Autophagy in the placenta of women with hypertensive disorders in pregnancy.

INTRODUCTION: Autophagy has not been studied extensively in the human placenta. This study was performed to determine whether autophagy is increased in the placentas of women with hypertensive disorders in pregnancy compared to normotensive pregnancies. METHODS: LC3-II and p62 protein expression were examined by quantitative Western blotting analysis in 40 placentas from women not experiencing labor pains. The 40 placentas were from 13, 8, and 19 women with preeclampsia, gestational hypertension, and normal pregnancy, respectively. Hypertensive disorders in pregnancy included preeclampsia and gestational hypertension. RESULTS: LC3-II expression was significantly increased, while that of p62 was significantly reduced in 21 placentas of women with hypertensive disorders compared to those with normal blood pressure irrespective of the presence or absence of fetal growth restriction (FGR). LC3-II expression was also significantly increased in 13 placentas of women with preeclampsia irrespective of the presence or absence of FGR. DISCUSSION: The results of this study suggested that autophagy is active in the placenta of hypertensive disorders even in the absence of FGR.

Pulp ablation therapy by inductive heating: heat generation characteristics in the pulp cavity.

OBJECTIVE AND METHODS: This study was performed to clarify the usefulness of inductive heating system for the new endodontic therapy. Dextran magnetite complex (DM) suspensions were injected into the root canal of a permanent tooth, and the tooth was heated up to about 55.0 degrees C by alternating-current magnetic field. RESULTS AND CONCLUSION: The time until the temperature in the pulp cavity reached 55.0 degrees C was 328 +/- 26 s (mean +/- s.d., n = 8) in the 56 mg as Fe ml(-1) of DM concentration. The temperature in the pulp cavity could be maintained at 53.5-59.0 degrees C for 1200 s by changing the magnetic field intensity safely, while temperature elevations of the dental surface on the coronal and apical sides were 4.9 degrees and 3.7 degrees C, respectively. Thus, this inductive heating system, which has the possibility of selective heating, might be useful for eliminating residues of pulp as a new ablation therapy.

A hydrogen peroxide-generating agent, 6-formylpterin, enhances heat-induced apoptosis.

The enhancement of heat-induced apoptosis by 6-formylpterin, an intra-cellular generator of hydrogen peroxide (H2O2), was examined in human myelomonocytic lymphoma U937 cells. The cells were treated with either 6-formylpterin alone at a nontoxic concentration of 300 microM (37 degrees C), heat shock (44 degrees C per 20 min) alone or a combination of the two, then incubated at 37 degrees C for 6 h. Assessments of apoptosis, mitochondrial membrane potential and caspase-3 activation were performed by flow cytometry. Moreover, caspase-8 activation and changes in the intra-cellular Ca2+ concentration ([Ca2+]i) were examined. Bax, Bcl-2, Bcl-XL, Bid, cytochrome c and PKCd were detected by Western blotting. The induction of heat-induced apoptosis evaluated by morphological observation and DNA fragmentation were promoted by the addition of 6-formylpterin. Mitochondrial membrane potential was decreased and the activation of caspase-3 and -8 was enhanced in the cells treated with the combination. A decreased-expression of Bid was noted, although no significant changes in Bax, Bcl-2 and Bcl-XL expression were observed after the combined treatment. Furthermore, both the release of cytochrome c from mitochondria to cytosol and the translocation of PKCd from cytosol to mitochondria, which were induced by heat shock, were enhanced by the addition of 6-formylpterin. The number of cells with a higher [Ca2+]i was also increased by the addition of 6-formylpterin. These findings suggest that the increase in [Ca2+]i, the activation of the mitochondria-caspase dependent pathway and the translocation of PKCd to mitochondria play principal roles in the enhancement of heat-induced apoptosis by 6-FP.

Bone mineral density of the lumbar spine is associated with TNF gene polymorphisms in early postmenopausal Japanese women.

