An innovative masticatory efficiency test using odour intensity in the mouth as a target marker: a feasibility study.

A large number of studies have reported the findings for masticatory efficiency tests; however, some objective masticatory efficiency tests have a drawback, in that subjects are required to spit out the test material. This study examined the possibility of a masticatory efficiency test that evaluates the intensity of odours released when chewing an odour compound-containing material. A total of 20 volunteers were used in this study. The odour intensity in the breath after chewing a gum was measured by portable odour sensor device. The odour intensity after chewing the gum was measured over four chewing durations and at four intervals between spitting out and measurement of the odour intensity. The volume of stimulated saliva was measured by calculating the difference in the weight of the gauze before and after chewing to examine the effect of saliva flow. With an increase in chewing duration, odour intensity reduced. The odour intensity was the highest immediately after chewing. There was a positive correlation between odour intensity and gummy jelly-related masticatory efficiency test value (G-METV), which was significant for 10-s chewing. The regression equation was calculated from three objective variables of odour intensity and G-METV as dependent variable. Pearson's correlation coefficient between G-METV and the odour intensity-related masticatory efficiency value (O-METV) was 0·68. The coefficient of variation of O-METV was significantly lower than that of G-METV. These results suggest that the masticatory performance can be estimated by measuring the odour intensity immediately after chewing food containing odour compounds for 10 s.

Reduced angiotensin II levels in the cerebrospinal fluid of patients with amyotrophic lateral sclerosis.

BACKGROUND: Recent studies suggest that angiotensin II, a major substrate in the renin-angiotensin system, protects neurons through stimulation of its type 2 receptors. However, quite a few clinical studies of angiotensin II levels have shown their relation to disease severity in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). AIMS OF THE STUDY: To clarify the significance of angiotensin II in ALS. METHODS: We assayed angiotensin II concentrations in cerebrospinal fluid (CSF) samples from 23 patients with ALS, nine patients with spinocerebellar degeneration (SCD) and 24 control individuals. We evaluated the disability levels of patients with ALS using the Revised ALS Functional Rating Scale (ALSFRS-R) and calculated the disease progression rate (DPR). RESULTS: CSF angiotensin II levels were significantly lower in the ALS group compared with that in the control group (P = 0.00864), and showed a significant positive correlation with scores on the ALSFRS-R, and a significant negative correlation with the DPR. CONCLUSIONS: In the present study, we reveal for the first time that angiotensin II levels in the CSF from patients with ALS are significantly reduced and significantly associated with disease severity and progression rate. These findings suggest that reduced levels of intrathecal angiotensin II may play a role in ALS.

Enzymatic properties and deduced amino acid sequence of a high-alkaline pectate lyase from an alkaliphilic Bacillus isolate.

A high-alkaline pectate lyase (pectate trans-eliminase, EC 4.2.2.2.) from alkaliphilic Bacillus sp. strain KSM-P7, designated Pel-7, was purified to homogeneity. The purified Pel-7 had a molecular mass of approximately 33 kDa as determined by SDS-polyacrylamide gel electrophoresis. The isoelectric point was close to or higher than pH 10.5. In the presence of Ca2+ ions, Pel-7 trans-eliminated polygalacturonate in random manner to generate oligogalacturonides; it exhibited optimal activity at pH 10.5 and around at 60 to 65 degrees C in glycine-NaOH buffer. Mn2+ and Sr2+ ions can serve as cofactors at almost the same level of Ca2+ ions. It also exhibited a protopectinase-like activity, liberating soluble pectin and/or oligogalacturonides from cotton fibers. The pel gene was cloned and sequenced, and the deduced amino acid sequence of mature Pel-7 (302 amino acids, 33, 355 Da) showed some conserved regions in Pel superfamily, although homology to amino acid sequences of known Pels with 27 to 32% identity. Furthermore, Pel-7 appears to have similar core structure of parallel beta-helix and active site topology with other Pels as revealed by secondary structure prediction in the Pel proteins. These results suggest that Pel-7 is basically grouped into Pel superfamily although the enzymatic and molecular properties are different.

Detection of K-ras gene mutations in plasma DNA of patients with pancreatic adenocarcinoma: correlation with clinicopathological features.

