The omohyoid muscle: a variation seen in radical neck dissection.
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Humans↗
Biomedical subjects
T Hagiwara
Publications and source records attributed to T Hagiwara.
Differentiation of Chlamydia species by combined use of polymerase chain reaction and restriction endonuclease analysis.
To differentiate Chlamydia spp., a primer pair designed to generate a genus-specific region of the major outer membrane protein (MOMP) gene was used in a PCR to amplify a single DNA fragment of 245-259 bp. In the PCR, the expected single DNA fragment was amplified from strains of Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, respectively. By restriction endonuclease analysis with AluI and PvuII, the amplified products exhibited four distinct patterns, corresponding to the four species. It is, therefore, concluded that one-step PCR followed by restriction endonuclease analysis as described in this study could be a valuable method for the detection and differentiation of Chlamydia species.
Chlamydia↗
Inhibition by midazolam of the adrenergic function in the isolated canine mesenteric vein.
BACKGROUND: Midazolam has been reported to cause hypotension or to depress sympathetic activity following intravenous injection. However, little information is available concerning the mechanism of these effects. The aim of the present study was to determine the effects of midazolam on release of noradrenaline (NA) at nerve terminals and on receptors in the venous smooth muscle. METHODS: The effect of midazolam at nerve terminals was examined by measuring the amount of NA release from superfused canine mesenteric vein helical strips during electrical stimulation (ES; 5 Hz, 2 ms, 9 V). The NA was quantified by high-performance liquid chromatography with electrochemical detection; tension development evoked by ES was also recorded simultaneously. In a separate series of experiments, ring preparations from the isolated vein were mounted in Krebs-Ringer solution for isometric tension recording to assess the effect of midazolam on alpha-adrenoceptors. RESULTS: Application of tetrodotoxin (10(-6) M) or replacement of superfusate with Ca(2+)-free solution decreased both the release of NA and the tension development evoked by ES. Yohimbine (5 x 10(-8) M) increased the ES-evoked release of NA, whereas it decreased tension development in the vein strips. Midazolam (10(-4) M) did not affect either the basal release of NA or the basal tension, but inhibited both the NA release (P < 0.01) and the tension development (P < 0.01) during ES; midazolam at 10(-5) M inhibited the tension development (P < 0.05) but had no effect on NA release. In the ring preparations, midazolam (10(-5) and 10(-4) M) attenuated responses to NA (a mixed alpha 1- and alpha 2-adrenoceptor agonist, 10(-8) to 10(-3) M), phenylephrine (the alpha 1-adrenoceptor agonist, 10(-8) to 10(-3) M) and 5-bromo-6-[2-imidazolin-2yl-amino]-quinoxaline (UK14304; the alpha 2-adrenoceptor agonist, 10(-7) to 10(-3) M) in a dose-dependent manner. CONCLUSION: The data obtained in the present study suggest that midazolam at 10(-4) M may reduce venous tone by inhibiting the release of NA from sympathetic nerve endings and both alpha 1- and alpha 2-adrenoceptor mediated smooth muscle contractions. It is also postulated that a stage of the post-receptor transduction mechanism linked to the venous smooth muscle contraction may be more sensitive to midazolam than the NA release mechanism at nerve terminals since midazolam at the low concentration tested inhibited ES-evoked tension development with no effect on the release of NA.
Adrenergic Antagonists↗
Detection of Chlamydia trachomatis in vaginal specimens from female commercial sex workers using a new improved enzyme immunoassay.
