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Biomedical subjects

H Ichikawa

Publications and source records attributed to H Ichikawa.

At least 127 records · Page 7Linked to original sources
PubMed1999

Osteocalcin-immunoreactive primary sensory neurons in the rat spinal and trigeminal nervous systems.

Osteocalcin-immunoreactivity (OC-ir) was examined in spinal and trigeminal primary sensory neurons of the adult rat. Sixteen percent of dorsal root ganglion (DRG) neurons were immunoreactive (ir) for this protein. These neurons were mostly large and measured 594-4583 microm(2) (mean+/-S.D.=2243+/-748 microm(2)). Thirty-four percent of DRG neurons >1200 microm(2) and 4% of those in the range 600-1200 microm(2) showed the ir. Virtually all DRG neurons <600 microm(2) were devoid of OC-ir. In the trigeminal ganglion (TG), 25% of neurons exhibited the ir. Such neurons were of various sizes (range=156-2825 microm(2), mean+/-S.D.=1234+/-543 microm(2)). Forty-five percent of TG neurons >800 microm(2) and 6% of those <400 microm(2) were immunoreactive for this protein. Twelve percent of TG neurons in the range 400-800 microm(2) showed the ir. In the mesencephalic trigeminal tract nucleus (Mes5), 63% of primary sensory neurons contained OC-ir. Virtually all OC-ir DRG and Mes5 neurons co-expressed parvalbumin-ir but not CGRP-ir. On the other hand, only 31% of OC-ir neurons co-expressed parvalbumin-ir and 10% co-expressed CGRP-ir in the TG. The present study indicates that DRG and Mes5 primary sensory neurons co-expressing OC- and parvalbumin-irs are spinal and trigeminal proprioceptors. OC-ir TG neurons which co-express parvalbumin- and CGRP-irs appear to include low-threshold mechanoreceptors and nociceptors, respectively.

Animals↗
PubMed1999

Microagglomeration of pulverized pharmaceutical powders using the Wurster process I. Preparation of highly drug-incorporated, subsieve-sized core particles for subsequent microencapsulation by film-coating.

A novel agglomeration process of pulverized pharmaceutical powders into subsieve-sized agglomerates (microagglomeration) was designed for manufacturing highly drug-incorporated core particles for subsequent microencapsulation by film-coating. The microagglomeration of pulverized phenacetin powder, whose mass median diameter was 9 microm, was performed by spraying an aqueous colloidal dispersion of acrylic polymer, Eudragit(R) RS30D, as a binding/coating agent using a spouted bed assisted with a draft tube (the Wurster process), and the effect of process variables was examined. An appropriate spray liquid flow rate made it possible to produce microagglomerates of 20-50 microm with 60% yield. However, 10% of the product still survived as particles smaller than 10 microm even at the elevated liquid flow rate. In contrast, the survived particles smaller than 10 microm tended to be predominantly reduced to 2%, while coarse agglomerates larger than 53 microm were not excessively produced, by additionally setting a fixed bed of glass beads in the spouted bed apparatus. The length of the draft tube influenced compaction of the agglomerates as well as their surface-smoothening. Equipping the fixed bed of the glass beads and the long draft tube in the spouted bed allowed us to prepare microagglomerates of 20-50 microm at yield of 55% applicable as highly drug-incorporated, free-flowing, surface-smoothed, narrowly size-distributed core particles for subsequent microencapsulation by film-coating.

Acrylic Resins↗
PubMed1999

Apoptotic cascade of neurons in the subcortical sensory relay nuclei following the neonatal infraorbital nerve transection.

