Use of sonographic elastography of the masseter muscles for optimizing massage pressure: a preliminary study.

To examine the stiffness of the masseter muscle using sonographic elastography and to investigate its relationship with the most comfortable massage pressure in the healthy volunteers. In 16 healthy volunteers (10 men and 6 women), the Masseter Stiffness Index (MSI) was measured using EUB-7000 real-time tissue elastography. They underwent massages at three kinds of pressures using the Oral Rehabilitation Robot (WAO-1). A subjective evaluation regarding the comfort of each massage was recorded on the visual analogue scale. Elastography was also performed in two patients with temporomandibular joint dysfunction with the myofascial pain. The mean MSI of the right and left muscles in the healthy volunteers were 0.85 +/- 0.44 and 0.74 +/- 0.35 respectively. There was no significant difference between the right and left MSI in the healthy volunteers. The MSI was related to massage pressure at which the healthy men felt most comfortable. The two temporomandibular disorder patients had a large laterality in the MSI. The MSI was related to the most comfortable massage pressure in the healthy men. The MSI can be one index for determining the massage pressure.

Nitrite reductase gene upregulated during conidiation is involved in macroconidium formation in Fusarium oxysporum.

Fusarium oxysporum produces three kinds of asexual spores, microconidia, macroconidia, and chlamydospores. We previously found that the transcript level of the nitrite reductase gene of F. oxysporum, named FoNIIA, was markedly upregulated during conidiation compared with during vegetative growth. FoNIIA was also found to be positively regulated by Ren1 that is a transcription regulator controlling development of microconidia and macroconidia. In this study, we analyzed the function of FoNIIA in conidiation of F. oxysporum. Conidiation cultures showed markedly higher level of accumulation of FoNiiA protein as well as FoNIIA mRNA than vegetative growth cultures. FoNIIA protein was significantly decreased in cultures of the REN1 disruption mutant compared with that of the wild type. These results confirmed that FoNIIA expression is upregulated during conidiation and is positively regulated by REN1. The FoNIIA disruption mutants produced microconidia, macroconidia, and chlamydospores, which were morphologically indistinguishable from those of the wild type. The mutants, however, produced significantly fewer macroconidia than the wild type, although the wild type and mutant strains produced similar numbers of microconidia and chlamydospores. These results demonstrate that nitrite reductase is involved in quantitative control of macroconidium formation as well as nitrate utilization in F. oxysporum.

In vitro synergism between nyasol, an active compound isolated from Anemarrhena asphodeloides, and azole agents against Candida albicans.

The antifungal activity of nyasol (NYS) alone or with various antifungal agents was measured in vitro against Candida albicans, Aspergillus fumigatus, and Trichophyton mentagrophytes. NYS is a compound recently purified from a medicinal plant, Anemarrhena asphodeloides. Among 12 agents, miconazole (MCZ), ketoconazole (KCZ), clotrimazole (CTZ), and cerulenin showed marked synergistic effects against C. albicans. The fractional inhibition concentration (FIC) indices against 4 strains of C. albicans were 0.067-0.31 for MCZ plus NYS, 0.078-0.31 for KCZ plus NYS, and 0.098-0.13 for CTZ plus NYS. These values indicate the possibility of using NYS as an adjuvant to azole agents in the chemotherapy of candidiasis.

Real-time monitoring of in vitro transcriptional RNA synthesis using fluorescence resonance energy transfer.

We have developed a novel method for real-time monitoring of RNA synthesis in in vitro transcription reactions using fluorescence resonance energy transfer (FRET). Two 15mer DNAs, either of which was labeled with Bodipy493/503 as a donor or Cy5 as an acceptor, were prepared. When the two fluorescent DNAs hybridized to adjacent locations on Xenopus: elongation factor 1-alpha (xelf1-alpha) RNA, the distance between the two fluorophores became very close, causing FRET to occur and resulting in changes in fluorescence spectra. A high accessibility 30mer site of xelf1-alpha RNA was found and excess amounts of a pair of donor and acceptor DNA probes that were complementary to the site were added to the in vitro transcription reaction solution. Changes in fluorescence spectra were observed in response to progression of xelf1-alpha RNA synthesis that showed that the fluorescent probes hybridized to the synthesized RNA. Furthermore, when probes hybridizing to the synthesized xelf1-alpha RNA with less efficiency were used to monitor the reaction, spectral changes in response to RNA synthesis were also observed. This result suggests that the probes hybridized to synthesizing RNA molecules before they folded to form secondary structure and that there is no need to select sites on the RNA for the probes, which is required for probes hybridizing to folded RNA molecules.