We investigated the presence of K-ras gene mutation in plasma DNA and assessed its clinical value in patients with pancreatic adenocarcinoma. Mutations in codon 12 of the K-ras gene were examined by mutant allele-specific amplification method using DNA extracted from surgical specimens and plasma samples of 21 patients with pancreatic adenocarcinoma. K-ras gene mutation was detected in 15 of 21 (71%) primary tumors. In 9 of 15 (60%) patients with K-ras gene mutation-positive tumors, an identical mutation was detected in the plasma DNA. None of four patients with chronic pancreatitis or five healthy subjects had such mutations in plasma DNA. Tumors positive for K-ras gene mutation in plasma DNA were significantly larger (P = 0.04) and less likely to result in a curative cure after surgical resection (P = 0.09) than those negative for the mutation. Other clinicopathological features, including age, sex, histological type, mode of invasion, and metastasis, did not correlate with K-ras gene mutations in plasma DNA. Treatment resulted in disappearance of K-ras gene mutations in plasma DNA in six of nine (67%) patients. Three patients with a persistently positive K-ras gene mutation in pre- and post-treatment plasma samples were likely to show early recurrence or have a progressive disease. Our findings suggest that K-ras gene mutation can be detected in plasma DNA of patients with pancreatic adenocarcinoma. Detection of K-ras mutations in plasma may be clinically useful for evaluating tumor burden and efficacy of treatment.

Outcome of pancreatic cancer patients based on genetic lymph node staging.

We prospectively assessed the clinical value of genetic staging of lymph node metastasis in patients with pancreatic adenocarcinoma who underwent curative surgery. K-ras gene mutations were detected in the primary tumors in 18 of 25 patients with pancreatic adenocarcinoma. Among these 18 patients, mutated K-ras gene was also found in at least one lymph node in 13 patients. Of these 13 patients, seven had no evidence of histological nodal involvement and six had histological lymph node metastasis. Although there was no significant difference in overall survival rates between the pathological node-negative and -positive patients, overall survival of the five patients with nodes-negative for the mutated K-ras gene were significantly better than that of the 13 patients with genetically metastasis-positive nodes (p<0.001). Furthermore, overall survival of the six patients with genetically metastasis-positive nodes limited to peripancreatic area was significantly better than that of seven patients with genetical metastasis in lymph nodes beyond the peripancreatic areas (p=0.018). These findings suggest that detection of K-ras gene mutations in lymph nodes may be clinically useful to assess the accurate tumor staging and to stratify the patient with pancreatic adenocarcinoma who are at high or low risk for recurrence after curative surgery.

Purification and characterization of glutaredoxin (thioltransferase) from rice (Oryza sativa L.).

We purified and characterized glutaredoxin (thioltransferase), which catalyzes thiol/disulfide exchange reaction, for the first time in plants. The purification procedure employed an immunoabsorbent, antiglutaredoxin-Sepharose. Glutaredoxin was purified about 2,200-fold from rice bran and it appeared to be homogeneous on SDS-PAGE. MALDI-TOF mass spectrometry revealed that the protein has a molecular mass of 11,097.9 Da. Rice glutaredoxin consists of 105 amino acid residues, containing the tetrapeptide -Cys-Phe-Pro (Tyr)-Cys-, which constitutes the active site of Escherichia coli and mammalian glutaredoxins. Inactivation assay also indicated that cysteine residues are responsible for enzyme activity. Kinetic analyses revealed that the enzyme did not exhibit normal Michaelis-Menten kinetics. The enzyme has an optimum pH of about 8.7 with 2-hydroxyethyl disulfide as a substrate. In addition, rice glutaredoxin has dehydroascorbate reductase activity, like mammalian glutaredoxin.

Evaluation of preoperative and intraoperative arterial stimulation and venous sampling for diagnosis and surgical resection of insulinoma.