We investigated the relationships between tumor necrosis factor (TNF) gene polymorphism, circulating TNF-alpha (TNF-alpha) concentrations, and bone mineral density (BMD) in the lumbar spine. TNF gene polymorphisms studied were the Nco I polymorphism within the first intron of TNF-beta (TNF-beta) and three single nucleotide polymorphisms in the promoter region of the TNF-alpha gene, at positions -857, -863, and -1031. Allelic variants of the TNF gene were identified using restriction fragment length polymorphism (RFLP) analysis in 177 postmenopausal Japanese women within 10 years after menopause, aged 56.4 +/- 4.5 years (mean +/- SD). A significantly higher prevalence of the alleles TNF-alpha-863A (20.3% versus 9.9%) and TNF-alpha-1031C (21.3% versus 12.4%) was seen in the low BMD group (Z-score < 0, n = 91) than in the high BMD group (0 < Z-score, n = 86). In genotype analysis, although difference did not reach a significant level, women with the rarest allelic variants, i.e., homozygous TNFbl, TNF-alpha-863A, and TNF-alpha-1031C, showed the lowest BMD Z-scores. Women with another rarest allelic variant, TNF-alpha-857T/T had significantly lower BMD Z-scores than did women with TNF-alpha-857C/T or -857C/C. The BMD Z-score decreased significantly with an increase in the total number of such rare alleles. Serum concentrations of TNF-alpha did not differ significantly among groups divided by genotypes. Multiple linear regression analysis revealed that the total number of rare alleles, in addition to the body mass index and the number of years since menopause, was an independent predictor of the BMD. These presumptive functional polymorphisms of the TNF gene may be associated with the lumbar spine BMD in early postmenopausal Japanese women.

Antitumor effect of new local hyperthermia using dextran magnetite complex in hamster tongue carcinoma.

OBJECTIVE: This study was performed to clarify the usefulness of Dextran magnetite (DM) for the oral cancer hyperthermia. METHODS: Tumors were induced in golden hamster tongue by 9,10-dimethyl 1-1,2-benzanthracene (DMBA) application. DM suspension was locally injected into the tumor-bearing tongue and tongues were heated up to 43.0-45.0 degrees C, by AC magnetic field of 500 kHz. RESULTS: The average time taken for the temperature to rise to 43.0 degrees C or above was 162 s (n = 17) at the margin of the tumor and 420 s (n = 17) at the center of the tumor. According to the tumor volume, the time required for an increase in the central temperature of tumor to 43.0 degrees C tended to be prolonged. Both temperatures could be maintained at approximately 43.0-45.0 degrees C for 30 min. The inhibition of the growth of tongue carcinoma in the four-time heating group was significantly greater than in the control group (P < 0.01). Moreover, the survival rate was significantly higher in the heated groups than in the control group (P < 0.01). Histological examination revealed a brown uniform DM accumulation at the stroma in the margin of the tumors. Many of tumor cells disappeared at the site adjacent to this accumulation. CONCLUSION: These results strongly suggest the usefulness of this local hyperthermic system in the oral region that is accessible to this treatment.

Characterization of heteroresistance to fluconazole among clinical isolates of Cryptococcus neoformans.

Strains of Cryptococcus neoformans expressing heteroresistance to fluconazole have been described previously. The present study was conducted to investigate the prevalence of heteroresistance among clinical isolates of C. neoformans and to characterize the heteroresistant phenotypes. A total of 107 clinical isolates of C. neoformans for which the MICs of fluconazole ranged from 0.25 to 32 microg/ml were selected. The isolates were chosen to represent a broad geographic distribution. Of the 107 C. neoformans isolates tested, 4 grew on medium containing fluconazole at concentrations that were four to eight times higher than the MICs for each strain. A fifth isolate, for which the fluconazole MIC was 32 microg/ml, grew on agar with 64 microg of fluconazole per ml. These five isolates (4.7% of the total number) were confirmed to exhibit heteroresistant compositions by population analysis. The degree and frequency of resistance varied among the isolates. Stepwise selection by exposure to fluconazole resulted in subclones of all five strains for which the fluconazole MIC was >64 microg/ml. Subclones of three strains demonstrated a homogeneous population of resistant cells on medium containing 64 microg of fluconazole/ml. The resistance was sensitive to incubation temperature, that is, heteroresistance was demonstrable only at 30 degrees C by agar-based tests, and was reversible through serial transfers on fluconazole-free medium over a period of 8 days. These results suggest that the fluconazole-heteroresistant phenotype of C. neoformans exists in a significant proportion of clinical isolates and that fluconazole resistance can be developed by selection from heteroresistant clones and induction by exposure to fluconazole.