OBJECTIVE: To evaluate the performance of a new improved enzyme immunoassay (EIA) kit for the detection of Chlamydia trachomatis in vaginal swab and endocervical swab specimens from female commercial sex workers, in comparison with a conventional EIA test and a polymerase chain reaction (PCR) assay. METHODS: A high risk group of 163 female commercial sex workers who visited a sexually transmitted disease (STD) clinic in order to undergo screening for major STDs, including chlamydial infection, were enrolled. A total of four swab specimens, including two vaginal and two endocervical specimens, were collected from each woman by a clinician. To identify C trachomatis, a new improved EIA kit (IDEIA PCE), a conventional EIA kit (IDEIA), and PCR assay (Amplicor) were used. Discrepancies in the results were resolved using supplementary PCR assay. A female patient was considered to be infected with C trachomatis if the IDEIA PCE test and PCR test for both sample sites (endocervical and vaginal) gave positive results. Following resolution of these discrepancies, relative sensitivity and specificity, confidence intervals, and predictive values for each type of specimen by each assay were calculated. RESULTS: Of the 163 women tested, 35 (21.5%) were shown to be infected with C trachomatis. The relative sensitivities in vaginal swab specimens were 88.8%, 68.6%, and 91.4% using IDEIA PCE, IDEIA, and PCR, respectively. The relative specificities in vaginal swab specimens were 99.2%, 99.2%, and 100%, respectively. The relative sensitivities in endocervical swab specimens were 85.7%, 77.1%, and 91.4% with IDEIA PCE, IDEIA, and PCR, respectively. The relative specificities in endocervical swab specimens were all 100%. CONCLUSIONS: The results obtained in this study suggest that the sensitivity and specificity of IDEIA PCE test on vaginal swab and endocervical swab specimens were similar to those of PCR assay on the two types of specimen. It is concluded that IDEIA PCE test on vaginal swab specimens is an acceptable, sensitive, and less invasive approach for the detection of C trachomatis in commercial sex workers with a high prevalence of C trachomatis infection.
Adult↗
Clinical study of the effectiveness of a dual amplified immunoassay (IDEIA PCE Chlamydia) for the diagnosis of male urethritis.
A clinical study of patients with male urethritis (n=316) was undertaken to determine the sensitivity potential for a new dual amplified immunoassay (IDEIA PCE Chlamydia). Increased sensitivity (98.8%, 84/85) was obtained for IDEIA PCE Chlamydia compared to a conventional antigen detection test (IDEIA Chlamydia, 81.2%, 69/85) when testing urine samples. In a smaller patient population (n=104) the positivity rate for the first-void urine tested with IDEIA PCE Chlamydia of 30.8% (32/104) was similar to the 27.9% (29/104) obtained from urethral swabs tested with a DNA probe assay (PACE 2). The increased sensitivity of the test was confirmed with a commercial PCR kit (Amplicor) and nested PCR. The IDEIA PCE Chlamydia kit has the sensitivity potential to be a clinically reliable alternative for detecting Chlamydia trachomatis.
Chlamydia Infections↗
Proliferative properties of human umbilical cord blood megakaryocyte progenitor cells to human thrombopoietin.
We evaluated the effects of human thrombopoietin (TPO) alone, or in combination with other several hematopoietic factors, on megakaryocyte colony growth from human umbilical cord blood CD34+ cells in serum-depleted agar cultures. The addition of TPO alone had a concentration-dependent effect on the number of pure megakaryocyte colonies and of megakaryocytes per colony (colony size). The maximally stimulating concentration of TPO generated a greater number of megakaryocyte colonies and larger megakaryocyte colony size compared with the stimulation observed with an optimal concentration of human interleukin-3 (IL-3) or stem cell factor (SCF). At the high concentration of TPO that yielded the maximum colony numbers, a substantial proportion of megakaryocyte colonies contained 100 or more cells per colony. The combination of TPO plus SCF resulted in a synergistic enhancement of both the number and size of megakaryocyte colonies. Among the combinations of TPO plus other cytokines tested, IL-3 plus TPO had a modest effect on megakaryocyte colony numbers. The generation of megakaryocyte colonies from subpopulations of CD34+ cells was further examined. The addition of TPO alone induced a greater number of megakaryocyte colonies from CD34+CD41+ cells compared with CD34+CD41- cells, and TPO plus IL-3 exerted a synergistic effect on the number of megakaryocyte colonies only from CD34+CD41- cells. In contrast to the effects on colony numbers, TPO induced larger megakaryocyte colony sizes from CD34+CD41- cells, compared with CD34+CD41+ cells. In the case of HLA-DR expression, TPO and IL-3, administered singly or in combination, induced similar megakaryocyte colony numbers and sizes from CD34+DR+ and CD34+DR- subpopulations. Ploidy analysis revealed that the majority of megakaryocytes generated from cord blood CD34+ cells in serum-free liquid cultures containing TPO displayed 2N and 4N values, suggesting that they were immature. These results indicate that, compared with IL-3 and SCF, TPO has more potent proliferative effect on human cord blood megakaryocyte progenitors, leading to greater numbers of megakaryocyte progenitors triggered for both growth and cell division, and synergizes with SCF to enhance megakaryocyte colony growth.