BACKGROUND: The precise intraoperative localization of insulinoma is essential for successful surgical treatment. In addition to various imaging modalities developed recently, arterial stimulation and venous sampling (ASVS) has also been used for tumor localization. METHODS: Preoperative and intraoperative ASVS procedures were performed in 6 patients with insulinoma. Intraoperative ASVS was performed before and after tumor resection. Immunoreactive insulin (IRI) concentrations and the IRI ratio (IRI concentration at each time interval after calcium injection/baseline IRI concentration) were determined by the conventional or a quick IRI method. RESULTS: The site of the tumor was identified preoperatively in all patients. The peak of the IRI ratio varied widely, but setting the cutoff value at 3.0 clearly differentiated peak IRA ratios in feeding arteries from those of nonfeeding arteries. Intraoperative ASVS showed a similar elevation of IRI levels, but the elevation disappeared after tumor resection in all but 1 patient. In 2 patients, resection of the tumor was confirmed during surgery by measuring IRI levels by the quick IRI method. CONCLUSIONS: A combination of ASVS and conventional imaging modalities is useful for precise localization of insulinoma. Resection of the tumor can be confirmed intraoperatively by comparing IRI levels associated with preoperative and postresective ASVS.

Anatomical reconstruction of carotid bifurcation with a branched non-reversed saphenous vein.

A large carotid body tumor surrounding the entire carotid bifurcation of a 17-year-old female was completely resected with the carotid bifurcation. Anatomical reconstruction was attempted, using a branched non-reversed saphenous vein, the valve cusps of which were incised by a valvulotome. Postoperative angiogram showed smooth, satisfactory flow from the common carotid artery to both internal and external carotid arteries. The postoperative course has been uneventful for the 16 months since the operation. We conclude that the branched non-reversed saphenous vein is an excellent graft for anatomical reconstruction of the carotid bifurcation.

[Mouth mask method for fiberoptic tracheal intubation in difficult intubations].

We tried 72 fiberoptic tracheal intubations (FTI) using a mouth mask in difficult intubation cases. In this method, ventilation is performed via only the mouth using a mask applied over the mouth (mouth mask) and FTI can be done via a nostril with no hindrance from the mask in anesthetized patients. We have been using an infant or child type Seal Mask (Gibeck Respiration) for the mouth mask or a specially made mouth mask. An oral airway is usually inserted and the nostril of one side is plugged with cotton. FTI is performed by another anesthesiologist. An endotracheal (ET) tube capped with a rubber diaphragm is passed through another nostril, and a fiberscope is inserted through the ET tube. The subsequent technique is the same as that of the usual FTI for awake patients. Intubations were successful in all cases except 2; in one, ventilation was impaired even with oral airway in place, and in the other, bleeding in upper airway due to jaw injury from traffic accident hindered the sight of the scope. Mouth mask method for FTI is safe, useful and practical in difficult intubations with little discomfort to the patient.

[The efficacy of transarterial immuno-embolization therapy in patients with unresectable hepatocellular carcinoma].

We devised a new therapeutic modality for multiple hepatocellular carcinoma (HCC) consisting of transarterial immuno-embolization (TIE) using OK-432 and fibrinogen, and applied the treatment to 22 patients with advanced HCC who had been insensitive to TAE, and 21 patients who had no treatment. Nineteen patients had a high AFP level of more than 200 ng/ml. The serum AFP level decreased in 15 patients after TIE, and in 7 patients the AFP level decreased to less than 30%. Furthermore, a marked reduction in tumor size was observed in 69% after TIE. We report 61% 1-year, and 3-year survival rate for cases who had been insensitive to TAE, and 86% 2-year survival rate for those who selected TIE as the first choice. A high fever of more than 38 degrees C occurred in all cases, and hypotension less than 80 mmHg was observed in 42% patients. No deterioration of liver function and no disturbance of the coagulation-fibrinolysis system due to TIE except in one patient with liver failure after TIE.

[A patient with pancreatic acinar cell carcinoma, effectively treated by the infusion chemotherapy through hepatic artery using 5-FU, CDDP and MMC].

Chemotherapy for pancreatic acinar cell carcinoma has not been well studied. We report herein one patient, in whom arterial chemotherapy with 5-FU, CDDP and MMC was extremely effective. He underwent distal pancreatectomy associated with partial resection of transverse colon. Hepatic recurrence was detected by MRI 8 months after the surgery. 5-FU (250 mg/day), CDDP (20 mg/day), and MMC (10 mg) was injected through a catheter introduced into the proper hepatic artery. His tumor became hardly detectable by MRI, and he was discharged about 1 month after admission. 5-FU (250 mg) and CDDP (20 mg) were injected every two weeks, and recurrence was not observed for 5 months.