Direct observation of specific messenger RNA in a single living cell under a fluorescence microscope.

We observed the expression of human c-fos mRNA in a living transfected Cos7 cell under a fluorescence microscope by detecting hybrid formed with two fluorescently labeled oligodeoxynucleotides (oligoDNAs) and c-fos mRNA in the cytoplasm. Two fluorescent oligoDNAs were prepared, each labeled with a fluorescence molecule different from the other. When two oligoDNAs hybridized to an adjacent sequence on the target mRNA, the distance between the two fluorophores became very close and fluorescence resonance energy transfer (FRET) occurred, resulting in changes in fluorescence spectra. To find sequences of high accessibility of c-fos RNA to oligoDNAs, several sites that included loop structures on the simulated secondary structure were selected. Each site was divided into two halves, and the pair of fluorescent oligoDNAs complementary to the sequence was synthesized. Each site was examined for the efficiency of hybridization to c-fos RNA by measuring changes in fluorescence spectra when c-fos RNA was added to the pair of oligoDNAs in solution. A 40 mer specific site was found, and the pair of oligoDNAs for the site were microinjected into Cos7 cells that expressed c-fos mRNA. To block oligoDNAs from accumulating in the nucleus, oligoDNA was bound to a macromolecule (streptavidin) to prevent passage of nuclear pores. Hybridization of the pair of oligoDNAs to c-fos mRNA in the cytoplasm was detected in fluorescence images indicating FRET.

Detection of antifungal activity in Anemarrhena asphodeloides by sensitive BCT method and isolation of its active compound.

Antifungal activity was detected from Anemarrhena asphodeloides by the Bio-Cell Tracer (BCT) method. An active fraction was separated by silica gel column chromatography and reverse-phase HPLC. The molecular weight was determined by GC-MS, and the molecular structure was analyzed by IR, (1)H NMR, and (13)C NMR. The isolated compound was found to be identical to nyasol, (Z)-1, 3-bis(4-hydroxyphenyl)-1,4-pentadiene, which formerly appeared in the literature without any remark on the antifungal activity. This compound showed antimicrobial activity against 38 strains of fungi and five strains of bacteria. The minimum inhibitory concentration (MIC) ranged from 12.5 to 200 microg mL(-)(1), except for two strains based on the broth dilution method.

Konjac mannan improves the vitamin B-6 status of rats fed a vitamin B-6-deficient diet.

To investigate how dietary fiber in the diet affects vitamin B-6 nutriture of rats which have been deprived of vitamin B-6, rats were made vitamin B-6-deficient by feeding a vitamin B-6-deficient 70% casein diet. They were fed 2% cellulose powder-based vitamin B-6-deficient diets supplemented with 3% of additional dietary fiber sources (agar, konjac mannan, pectin and cellulose powder) for subsequent 18 days. Vitamin B-6 status was evaluated according to several biological criteria (weight gain, urinary excretion of xanthurenic acid after tryptophan loading, plasma pyridoxal 5'-phosphate, apparent pyridoxal 5'-phosphate-saturation of liver kynureninase, urinary excretion of 4-pyridoxic acid and fecal output of vitamin B-6). Vitamin B-6 status evaluated by these criteria was considerably improved in the konjac mannan-fed group, when compared with the respective data of the vitamin B-6 supplemented group. The relative mean effect of the konjac mannan diet was about 40% of the vitamin B-6 supplemented diet. In conclusion, konjac mannan was effective for improving the vitamin B-6 nutritional state in vitamin B-6-deprived rats.

[Sensitive screening of antifungal compounds from acetone extracts of medicinal plants with a Bio-Cell Tracer].

Antifungal activity of medicinal plants against Aspergillus niger was evaluated using Bio-Cell Tracer (BCT). By this system enabled the real time determination of hyphal growth rate could be done in the presence or absence of Chinese herbal extracts. Of the 41 herbal extracts with 60% acetone extracts tested by the present method, 26 were found to contain active components against A. niger hyphal growth. In contrast, by conventional methods, no active component was detected from every herbal extract except 3 herbal extracts. From the extract of Anemarrhena asphodeloides, one of active components was isolated and its structure determined by NMR, UV, and mass spectroscopy. The compound was identified as broussonin A (2-3-(4-hydroxyphenyl)propyl)-5-methoxy-phenol) which was formerly reported as a phytoalexin of Broussonetia papyrifera Vent.

