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Biomedical subjects

S Nishikawa

Publications and source records attributed to S Nishikawa.

At least 253 records · Page 14Linked to original sources
PubMed1994

Serial water changes in human skeletal muscles on exercise studied with proton magnetic resonance spectroscopy and imaging.

In vivo 1H-magnetic resonance imaging (MRI) enabled us to study the distribution of water in living tissues and to document changes in human skeletal muscles during physical exercise. The purpose of the present study was to determine the total muscle water changes after exercise using water in 1H-MR spectroscopy and to compare these changes to the signal intensity change on T2*-weighted images and/or to the T2 value change. Seven young male volunteers were positioned in a 1.5 T Philips MR imaging system. They were then asked to dorsiflex their ankle joint against a 2 kg weight once every 2 seconds for 2 minutes. The peak height of water declined according to the clearance curve after exercise in all seven cases with the 1H-MRS similar to the signal intensity. The increasing rate at peak height of total muscle water exceeded both the signal intensity and the T2 value because the water peak height on the 1H-MRS included the extracellular water. In addition, we measured the changes in signal intensity in both calf muscles after walking race exercise. The time intensity curves were used to draw a clearance curve for each muscle group after exercise. It was possible to discern which muscle was used most from the T2*-weighted image that was obtained once after exercise.

Adult↗
PubMed1994

1 alpha,25-dihydroxyvitamin D3 stimulation of osteopontin expression in rat clonal dental pulp cells.

Osteopontin (OPN) is a major phosphorylated non-collagenous protein isolated from bone. Rat clonal dental-pulp cell lines RPC-C2A and RDP4-1 produce and secrete OPN as a principal phosphoprotein. 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] is a potent calcitropic hormone which regulates calcified tissue metabolism including the synthesis of extracellular matrix proteins. The effects of 1,25(OH)2D3 on the expression of OPN mRNA and the synthesis of OPN protein by pulp cells in vitro were investigated. In RPC-C2A cells, 1,25(OH)2D3 markedly stimulated synthesis of both [32PO4]- and [35S]-methionine-labelled OPN. Phosphorylated OPN synthesis increased dose-dependently and showed a maximum level at 48 h after addition of 10(-11)-10(-7) M 1,25(OH)2D3. Similar stimulation was also observed in RDP4-1 cells. Northern hybridization analysis revealed that 1,25(OH)2D3 greatly increased the level of OPN mRNA in both pulp cell lines. Examination of the time course of the effects of 1,25(OH)2D3 on the level of OPN mRNA in RPC-C2A cells by dot-blot analysis showed that stimulation was detectable at 24 h and reached a maximum at 48 h after exposure to 10(-7)M 1,25(OH)2D3. These findings indicate that 1,25(OH)2D3 stimulates the production of dental-pulp OPN by a mechanism that involves de novo synthesis and transcriptional control.

Animals↗
PubMed1994

Stromal cell-dependent bone marrow culture with a nearly protein-free defined medium.

We examined the ability of a defined medium mSFO2 to support stromal cell-dependent B lymphopoiesis and myelopoiesis. Both ST2 and PA6 stromal cell lines were able to form monolayers in the presence of mSFO2 alone. While the ST2 monolayer could last for long period of time, either bFGF or EGF were required for the maintenance of the PA6 monolayer with mSFO2. mSFO2 did support the generation of distinct stages of B precursors on the ST2 layer and this culture condition was efficient enough to be used for limiting dilution assay of in vitro clonable B progenitors. While ST2 cell line failed to support long-term myelopoiesis with mSFO2, PA6 in combination of bFGF was able to support sustained generation of various hematopoietic progenitors. Indeed, 20 times increase of IL-3 reactive colony-forming cells was induced upon culturing the normal bone marrow cells on the PA6 layer for 8 days with mSFO2. Because total protein concentration of mSFO2 is only 10 micrograms/ml consisting of transferrin and insulin, the present result that mSFO2 is able to support the proliferation of normal hematopoietic progenitor cells would make an important step towards the standardization of bone marrow culture.

