Efferent pathways involved in the running activity originate in the ventromedial hypothalamus of the rat.
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2-Amino-5-phosphonovalerate↗
Biomedical subjects
T Higuchi
Publications and source records attributed to T Higuchi.
Direct evidence of NO production in rat hippocampus and cortex using a new fluorescent indicator: DAF-2 DA.
The biological functions of nitric oxide in the neuronal system remain controversial. Using a novel fluorescence indicator, DAF-2 DA, for direct detection of NO, we examined both acute rat brain slices and organotypic culture of brain slices to ascertain NO production sites. The fluorescence intensity in the CA1 region of the hippocampus was augmented, especially after stimulation with NMDA, in acute brain slices. This NO production in the CA1 region was also confirmed in cultured hippocampus. This is the first direct evidence of NO production in the CA1 region. There were also fluorescent cells in the cerebral cortex after stimulation with NMDA. Imaging techniques using DAF-2 DA should be very useful for the clarification of neuronal NO functions.
Animals↗
Thrombopoietin enhances the production of myeloid cells, but not megakaryocytes, in juvenile chronic myelogenous leukemia.
We previously reported the aberrant growth of granulocyte-macrophage (GM) progenitors induced by a combination of stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in juvenile chronic myelogenous leukemia (JCML). We examined here the effects of thrombopoietin (TPO) on the proliferation and differentiation of hematopoietic progenitors in JCML. In serum-deprived single-cell cultures of normal bone marrow (BM) CD34+CD38high cells, the addition of TPO to the culture containing SCF + GM-CSF resulted in an increase in the number and size of GM colonies. In the JCML cultures, in contrast, the number of SCF + GM-CSF-dependent GM colonies was not increased by the addition of TPO. However, the TPO addition caused an enlargement of GM colonies in cultures from the JCML patients to a significantly greater extent compared with the normal controls. There was no difference in the type of the constituent cells of GM colonies with or without TPO grown by JCML BM cells. A flow cytometric analysis showed that the c-Mpl expression was found on CD13+ myeloid cells generated by CD34+CD38high BM cells from JCML patients, but was at an undetectable level in normal controls. The addition of TPO to the culture containing SCF or SCF + GM-CSF caused a significant increase in the production of GM colony-forming cells by JCML CD34+CD38neg/low population, indicating the stimulatory effects of TPO on JCML primitive hematopoietic progenitors. Normal BM cells yielded a significant number of megakaryocytes as well as myeloid cells in response to a combination of SCF, GM-CSF, and/or TPO. In contrast, megakaryocytic cells were barely produced by the JCML progenitors. Our results may provide a fundamental insight that the administration of TPO enhances the aberrant growth of GM progenitors rather than the recovery of megakaryocytopoiesis.
Cell Differentiation↗
Physical and serologic examinations of foals at 30 and 45 days of age for early diagnosis of Rhodococcus equi infection on endemically infected farms.
OBJECTIVE: To evaluate results of physical and serologic examinations of foals at 30 and 45 days of age on 3 types of farms with various prevalences of clinical disease (endemic, sporadic, none) caused by Rhodococcus equi and to determine whether evaluations were helpful in early diagnosis and control of the disease. DESIGN: Prospective cohort study. ANIMALS: 144 foals at 30 and 45 days of age. PROCEDURE: During a 2-year period, 36 foals on farms at which R equi infection was endemic, 71 foals on farms at which the disease was sporadically detected, and 37 foals on farms without the disease were examined by means of auscultation of lungs, serum biochemical and hematologic analyses, and determination of antibody titers against R equi, using ELISA. Transtracheal aspirates were obtained from 14 of 32 foals that had clinical signs of disease and 7 of 41 seropositive foals that did not have clinical signs of disease. RESULTS: Prevalences of respiratory tract disease and seropositive conversion rates for 45-day-old foals on endemically and sporadically infected farms were significantly higher than on farms without the disease. Rhodococcus equi was isolated from tracheal aspirates of seropositive foals, even when clinical signs were not evident. CLINICAL IMPLICATIONS: Physical and serologic examinations of foals at 30 and 45 days of age were useful for early diagnosis of R equi infection, especially for foals on farms at which the disease was endemic.
Actinomycetales Infections↗
Detection of virulent Rhodococcus equi in tracheal aspirate samples by polymerase chain reaction for rapid diagnosis of R. equi pneumonia in foals.