Functional quantitative analysis of the genome in cultured human mesangial cells. Technical note.

For normal physiological function, each cell tightly regulates gene expression in a specific fashion so that critical proteins are synthesized in a well-coordinated manner. Therefore, it is very important to uncover which genes are expressed in specific cells. Recent technological advances combined with rapid large-scale DNA sequencing and computerized data processing have allowed us to investigate the expression levels of a variety of transcripts in the mesangial cells, a target of injury in many forms of glomerulonephritis. Utilizing a large scale sequencing of a 3'-directed cDNA library, which allows us to avoid variable cloning efficiencies reflecting the size of cDNA, we investigated expression profiles of various molecules in cultured human mesangial cells. Among the 1,193 sequenced clones, 688 (57.7%) appeared more than once (redundant sequence group), representing 203 different species. Thirty-nine of these appeared more than three times. The most abundant mRNA was that of fibronectin, which consisted of 3.9% of the total mRNA population. Except for mitochondrial or ribosomal genes, calcyclin came next (2.5%), followed by two cytoskeletal genes, gamma-actin gene and calpactin 1 light chain gene, in addition to an amyloid precursor protein homolog (0.7%). In conclusion, we performed a molecular biological quantification of transcripts in mesangial cells. Fibronectin was the most abundantly expressed, followed by calcyclin, gamma-actin, calpactin 1 light chain, and an amyloid precursor protein homolog. We also discovered some candidate genes specific for human mesangial cells. The expression profile of the transcripts serves as an important tool in understanding the biological properties of mesangial cells.

Diagnostic value of technetium-99m methoxyisobutyl isonitrile (99mTc-MIBI) scintigraphy in detecting thyroid cancer metastases: a critical evaluation.

Technetium-99m methoxyisobutyl isonitrile (99mTc-MIBI) scintigraphy has recently been used in clinical application for detecting thyroid cancer metastases, its role being considered supplementary to serum thyroglobulin (Tg) measurements and radioactive iodine (131I) whole-body scans (WBS). The present retrospective study was designed to elucidate the role of 99mTc-MIBI scans in localizing metastatic lesions by assessing sensitivity and specificity of the scan results obtained in a group of 68 thyroidectomized thyroid cancer patients. Presence or absence of thyroid cancer was judged with other diagnostic modes including serum Tg measurements, 131I WBS, bone scans, chest x-rays, computed tomography (CT), ultrasonography, histopathology, and evolution of disease during follow-up. All scans were read on lesion basis for detecting neck, lung, and bone metastases and also on region basis, namely head-neck, chest, and abdomen-pelvis-extremities (ab-p-ex) areas. The sensitivity of detection was 94.4% (17/18) for neck, 78.4% (40/51) for lung, and 92.8% (64/69) for skeletal lesions. Positive predictive value (PPV) and negative predictive value (NPV) were 96.3% (26/27) and 97.7% (43/44) for head-neck; 94.7% (71/75) and 50.0% (12/24) for chest; 100.0% (25/25) and 93.1% (54/58) for ab-p-ex regions, respectively. For all scan sites taken together, PPV and NPV were 96.1% (122/127) and 86.5% (109/126), respectively. In conclusion, the present study reveals that 99mTc-MIBI can be proposed as a first-line diagnostic agent for the follow-up protocol of thyroid cancer patients, although the ability to detect small lung metastases is somewhat limited.

Acute hepatic failure following transcatheter arterial embolization for the treatment of hepatocellular carcinoma.

We conducted a retrospective analysis to evaluate the risk factors associated with the occurrence of acute hepatic failure following transcatheter arterial embolization (TAE) for hepatocellular carcinoma. From 1984 to 1993 we performed a total of 623 embolization procedures in 369 patients with both hepatocellular carcinoma and chronic liver disease. Within 2 weeks after TAE, 13 patients (2.1%) experienced hepatic failure as characterized by a rapid increase in serum bilirubin levels and the development of hepatic encephalopathy of grade 2 or higher. These results indicated that the following are risk factors for acute hepatic failure after TAE: poor hepatic functional reserve; high-dose infusion of chemotherapeutic agents, and a history of multiple embolization procedures.

Rapid and sensitive screening of antifungal activity in medicinal plants by a single-cell biosensing system.

