal growth. Our aim is to propose an advisable and practically feasible nutritional intake for ELBW infants. METHODS: We retrospectively investigated the nutritional intake in 16 ELBW infants (gestational age 26.7 weeks, birthweight 879.6 g), who exceeded the standard growth curves for Japanese infants of ELBW. We also analyzed the nutritional composition of human milk from 15 mothers delivering before term (PT milk) until the 12th week of lactation. The nutritional intake was calculated according to the composition of the parenteral and enteral regimen. RESULTS: The infants tolerated more than 100 mL/kg per day of milk until the fourth week of life. An average weight gain of more than 15 g/kg per day was achieved by the sixth week. An advisable nutritional intake capable of sustaining ELBW infants in a stable growing phase was calculated as the sum of the mean plus 1SD of the daily nutritional intake. CONCLUSIONS: To exceed the ordinary growth curves for ELBW Japanese infants, it was suggested that more than 100 mL milk/kg per day could be fed until the fourth week of life, while the advisable daily intake of nutrients after six weeks of life should be as follows: water 160 mL/kg, protein 3.2 g/kg, fat 7.4 g/kg, carbohydrate 12.8 g/kg, energy 544 kJ/kg, the following minerals in mg/kg: sodium 45, chloride 82, potassium 110, calcium 140, phosphorus 70, magnesium 11, zinc 0.5 and copper at 60 micrograms/kg. Further studies will be needed to confirm the safety and effectiveness of this advisable intake for ELBW infants.

Birth Weight↗
PubMed1999

[New operative technique to resect of peripheral bronchogenic carcinoma with chest wall invasion using Kent's retractor].

We report the new operative technique for resection of peripheral bronchogenic carcinoma with chest wall invasion using Kent's retractor and hook suspender. Standard muscle sparing thoracotomy cannot obtain the adequate operative field in the posterior chest wall under the scapula because of sparing the latissimus dorsi muscle. To obtain a sufficient operative field at the operation for en bloc chest wall resection, especially with the area of the posterior chest wall under the scapula, resection of the trapezius muscle, rhomboideus major muscle, and latissimus dorsi muscle has been added to muscle-sparing thoracotomy. Through our new technique using Kent's retractor and hook suspender that has been used in the abdominal surgery, assistant surgeon can be eliminated in favor of hanging up the scapula, which is able to be performed much easier to obtain a good operative field. To use the Kent's retractor and hook suspender, the scapula is lifted upward and cranially. With these procedures, the proximal portion of the first, second and third ribs in the posterior chest wall covering by the scapula is able to resect from vertebrocostal junction with muscle-sparing thoracotomy. We examined the apparatus for a case of the right upper lung carcinoma with invasion to the posterior chest wall. We performed right upper lobectomy with chest wall in the area of the second, third and forth ribs with suspending the scapula by Kent's retractor after muscle-sparing thoracotomy.

Adenocarcinoma↗
PubMed1998

Promoter structure of human sonic hedgehog gene.

Sonic hedgehog (Shh) is a secreted signal transducer responsible not only for patterning of the anterior-posterior axis during early limb and neuronal development, but also for generating cell-type diversity at later developmental stages. To elucidate the mechanism regulating human Shh gene expression, we cloned the 5'-flanking region of the human Shh gene and characterized it by transient transfection studies. Two transcription start sites were identified by primer extension analysis. Two TATA-boxes, a CCAAT-box and a palindrome-like structure constituted the basic promoter structure. Furthermore, two continuous E-boxes and a putative homeodomain containing an ATTA-box were located around 350-450 bp upstream of the upper TATA-box. Consensus binding sites of the RA, estrogen, D3 and glucocorticoid/progesterone receptors were not found within the cloned sequence. Short-term treatment with TPA increased luciferase activity up to 2.1-fold; on the other hand, treatment with dibutyryl-cyclic AMP decreased it to 0.8-fold. Retinoic acid (RA), vitamin D3, dexamethazone (DEX) and estradiol (E2) had no effect on the luciferase activity. Since the zebrafish Shh promoter contains two closely spaced axial (HNF3beta) binding sequences on its basic promoter, the palindrome-like structure located in the corresponding site of the human Shh promoter may be a crucial binding domain regulating human Shh gene expression.

Base Sequence↗
PubMed1998

Analysis of 5'-flanking region of human Smad4 (DPC4) gene.