Polymerase chain reaction (PCR)-based assays were developed to detect virulent Rhodococcus equi in transtracheal aspirate samples from sick foals showing respiratory signs. An oligonucleotide primer pair from the sequence of the virulence-associated 15- to 17-kDa antigen gene of the virulence plasmid in virulent R. equi was used to amplify a 564 bp region by PCR, and the result was confirmed by Southern blot hybridization. No positive reaction was seen in DNA from 13 different microorganisms typically found in the respiratory tract. In tracheal aspirates seeded with virulent R. equi, a visible band could detect 10 to 10(2) bacteria per PCR assay (10(3) to 10(4)/ml of the aspirate). Virulent R. equi was demonstrated in 31 of 42 transtracheal aspirates by culture and colony blot analysis, whereas a positive PCR result was observed in only 12 of the 31 culture positive samples. To prevent false-negative results, two methods were developed: a nested PCR and a PCR in combination with enrichment cultures of aspirates in the selective medium to increase the number of bacteria to 10(4)/ml or more. All of the PCR-negative and culture-positive samples were positive by the two methods. These results indicated that PCR-based assays provide a specific and sensitive means to detect virulent R. equi in tracheal aspirates of foals, and they are more rapid than the routine culture procedures for the diagnosis of R. equi pneumonia in foals.
Actinomycetales Infections↗
Perivascular localization of nitric oxide synthase in the rat adenohypophysis: potential implications for function and cell-cell interaction.
The possible localization of nitric oxide (NO) synthase (NOS) in proximity to the microvasculature was examined in the rat adenohypophysis using immunohistochemistry and nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. A population of NOS-positive cells was localized in very close contact with the sinusoidal capillaries. The pattern of this perivascular localization was either unicellular, bicellular or multicellular. These observations suggest that, at least, some actions of NO in the adenohypophysis can be accounted for by a local regulation of the glandular microvasculature.
Animals↗
Disseminated intravascular coagulation in acute lymphoblastic leukemia at presentation and in early phase of remission induction therapy.
A high frequency of disseminated intravascular coagulation (DIC) in adult acute lymphoblastic leukemia (ALL) has been reported; however, its clinical relevance and characteristics have not been fully determined. We studied 67 adults with newly diagnosed ALL between 1982 and 1996 to clarify these questions. DIC was diagnosed in ten of 64 patients (16%) who underwent coagulation study at presentation and in 14 of 40 patients (35%) screened for DIC within 7 days after starting remission induction therapy. Overall, 24 of 67 patients (36%) had DIC during this period. Hemorrhagic symptoms were generally mild, while two patients required red blood cell transfusions. Patients who developed DIC had higher white blood cell counts and more frequently a palpable spleen than those who did not. There was no difference in age, French-American-British subtype, karyotype, immunophenotype, lactate dehydrogenase level, percentage of blasts in bone marrow, or frequency of lymphadenopathy or hepatomegaly between patients who had DIC and those who did not. Fibrinolysis tended to be milder in DIC complicating ALL than in that complicating acute promyelocytic leukemia; however, there was no difference in other coagulation parameters between these two subtypes. An etiological link between CD34 expression in common ALL patients and DIC was suggested.
Adolescent↗
A new assisted hatching technique using a piezo-micromanipulator.
OBJECTIVE: To estimate the efficacy of a new assisted hatching technique using a piezo-micromanipulator to support embryonic implantation. DESIGN: Sibling spare embryos from human cycles of IVF were allocated to either a treated group for assisted hatching by a piezo-micromanipulator or a nontreated control group. SETTING: The Infertility and IVF unit of the Kyoto University Hospital. PATIENT(S): Sixty-eight women undergoing conventional IVF treatment. INTERVENTION(S): One hundred ten spare 4- or 8-cell embryos from 68 patients undergoing IVF were treated with the new assisted hatching technique, and the results were compared with those obtained for 112 sibling embryos without the treatment. In the assisted hatching procedure, zona thinning combined with drilling was performed by the vibration of a microneedle produced by the piezo-micromanipulator unit. MAIN OUTCOME MEASURE(S): The rates of partial hatching and completely hatched blastocysts. RESULT(S): The rates of hatching and of hatched blastocysts per total developing blastocysts were significantly higher in the treated group (86.7% and 33.3%, respectively) than in the control group (15.3% and 2.8%, respectively). CONCLUSION(S): These results demonstrate that the newly devised zona thinning and drilling technique using a piezo-micromanipulator is useful for assisted hatching.