Antigens, CD34↗
Investigation of morphological changes in absorptive cells in young adult and infant mice fed different amounts of iron for a long-term.
The changes in fine structure of the intestinal tract in young adult (4 week-old) and infant (2 week-old) mice fed a diet containing different amounts of iron salt (Fe-0, Fe-2.5, Fe-25: 0, 2.5 and 25 mg Fe/100 g diet, respectively) for a long-term (1 or 2 weeks) were investigated. The hepatic iron levels in infant mice fed Fe-25 for 2 weeks were significantly higher than those observed after 1 week of feeding, but there was no such increase in young adult mice during the feeding period. Observations of fine structure indicated typical signs of impairment of enterocytes due to excess iron such as the opening of intercellular junctions between adjacent epithelial cells and the marked appearance of eosinophilic leukocytes outside the basement membrane in young adult and infant mice fed Fe-25. The frequency of the opening in intercellular junctions increased in young adult mice fed Fe-25 for 2 weeks, but decreased in infant mice. On the contrary, under iron-deficient conditions, the frequency in infant mice was higher than that in young adult mice. The appearance of eosinophilic leukocytes indicated that some immunological reaction was elicited in both groups of mice fed Fe-25 for 2 weeks.
Animal Feed↗
[A rapid particle-counting method for chlamydial elementary bodies using the fluorescence microscope].
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Bacteriological Techniques↗
Action of thrombopoietin at the megakaryocyte progenitor level is critical for the subsequent proplatelet production.
Formation of proplatelets from megakaryocytes is believed to be the first step of platelet production in vitro. In this study, we evaluated the effects of recombinant human thrombopoietin (hTPO) on the development of proplatelets from a GpIIb/IIIa+ population of rat bone marrow cells highly enriched for late megakaryocyte progenitors (GpIIb/IIIa+ CFU-MK) that we recently found to be a primary target population of TPO. Quantitative measurement of hTPO-induced proplatelet formation was performed in liquid cultures. Proplatelet formation from megakaryocytes derived from GpIIb/IIIa+ CFU-MK in the presence of hTPO began on day 4 of culture and peaked the following day. On day 5 of culture, lower concentrations of hTPO expanded the number of megakaryocytes, increased the number of proplatelets and the percentage of proplatelet-developing megakaryocytes. Increasing hTPO concentrations resulted in a modest decrease in proplatelet development. We next used hTPO to derive immature or mature megakaryocytes from GpIIb/IIIa+ CFU-MK. These populations of cultured megakaryocytes spontaneously formed proplatelets when recultured in the absence of exogenous hTPO. The addition of hTPO at higher concentrations modestly augmented proplatelet production from immature megakaryocytes derived from 2-day liquid cultures. However, either murine interleukin-6 (IL-6) or human IL-11, but not rat IL-3, was more potent than hTPO in augmenting proplatelet formation from immature megakaryocytes. Each of these four cytokines had an inhibitory effect on proplatelet formation from more differentiated megakaryocytes derived from 3-day liquid cultures. These results indicate that TPO enhances proplatelet production primarily by stimulating CFU-MK to increase the number of proplatelet-forming megakaryocytes and that its action is clearly different from those of other cytokines that also stimulate megakaryocytopoiesis.