Leiomyosarcoma in the maxilla: a case report.

We describe a rare case of leiomyosarcoma of the maxilla arising in a 71-year-old Japanese woman. The lesion was treated successfully by surgical resection with external irradiation of 70 Gy. No sign of recurrence or distant metastasis was present 3 years after operation. Histogenetically, this maxillary leiomyosarcoma apparently arose from the medial muscle of blood vessels or from primitive mesenchyme in the maxilla.

Increased apoptosis of human fetal membranes in rupture of the membranes and chorioamnionitis.

Apoptosis is thought to participate pathophysiologically in the rupture of human fetal membranes (ROM). The aim of this study was to assess apoptosis of the amnion and the chorion in relation to ROM and chorioamnionitis (CAM). The amnion and chorion at the position of the cervical os and fundus of the uterus were obtained from 44 patients. Apoptotic DNA fragmentation was densitometrically determined, and the relative ratio was used for the quantitative evaluation. Among patients without CAM, the relative ratios of apoptosis in the amnion from patients with ROM were higher than those in patients without ROM (P< 0.05). Among patients without ROM, the apoptotic levels in the amnion from patients with CAM were higher than those in patients without CAM (P< 0.05). These were the cases with the amnion at the position of cervical os and fundus, but not with the chorion. The highest ratio of apoptosis was seen in the amnion from patients with CAM and ROM. Among patients with ROM and no CAM, the apoptotic levels at the cervical os in the amnion (P=0.059) and chorion (P< 0.05) was higher than those at the fundus. The increased apoptosis of human fetal membranes was related to ROM and CAM. Apoptosis plays a role in the pathophysiology of ROM.

Determination of vancomycin in human serum by micellar electrokinetic capillary chromatography with direct sample injection.

BACKGROUND: Vancomycin (VCM) has a bacteriostatic effect on gram-positive bacteria such as the methicillin-resistant Staphylococcus aureus. METHODS: A new assay for measuring vancomycin concentration by micellar electrokinetic capillary chromatography using direct serum injection was developed. A borate buffer (pH 10.0) containing 100 mmol/l sodium dodecyl sulfate was used as an electrophoresis buffer, and the detection was at 210 nm. The migration time of vancomycin was approximately 7 min. RESULTS: The linearity was from 0 to 100 microg/ml, with the limit of detection of 1.0 microg/ml (S/N=3). The within-run CV was 3.99-5.53%, and the recovery rate was 91-103% for a concentration range of 6.5-45.5 microg/ml. The between-day CV was 6.76% at 22.2 microg/ml. There was no interference from 32 other antibiotics. The correlation coefficient between the assay and fluorescence polarization immunoassay and direct injection HPLC was 0.982 and 0.985, respectively. The assay required no sample preparation of serum and used only microquantities of an electrophoresis buffer and samples. CONCLUSIONS: This assay is cost-effective and suitable for routine clinical use.

[Antimicrobial susceptibility pattern and epidemiological investigation of the clinical isolates of Pseudomonas aeruginosa in 1999and 2000].

During 1999-2000, a total of 1,215 isolates of Pseudomonas aeruginosa (P. aeruginosa) were analyzed by wards, type of clinical specimens and serotypes. Antimicrobial susceptibility was also examined for 10 agents (PIPC, CAZ, LMOX, IPM-CS, GM, AMK, MINO, LVFX, NFLX, and ST) with criteria by NCCLS (M7-A5). Sputum (33%) was most common source of P. aeruginosa followed by urine (17) and wound discharge (14%). According to wards, the isolation rate at the critical care unit was the highest (40%) in 1999 but decreased to 14% in 2000. In the isolates from inpatients (1999, 2000) the resistance rate was 96.8-98.9% for MINO and ST, 23.5-30.8% for IPM-CS, 27.2-28.9% for LMOX, 12.5-20.1% for GM, and 6.7-11.8% for NFLX. Comparison between the two years showed increases in isolates resistant to GM (p<0.01) and NFLX (p<0.05) but a decrease in IPM-CS resistant isolates (p<0.01). According to serotypes, the E and G type were most frequently observed (242 isolates each, 21.8%) followed in order by F type (127 isolates, 11%) and the B type (124 isolates, 11%). The incidence of the F type was high compared with other medical institutions. There was a reduction in the rates of susceptibility of serotype E isolates to PIPC (74.4%) and GM (60.3%), and of serotype F isolates to IPM-CS (52%). Multidrug-resistant P. aeruginosa (MDR-PSA) that were resistant to beta-lactams, aminoglycosides, and new quinolons accounted for 3%.

