Clinical evaluation of a new hand-held impedance audiometer

In order to evaluate the accuracy and clinical availability of MicroTymp (Welch Allyn) which was new, portable, cordless, and hand-held impedance audiometer, 64 normal subjects and 52 patients with otitis media with effusion were examined by use of MicroTymp and Amplaid 702 (Dana Japan). The types of tympanogram were agreed with each other in 87% of ears. Six cases of type C tympanograms in MicroTymp were shown to be type A by Amplaid 702, and the average of peak pressure of MicroTymp was more negative than Amplaid 702. The difference of peak pressure seemed to be influenced by the speed of ear canal pressure change, because the speed of MicroTymp was considerably higher than Amplaid 702; -200mmH2O/sec in MicroTymp, and -50mmH2O/sec in Amplaid 702. In the experimental study on 7 normal ears, negative shift of peak pressure was observed by increasing the rate of ear canal pressure change from -25mmH2O/sec to -50mmH2O/sec in Amplaid 702. These findings suggest that one should note in the judgment of type C tympanogram in MicroTymp having the peak pressure close to -100mmH2O.     

Peripheral large granular lymphocytes in normal pregnant and postpartum women: decrease in late pregnancy and dynamic change in the puerperium

Large granular lymphocytes (LGLs) have a variety of cytotoxic activities of NK, K and cytotoxic T lymphocytes, suggesting that their morphology is indicative of lytic function. In non-pregnant normal control women (n = 48), the number of LGLs was 0.30 +/- 0.14 x 10(9)/l and the proportion of LGLs in their peripheral lymphocyte fraction was 14.0 +/- 5.4%. The number and proportion of LGLs were significantly decreased in the third trimester of pregnancy (n = 32; 0.19 +/- 0.08 x 10(9)/l, P less than 0.01, and 11.7 +/- 3.8%, P less than 0.05), although an unexpected increase in the proportion of LGLs was observed in the first trimester of pregnancy (n = 24; 17.5 +/- 6.5%, P less than 0.05). After delivery, the number and proportion of LGLs increased rapidly to restore the non-pregnant levels and showed a marked increase in LGL count 4 months postpartum. These data suggest that lymphocyte-mediated cytotoxicity decreases in late pregnancy and increase dynamically after delivery to restore the non-pregnant state.     

Onset of vecuronium-induced neuromuscular block after a long priming interval

Purpose. We examined whether a new application of the priming principle, i.e., having the priming dose of vecuronium administered before the insertion of the epidural catheter, would hasten the onset of the neuromuscular block induced by the intubating dose of vecuronium. Methods. Forty-five adult female patients scheduled for general anesthesia combined with epidural anesthesia were studied. In group A (n = 15), the priming dose of vecuronium, 0.01 mg·kg⁻¹, was administered before insertion of the epidural catheter. The intubating dose of vecuronium, 0.09 mg·kg⁻¹, was given after the insertion of the epidural catheter. In group B (n = 15), the priming dose of vecuronium, 0.01 mg·kg⁻¹, was given 4 min before the intubating dose of vecuronium, 0.09 mg·kg⁻¹. In the control group (n = 15), no priming dose was given, and only the intubating dose of vecuronium, 0.10 mg·kg⁻¹, was administered. In all three groups, general anesthesia was induced with propofol 2.5 mg·kg⁻¹, and the trachea was intubated when T1/control value (control twitch height in response to train-of-four stimuli) was less than 0.1. Results. In group A, the priming dose was given 16 ± 3 min (mean ± SD) before the administration of the intubating dose. The times to onset of neuromuscular block in groups A and B, and the control group were: 145 ± 30, 184 ± 45, and 219 ± 23 s, respectively (P < 0.05 among the three groups). In all three groups, intubating conditions (graded on a four-point scale) were excellent (P = 0.59). Before the induction of anesthesia, symptoms of paralysis were observed in 5, 4, and 0 patients in groups A and B and the control group, respectively (P < 0.05 between group A or B vs control group). Conclusions. If the priming dose of vecuronium is given after a long priming interval (16 ± 3 min), the time to onset of the neuromuscular block caused by the intubating dose of vecuronium is markedly shorter than when the conventional priming interval of 4 min is employed. Received: March 5, 2001 / Accepted: October 4, 2001     

Effective transfection of a cis element "decoy" of the nuclear factor-kappaB binding site into the experimental choroidal neovascularization

PURPOSE: To evaluate the efficacy of the gene transfer of a double-stranded phosphorothioate oligonucleotides (ODNs), called a "decoy", against the NF-kappaB binding site into cells of an experimentally-induced choroidal neovascularization. METHODS: FITC-labeled decoy was injected into the subretinal space of rat eyes by the HVJ-liposome delivery system, and 3 days later, choroidal neovascularization was induced by laser photocoagulation. The eyes were removed and the transfected cells were detected by fluorescence microscopy and also detected by immunohistochemistry. The degree of neovascularization was evaluated by fluorescein angiography. RESULTS: The decoy was transfected into the retinal pigment epithelial (RPE) cells, inner and outer segment of the photoreceptors at 3 days after the injection. When choroidal neovascularization was induced, highly effective transfection of the decoy was observed 3 to 14 days after photocoagulation, after which the level decreased. Decoys were transfected into the RPE cells and macrophages in the choroidal neovascularization. The eyes transfected with NF-kappaB decoy showed a weaker leakage in fluorescein angiograms than that of the control eyes transfected with scrambled decoy. CONCLUSIONS: A decoy can be transfected into retinal cells and cells within a choroidal neovascularization by the HVJ-liposome method. The transferred NF-kappaB decoy reduced the degree of choroidal neovascularization. Decoy targeted against NF-kappaB may be considered as a potential therapy for neovascularization.     