A terminal transferase-mediated dUTP nick end labeling (TUNEL) method was utilized for detection of neuronal death in the subcortical relay nuclei of the trigeminosensory system following the infraorbital nerve transection in newborn rats. At 18-24 h after injury, numerous TUNEL-positive profiles were found within the ventroposteromedial thalamic nucleus (VPM) contralateral to the injury, whereas the VPM on the ipsilateral side and of the age-matched normal control contained only a few profiles per section. Electron microscopy revealed that the TUNEL-positive profiles were apoptotic neurons. The ventral part of the ipsilateral brainstem sensory trigeminal nuclear complex (the nucleus principalis, and the subnuclei oralis and interpolaris) exhibited statistically significant 65-70% increase in number of apoptotic neurons compared to the contralateral side. Taken together with our previous study [T. Sugimoto, C. Xiao, H. Ichikawa, Neonatal primary neuronal death induced by capsaicin and axotomy involves an apoptotic mechanism, Brain Res. 807 (1998) 147-154], the present results demonstrated a cascade of apoptosis in the primary, secondary and tertiary order sensory neurons along the neuroaxis.

Animals↗
PubMed1999

Negative regulation by the Bacillus subtilis GerE protein.

GerE is a transcription factor produced in the mother cell compartment of sporulating Bacillus subtilis. It is a critical regulator of cot genes encoding proteins that form the spore coat late in development. Most cot genes, and the gerE gene, are transcribed by sigmaK RNA polymerase. Previously, it was shown that the GerE protein inhibits transcription in vitro of the sigK gene encoding sigmaK. Here, we show that GerE binds near the sigK transcriptional start site, to act as a repressor. A sigK-lacZ fusion containing the GerE-binding site in the promoter region was expressed at a 2-fold lower level during sporulation of wild-type cells than gerE mutant cells. Likewise, the level of SigK protein (i. e. pro-sigmaK and sigmaK) was lower in sporulating wild-type cells than in a gerE mutant. These results demonstrate that sigmaK-dependent transcription of gerE initiates a negative feedback loop in which GerE acts as a repressor to limit production of sigmaK. In addition, GerE directly represses transcription of particular cot genes. We show that GerE binds to two sites that span the -35 region of the cotD promoter. A low level of GerE activated transcription of cotD by sigmaK RNA polymerase in vitro, but a higher level of GerE repressed cotD transcription. The upstream GerE-binding site was required for activation but not for repression. These results suggest that a rising level of GerE in sporulating cells may first activate cotD transcription from the upstream site then repress transcription as the downstream site becomes occupied. Negative regulation by GerE, in addition to its positive effects on transcription, presumably ensures that sigmaK and spore coat proteins are synthesized at optimal levels to produce a germination-competent spore.

Bacillus subtilis↗
PubMed1999

Peptide 19 in the dorsal root ganglion and the mesencephalic trigeminal tract nucleus of the adult rat.

Peptide 19-immunoreactivity (PEP 19-ir) was examined in the dorsal root ganglion (DRG) and the mesencephalic trigeminal tract nucleus (Mes5) of the adult rat. Thirty-eight percent of DRG cells were immunoreactive (ir) for PEP 19. These neurons were small to large and measured 167-4583 micron2 (mean+/-S.D.=2048+/-913 micron2). Seventy-five percent of DRG cells >2000 micron2 and 15% of those <1000 micron2 exhibited PEP 19-ir. Thirty-six percent of DRG neurons in the range 1000-2000 micron2 showed the ir. In the Mes5, 87% of primary neurons were ir for this peptide. Muscle spindles in the soleus and masseter muscles contained PEP 19-ir spiral axon terminals. Double immunofluorescence methods revealed the co-expression of PEP 19 and calcium binding proteins. Eighty-six percent of parvalbumin-ir neurons exhibited PEP 19-ir. Conversely, 60% of PEP 19-ir neurons showed parvalbumin-ir. The cell size analysis revealed that 55% of PEP 19-ir neurons >600 micron2 showed parvalbumin-ir and that all PEP 19-ir neurons <600 micron2 were devoid of it. Ninety percent of PEP 19-ir DRG neurons showed S100-ir, whereas 60% of S100-ir ones co-expressed PEP 19-ir. In the Mes5, virtually all PEP 19-ir primary neurons exhibited parvalbumin-ir. The co-expression of PEP 19 and S100 could not be observed in the nucleus. The present study indicates that PEP 19-ir neurons which co-express parvalbumin-ir are proprioceptors in the spinal and the trigeminal systems. PEP 19-ir small DRG neurons without S100-ir are probably exteroceptors and may include unmyelinated nociceptors.