Blastocyst↗
Prolonged warm ischemia affects long-term prognosis of kidney transplant allografts from non-heart-beating donors.
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Adolescent↗
Cytogenetic clonality analysis in monosomy 7 associated with juvenile myelomonocytic leukemia: clonality in B and NK cells, but not in T cells.
It remains unclear which lymphoid lineages are involved in juvenile myelomonocytic leukemia (JMML). We report a JMML patient who acquired monosomy 7 after intensive chemotherapy. In this case, the expression of monosomy 7 was analyzed in T, B and natural killer (NK) cells highly purified from peripheral blood mononuclear cells of the patient. The fluorescence in situ hybridization method revealed the expression of monosomy 7 in B cells, but not T cells. Half of the NK cells expressed monosomy 7; when NK cells were divided into CD2- and CD2+ populations, this abnormality was positive in 91.1% of CD2- NK cells but in only 14.7% of CD2+ NK cells. These results suggest that, in this JMML patient who acquired monosomy 7 after intensive chemotherapy, B cells and half of NK cells, but not T cells, have monosomy 7.
B-Lymphocytes↗
Establishment of a GM-CSF-dependent megakaryoblastic cell line with the potential to differentiate into an eosinophilic lineage in response to retinoic acids.
We recently established a human granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell line (HML) from colony-constituent cells grown by peripheral blood cells of a patient with acute megakaryoblastic leukaemia. The HML cells possessed megakaryocytic features, as determined by cytochemical, electron microscopic and flow cytometric analysis. In the present study we examined the effects of retinoic acid (RA) on the development of HML cells. All-trans-RA, 13-cis-RA and 9-cis-RA at 10(-8) mol/l to 10(-5) mol/l inhibited the GM-CSF-dependent cell growth. Some of the RA-treated cells contained prominent azurophilic granules and were positive for peroxidase. They also reacted with Biebrich scarlet, Luxol fast blue and a monoclonal antibody against eosinophil peroxidase. In addition, exposure to RA increased the frequency and the intensity of major basic protein-positive cells. However, eosinophil-derived neurotoxin and eosinophil cationic protein were not detected or were only detected at a low level in the lysates of the HML cells treated with RA. Although IL-5 alone could not stimulate cell growth, the addition of IL-5 to the cultures containing stem cell factor + all-trans-RA was required for the expression of the eosinophilic phenotype. These results suggest that the HML cell line is a megakaryoblastic cell line with the potential to differentiate into the eosinophilic lineage. HML cells may be a useful model for elucidating the eosinophilic differentiation programme.
Cell Differentiation↗
Imaging tumor folate receptors using 111In-DTPA-methotrexate.
It is known that membrane folic acid receptors are responsible for cellular accumulation of folate and folate analogs, such as methotrexate, and overexpressed on various tumor cells. This study was aimed to develop an 111In labelled DTPA-methotrexate (DTPA-MTX) to image tumor folate receptors in vivo. DTPA-MTX was synthesized by reacting ethylenediamine with MTX. The resulting amino analogue of MTX was reacted with DTPA dianhydride in basic aqueous solution followed by dialysis. Tissue distribution was determined in breast tumor-bearing rats at 0.5, 2, 24, and 48 h (n = 3/time interval). To determine receptor-mediated process 111In-DTPA-MTX was co-administrated with varying blocking doses of cold folate to tumor-bearing rats. Planar imaging and whole-body autoradiograms were performed. The data was compared to that using 111In-DTPA. In animal studies, tumor/blood count density ratios at 0.5-48 h gradually increased from 0.8 +/- 0.32 to 2.2 +/- 0.41 with 111In-DTPA-MTX. Conversely, these values showed time-dependent decrease from 1.19 +/- 0.69 to 0.56 +/- 0.10 with 111In-DTPA in the same time period. Tumor/muscle and tumor/blood count density ratios significantly decreased with high doses of folic acid co-administration. Planar images and autoradiograms confirmed that the tumors could be visualized acceptably with 111In-DTPA-MTX. The results indicate the feasibility of using 111In-DTPA-MTX to image tumors through a folate receptor-mediated process.
Animals↗
99mTc-ethylenedicysteine-folate: a new tumor imaging agent. Synthesis, labeling and evaluation in animals.