Animals↗
Involvement of beta-glucuronidase in intestinal microflora in the intestinal toxicity of the antitumor camptothecin derivative irinotecan hydrochloride (CPT-11) in rats.
Irinotecan hydrochloride (CPT-11), an antitumor camptothecin derivative, causes severe forms of diarrhea clinically. We characterized CPT-11-induced diarrhea histologically and enzymologically and assessed the relationships between intestinal toxicity and the activity of the enzymes that play a key role in the major metabolic pathway of CPT-11 in rats. CPT-11 (60 mg/kg i.v. for 4 days) induced intestinal toxicity characterized by severe chronic diarrhea, loss of body weight, and anorexia. Histological damage was most severe in the cecum. The segmental difference in the degree of the damage showed good correlation with the beta-glucuronidase activity in the contents of the lumen in each case, but not with the intestinal tissue carboxylesterase activity, which converts CPT-11 to its active form (7-ethyl-10-hydroxycamptothecin). Inhibition of the beta-glucuronidase activity in the intestinal microflora by antibiotics (1 mg penicillin and 2 mg streptomycin per ml of drinking water) markedly ameliorated the diarrhea and reduced cecal damage. Analysis of CPT-11 and its metabolites in the feces indicated that antibiotics completely inhibited the deconjugation of the glucuronic conjugate of 7-ethyl-10-hydroxycamptothecin by beta-glucuronidase. It is suggested that CPT-11-induced diarrhea would be attributable to the damage to the cecum, and that the inhibition of the beta-glucuronidase activity in the intestinal microflora is a major protective effect of antibiotics.
Animals↗
Establishment and characterization of the thrombopoietin-dependent megakaryocytic cell line, UT-7/TPO.
UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombopoietin (TPO), the ligand for the receptor encoded by c-mpl proto-oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7/GM, which is a subline of UT-7, but neither UT-7 nor UT-7/EPO, can proliferate in response to TPO. The subline, UT-7/TPO, was established from UT-7/GM by culture at lower concentrations of TPO. UT-7/TPO cells had morphologically mature megakaryocytic characteristics such as developed demarcation membrane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor-4 and glycoprotein IIb at the mRNA levels and by the high level of DNA content. UT-7/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase. In the absence of TPO, the majority of the cells died within a few days. Thus, UT-7/TPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c-mpl was detected in UT-7/TPO and, to a lesser degree, in UT-7/GM. The mRNA level of NF- E2 p45, reported to be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down-regulated in the erythroid subline, UT-7/EPO. There were no significant differences in GATA-1 and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine kinase and STAT5-related protein. These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation-associated proteins and to study the specific actions of TPO.
Cell Differentiation↗
Genomic organization, promoter analysis, and chromosomal localization of the gene for the mouse glial high-affinity glutamate transporter Slc1a3.
The mouse gene encoding glial high-affinity, Na+-dependent glutamate transporter Slc1a3 (GluT-1/GLAST) was isolated, and its structural organization was characterized. The gene appeared to exist as a single copy in the mouse genome and comprised 10 exons spanning more than 56 kilobases. The transcription initiation sites were mapped to positions 503, which is the first transcriptional point (defined as +1), 128 (+376), and 64 (+440) basepairs upstream of the 3'-end of exon 1 by primer extension. The 5'-flanking region of the mouse GluT-1 gene had a typical CCAAT box and a GC box but lacked a TATA box. These features of the promoter region were characteristic of housekeeping genes. The fusion plasmids containing approximately 4 kb of the 5'-flanking region (-3830 to +450) and the firefly luciferase gene induced a significant luciferase activity when transfected into COS-1 cells. Distal deletion of the 5'-flanking region, leaving 619 bp (-169 to +450), resulted in a marked decrease in luciferase activity in COS-1 cells, suggesting that a CCAAT box, which was positioned at -200, is necessary for the expression of this gene. In situ hybridization localized this gene to mouse chromosome 15A2. These structural features will lead to a better understanding of the regulatory mechanism of the expression of the GluT-1 gene by ischemia and will also provide a basis for future evolutionary comparisons with other neurotransmitter transporters.