Comparison of the Vitek gram-positive susceptibility 106 card, the MRSA-Screen latex agglutination test, and mecA analysis for detecting oxacillin resistance in a geographically diverse collection of clinical isolates of coagulase-negative staphylococci.

The Vitek automated susceptibility testing system with a modified gram-positive susceptibility (GPS) 106 card (bioMerieux Vitek, Inc., Hazelwood. Mo.) and a rapid slide latex agglutination test (MRSA-Screen test; Denka Seiken Co., Ltd., Tokyo, Japan) were evaluated for their abilities to detect oxacillin resistance in coagulase-negative staphylococci (CoNS). The reference broth microdilution method and the detection of the mecA gene by PCR ("gold standard" reference result) were used to compare the results obtained with the commercial products. A total of 123 clinical isolates consisting of eight species were selected from U.S. surveillance collections. Among the mecA-positive isolates (95 strains), 30 isolates were initially negative on the MRSA-Screen test read at 3 min. When the agglutination reaction was extended for 10 min, 26 of the 30 isolates became positive. For a different four isolates, the oxacillin MIC was < or =0.25 microg/ml on the Vitek GPS 106 card. Among the mecA-negative isolates (28 strains), for two Staphylococcus warneri, two S. lugdunensis, and two S. saprophyticus strains MICs were > or =0.5 microg/ml by the reference broth microdilution method. Four of these isolates were also categorized as resistant with the Vitek GPS 106 card and two isolates were positive by the MRSA-Screen test. Overall, the MRSA-Screen test, GPS 106 card, and reference broth microdilution method had sensitivities of 95.7 (result at 10 min), 95.7, and 100%, respectively, and specificities of 92.8, 85.7, and 78.5%, respectively. Although the MRSA-Screen test required a slight procedural modification, both commercial methods achieved a sensitivity and specificity at detecting oxacillin resistance in CoNS at a level that was acceptable for clinical laboratory use.

New local hyperthermia using dextran magnetite complex (DM) for oral cavity: experimental study in normal hamster tongue.

The possibility of dextran magnetite complex (DM) as a new hyperthermic material was examined in this study. DM suspension of 56 mg ml(-1) iron concentration was locally injected into the normal tongue of golden hamster. DM injected tongues were heated by 500 kHz alternating current (AC) magnetic field and its serial changes in temperature were recorded at 30-s intervals. The temperature of DM injected tongue was maintained at about 43.0-45.0 degrees C for 30 min by changing the AC magnetic field intensity. While temperature elevations of the contralateral tongue and the rectum were only of minor degree. In experiment on the extent of heating area, there was correlation between volume of black stain area and amount of the injected DM suspension (Y = - 18.1 + 1.94X, r = 0.931, P < 0.0001, n = 9 ). Histological examination after heating revealed brown uniform DM accumulation in the connective tissue between fibers of the tongue muscle. Except for vascular dilatations, no tissue damage was seen in the heated tongue. Thus, DM which has the possibility of selective and uniform heating in local hyperthermia might be useful for oral cancer therapy.

Persistent detection of alfa-fetoprotein in the vagina without overt preterm premature rupture of the membranes. Clinical and chemical characterizations.

