Relationship between perceived softness of bilayered skin models and their mechanical properties measured with a dual-sensor probe.

The development of a sensor system that can predict the subjective softness of human skin is an important goal for the cosmetics industry. Here, we first carried out a subjective softness evaluation test using 65 skin models consisting of polyurethane bilayers with different thickness of the superficial layer and different degree of cross-polymerization of the basal layer. The results showed that perceived softness was dependent on the mechanical properties of both the superficial and basal layers. Then, we used a recently developed tactile sensor system composed of a piezoelectric tactile sensor and a load cell to measure mechanical softness parameters of the superficial layer and the whole model, respectively. Statistical analysis showed that the data obtained from these two sensors were well correlated with the perceived softness of the prepared models. These results suggest that it may be feasible to predict the subjective softness of human skin in vivo from non-invasive mechanical softness measurements of the superficial skin layer and whole skin obtained with our new dual-probe sensor system.

Methylation in p14(ARF) is frequently observed in colorectal cancer with low-level microsatellite instability.

Colorectal cancer (CRC) can be classified as high-level microsatellite instability (MSI-H), low-level MSI (MSI-L) and microsatellite stable (MSS) depending on levels of MSI. MSI-H CRC relies on a distinct molecular pathway due to the mismatch repair (MMR) deficiency and shows methylation in multiple gene promoters. The genetic pathway leading to MSI-L is unknown, although higher levels of promoter methylation are observed in this group compared with MSS CRCs. This study explored how promoter methylation affects MSI phenotype, by analysing the methylation status of eight CRC-related promoters, MSI phenotype and KRAS/BRAF mutations in a series of 234 CRCs. Promoter methylation of p14(ARF) was significantly related to MSI-L CRC with KRAS mutation. The MSI-H phenotype was related to methylation of MLH1 as expected, while the MSS phenotype was related to methylation of p16(INK4a) and O(6)-methylguanine-DNA methyltransferase, although this was not statistically significant. Thus, promoter methylation of p14(ARF) could be a significant alteration leading to CRC with MSI-L.

No duplicate KRAS mutation is identified on the same allele in gastric or colorectal cancer cells with multiple KRAS mutations.

Codon 12 and 13 mutations in 170 colorectal cancer (CRC) and 66 gastric cancer (GC) specimens were analysed by an 'enriched' polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. All identified mutations were verified by direct sequencing of the second PCR products. Among the 170 CRC specimens, mutations were identified in 47 (28%) and 13 (7.6%) cases in codons 12 and 13, respectively. In the 66 GC specimens examined, however, mutations in codons 12 and 13 were only detected in two (3.0%) and one (1.5%) cases, respectively. Mutations in both codon 12 and 13 were found in 3/170 (1.8%) CRCs and 1/66 (1.5%) GCs. Duplicate mutations were never identified in the same allele, which was confirmed by direct sequencing of the second amplified products. The majority of colorectal and gastric cancer cells with KRAS mutations are homogeneous because they have the same KRAS mutation. A few colorectal or gastric cancers, however, showed heterogeneity, as verified by the fact that single mutations were identified in the same allele.

Oesophageal squamous cell carcinoma may develop within a background of accumulating DNA methylation in normal and dysplastic mucosa.

BACKGROUND: Oesophageal squamous cell carcinoma (OSCC) often arises from preceding dysplastic lesions in the oesophageal epithelium. However, the molecular changes occurring in premalignant lesions are not well understood. An epigenetic change is an example of OSCC that may occur within the epithelium. AIM: To investigate the methylation status of multiple promoters in cancer-derived DNA, as well as in the background epithelium of OSCC, including dysplastic lesions and non-neoplastic mucosa. The normal epithelium from patients without cancer was also examined. The findings were correlated with the mutational status of p53. PATIENTS AND METHODS: 56 patients with advanced OSCC, 21 patients with intraepithelial neoplasia (IEN), 56 patients with a background of non-neoplastic epithelium, adjacent to the OSCC, and 42 normal control epithelia from healthy volunteers were studied. The promoter methylation status of SFRP1, SFRP2, DCC, APC, p16(INK4a), p14(ARF), MINT1, MINT2, MINT31, CACNA1G, COX2, DAPK, hMLH1 and MGMT was examined by methylation-specific single polymerase chain reaction or combined bisulphite restriction analysis. The mutation of p53 by direct sequencing was assessed. RESULTS: DNA methylation was observed in OSCC and in its background epithelium. The frequency of CpG island methylation increased from a baseline level in the background non-neoplastic epithelium, through IEN, to advanced OSCC. However, mutations in p53 were almost exclusively observed in IEN and OSCC. More extensive DNA methylation was seen in the neoplastic lesions (OSCC or IEN) having a p53 mutation than in those with wild-type p53. CONCLUSION: DNA methylation is present at low levels in the non-neoplastic oesophageal epithelium and appears to contribute to the progression of the dysplasia-carcinoma sequence in OSCC carcinogenesis.