ATP-Binding Cassette Transporters↗
Inhibition of bacterial translocation from the gastrointestinal tract of mice injected with cyclophosphamide.
Effects of intraperitoneal injection of cyclophosphamide, an immunosuppressant, on the degree of bacterial translocation and morphological changes of Peyer's patches (PP) in the intestine were investigated with antibiotic-decontaminated SPF mice and germfree mice monoassociated with Escherichia coli C25. It has been reported that treatment with cyclophosphamide induces bacterial translocation. Cyclophosphamide treatment in this study, however, significantly decreased E. coli C25 translocation from the gastrointestinal tract to the mesenteric lymph nodes (MLN), although the numbers of lymphoid cells, especially B cells, in the PP, MLN, and spleen were remarkably reduced. Four injections of cyclophosphamide at a dose of 100 mg/kg inhibited bacterial translocation more than one injection at a dose of 200 mg/kg in SPF mice. Germfree mice, however, treated with one dose of 200 mg/kg showed the same inhibition of bacterial translocation as those given 100 mg/kg four times. In cyclophosphamide-treated mice, lymph follicles in the PP were obviously smaller than those in control mice, M-cells were similar in appearance to absorption epithelial cells except for short microvilli, and immune cells among the M-cells had disappeared. These data suggested that inhibition of bacterial translocation in mice treated with cyclophosphamide may be the result of morphological and physiological changes of epithelial cells in the gastrointestinal tract, especially M-cells, as a point of entry of invading bacteria, independent of the changes in immunological function.
Animals↗
The requirement for and mobilization of calcium during induction by sodium ascorbate and by hydrogen peroxide of cell death.
The requirement for and mobilization of Ca2+ ions during induction of cell death by sodium ascorbate were compared with those during induction of cell death by hydrogen peroxide. When HL-60 cells were incubated with sodium ascorbate, a rapid increase in the intracellular concentration of Ca2+ ions and subsequent apoptotic cell death, characterized by cell shrinkage, nuclear fragmentation and cleavage of internucleosomal DNA to yield fragments that were multiples of 180-200 base pairs, were induced. However, these effects of sodium ascorbate were significantly reduced in Ca2+-depleted medium. By contrast, hydrogen peroxide induced similar apoptosis associated phenomena in the presence and in the absence of extracellular Ca2+ ions. The intracellular concentration of the reduced form of glutathione was not significantly affected and glutathione disulfide was undetectable during the early stages of apoptosis. These data suggest that sodium ascorbate and hydrogen peroxide initiate cell death by different mechanisms.
Apoptosis↗
Interferon-gamma promotes the survival and Fc epsilon RI-mediated histamine release in cultured human mast cells.
We examined the effects of interferon-gamma (IFN-gamma) on 100% pure human mast cells generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of stem cell factor (SCF) and interleukin-6 (IL-6). When mast cells were suspended in serum-free medium without any cytokine after the withdrawal of SCF and IL-6, they died over a period of 5 days because of apoptosis. IFN-gamma in the cultures suppressed apoptosis and prolonged their survival in a dose-dependent manner. This survival-promoting effect of IFN-gamma was blocked by neutralizing antibodies to IFN-gamma or to IFN-gamma receptor (IFN-gamma R). When mast cells were incubated with IFN-gamma in serum-free medium for more than 4 hr during sensitization, immunoglobulin E (IgE)/anti-IgE antibody-induced histamine release was effectively enhanced. Polymerase chain reaction (PCR) amplification of the alpha-chain of IFN-gamma R (IFN-gamma R alpha) yielded products of the correct size predicted from the sequence of the receptor. In addition, flow cytometry using anti-IFN-gamma R monoclonal antibodies (mAbs) indicated that these mast cells bear IFN-gamma R on their surface. These findings suggested that IFN-gamma activates human mast cells via specific receptors in certain aspects of inflammatory reactions.