OBJECTIVES: The objectives of this study were to clinically characterize and seek risk factors for the patients with persistently detected alfa-fetoprotein (AFP) in the vagina without showing overt preterm premature rupture of the membranes (PROM). STUDY DESIGN: The cohort study was undertaken using vaginal-cervical specimens collected consecutively between 21 and 40 weeks of gestation from 25 consenting women. Demographic, obstetric, neonatal, and laboratory data from patients with persistent positive results of AFP kit tests without showing overt leakage of amniotic fluid were compared to those from controls. Cervical levels of interleukin-1 alpha, interleukin-1 beta, interleukin-6, interleukin-8, and granulocyte elastase were measured by immunoassay, and the uterine cervix was assessed by transvaginal ultrasonography. Statistical analysis involved Fisher's exact test and Mann-Whitney U test. RESULTS: The incidence of overt preterm PROM was significantly higher in the patients with persistently detected AFP in preterm (3 in 4 cases) than in controls (3 in 21 controls). At sampling before determination of persistently detected AFP, patients with persistently detected AFP had significantly higher cervical levels of interleukin-6 and significantly shorter cervical length than controls. CONCLUSIONS: Patients with persistently detected AFP in the vagina without showing overt preterm PROM have the risk of overt preterm PROM. Increased levels of interleukin-6 in cervical specimens and short cervical length may be risk factors for the occurrence of persistent detection of AFP in the preterm.

Calmin, a protein with calponin homology and transmembrane domains expressed in maturing spermatogenic cells.

A cDNA named calmin of approximately 3.2 kb was isolated by RNA differential display applied to developing mouse skin. Calmin cDNA encodes 1021 amino acids with two calponin homology (CH) domains in tandem on the N-terminal side and a transmembrane domain on the C-terminal side. The region covering the CH domains showed a high level of homology with beta-spectrin, alpha-actinin, and dystrophin. Among the proteins with the tandem CH domains, calmin is unique in having a transmembrane domain. Three alternative splicing sites were identified at the 3'-side of calmin, giving rise to polymorphic protein products with or without the transmembrane domain. The calmin transcript was detected in adult testis, liver, kidney, and large intestine; the expression in testis was far stronger than that in the other tissues. In situ hybridization and immunostaining revealed that calmin was expressed in maturing spermatogenic cells at later stages. Human calmin cDNA was also isolated, and its exon/intron organization was determined.

Suppression of hair follicle development inhibits induction of sonic hedgehog, patched, and patched-2 in hair germs in mice.

Embryonic induction of hair follicles is a fascinating model of localized morphogenesis from a simple homogeneous epithelial cell sheet. Accumulating evidence indicates that Sonic hedgehog (Shh) signaling plays a central role in hair follicle formation. We quantitated the expression levels of Shh and its receptor genes, Patched (Ptc) and Patched-2 (Ptch2), in two distinct experimental systems in which the development of hair follicles was suppressed. Shh, Ptc, and Ptch2 were induced about six- to tenfold in normal embryonic hair germs in vivo as well as in developing skin tissue maintained in organ culture. This induction was almost completely inhibited both in the developing skin tissue of ICR mice cultured with 30ng/ml epidermal growth factor and in embryos of Tabby mutant mice (a model of hypohidrotic ectodermal dysplasia) at 14.5-15.5 days postcoitus. Expression of Shh, Ptc and Ptch2 was induced in the Tabby embryos at 16.5 days postcoitus, indicating that Shh signaling may be involved in the formation not only of the well-studied guard hair but also of the awl hair. The potential of the two biological systems for studying molecular mechanisms in hair follicle formation, particularly at an early phase including Shh signaling, is discussed.

[Quantitative determination of lipoprotein by agarose gel electrophoresis and enzymatic staining of cholesterol and triglyceride: effects of age and gender].

290 normal duplicate samples (152 males and 138 females), were subjected to lipoprotein analysis by agarose gel electrophoresis to yield HDL, VLDL and LDL fractions which were respectively stained with Cholesterol and Triglyceride reagents. HDL-Cholesterol were significantly higher in female than male, and VLDL-TG were significantly higher in male than female. Assay C.V.'s varied from 0.96 to 5.75 for cholesterol fractions and 2.00 to 4.34 for triglyceride fractions. Comparison of electrophoretic HDL-Cholesterol and LDL-Cholesterol concentrations with the results of a direct method (HDL-EX and LDL-EX, Denka Seiken) gave correlation coefficients of 0.967 and 0.952 respectively. This method is simple, rapid and can provide the simultaneous assessment of the cholesterol and triglyceride component in each Lipoprotein fraction. Additionally, the method is useful for evaluating lipoprotein assays.