Animals↗
PubMed1999

Calmodulin functions as an activator of Pur alpha binding to single-stranded purine-rich DNA elements (PUR elements).

Pur alpha is a single stranded DNA-binding protein and binds to a consensus sequence (GGN)n. We have reported that the DNA-binding activity of a single stranded cyclic AMP response element-binding protein (ssCRE-BP) is suppressed in cerebellum treated chronically with morphine, ssCRE-BP is identical to Pur alpha and the DNA binding activity of Pur alpha is markedly enhanced by a heat stable activator in the nuclear extract. In this report, we purified this activator. The amino acid composition and partial amino acid sequence were determined to be identical to those of calmodulin (CaM), which enhanced the binding of GST-Pur alpha to various PUR elements in the 5' non-coding regions of the neuropeptide Y, myelin basic protein and nicotinic Ach receptor beta 4 subunit genes. The data suggest a novel gene expression pathway mediated by Ca/CaM-Pur alpha which may regulate a variety of genes in addition to those regulated through the CREB pathway.

Animals↗
PubMed1999

Electron microscopic demonstration of nick end-labeled DNA fragments during capsaicin-induced apoptosis of trigeminal primary neurons in neonatal rats.

DNA fragmentation was induced in the trigeminal ganglion of newborn rats by subcutaneous capsaicin injection (50 mg/kg). Twenty-four hours later, numerous roundish profiles were intensely labeled by both a DNA polymerase I-mediated nick translation method and a terminal transferase-mediated tailing method. Direct electron microscopic examination of labeled profiles indicated that the labeled profiles were neurons at earlier stages of apoptosis. DNA fragmentation signal was first detected in the nucleoplasm and later spread to the cytoplasm. The cell finally disintegrated forming many small apoptotic bodies. DNA fragmentation signal in the apoptotic bodies was readily labeled by the tailing but not the translation method.

Analysis of Variance↗
PubMed1999

Characterization of auxin-binding protein 1 from tobacco: content, localization and auxin-binding activity.

There is evidence that auxin-binding protein 1 (ABP1) is an auxin receptor on the plasma membrane. Maize (Zea mays L.) possesses a high level of auxin-binding activity due to ABP1, but no other plant source has been shown to possess such an activity. We have analyzed the ABP1 content of tobacco (Nicotiana tabacum L.) to examine whether or not the ABP1 content of maize is exceptionally high among plants. The ABP1 content of tobacco leaves was shown by quantitative immunoblot analysis to be between 0.7 and 1.2 microg ABP1 per gram of fresh leaf. This value is comparable to the reported value in maize shoots, indicating that ABP1 is present at a similar level in both monocot and dicot plants. The ABP1 content of tobacco leaves was increased up to 20-fold by expression of a recombinant ABP1 gene, and decreased to half of the original value by expression of the antisense gene. Although ABP1 was found mainly in the endoplasmic reticulum fraction, a secreted protein showing a molecular size and epitopes similar to intracellular ABP1 was also detected in the culture medium of tobacco leaf disks. The secretion of this protein was dependent on the expression level of the ABP1 gene.

Animals↗
PubMed1999

Myocardial protection with endogenous overexpression of manganese superoxide dismutase.

BACKGROUND: Superoxide dismutase (SOD) is a potent candidate for myocardial protection against ischemia-reperfusion injury; however, its clinical significance by means of exogenous administration remains controversial. METHODS: To determine a role of endogenously overexpressed manganese SOD (Mn-SOD) in myocardial tolerance, rat hearts were transfected with Mn-SOD gene (group M) or no gene (group C) through intracoronary infusion of hemagglutinating virus of Japan (HVJ) liposome. Each group was divided into two subgroups to be subjected to ischemia-reperfusion using Langendorff apparatus with (subgroups M+ and C+) or without (M- and C-) administration of recombinant SOD. RESULTS: Mn-SOD overexpression was confirmed in M with ELISA, activity measurement, and immunohistochemistry. The highest recoveries of maximum and minimum dp/dt and the least creatine phosphokinase (CPK) leakage were observed in M+. These recoveries were higher in M- than in C- and C+. CONCLUSIONS: Thus, endogenous overexpression of Mn-SOD improved myocardial tolerance and its protective effect was enhanced by exogenous administration of SOD. These results suggest a possible strategy for myocardial protection with SOD: a combination of endogenous introduction through gene transfer with exogenous administration.