Animals↗
PubMed1994

Inhibition of endoplasmic reticulum (ER)-to-Golgi transport induces relocalization of binding protein (BiP) within the ER to form the BiP bodies.

Immunofluorescence staining of yeast cells with anti-binding protein (BiP) antibodies shows uniform staining of the endoplasmic reticulum (ER). We have found that overproduction of Sec12p, an ER membrane protein, causes a change of BiP distribution within the cell. Upon induction of Sec12p by the GAL1 promoter, the staining pattern of BiP turns into bright dots scattering in the cell, whereas the staining of Sec12p remains to be the typical ER figure. Overproduction of other ER membrane proteins, HMG-CoA reductase or Sed4 protein, does not induce such relocalization of BiP. Pulse-chase experiments and electron microscopy have revealed that the overproduction of Sec12p inhibits protein transport from the ER to the Golgi apparatus. When the transport is arrested by one of the sec mutations that block the ER-to-Golgi step at the restrictive temperature, the BiP staining also changes into the punctate pattern. In contrast, the sec mutants that block later or earlier steps of the secretory pathway do not induce such change of BiP localization. These observations indicate that relocalization of BiP is caused by the inhibition of ER-to-Golgi transport. Using immunoelectron microscopy, we have found that the punctate staining is because of the accumulation of BiP in the restricted region of the ER, which we propose to call the "BiP body." This implicates existence of ER subdomains in yeast. A vacuolar protein, proteinase A, appears to colocalize in the BiP body when the ER-to-Golgi transport is blocked, suggesting that the BiP body may have a role as the site of accumulation of cargo molecules before exit from the ER.

Base Sequence↗
PubMed1994

Implication of the common gamma chain of the IL-7 receptor in intrathymic development of pro-T cells.

The effects of IL-7 on the growth and differentiation of thymocytes were analyzed using murine fetal thymus organ cultures (FTOC) in the presence of mAbs specific for the conventional IL-7 receptor (IL-7R) and for the common gamma (gamma c) chain. In FTOC, the development of CD4-CD8- double-negative thymocytes to CD4+CD8+ double-positive (DP) and CD4+ or CD8+ single-positive (SP) cells was not completely blocked by adding these mAbs, although cell growth was reduced by the treatment. To define a developing stage sensitive to the mAbs, most immature thymocytes, Pgp-1+ c-kit+ cells, were cultured in 2-deoxyguanosine treated fetal thymus. In the presence of both mAbs in the culture, neither DP nor SP thymocytes developed whereas either of the mAbs partially blocked their development. These results indicate that the gamma c chain is involved in early T cell development as an indispensable subunit of the functional IL-7 receptor complex.

Animals↗
PubMed1994

Mutational analysis of the Sar1 protein, a small GTPase which is essential for vesicular transport from the endoplasmic reticulum.

SAR1 encodes a 21-kDa GTPase, which is required for vesicle formation from the endoplasmic reticulum in yeast. Although it belongs to the expanding small GTPase superfamily, there are interesting structural features that are unique to the Sar1 protein. We performed a site-directed mutational study to identify the amino acid residues that are essential for the Sar1p function. Among seven mutants we constructed, four are functionless by themselves, while two confer temperature sensitivity to cells. When the mutant proteins are overproduced in wild-type cells, all of these six show a dominant negative effect on cell growth. The replacement by serine of the only cysteine residue present in Sar1p caused no significant change in the growth phenotype. These findings are not only important for analyzing the mechanism of the Sar1p action in yeast, but will also be very useful for studying the function of Sar1p counterparts in higher eukaryotes.

Amino Acid Sequence↗
PubMed1994

Clinical evaluation of a potassium nitrate dentifrice for the treatment of dentinal hypersensitivity.