Rapid serum vancomycin assay by high-performance liquid chromatography using a semipermeable surface packing material column.

A new isocratic high-performance liquid chromatographic (HPLC) assay has been developed for vancomycin that uses direct injection of microquantities of serum into a separation column filled with octyl-C(8) silica support that has a semipermeable surface. A mixture of disodium hydrogen phosphate buffer (pH 7.0) and acetonitrile is used as the mobile phase, and vancomycin is directly detected at 240 nm. The minimum limit of detection was 0.5 microg/ml at a signal-to-noise ratio of 3:1. Linearity was established from 0 to 100 microg/ml. The coefficient of variation for within-run reproducibility was 1.1-2.7% for a concentration range of 2.9-52.5 microg/ml; for day-to-day reproducibility it was 4.0% and 3.1% for a concentration range of 5.8-26.4 microg/ml, and the recovery rate was 94-105%. There was no interference from 41 antibiotics or other drugs currently in use. The correlation coefficient between the fluorescence polarization immunoassay (x) and this method (y) was 0.995 with a linear equation, y = 1.06x - 0.924. This method is simple, rapid, and provides an economical quantification of serum vancomycin.

Thrombospondin-1 expression in oral squamous cell carcinomas: correlations with tumor vascularity, clinicopathological features and survival.

Thrombospondin-1 (TSP-1) is a 450 kd glycoprotein synthesized and incorporated into the extracellular matrix by numerous cell types and reported to suppress tumor growth and progression by its inhibition of angiogenesis. In order to clarify the biological role of TSP-1 and determine its clinicopathological significance in oral squamous cell carcinomas (SCCs), we identified TSP-1 protein expression in 54 oral SCCs by immunohistochemistry and correlated it with microvessel density (MVD), clinicopathological features and patient's survival. Thirty-two out of 54 carcinomas (59%) were identified as having a low level of TSP-1 expression (TSP-1-L), and 22/54 (41%) carcinomas identified as having a high level of TSP-1 expression (TSP-1-H). The MVD counts (mean+/-S.D.=9.0+/-4.9) in TSP-1-H tumors was significantly lower than that (mean+/-S.D.=12.7+/-4.4) in TSP-1-H tumors (P=0.0065). The level of TSP-1 expression was not correlated with T category and histologic grade, but inversely correlated with the pattern of tumor invasion (P=0.0136) and with lymph nodal status (P=0.0119). Furthermore, Kaplan-Meier analysis showed that the 5-year survival rate of TSP-1-H group was significantly higher than that of TSP-l-L group. Our results suggested that TSP-1 expression exerts an inhibitory effect on tumor vascularity, and that it has value in assessment of aggressiveness and prognosis of oral SCCs.

Effects of inflammatory cytokines on prostaglandin E(2) production from human amnion cells cultured in serum-free condition.

The effects of five inflammatory cytokines, i.e. interleukin(IL)-1alpha, IL-1beta, IL-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha) on prostaglandin E(2) (PGE(2)) production from amnion cells cultured in a serum-free condition was evaluated. After human amnion cells obtained from term placenta were incubated with the inflammatory cytokines at various concentrations, PGE(2) production in the culture supernatant was determined using an enzyme immunoassay method. Under a serum-free culture condition, an increase in PGE(2) production by IL-1alpha and IL-1beta was observed at concentrations of 10 and 100 ng/ml compared to control cultures. However, the increases in PGE(2) production by IL-6 and IL-8 were found at relatively high concentrations, i.e. at 100 and 200 ng/ml. TNF-alpha induced a significant increase in PGE(2) production at 50 and 100 ng/ml, but not at 200 ng/ml. These data suggest that these inflammatory cytokines directly stimulate PGE(2) production from amnion cells and may initiate premature labor if amniotic inflammatory cytokines are elevated, e.g. following intrauterine infection.