Animals↗
PubMed1999

Development of a non-radioactive endpoint in a modified local lymph node assay.

A murine local lymph node assay (LLNA) has been developed as an alternative to guinea pig models for contact sensitization testing. Although the LLNA appears to be a little less sensitive than the most stringent of guinea pig assays, it provides a rapid, objective, quantitative and cost-effective method for screening strong contact sensitizers and has advantages with respect to animal welfare. However, a potential disadvantage is the need for the use of radioactive material. We have reported previously that an ex vivo assay based on similar principles to the original in vivo LLNA, but using a non-radioactive endopoint, was valid for the prediction of strong sensitizers. This ex vivo assay was not sensitive enough to allow prediction of moderately potent ones. In this study, we propose a new parameter, Corrected IL-2 Index (CII), for the prediction of moderate sensitizers. To obtain CII the IL-2 release in the supernatant of the cell culture is corrected for lymph node weight ratio and ratio of CD4-positive subset. We found that CII predicted the allergenicity of moderate sensitizers, including the ones recommended by the OECD in guideline 406, such as mercaptobenzothiazole and hexyl cinnamic aldehyde. The allergenicity of metal salts, such as potassium dichromate, ammonium tetrachloroplatinate and cobalt chloride, was also predicted by the CII. We conclude that the use of CII as an index significantly increases the sensitivity of the ex vivo method so that moderate sensitizers may also be detected.

Allergens↗
PubMed1999

Chitosan-gadopentetic acid complex nanoparticles for gadolinium neutron-capture therapy of cancer: preparation by novel emulsion-droplet coalescence technique and characterization.

PURPOSE: The gadopentetic acid (Gd-DTPA)-loaded chitosan nanoparticles (Gd-nanoCPs) were prepared for gadolinium neutron-capture therapy (Gd-NCT) and characterized and evaluated as a device for intratumoral (i.t.) injection. METHODS: Gd-nanoCPs were prepared by a novel emulsion-droplet coalescence technique. The effects of the deacetylation degree of chitosan and Gd-DTPA concentration in chitosan medium on the particle size and the gadolinium content in Gd-nanoCPs were examined. In vitro Gd-DTPA release from Gd-nanoCPs was evaluated using an isotonic phosphate-buffered saline solution (PBS, pH 7.4) and human plasma. In vivo Gd-DTPA retention in the tumor after i.t. injection of Gd-nanoCPs was estimated on mice bearing s.c. B16F10 melanoma. RESULTS: Gd-nanoCPs with the highest Gd content, which were obtained using 100% deacetylated chitosan in 15% Gd-DTPA aqueous solution, were 452 nm in diameter and 45% in Gd-DTPA content. A lower deacetylation degree of chitosan led to an increase in particle size and a decrease in Gd-DTPA content in Gd-nanoCPs. As Gd-DTPA concentration in the chitosan solution increased, Gd-DTPA content in Gd-nanoCPs increased but the particle size did not vary. Gd-DTPA loaded to Gd-nanoCPs was hardly released over 7 days in PBS (1.8%) despite the high water solubility of Gd-DTPA. In contrast, 91% of Gd-DTPA was released in plasma over 24 hours. When Gd-nanoCPs were i.t. injected, 92% of Gd-DTPA injected effectually without outflow was held in the tumor tissue for 24 hours, which was different from the case of gadopentetate solution injection (only 1.2%). CONCLUSIONS: Gd-nanoCPs highly incorporating Gd-DTPA were successfully prepared by the emulsion-droplet coalescence technique. Their releasing properties and their ability for long-term retention of Gd-DTPA in the tumor indicated that Gd-nanoCPs might be useful as an i.t. injectable device for Gd-NCT.