[The change of hepatic energy status after transcatheter arterial embolization (TAE) for hepatocellular carcinoma--a study using 31P-MRS].

In six patients with hepatocellular carcinoma, adenosine triphosphate (ATP) phosphomonoester (PME), phosphoric acid (Pi) and pH were measured before and after transcatheter arterial embolization (TAE), using 31P-magnetic resonance spectroscopy (MRS). The effectiveness of TAE was judged by the extent of lipiodol accumulation within the tumor and change in serum alphafetoprotein level. The pH within the tumor decreased 2-9 days after TAE in all the cases, and beta ATP/Pi ratio decreased when TAE was effective, whereas the ratio did not decrease when TAE was ineffective. The beta ATP/Pi ratio measured by 31P-MRS seems to be a useful, non-invasive marker of the anti-tumor effect of TAE, although further investigation is needed.

[A patient with HCC successfully treated by ethanol injection therapy with etoposide].

A patient with hepatocellular carcinoma (HCC) who underwent successfully percutaneous ethanol injection therapy (PEIT) with etoposide was reported. A 72-year-old Japanese man with positive HCV-Ab developed multiple tumors of HCC. He underwent transcatheter arterial embolization (TAE) four times and PEIT three times, but the growth of tumors (particularly, S8) could not be controlled by these modalities. Then, the mixture of etoposide (20 mg), ethanol (8.5 ml) and lipiodol (1.5 ml) was injected into S6 tumors. Consistent with the normalization of serum AFP level, CT scan revealed the marked remaining lipiodol in these tumors. PEIT with etoposide is the treatment of choice in patients with HCC refractory to treatment by conventional TAE or PEIT.

Angiotensin-converting enzyme (ACE) and ACE2 levels in the cerebrospinal fluid of patients with multiple sclerosis.

BACKGROUND: We reported a reduction in the levels of angiotensin II in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS). OBJECTIVE AND METHODS: To clarify the mechanism underlying this reduction, we assayed angiotensin-converting enzyme (ACE) and ACE2 concentrations along with angiotensin II concentrations in CSF samples from 20 patients with MS and 17 controls with non-neurological diseases. RESULTS: ACE levels were significantly elevated in patients with MS compared with controls (48.42 +/- 4.84 vs 44.71 +/- 3.9 pg/mL), whereas ACE2 levels were significantly reduced (2.56 +/- 0.26 vs 2.78 +/- 0.24 pg/mL), acting toward a normalization of angiotensin II levels. CONCLUSION: These results further indicate an alteration of the intrathecal renin-angiotensin system in patients with MS.

Detection of carbon-phosphorus lyase activity in cell free extracts of Enterobacter aerogenes.

The bacterium Enterobacter aerogenes could grow on a medium containing alkylphosphonic acid as a phosphorus source. The extracts prepared from the cells grown on phosphonoacetic acid as a sole source of phosphorus showed an activity of carbon-phosphorus lyase and hydrolyzed methyl-phosphonic acid, phosphonoacetic acid and phenylphosphonic acid with a liberation of inorganic phosphates.

A microbial carbon-phosphorus bond cleavage enzyme requires two protein components for activity.

Enterobacter aerogenes IFO 12010 contains a carbon-phosphorus (C-P) bond cleavage enzyme catalyzing the liberation of inorganic phosphate from various alkyl- and phenylphosphonic acids. The enzyme in the bacterium was found to be composed of two physically different protein components, E2 and E3. The molecular weights of E2 and E3 were 560,000 and 110,000, respectively, and E3 was resolved into two apparently homogeneous subunits. Neither component alone could catalyze the C-P bond cleavage reaction, but the reaction was efficiently catalyzed when the components were mixed.

Pros and cons of chest wall reconstruction using prosthetic meshes in a two-layer fashion.

A 46-year-old woman with giant chondrosarcoma of the sternum underwent wide full-thickness resection of the anterior chest wall, which included the pericardium and lung. The free rectus abdominus musculocutaneous flap was transplanted onto prosthetic meshes placed in two layers. While stability and esthetic effect were both good, the subsequent infection in the space between the two meshes prolonged for one month. As a result, the space was closed with an omentum flap. As a consequence, we recommend one-stage omentopexy to prevent the space problem between the two meshes.