It is known that membrane folic acid receptors are responsible for cellular accumulation of folate and folate analogs such as methotrexate and overexpressed on various tumor cells. However, these receptors are highly restricted in normal differentiated tissues. Results of limited in vitro and in vivo animal studies suggest that folate receptors could be a potential target for tumor imaging. This study aimed to develop a 99mTc-labeled folic acid using ethylenedicysteine (EC) as a chelator and evaluate its labeling efficiency and potential use as a tumor seeking agent. Tissue distribution of 99mTc-EC-folate was determined in breast tumor-bearing rats at 20 min, 1, 2, and 4 h (n = 3/time interval, 370 KBq/rat, i.v.). Blocking study was employed to determine receptor-mediated process; 99mTc-EC-folate was co-administrated with 50 and 150 mumol/kg of cold folic acid to tumor-bearing rats. Planar imaging and whole-body autoradiograms were performed. The data was compared to that using 99mTc-EC (control). In animal studies, tumor/blood count density ratios at 20 min-4 h increased from 0.81 +/- 0.09 to 1.23 +/- 0.13 with 99mTc-EC-folate. Conversely, these values showed time-dependent decrease from 0.77 +/- 0.32 to 0.65 +/- 0.01 with 99mTc-EC in the same time period. Tumor/muscle and tumor/blood count density ratios significantly decreased with folic acid co-administrations. Planar images and autoradiograms confirmed that the tumors could be visualized clearly with 99mTc-EC-folate.
Animals↗
Administration of thymocytes derived from non-pregnant mice induces an endometrial receptive stage and leukaemia inhibitory factor expression in the uterus.
We have previously reported that intravenous administration of splenocytes prepared from mice in the early stages of pregnancy promoted embryo implantation in pseudopregnant mice. Since a T-lymphocyte-rich, but not a monocyte-rich preparation from splenocytes enhanced embryo implantation, similar effects of thymocytes from non-pregnant mice on implantation were examined in this study. Thymocytes were prepared from immature 21 day old ICR female mice and the supernatant of a thymocyte suspension (Th-sup) was used as the control. Thymocytes or Th-sup were injected into the caudal vein of recipient mice on pseudopregnancy day 2, and blastocysts were transferred into the endometrial lumen. The implantation rates per recipient were significantly higher in the thymocyte-treated group. ICR mice were then oophorectomized on pseudopregnancy day 3. After 3-day progesterone supplementation, blastocysts were transferred with intravenous injection of thymocytes or Th-sup. Under progesterone supplementation, successful implantations were observed in the thymocyte-treated group, but not in the Th-sup-treated group. Reverse transcriptase-polymerase chain reaction analysis revealed that mRNA expression of leukaemia inhibitory factor in the uterus was induced by thymocyte administration, but not by Th-sup. Thymocytes were divided into two populations, CD4(+/-)CD8(-) group and CD4(-)CD8(+/-) group, by separation columns. On pseudopregnancy day 2, the separated thymocytes in each group or their supernatant were injected into the endometrial stroma of the recipient mice, and blastocysts were transferred into the endometrial lumen. The administration of CD4(+/-) CD8(-) lymphocytes significantly promoted implantation rates, but no effect was observed in the CD4(-) CD8(+/-) group. These findings showed that thymocytes, especially CD4-positive lymphocytes, facilitate embryo implantation, probably by regulating endometrial differentiation.
Animals↗
A monoclonal antibody, HCL-2, raised against human luteal cells reacts with apolipoprotein-B and detects the uptake of low density lipoprotein by luteinizing granulosa cells.
A monoclonal antibody, HCL-2, was raised by immunizing mice against human luteal cells. HCL-2 reacted with luteal cells and villous trophoblasts. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis profile of immunopurified antigens from corpus luteum, chorionic villi, and placenta showed the same main protein band, the molecular mass of which is >200 kDa. The sequence of a portion of the N-terminal region of the antigenic protein purified from placenta was identical to that of apolipoprotein-B. The antigen purified from human serum and low density lipoprotein (LDL) using HCL-2 showed the same protein band as that from corpus luteum. Furthermore, the amino acid sequence (20 amino acids) of the protein purified from serum was also identical to that of apolipoprotein-B. Thus, we concluded that HCL-2 antigen is apolipoprotein-B. Human luteinizing granulosa cells isolated from the patients undergoing in-vitro fertilization treatment were cultured in the medium containing lipoprotein-deficient serum with or without supplementation of LDL. Using HCL-2, apolipoprotein-B was immunocytochemically detected on granulosa cells only in the presence of LDL. These findings showed that the uptake of LDL by granulosa cells was detected by immunocytochemical staining of apolipoprotein-B, indicating that HCL-2 is useful for analysing dynamic utilization of LDL by ovarian cells.