Among the transducers of the transforming growth factor (TGF)-beta/bone morphogenetic protein (BMP) receptor signaling proteins, Smad4 and Smad1 act together in BMP 2/4 signaling pathways, and Smad4 and Smad2 act in TGF-beta/activin signaling. To investigate how the Smad4 gene is regulated at the transcriptional level, we cloned and characterized its 5'-flanking region. The major transcription start site mapped by primer extension analysis was 132 bp upstream of the translation start site. The promoter region lacked canonical TATA and GC boxes; it did, however, contain a TATA-like structure (TAAAAT) 32 bp upstream of the transcription start site. A consensus sequence for homeoprotein HoxA-5 (TTTAAAAATTA) was identified at 171 bp upstream of the transcription start site. Within 600 bp upstream of the transcription start site, two Pit-1 and four F2F binding sites were found. One putative AP-1 site was located at -1129. These findings suggest that these homeoproteins could conduct their signals specifically by controlling both inter- and intracellular signal transduction pathways.

Base Sequence↗
PubMed1998

Sequential hemodynamic activation of motor areas and the draining veins during finger movements revealed by cross-correlation between signals from fMRI.

Activity in the human supplementary motor area (SMA), primary motor cortex (M1), and the draining vein of the motor cortex during a visually triggered finger opposition task was measured by functional magnetic resonance imaging with a repetition time of 1 s. Sequential hemodynamic activation in these areas was revealed by cross-correlating a signal sequence in M1 directly with signal sequences from SMA and the draining vein, and applying polynomial fitting with the aid of Akaike's information criterion. We succeeded in detecting a time delay of approximately 0.5 s between the activations of SMA and M1, and a delay of nearly 1.3 s between the activations of M1 and its draining vein. The new combination of fMRI and data analysis techniques has attained a time resolution comparable to those in preceding studies that used shorter fMRI repetition times of 100-200 ms.

Adult↗
PubMed1998

Cerebellar complex spikes encode both destinations and errors in arm movements.

Purkinje cells of the cerebellum discharge complex spikes, named after the complexity of their waveforms, with a frequency of approximately 1 Hz during arm movements. Despite the low frequency of firing, complex spikes have been proposed to contribute to the initiation of arm movements or to the gradual improvement of motor skills. Here we recorded the activity of Purkinje cells from the hemisphere of cerebellar lobules IV-VI while trained monkeys made short-lasting reaching movements (of approximately 200 milliseconds in duration) to touch a visual target that appeared at a random location on a tangent screen. We examined the relationship between complex-spike discharges and the absolute touch position, and between complex-spike discharges and relative errors in touching the screen. We used information theory to show that the complex spikes occurring at the beginning of the reach movement encode the absolute destination of the reach, and the complex spikes occurring at the end of the short-lasting movements encode the relative errors. Thus, complex spikes convey multiple types of information, consistent with the idea that they contribute both to the generation of movements and to the gradual, long-term improvement of these movements.

Action Potentials↗
PubMed1998

Serum matrix metalloproteinase-2 and its density in men with prostate cancer as a new predictor of disease extension.

We examined whether the serum matrix metalloproteinase-2 (MMP-2) level and MMP-2 density could be predictors of the development and extension of prostate cancer. Serum samples were collected before any clinical treatment from 98 patients with prostate cancer and from 76 patients with benign prostatic hyperplasia (BPH). Control sera were obtained from 70 healthy men. The serum level of MMP-2 was determined by 1-step enzyme immunoassay. A newly defined MMP-2 density parameter was determined by dividing the serum level of MMP-2 by the prostate volume, which was measured by ultrasonography. The mean serum level of MMP-2 in prostate cancer patients was significantly higher than in the control and BPH groups. Furthermore, the serum MMP-2 levels in prostate cancer patients with metastasis were highly elevated compared with those without metastases. The MMP-2 density in pathologically organ-confined prostate cancer was significantly higher than that in BPH. There was a statistically significant difference in the MMP-2 density between pT2N0M0 and pT1N0M0 prostate cancers. Moreover, the serum MMP-2 level correlated well with the clinical course of prostate cancer with bone metastasis. Our results suggest that MMP-2 plays an important role in the development and extension of prostate cancer and that the serum level of MMP-2 and the MMP-2 density indicate prostate cancer extension and are, therefore, useful for the followup of prostate cancer patients.

Aged↗
PubMed1998

Molecular cloning and analysis of the 5'-flanking region of the human bone morphogenetic protein-6 (BMP-6).

We cloned a genomic fragment of the 5'-flanking region of the gene encoding bone morphogenetic protein-6 (BMP-6) and assessed its promoter activity. Primer extension revealed the presence of one major transcription start site 178 bp upstream of the translation start site. The promoter region lacked a canonical TATA box but did contain a GC-rich region. A putative tramtrack responsive element, a Drosophila transcriptional factor regulating the segment polarity, was found in the promoter region. Known steroid hormonal responsive elements, however, were not found. Although BMP-6 is classified as a member of the vgr-1 family, the structure of the promoter was similar to that of BMP-2 and 4.