The effectiveness of a 5% potassium nitrate dentifrice as a daily home treatment for dentinal hypersensitivity was evaluated in a double-blind study in 36 Japanese subjects who complained of cold and/or tactile hypersensitivity. The subjects were divided into 2 groups, with 18 being given a 5% potassium nitrate dentifrice (treated group) and the other 18 a vehicle paste (control group). Both groups were instructed to brush their teeth 2 x a day. The hypersensitivity levels of the affected teeth were assessed by 2 stimuli, one tactile and the other cold air, and by the perception of pain. The results of all 3 assessment methods indicated that the potassium nitrate dentifrice significantly decreased the level of hypersensitivity at weeks 4, 8, and 12. In the treated group, a rapid decrease of positive scores for both the cold air stimulus and the subjective symptoms appeared from week 2. Although a significant decrease of the assessment score was also observed in the control group, the reduction rate of the score was much greater in the treated group by all 3 assessment methods at weeks 4, 8, and 12. Complete relief of subjective symptoms throughout the 12 weeks' examination was noted in 67% of the subjects in the treated group, but in only 6% in the control group. These results suggest the usefulness of a 5% potassium nitrate dentifrice in Japanese patients with dentinal hypersensitivity.

Adult↗
PubMed1994

Progesterone and EGF inhibit mouse mammary gland prolactin receptor and beta-casein gene expression.

Regulation of mouse mammary gland long-form prolactin receptor (PRL-RL) mRNA levels by progesterone and epidermal growth factor (EGF) and the relationship between PRL-RL and beta-casein gene expression were examined in vivo and in vitro. PRL-RL and beta-casein mRNA levels increased approximately 6- and 15-fold from the pregnant to the lactating period, respectively, when normalized to the level of beta-actin mRNA. Ovariectomy of pregnant mice rapidly reduced the serum concentration of progesterone and increased the level of PRL-RL and beta-casein mRNAs approximately three- and fourfold compared with sham-operated animals 24 h after the operation. Injection of progesterone, but not estrogen, inhibited the increase in both mRNA levels. PRL-RL and beta-casein mRNA levels in cultured mammary epithelium increased in response to insulin, hydrocortisone, and prolactin, whereas progesterone or EGF caused inhibition. The combination of EGF and progesterone produced a greater inhibition than either hormone alone. These results indicate that both progesterone and EGF serve as negative regulators of lactogenesis.

Animals↗
PubMed1994

Involvement of the c-kit receptor in the adhesion of hematopoietic stem cells to stromal cells.

The proto-oncogene c-kit, encoding a receptor-type tyrosine kinase, is allelic with the W locus of the mouse. The stromal cell line OP9, capable of supporting long-term hematopoiesis, was newly established from a newborn B6C3F1-op/op mouse calvaria. When bone marrow cells of WBB6F1-W/Wv mice were cocultured with the OP9 cells in liquid medium, hematopoiesis declined to a level one-thousandth of that in the cocultures of bone marrow cells of WBB6F1-+/+ mice and stromal cells by day 21. In contrast, when bone marrow cells of W/Wv mice were cocultured with OP9 cells in semisolid medium, at least 61% of the number of colonies were detected until the end of our observation period of 42 days when compared with that in control cocultures, although colonies formed by hematopoietic stem cells of W/Wv mice were significantly smaller than those of normal stem cells. After a 24-hour incubation with OP9 cells, fewer stem cells of W/Wv mice than normal ones adhered to the stromal cells. Adhesion of normal stem cells to stromal cells was inhibited by the addition of an antagonists anti-c-kit monoclonal antibody, ACK2. These results demonstrate that the c-kit receptor plays an important role not only in the proliferative response of hematopoietic stem cells but also in their adhesion to stromal cells.

Animals↗
PubMed1994

A transient expression of alpha B-crystallin in the developing rat retinal pigment epithelium.

PURPOSE: To investigate the expression of alpha B-crystallin in the developing rat retina by immunohistochemical techniques. METHODS: Rat eyes were enucleated on embryonic day 18 and on postnatal days 1, 4, 9, 15, 19, and 90. The avidin-biotin-peroxidase technique was applied to show alpha B-crystallin immunostaining. After the cornea and lens were removed, the developing rat eyeballs were solubilized by 1% sodium dodecyl sulfate. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) were performed to detect alpha B-crystallin in the extracts. RESULTS: From postnatal day 4 to postnatal day 9, alpha B-crystallin was present in retinal pigment epithelium, but it disappeared after postnatal day 15. alpha A-crystallin immunoreactivity was negative in retina and retinal pigment epithelium throughout development. The results of Western blot analysis and ELISA coincided with those of the immunostaining. CONCLUSIONS: These results show that retinal pigment epithelium in mammalian rat retina demonstrates alpha B-crystallin expression in the early developmental stages but loses it after 15 days of age.