E-Cell 2: multi-platform E-Cell simulation system.

A new version of the E-Cell simulation system,which runs on Windows as well as Linux, has been released as free software under the terms of the GNU General Public License.

E2F-4 mutation in hereditary non-polyposis colorectal cancer.

Defects in the DNA mismatch repair function are known to cause microsatellite instability (MSI) in hereditary non-polyposis colorectal cancer (HNPCC) as well as in a subset of sporadic colorectal cancer (CRC). We previously reported that the E2F-4 gene, which encodes an important transcription factor in cell cycle control, had frequent tumor-specific mutations at a coding region of trinucleotide microsatellite (CAG)n in a subset of human sporadic CRC with high-frequency MSI (MSI-H). In this study, we assessed mutations of E2F-4 in HNPCC as well as other target genes of defective DNA mismatch repair function. Eighteen colorectal cancer (CRC) patients from 13 kindreds meeting the Amsterdam criteria for HNPCC were analyzed and compared to sporadic CRC patients with MSI-H. We detected mutations of E2F-4 at the same repeat sequence in HNPCC. The frequency of the E2F-4 mutation in HNPCC was comparable with that in sporadic CRC with MSI-H. E2F-4 was considered to be one of the important target genes responsible for the carcinogenesis of HNPCC.

Reduction of incretin-like salivatin in saliva from patients with type 2 diabetes and in parotid glands of streptozotocin-diabetic BALB/c mice.

AIM: Diabetic xerostomia is a typical syndrome in diabetic complication. We have reported that salivatin (salivary peptide P-C) derived from human saliva potentiates glucose-stimulated insulin release and inhibits arginine-stimulated glucagon release. The present study is aimed to gain further evidence on the physiological role by investigating the diabetic state-induced change in the amount of salivatin. METHODS: The amount of salivatin was measured in saliva taken from patients with type 2 diabetes with ELISA and with rabbit antiserum against human salivatin immunocytochemically in sections of parotid glands from streptozotocin-diabetic BALB/c mice. RESULTS: The amount of salivatin after a meal was reduced by diabetes in both human saliva and in the serous secretory granule of mouse parotid gland acinar cells. CONCLUSIONS: The above results suggest that salivatin lowers hyperglycaemia after meal and sustains the normal blood glucose levels by incretin-like mechanisms. The function may be damaged by diabetes, and this in turn might make the diabetes worse.

[Long-term survival of a patient with postoperative liver metastasis of stage IVa gallbladder cancer responding to hepatic arterial infusion chemotherapy].

A 58-year-old man was diagnosed as having advanced gallbladder cancer (T4, P0, H0, N0, stage IVa) with direct invasion to the liver, transverse colon and duodenum. Therefore, extended cholecystectomy and bile duct resection with a partial resection of the transverse colon and the duodenum were performed in March 1992. Histopathological examination revealed moderately differentiated tubular adenocarcinoma of si, ly1, v1, hinf3, binf3, n0. Three years and eight months after the operation, multiple liver metastases were diagnosed by abdominal CT. Repeated hepatic arterial infusion chemotherapy with 5-FU 500 mg/body/w, MMC 4 mg/body/2w and EPI 40 mg/body/4w was performed starting in January 1996. Four months later, the lesions in the liver were reduced in size, and abdominal CT 10 months after the chemotherapy showed a partial response. However, the liver metastasis of the right lobe was enlarged on an abdominal CT in August 1997. Repeated hepatic arterial infusion chemotherapy with the same regimen was performed again starting in March 1998. Ten months later, the liver metastasis was slightly enlarged, but the greater part of the metastasis showed necrosis on an abdominal CT in January 1999. However, peritonitis carcinomatosa was observed later and the patient died 8 years after the operation.