Base Sequence↗
PubMed1998

Undifferentiated HL-60 cells internalize an antitumor alkyl ether phospholipid more rapidly than resistant K562 cells.

In this study, we confirmed a previous finding that 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (methyl-PAF) expresses higher antineoplastic activity against the promyelocytic leukemia cell line HL-60, than against the erythroleukemic cell line K562, and intended to clarify the reason for this. Using an albumin back-exchange method, we measured the rates of binding and internalization of [3H]methyl-PAF by HL-60 and K562 cells. We found that methyl-PAF associated very rapidly and to similar extents with the two types of cells at low concentrations of extracellular bovine serum albumin, but that when bound to the cell surface, it was internalized into HL-60 cells faster than into K562 cells. The internalization of methyl-PAF by HL-60 cells was concentration-independent, intracellular ATP-independent and susceptible to thiol group-modifying reagents and cytochalasin B. Thus the inward transbilayer movement of methyl-PAF seems to occur by cytochalasin B-sensitive protein-mediated mechanism based on passive diffusion not requiring energy, in which SH-groups of protein play a critical role. We also found that the internalization of 1-hexadecanoyl-2-(4,4-difluoro-5,7- dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (Bodipy-C5-PC), whose structure resembles that of methyl-PAF, into HL-60 cells was faster than that into K562 cells. Using a combination of an albumin back-exchange method and observation by confocal laser scanning microscopy, we next examined the intracellular distribution of this fluorescent phospholipid probe after its internalization. Intracellular membranes, especially those peripheral to nuclei, were fluorescence-labeled in both HL-60 and K562 cells, but fluorescence of the nuclear membranes was weak, suggesting that this probe seems mainly to accumulate in intracellular granules, and may interact directly with several key enzymes for phospholipid metabolism, leading to cell injury. Because the difference between the internalization rates of methyl-PAF in HL-60 and K562 cells was correlated with their different susceptibilities to the cytotoxic effect of methyl-PAF, we suggest that the capacities for uptake of methyl-PAF and its accumulation in intracellular membranes are critical factor for its induction of apoptosis. (c) 1998 Elsevier Science B.V.

Adenosine Triphosphate↗
PubMed1998

Type IIa early gastric cancer with proliferation of xanthoma cells.

We report a type IIa early gastric cancer associated with xanthoma cell proliferation in a 61-year-old man. The patient was admitted to our hospital because of a gastric polyp detected at a medical checkup. An irregular protruding lesion with xanthoma cell proliferation was detected endoscopically. Histological examination showed a well differentiated tubular adenocarcinoma in the mucosa associated with xanthoma cell proliferation. The distribution of the xanthoma cells in the stroma corresponded closely with that of the cancer cells. Neither atypism nor mitotic figures were recognized in the xanthoma cells. In an immunohistochemical study, almost all the xanthoma cells were stained positive for alpha 1-antitrypsin, while relatively few exhibited positive S-100 protein staining. Specific monocyte chemotactic and activating factor immunoreactivity was present only in the xanthoma cells, and not in the cancer cells. On the basis of these findings, it was speculated that the gastric cancer cells may have caused the xanthoma cell proliferation via an autocrine mechanism i.e., by a chemical mediator acting in a paracrine or juxtacrine manner.

Adenocarcinoma↗
PubMed1998

Conservative management of post-operative peritoneal cysts associated with endometriosis.

OBJECTIVE: To describe the usefulness of fine-needle aspiration cytology and drainage, followed by long-acting GnRH-agonist therapy in the management of post-operative peritoneal cysts with endometriosis. METHODS: In six women who were diagnosed as having post-operative peritoneal cysts with endometriosis, fine-needle aspiration cytology and drainage was performed. Thereafter, four patients were treated with long-acting GnRH-agonists for 6 months. Two patients refused the treatment. RESULTS: In all patients fine-needle aspiration yielded specimens which consisted of a population of mesothelial cells. The mean follow-up time was 4 years (range 3.5-5). The four patients treated with long-acting GnRH-agonists show no evidence of recurrence. In two patients who had no additional treatment, the recurrence of the cyst was noted 2 months and 5 months after the drainage. CONCLUSION: Combination of fine-needle aspiration cytology and drainage and subsequent long-acting GnRH-agonist therapy can be a useful conservative management of post-operative peritoneal cysts associated with endometriosis.

Adult↗