Antibodies↗
Fractal analysis of aggregates formed by heating dilute BSA solutions using light scattering methods.
The structure of aggregates formed by heating dilute BSA solution was analyzed with fractal concept using light scattering methods. BSA was dissolved in HEPES buffer on pH 7.0 and acetate buffer of pH 5.1 to 0.1% and 0.001% solutions, respectively, and heated at 95 degrees C, varying the heating time ta. The fractal dimension Df of the aggregate in the solution was evaluated from static light scattering experiments. The polydispersity exponent tau and the average hydrodynamic radius of the aggregates were calculated from dynamic light scattering experiments using master curves obtained by Klein et al. The values of Df and tau of heat-induced aggregates of BSA at pH 7.0 were about 2.1 and 1.5, respectively, the values of which agreed with those predicted by the reaction-limited cluster-cluster aggregation (RLCCA) model. On the other hand, Df of heat-induced aggregates at pH 5.1 was about 1.8, which agreed with that predicted by the diffusion-limited cluster-cluster aggregation (DLCCA) model. The dependence of for the sample of pH 7.0 on ta was similar to that of the polystyrene colloids reported previously.
Animals↗
Expression of adhesion molecules on cytoplasmic processes of human megakaryocytes.
Megakaryocytes generate cytoplasmic processes (CP) that penetrate endothelial cells in the bone marrow sinus, and these processes may release platelets into the circulation at their terminal stage. Adhesion between the CP and endothelial cells may be important during the extension of CP. We examined the expression of adhesion molecules of the integrin family (CDw49b, CDw49d, CDw49e, CDw49f, CD18, CD11a CD11c, and CD11b), the immunoglobulin superfamily (CD54, CD56, CD58, and CD31), the selectin family (ELAM-1, LECAM-1, and CD62), and CD44, CD41b, and CD42b on platelets, megakaryocytes, and megakaryocytes with CP. No specific adhesion molecules were observed on the megakaryocytes with CP. Three staining patterns of adhesion molecules-homogeneous, speckled, and accumulated-were observed on the megakaryocytes with CP, but not on those without CP. Platelet integrins (i.e., CD41a, CDw49b, CDw49e and CDw49f) and GPIb (CD42b) were strongly and homogeneously stained on the CP. GMP-140 CD62) was weakly stained, in a speckled pattern. CD31 (PECAM-1) was also weakly stained but accumulated selectively on the tip of the CP. ANTI-CD31 suppressed CP formation of megakaryocytes. We speculate that the homodimerization of CD31 expressed on the tips of CP and endothelial cells is important for the extension of the processes and for the migration of megakaryocytes.
Antigens, CD↗
[Mutation detection enhancement (MDE) gel electrophoresis method for analysis of the von Willebrand factor gene].
We conducted electrophoresis using Mutation Detection Enhancement (MDETM) gel (AT Biochem) on the von Willebrand factor (vWF) gene from three unrelated patients with type 2A von Willebrand disease and one type 3 patient whose point mutations we had identified earlier. The mutations were located on the 3'region of exon 28 in the vWF gene in which three known polymorphisms were included. To eliminate any influence of the polymorphisms, 671bp including two AluI sites amplified by polymerase chain reaction (PCR) from the region of the vWF gene was digested with the restriction enzyme AluI and three fragments (283, 148, and 240bp) were obtained. Three polymorphisms were contained in the 283bp fragment and four mutations were found in the remaining fragments. Following MDETM gel electrophoresis, three of the four cases showed heteroduplex formations. One mutation was not detected, although the mutation was found to be heterozygous by the restriction analysis of MspI. Failure to detect this mutation may be attributed to the fact that it was located on the extreme end of the 148bp fragment of the 5' region. The results of this study suggest the MDETM gel electrophoresis method is useful in detecting gene mutations or polymorphisms in heterozygous subjects. Furthermore, this technique is more convenient than other methods for a base mismatch detection since specific apparatus or radioisotopes are not required.
Electrophoresis↗