Animals↗
PubMed1999

Probiotic bacteria stimulate gut epithelial cell proliferation in rat.

Probiotics are used for various intestinal diseases. However, their effects on gut epithelial cell proliferation have not been investigated. We administered 10(7) colony-forming units of Lactobacillus casei or Clostridium butyricum, or no probiotics (control) by gastric intubation once a day for seven days to rats fed an elemental diet. We estimated the crypt cell production rate of the jejunum, ileum, cecum, and distal colon. We also quantified cecal bacteria. Both probiotics increased the crypt cell production rate of the jejunum and ileum by 25-40%, of the cecum by 70%, and of the distal colon by more than 200% compared with control. Only minor variance in the cecal bacterial composition existed among the three groups. Probiotics enhanced gut epithelial cell proliferation in rats fed an elemental diet.

Animals↗
PubMed1999

Control of sigma factor activity during Bacillus subtilis sporulation.

When starved, Bacillus subtilis undergoes asymmetric division to produce two cell types with different fates. The larger mother cell engulfs the smaller forespore, then nurtures it and, eventually, lyses to release a dormant, environmentally resistant spore. Driving these changes is a programme of transcriptional gene regulation. At the heart of the programme are sigma factors, which become active at different times, some only in one cell type or the other, and each directing RNA polymerase to transcribe a different set of genes. The activity of each sigma factor in the cascade is carefully regulated by multiple mechanisms. In some cases, novel proteins control both sigma factor activity and morphogenesis, co-ordinating the programme of gene expression with morphological change. These bifunctional proteins, as well as other proteins involved in sigma factor activation, and even precursors of sigma factors themselves, are targeted to critical locations, allowing the mother cell and forespore to communicate with each other and to co-ordinate their programmes of gene expression. This signalling can result in proteolytic sigma factor activation. Other mechanisms, such as an anti-sigma factor and, perhaps, proteolytic degradation, prevent sigma factors from becoming active in the wrong cell type. Accessory transcription factors modulate RNA polymerase activity at specific promoters. Negative feedback loops limit sigma factor production and facilitate the transition from one sigma factor to the next. Together, the mechanisms controlling sigma factor activity ensure that genes are expressed at the proper time and level in each cell type.

Adenosine Triphosphate↗
PubMed1999

Quantitative polymerase chain reaction using an external control mRNA for determination of gene expression in a heterogeneous cell population.

Gene expression can be evaluated quantitatively by conventional RT-PCR or Northern blotting with the aid of a correction based on the expression of an internal control gene. However, this approach is not suitable for quantitating gene expression in a group of heterogeneous cell subsets, because the internal control gene expression may vary among the subsets. Therefore, we developed a new method for quantitative PCR using rat poly(A)+ RNA as an external control. We used this method to investigate cytokine gene expression in lymph node cells from mice during the induction of contact hypersensitivity. Expression of the murine glyceraldehydephosphate dehydrogenase (GAPDH) gene, a candidate internal control, was not constant in cells from trinitrochlorobenzene- and vehicle-applied animals, suggesting that GAPDH gene expression changes in heterogeneous lymph node-cell subsets during induction of contact hypersensitivity. Therefore, we decided to use rat GAPDH mRNA as an external control. Cytokine gene expression was measured by quantitative PCR and was corrected based on external rat GAPDH cDNA. The reliability of this quantitative PCR was superior to that of the conventional method with an internal control.

Animals↗
PubMed1999

Dual transforming activities of the FUS (TLS)-ERG leukemia fusion protein conferred by two N-terminal domains of FUS (TLS).