Animals↗
PubMed1994

[IL-7].

IL-7, a 25-kDa glycoprotein was cloned as a cytokine derived from bone marrow stromal cells, which induces proliferation of pre-B cells. IL-7 was found to be produced by the thymus, spleen, kidney and bone marrow stromal cells and induces proliferation of immature B and T cells. It has no effects on mature B cells but induce proliferation of mature T cells IL-7 R is expressed during the stages; B 220+ sIgM- of pre-B cells but not sIgM+ mature B cells, and also expressed CD4-8-, CD4+8- CD4-8+ T cells. It is shown that IL-7 can act only on these IL7R+ cells functionally in vitro and in vivo. We and other group demonstrated that IL-7, when used in combination with stromal cells or SCF can induce proliferation of pre-B cells. Recently we generated successfully SCF and IL-7 dependent pre-B cell lines, and now have used them for cell cycle analysis. To date, it was found that IL-7 is required for G1 to S transition. IL-7 also acts as a co-stimulus for the proliferation of antigen- or mitogen-activated T cells and can induce CTL response and LAK activity.

Animals↗
PubMed1993

Enhancement of the cleavage rates of DNA-armed hammerhead ribozymes by various divalent metal ions.

In order to characterize structure-function relationships, the kinetic behavior of chimeric RNA/DNA ribozyme was compared with that of all RNA ribozyme. Determined kcat values were proven to represent the chemical-cleavage step and not the product-dissociation step. In agreement with the finding by Dahm and Uhlenbeck [Biochemistry 30, 9464-9469 (1991)], various metal ions, including Co2+ and Ca2+ with the ionic radius of 0.65 and 1.0 A, respectively, could support hammerhead cleavage for both types of ribozyme. Measurements of kinetic parameters in the presence of various divalent metal ions revealed that DNA arms always enhanced kcat values. Chemical-probing data using dimethylsulfate indicated that the catalytic-loop structures of all-RNA and chimeric ribozymes were nearly identical with the exception of enhanced termination of primer extension reactions at C3 in the case of the chimeric ribozyme. These observations and others demonstrate that DNA substitution in non-catalytic-loop regions increases chemical-cleavage activity, possibly with an accompanying very subtle change in the structure.

Base Sequence↗
PubMed1993

High-resolution NMR study of a synthetic oligoribonucleotide with a tetranucleotide GAGA loop that is a substrate for the cytotoxic protein, ricin.

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Its substrate domain forms a double helical stem and a 17-base loop that includes the sequence GAGA, the second adenosine of which corresponds to A4324. Recently, studies of mutant RNAs have shown that the four-nucleotide loop, GAGA, can function as a substrate for ricin. To investigate the structure that is recognized by ricin, we studied the properties of a short synthetic substrate, the dodecaribonucleotide r-CUCAGAGAUGAG, which forms a RNA hairpin structure with a GABA loop and a stem of four base pairs. The results of NMR spectroscopy allowed us to construct the solution structure of this oligonucleotide by restrained molecular-dynamic calculations. We found that the stem region exists as an A-form duplex. 5G and 8A in the loop region form an unusual G:A base pair, and the phosphodiester backbone has a turn between 5G and 6A. This turn seems to help ricin to gain access to 6A which is the only site of depurination in the entire structure. The overall structure of the GAGA loop is similar to those of the GAAA and GCAA loops that have been described but that are not recognized by ricin. Therefore, in addition to the adenosine at the depurination site, the neighboring guanosine on the 3' side (7G) may also play a role in the recognition mechanism together with 5G and 8A.

Base Composition↗
PubMed1993

Importance of independence in ribozyme reactions: kinetic behavior of trimmed and of simply connected multiple ribozymes with potential activity against human immunodeficiency virus.