Adult↗
Stage-specific uptake of apolipoprotein-B in ovarian follicles and corpora lutea of the menstrual cycle and early pregnancy.
A monoclonal antibody (mAb), HCL-2, was raised which reacts with apolipoprotein-B, and it was shown by immunohistology that HCL-2 can be used to analyse the uptake of apolipoprotein-B by steroid-producing cells in vivo. In this study we have investigated the dynamic utilization of low density lipoprotein (LDL) in human ovary by immunohistological localization of apolipoprotein-B and LDL receptors using HCL-2 and anti-LDL receptor mAb. In antral follicles, including those of <1 mm in diameter, both apolipoprotein-B and LDL receptors were localized to theca interna cells, but not granulosa cells. In pre-ovulatory follicles, the LDL receptor was expressed on all granulosa cells. Apolipoprotein-B was also detected in granulosa cells located at the basal layer, suggesting that they utilize LDL through the basal lamina before ovulation. In mid-luteal phase, large luteal cells seemed to stain more intensely for apolipoprotein-B than did small luteal cells, suggesting that large lutal cells are the main sites of LDL utilization. In regressing corpora lutea, the expression of LDL receptor was weak, and apolipoprotein-B was rarely detected. In corpora lutea of early pregnancy, LDL receptor and apolipoprotein-B were localized to both luteal cells. These findings show the precise dynamic changes in LDL uptake by human ovarian cells during their differentiation in vivo.
Adolescent↗
Undifferentiated pancreatic cancer associated with humoral hypercalcemia of malignancy.
We present a case of undifferentiated pancreatic cancer associated with humoral hypercalcemia of malignancy (HHM) in which parathyroid hormone-related protein (PTH-rP) is identified as the causative factor of hypercalcemia. A 61-year-old man was hospitalized with right hypochondralgia. Ultrasound examination and computed tomography demonstrated a large mass in the pancreatic head with liver metastases. Biopsy of the pancreatic tumor demonstrated undifferentiated carcinoma. Serum calcium level and PTH-rP were elevated. Bone scan with technetium-99 demonstrated no accumulation in the bones. Immunohistochemical staining for PTH-rP was weakly positive in the tumor cells. We considered that PTH-rP was the causative factor of HHM in this case from laboratory data and immunohistochemical findings. This rare case was successfully treated with pamidronate disodium, which is a type of bisphosphonate derivative. We compared this case with previously reported cases.
Antineoplastic Agents↗
Membrane-bound carboxypeptidase-M is expressed on human ovarian follicles and corpora lutea of menstrual cycle and early pregnancy.
We have previously reported that membrane-bound aminopeptidases were expressed on human follicles and corpora lutea (CL) and we showed that these aminopeptidases are involved in follicular growth, probably by regulating extracellular peptide concentrations. In this study, the expression of membrane-bound carboxypeptidase-M (CP-M), which cleaves carboxyl-terminal amino acids from peptides extracellularly, on human follicles and CL was examined. In growing and pre-ovulatory follicles, CP-M was immunohistochemically detected with weak or moderate intensity on theca interna cells. Although CP-M was not detected on granulosa cells in growing and pre-ovulatory follicles, it was strongly detected on the cell surface of luteinizing granulosa cells isolated from patients undergoing in-vitro fertilization treatment, indicating that CP-M was rapidly expressed on granulosa cells during ovulation. In menstrual and pregnant CL, CP-M was clearly detected on luteal cells. In menstrual CL, the expression of CP-M mRNA was observed by reverse transcription-polymerase chain reaction (RT-PCR). Western blotting analysis revealed that the molecular mass of the CP-M extracted from mid-luteal CL was 62 kDa. These results indicate that CP-M is a cell surface differentiation-related molecule of human granulosa, theca, and luteal cells. The rapid expression on granulosa cells during ovulation strongly suggests the involvement of CP-M in the ovulation and CL formation processes.
Blotting, Western↗