A triangle lattice model that predicts transmembrane helix configuration using a polar jigsaw puzzle.

We developed a method of predicting the tertiary structures of seven transmembrane helical proteins in triangle lattice models, assuming that the configuration of helices is stabilized by polar interactions. Triangle lattice models having 12 or 11 nearest neighbor pairs were used as general templates of a seven-helix system, then the orientation angles of all helices were varied at intervals of 15 degrees. The polar interaction energy for all possible positions of each helix was estimated using the calculated polar indices of transmembrane helices. An automated system was constructed and applied to bacteriorhodopsin, a typical membrane protein with seven transmembrane helices. The predicted optimal and actual structures were similar. The top 100 predicted helical configurations indicated that the helix-triangle, CFG, occurred at the highest frequency. In fact, this helix-triangle of bacteriorhodopsin forms an active proton-pumping site, suggesting that the present method can identify functionally important helices in membrane proteins. The possibility of studying the structure change of bacteriorhodopsin during the functional process by this method is discussed, and may serve to explain the experimental structures of photointermediate states.

Immunocytochemical localization of salivary peptide P-C in human submandibular gland.

Human saliva contains a proline-rich polypeptide, salivary peptide P-C, which potentiates insulin release and reduces glucagon release from perfused rat pancreas to decrease blood glucose level. To elucidate the process of secretion into humoral fluid of this peptide morphologically, we investigated ultrastructural localization of P-C in human submandibular gland by immunogold technique with anti-peptide P-C whose specificity to P-C was confirmed by immunoblotting. The labeling with gold particles which represents the distribution of P-C-like-immunoreactivity (P-C-LI) was detected in the secretory granules and rough endoplasmic reticula of the acinar serous cells and in few mucosa cells. P-C-LI was also observed in the lumen of striated duct but not intracellularly in the ductal cells themselves, indicating that P-C is not probably reabsorbed there. These results suggest that salivary peptide P-C is present in acinar serous cells, is secreted into the oral cavity, and may be reabsorbed through the digestive tract to modulate the blood glucose level after feeding.

Plantlet regeneration from mesophyll protoplasts ofBetula platyphylla var.japonica.

InBetula platyphylla var.japonica, colonies were induced efficiently from mesophyll protoplasts cultured in half strength MS (1/2MS) liquid medium containing 0.6 M mannitol, 0.09M sucrose and 1 µM 4-PU and 1 µM NAA at a cell density of 5 × 10(4)/ml. The colonies grew actively and developed into callus after 3 months of culture.Roots differentiated from the protoplast-derived white calluses cultured on the 1 /2MS solid media supplemented with 0.1-1 µM 4-PU and 1 µM NAA, and 10 µM zeatin with no supplementation of NAA. Furthermore, the protoplast-derived green callus differentiated shoots with 1/2MS solid medium containing 1 µM 4-PU or 10 µM zeatin with no supplementation of NAA. When shoots obtained were cultured on the cytokinin-free MS solid medium with 2.5 µM IBA and 0.1 µM NAA, they rooted and developed into plantlets after one month of culture.The phenylurea-type cytokinin, 4-PU, was effective for plantlet regeneration from the mesophyll protoplasts ofB. platyphylla var.japonica. This suggests that there is potential for the use of 4-PU in the culture of protoplasts in many forest tree species.

Extracellular production of mouse interferon beta by the Bacillus subtilis alpha-amylase secretion vectors: antiviral activity and deduced NH2-terminal amino acid sequences of the secreted proteins.

Using two Bacillus subtilis alpha-amylase secretion vectors, mouse interferon-beta (IFN-beta) cDNA was expressed. Ten kinds of mature and hybrid forms of biologically active mouse IFN-beta proteins were synthesized in B. subtilis and were secreted into the culture medium. Similar amounts of proteins, which were determined by immunoblot analysis, were secreted from the B. subtilis transformants, while the antiviral activity in the medium was markedly different in each one of them. The highest antiviral activity was observed in a hybrid protein, in which eleven amino acids consisting of the prosequence of alpha-amylase (eight amino acids) and three amino acids encoding in the HindIII linker DNA were assumed to be extended in front of the Leu residue at position 6 of the mature form of mouse IFN-beta. Its activity was higher than that of the mature form of the mouse IFN-beta secreted from a B. subtilis transformant. These results indicate that the amino acid sequence around the NH2-terminal region of the mouse IFN-beta is closely related to the antiviral activity, but the NH2-terminal amino acid is not. The hybrid proteins of mouse IFN-beta show no interferon activity against a human cell line, HEL.