Identification of silencing of nine genes in human gastric cancers

Aberrant methylations in human gastric cancers were searched for by a genome scanning technique, methylation-sensitive representational difference analysis. Nine CpG islands (CGIs) in the 5' regions of nine genes, LOX, HRASLS, bA305P22.2.3, FLNc (gamma-filamin/ABPL), HAND1, a homologue of RIKEN 2210016F16, FLJ32130, PGAR (HFARP/ANGPTL4/ARP4), and thrombomodulin, were found to be methylated in two gastric cancer cell lines, MKN28 and MKN74, but unmethylated in the normal sample. Their expressions were lost in the two cell lines, and the causal roles of the methylations in gene silencing were shown by treatment with 5-aza-2'-deoxycytidine. In 41 primary gastric cancers, methylations of these CGIs in association with reduced expressions were observed at high incidences (29-41%) for five genes, including LOX and HRASLS, which have been reported to have tumor-suppressive activities. The other four genes were methylated at low incidences (0-7%). By analysis of the numbers of aberrant methylations in individual cancers, a subset of cancers with high prevalence of aberrant methylations was identified, and all of the 11 cancers in the subset were diffuse type. To analyze the possible involvement of decreased fidelity of maintenance methylation in this subset of cancers, aberrant hypomethylations of three normally methylated CGIs were examined. Cancers with high prevalence of hypomethylations of normally methylated CGIs, however, constituted a different subset. It is expected that these nine genes may include important genes in gastric cancer development and would be useful to identify a distinct subset of gastric cancers.     

Cloning of the 5' upstream region of the rat p16 gene and its role in silencing.

Hypermethylation of the 5' upstream region (5' region) of the human p16(CDKN2A) (p16) gene is known to cause silencing, which is involved in a wide range of human cancers. For the rat p16 gene, its 5' region has not been cloned, and it is uncertain whether surrogate use of exon 1 alpha is adequate for analysis of p16 silencing. In this study, we observed that methylation analysis of exon 1 alpha gave false positive results in three samples of normal rat mammary epithelia and in two of six primary mammary carcinomas. Therefore, we determined the nucleotide sequence of the 5' region of the rat p16 gene. To confirm that methylation status of the 5' region is correlated with p16 expression, the methylation status was analyzed by bisulfite sequencing and methylation-specific PCR in three samples of normal mammary glands, six samples of mammary carcinomas and four cell lines. The 5' region was demethylated in all of the three normal and six carcinoma samples that fully expressed p16. On the other hand, the 5' region was highly methylated in the 3Y1 cell line, which lacked p16 expression, but without deletion. These results showed that the methylation status of the 5' region was more closely correlated with p16 expression than that of the exon 1 alpha and analysis of the methylation status is useful in examining p16 silencing in various rat tumors.     

Phosphorylation of p38 mitogen-activated protein kinase downstream of bax-caspase-3 pathway leads to cell death induced by high D-glucose in human endothelial cells

Because high D-glucose significantly stimulates endothelial cell death, we examined the molecular mechanisms of high D-glucose-induced endothelial apoptosis. Treatment of human aortic endothelial cells with high D-glucose (25 mmol/l), but not mannitol and L-glucose, resulted in a significant decrease in cell number and a significant increase in apoptotic cells as compared with a physiological concentration (5 mmol/l). Interestingly, high D-glucose treatment significantly increased bax protein, accompanied by translocation of bax protein from cytosol to mitochondria-enriched heavy membrane fraction. In contrast, the expression and distribution of bcl-2 protein were not altered by high D-glucose. In addition, the activity of caspase-3 proteases was increased after exposure to high glucose, whereas caspase inhibitors prevented endothelial cell death induced by high D-glucose. On the other hand, p38 mitogen-activated protein kinase (MAPK) was markedly phosphorylated and showed sustained phosphorylation after stimulation. A specific inhibitor of p38 MAPK, SB 203580, and the overexpression of kinase-inactive p38 MAPK significantly attenuated cell death induced by high D-glucose in human aortic endothelial cells, whereas at 6 h after high D-glucose treatment, SB 203580 and overexpression of kinase-inactive p38 MAPK did not attenuate caspase-3 activation induced by high D-glucose. Importantly, caspase inhibitors significantly attenuated the sustained phosphorylation of p38 MAPK induced by high D-glucose. Thus, we finally focused the MAPK kinase (MEK) kinase 1 (MEKK1) to further examine the cross-talk between p38 MAPK and the bax-caspase proteases pathway. High D-glucose treatment induced MEKK1 cleavage, whereas caspase inhibitors significantly attenuated the cleavage. Importantly, kinase-inactive MEKK1 also blocked the phosphorylation of p38 MAPK induced by high D-glucose. Here, we demonstrated that high D-glucose induced apoptosis in human endothelial cells through activation of the bax-caspase proteases pathway and through phosphorylation of p38 MAPK mediated by MEKK1. Phosphorylation of p38 MAPK downstream of the bax-caspase pathway may play a pivotal role in endothelial apoptosis mediated by high D-glucose.