The kinetic behavior of ribozymes derived from two types of multiple-ribozyme expression vector were examined. In some cases, multiple ribozymes were expressed as a single RNA molecule and all the ribozymes were simply connected in tandem (connected type). In other cases, multiple ribozymes were flanked by cis-acting ribozymes at both their 5' and 3' ends so that, upon transcription, multiple ribozymes were trimmed at both their 5' and 3' ends, with resultant liberation of multiple independent ribozymes (shotgun type). When levels of ribozyme expression were examined for the shotgun-type vector, the level of the ribozyme transcript was found to be proportional to the number of units (n) connected in tandem. Accordingly, the activities of the shotgun-type ribozymes, in terms of the cleavage of HIV-1 RNA in vitro, were also found to be proportional to the number of units connected in tandem (n). By contrast, the activities of the connected-type ribozymes reached plateau values at around n = 3. These results indicate that, when the shotgun-type expression system is used, it is theoretically possible to generate various independent ribozymes, each specific for a different target site, without sacrificing the activity of any individual ribozyme.

Antiviral Agents↗
PubMed1993

Stage-specific expression of c-kit protein by murine hematopoietic progenitors.

We have analyzed c-kit expression by hematopoietic progenitors from normal and 5-fluorouracil (5-FU)-treated mice by staining with monoclonal anti-c-kit antibody ACK-4. Marrow cells that were enriched for progenitors by a combination of metrizamide density separation and negative immunomagnetic selection with lineage-specific monoclonal antibodies (MoAbs) were separated into three populations based on the level of c-kit expression, c-kit(high), c-kit(low), and c-kit-. The majority of colony-forming cells from normal mice were in c-kit(high) population, whereas most of the progenitors from 5-FU-treated mice were in the c-kit(low) population. Optimal colony formation from c-kit(low) cells from 5-FU-treated mice required the interactions of at least two factors among interleukin-3 (IL-3), IL-11 and steel factor (SF) whereas colony formation from c-kit(high) cells of normal mice was supported well by IL-3 alone. Blast cells that were derived from 5-day culture of c-kit(low) post 5-FU cells were c-kit(high). These observations suggest that the primitive hematopoietic progenitors in cell cycle dormancy are c-kit(low) whereas actively cell cycling maturer progenitors are c-kit(high). Mature cells, with the exception of mast cells, derived from secondary culture of the c-kit(high) blast cells expressed little, if any, c-kit. These results are consistent with a model in which c-kit expression progresses from low levels on primitive, dormant multipotent progenitors to high levels on later, actively cycling progenitors, and finally, decreases to very low or undetectable levels on most mature blood cells, with the exception of mast cells.

Animals↗
PubMed1993

Expression and function of the interleukin 7 receptor in murine lymphocytes.

A monoclonal antibody, A7R34, that recognizes the high-affinity interleukin 7 receptor (IL-7Ra) and blocks the binding between IL-7 and IL-7Ra has been produced. Cell surface staining with A7R34 demonstrated that IL-7Ra is expressed in both B- and T-cell lineages. In the bone marrow, immature B-lineage cells that do not express surface IgM were IL-7Ra+. In the thymus, IL-7Ra was detected in CD4-8- T cells and also in CD4 or CD8 single-positive cells but not in CD4+8+ double-positive cells. In the peripheral lymphoid tissues, both CD4 and CD8 single-positive cells were the major cell types that express IL-7Ra. Addition of A7R34 to a long-term B-precursor-cell culture inhibited proliferation of the B-lineage cells, indicating that IL-7 is an absolute requirement for in vitro B-cell genesis. Consistent with this in vitro result, continuous injection of A7R34 into an adult mouse resulted in a decrease of B-precursor cells and also of thymocytes, whereas a considerable fraction of mature B and T cells in the peripheral tissues persisted over 2 weeks of the experiment. When A7R34 injection is started from day 14 of gestation, it is possible to produce mice that lack B cells. These results indicate that IL-7 is an essential molecule for generation of both B and T cells in murine bone marrow and thymus, respectively. Moreover, IL-7Ra would be the sole receptor system regulating these processes.

Animals↗