The FUS (TLS)-ERG chimeric protein associated with t(16;21)(p11;q22) acute myeloid leukemia is structurally similar to the Ewing's sarcoma chimeric transcription factor EWS-ERG. We found that both FUS-ERG and EWS-ERG could induce anchorage-independent proliferation of the mouse fibroblast cell line NIH 3T3. However, only FUS-ERG was able to inhibit the differentiation into neutrophils of a mouse myeloid precursor cell line L-G and induce its granulocyte colony-stimulating factor-dependent growth. We constructed several deletion mutants of FUS-ERG lacking a part of the N-terminal FUS region. A deletion mutant lacking the region between amino acids 1 and 173 (exons 1 to 5) lost the NIH 3T3-transforming activity but retained the L-G-transforming activity. On the other hand, a mutant lacking the region between amino acids 174 and 265 (exons 6 and 7) lost the L-G-transforming activity but retained the NIH 3T3-transforming activity. These results indicate that the N-terminal region of FUS contains two independent functional domains required for the NIH 3T3 and L-G transformation, which we named TR1 and TR2, respectively. Although EWS intrinsically possessed the TR2 domain, the EWS-ERG construct employed lacked the EWS sequence containing this domain. Since the TR2 domain is always found in chimeric proteins identified from t(16;21) leukemia patients but not in chimeric proteins from Ewing's sarcoma patients, it seems that the TR2 function is required only for the leukemogenic potential. In addition, we identified three cellular genes whose expression was altered by ectopic expression of FUS-ERG and found that these are regulated in either a TR1-dependent or a TR2-dependent manner. These results suggest that FUS-ERG may activate two independent oncogenic pathways during the leukemogenic process by modulating the expression of two different groups of genes simultaneously.

3T3 Cells↗
PubMed1999

Effect of selective proteasome inhibitors on TNF-induced activation of primary and transformed endothelial cells.

The objective of this study was to assess the effects of two structurally distinct yet selective proteasome inhibitors (PS-341 and lactacystin) on leukocyte adhesion, endothelial cell adhesion molecule (ECAM) expression, and nuclear factor-kappaB (NF-kappaB) activation in tumor necrosis factor (TNF)-alpha-stimulated human umbilical vein endothelial cells (HUVEC) and the transformed, HUVEC-derived, ECV cell line. We found that TNF (10 ng/ml) significantly enhanced U-937 and polymorphonuclear neutrophil (PMN) adhesion to HUVEC but not to ECV; TNF also significantly enhanced surface expression of vascular cell adhesion molecule 1 and E-selectin (in HUVEC only), as well as intercellular adhesion molecule 1 (ICAM-1; in HUVEC and ECV). Pretreatment of HUVEC with lactacystin completely blocked TNF-stimulated PMN adhesion, partially blocked U-937 adhesion, and completely blocked TNF-stimulated ECAM expression. Lactacystin attenuated TNF-stimulated ICAM-1 expression in ECV. Pretreatment of HUVEC with PS-341 partially blocked TNF-stimulated leukocyte adhesion and ECAM expression. These effects of lactacystin and PS-341 were associated with inhibitory effects on TNF-stimulated NF-kappaB activation in both HUVEC and ECV. Our results demonstrate the importance of the 26S proteasome in TNF-induced activation of NF-kappaB, ECAM expression, and leukocyte-endothelial adhesive interactions in vitro.

Acetylcysteine↗
PubMed1999

Immunohistochemical demonstration of neuropeptides in the articular disk of the human temporomandibular joint.

The distribution of calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) was examined in the human temporomandibular articular disk using an indirect immunofluorescent method. All examined neuropeptides were observed in nerve fibers in the disk. These fibers were mostly located in the loose fibrous tissue of the posterior part (posterior attachment) and in the lateral region of the anterior part (anterolateral loose fibrous tissue) of the disk. The posterior attachment and anterolateral loose fibrous tissue occasionally contained isolated nerve fibers which usually showed immunoreactivity (IR) for CGRP or SP. In this tissue, perivascular fibers contained VIP or NPY, whereas isolated fibers and free nerve endings showed the IR for CGRP or SP. These neuropeptides may be involved in the regulation of blood flow and/or pain sensation in the human articular disk of the temporomandibular joint.

Aged↗