A biosensing system based on the response of fungal cells was used for the evaluation of antifungal activity of medicinal plants against Aspergillus niger. This system measured the hyphal growth rate in real time in the presence or absence of Chinese herbal extracts. The sensitivity of this system was 100-fold higher than that of conventional methods, and is advisable for the screening of antifungal compounds.

Ginseng pretreatment protects against transient global cerebral ischemia in the rat: measurement of local cerebral glucose utilization by [14C]deoxyglucose autoradiography.

The effect of ginseng on brain glucose metabolism has been determined in normal and transient cerebral ischemic rat using the autoradiographic [14C]2-deoxyglucose method. Ginseng had no effect on local cerebral glucose utilization (LCGU) in normal rat. By contrast, in rats subjected to 30 min of four vessel occlusion, a significant reduction of LCGU was shown in parts of the cortex and striatum in comparison to the control group. One-week administration of ginseng at 200 mg/kg/d before the occlusion produced an improvement of LCGU compared with the untreated group. These findings indicate that ginseng has some protective effects against brain damage in transient global cerebral ischemia.

Initial clinical experiences with dopamine D2 receptor imaging by means of 2'-iodospiperone and single-photon emission computed tomography.

Dopamine D2 receptor imaging was performed with 123I labeled 2'-iodospiperone (2'-ISP) and single-photon emission computed tomography (SPECT) in 9 patients: 4 with idiopathic Parkinson's disease, 2 with parkinsonism, 1 with Wilson's disease and 2 with pituitary tumor, and the results were compared with the data for 9 normal subjects. Following an intravenous injection of 123I-2'-ISP, early (within 30 min) and late (between 2 and 4 hr) SPECT images were obtained by means of a multi-detector SPECT scanner or a rotating gamma camera. In normal subjects, early SPECT images demonstrated uniform distribution of radioactivity in the cerebral gray matter and cerebellum reflecting regional cerebral blood flow, whereas late SPECT images showed high radioactivity only in the basal ganglia. All the patients with Parkinson's disease also demonstrated symmetrical basal ganglia uptake in the late SPECT images, but it was diminished in parkinsonism and Wilson's disease. One patient with a growth hormone-producing pituitary tumor had a positive uptake in the tumor. These preliminary clinical data demonstrated that 2'-ISP can be used for SPECT imaging of D2 dopamine receptors and may be of clinical value for the diagnosis and planning of the treatment of neurological diseases.

Bowel preparation for the total colonoscopy by 2,000 ml of balanced lavage solution (Golytely) and sennoside.

One of disadvantages of the Golytely preparation is that examinees have to drink as much as 4,000 ml of Golytely. To overcome this disadvantage, we designed a modified preparation regimen in which examinees have to drink only 2,000 ml of Golytely by taking sennoside orally. Bowel preparation was carried out in 297 examinees by this modified method. Examinees ate their usual diet and took 36 mg of sennoside orally on the night before the examination. On the day of the examination, the examinees drank a total of 2,000 ml of Golytely. No severe complications were noted and 97% of the examinees were able to drink the dose of 2,000 ml. Subjects who had also experienced bowel preparation by the modified method of Brown were asked to compare the two regimens, and only 1% preferred Brown's method while 73% preferred bowel preparation by our Golytely method. The result of bowel preparation by this method was excellent or good in 90 to 97% of the subjects at all sites in the colon and rectum. We conclude that bowel preparation for total colonoscopy using 2,000 ml of Golytely and sennoside is superior because it is highly acceptable to the examinees and provides excellent gut irrigation.

Nucleotide sequences of the 5.8S rRNA gene and internal transcribed spacer regions in carrot and broad bean ribosomal DNA.

Nucleotide sequences of the first and second internal transcribed spacers (ITS1 and ITS2, respectively) of ribosomal DNA (rDNA) from two dicot plants, carrot and broad bean, were determined. These sequences were compared with those of rice, a monocot plant, and other eukaryotic organisms. Both types of ITS region in some species of Angiospermae were the shortest among all eukaryotes so far examined and showed a wide range of variation in their G+C content, in contrast to a general trend toward very high G+C content in animals. Phylogenetic relationships of plants with animals and lower eukaryotes were considered using the nucleotide sequences of carrot and broad bean 5.8S rDNA that were determined in the present study, together with that of wheat 5.8S rRNA, which has been reported previously.