Purification and properties of a galacturonic acid-releasing exopolygalacturonase from a strain of Bacillus.

An exopolygalacturonase [exo-PGase; poly (1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67] was found to be extracellularly produced by Bacillus sp. strain KSM-P443. The exo-PGase was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, through sequential column chromatographies. The enzyme had a molecular weight of approximately 45,000 and an isoelectric point of pH 5.8. The N-terminal sequence was Ser-Met-Gln-Lys-Ile-Lys-Asp-Glu-Ile-Leu-Lys-Thr-Leu-Lys-Val-Pro-Val-Phe and had no sequence similarity to those of other pectinolytic enzymes reported to date. Maximum activity toward polygalacturonic acid (PGA) was observed at 60 degrees C and at pH 7.0 in 100 mM Tris-HCl buffer without requiring any metal ions. When the chain length of oligogalacturonic acids increased, the apparent Km for them decreased, but the kcat values increased. This is the first bacterial exo-PGase that releases exclusively mono-galacturonic acid from PGA, di-, tri-, tetra-, and penta-galacturonic acids.

Purification and properties of a high-molecular-weight, alkaline exopolygalacturonase from a strain of Bacillus.

1An exopolygalacturonase [exo-PG; poly (1,4-alpha-D-galacturonide) digalacturonohydrolase, EC 3.2.1.82] was found in a culture of Bacillus sp. strain KSM-P576. The purified exo-PG had a molecular weight of approximately 115,000 and an isoelectric point of pH 4.6. The N-terminal amino acid sequence was Thr-Glu-Val-Ser-Pro-Lys-Ser-Pro-Ala-Ser-Pro-Val. Maximum activity toward polygalacturonic acid (PGA) was observed at 55 degrees C and pH 8.0 in 100 mM Tris-HCl buffer. The exo-PG was quite stable in various pH buffers between pH 6 and 12 when incubated at 30 degrees C for 1 h. Mg(2+,) Mn(2+,) Pd(2+) and Ca(2+) ions stimulated the enzyme activity. The exo-PG released digalacturonic acid from PGA, tri-, tetra-, and penta-galacturonic acids. The apparent K(m) values for oligogalacturonic acids were almost identical, and k(cat) values increased with the chain length of the substrates.

Maintenance of connexin 32 and 26 expression in primary cultured rat hepatocytes treated with 3-acetylpyridine.

BACKGROUND AND AIM: We recently reported that primary rat hepatocytes treated with 3-acetylpyridine (3-AP), an analog of nicotinic acid, could maintain hepatic differentiated functions such as albumin, tryptophan 2,3-dioxygenase, and connexin 32 (Cx32) mRNA expressions for more than a week. In the present experiment, we investigated the expression of not only Cx32, but also Cx26 in cells treated with 10 mmol/L 3-AP in detail. METHODS: We examined the expression of Cx32 and Cx26 in primary rat hepatocytes by using the methods of immunocytochemistry, immunoelectron microscopy, northern blotting, and dye-transfer. RESULTS: The hepatocytes treated with 3-AP were polygonal with a large cytoplasm from day 3, and were maintained for approximately 2 weeks, whereas the cells without 3-AP began to die from day 4. Immunocytochemically in the cells with 3-AP, many Cx32- and Cx26-positive spots were observed between most adjacent cells, and the intensity of positive spots increased with time in culture, whereas in the cells without 3-AP, Cx32- and Cx26-positive spots disappeared at day 4. Furthermore, most Cx26-positive spots were colocalized with Cx32-positive ones. The amounts of Cx32 and Cx26 mRNA transcripts in the cells with 3-AP at day 14 were more than 80% and approximately 30% of those of Cx32 and Cx26 mRNA transcripts in the cells at day 1, respectively. Gap junctional intercellular communication was maintained in the cells treated with 3-AP at day 8, although it was lost in the cells without 3-AP. CONCLUSION: Thus, the addition of 10 mmol/L 3-AP to the medium enhanced the maintenance of Cx32 and Cx26 expression, which is one of the hepatic differentiated functions, in primary rat hepatocytes for a long time.