Signal peptide of Bacillus subtilis alpha-amylase.

Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by the removal of the NH2-terminal 41 amino acid sequence (41 amino acid leader sequence). DNA fragments coding for short sequences consisting of 28 (Pro as the COOH terminus) 29 (Ala), 31 (Ala), and 33 (Ala) amino acids from the translation initiator, Met, in the leader sequence were prepared and fused in frame to the DNA encoding the mature alpha-amylase. The secretion activity of the 33 amino acid sequence was nearly twice as high as that of the parental 41 amino acid sequence, whereas the activity of the 31 amino acid sequence was 75% of that of the parent. In contrast, almost no secretion activity was observed with the 28 and 29 amino acid sequences. The signal peptide cleavage site of the precursor expressed from the plasmid encoding the 33 amino acid sequence was located between Ala and Leu at positions 33 and 34 and that from the 31 amino acid sequence between Thr and Ala at positions 33 and 34. The NH2-terminal amino acid from the latter corresponded to the 3rd amino acid of the mature enzyme. These results indicated that the functional signal peptide of the B. subtilis beta-amylase consists of the first 33 amino acids from the initiator, Met.

[Efficacy of etoposide in bone metastasis of breast cancer].

We have used ADM, MMC, CDDP and other drugs for a case of bone metastasis of breast cancer, but the bone destruction was advanced and she could not walk. We have also used etoposide, a new chemotherapeutic drug, for the same case. Two months later bone sclerosis was seen by X-ray film and pain disappeared. Bone sclerosis then advanced after 6 months, she has begun to stand, and after 8 months she has been able to walk with a cane. There was no severe side effect. Etoposide was very effective for bone metastasis of the breast cancer.

Active forms of chymotrypsin C isolated from autolyzed porcine pancreas glands.

Four active forms of chymotrypsin C (C1, C2A, C2B, and C3) were isolated from the autolyzed porcine pancreas glands. Their molecular weights were estimated by SDS-polyacrylamide gel electrophoresis to be 29 100 for C1, 26 300 for C2A and C3, and 25 500 for C2B. The kinetic analyses of esterase activity of the enzymes toward Ac-LLeu-OEt and Ac-LPhe-OEt showed that chymotrypsin C1 hydrolyzed the two substrates more efficiently than did chymotrypsin C3. Chymotrypsin C1 consisted of chain A (H-Cys-...-Asn-OH, Mr 886) and chain BC (H-Val-...-Lys-OH, Mr 28 200). Chymotrypsin C3 consisted of the two components of C3L and C3S that could be dissociated in the presence of 2.3% SDS. C3L consisted of the chain A and the chain C (H-Ser-...-Lys-OH, Mr 13 600). C3S was the chain B (H-Val-...-Lys-OH, Mr 11 800). These kinetic and chemical analyses show that chymotrypsins C1 and C3 correspond to chymotrypsin A delta and A alpha, respectively.

[Floating knee fracture (ipsilateral fracture of the femur and tibia)--treatment by closed Ender nailing].

Fourteen cases of ipsilateral fractures of the femur and tibia (floating knee fracture) were reviewed and patients were graded according to the type of fracture and the method of treatment. Using the criteria for assessment described by Karlström and Olerud, patients were graded as excellent, good, fair and poor. Excellent results were achieved in five patients, good in three and fair in six. When the fracture extended into the knee, it caused knee problem in three out of five. In ten cases the femoral shaft fractures were nailed and in five both bones were nailed. Nailing of both femur and tibia gave good or excellent result in all five cases. As the comminution is often severe in the cases of floating knee fracture, intramedullary nailing by Küntscher may be difficult. We have introduced a closed Ender nailing for femoral and tibial shaft fractures and we have found that this method is also useful for the floating knee fracture. It is advantageous for this type of fracture, because it is technically simple, it has wide indications and it results in rapid bone union without knee disturbance.

Effect of Culture Conditions on DOPA Accumulation in a Callus Culture of Stizolobium hassjoo.

Effects of passage of subculture and several culture conditions, nutritional, hormonal and light conditions on 3,4-dihydroxyphenylalanine (DOPA) content were examined in callus of STIZOLOBIUM HASSJOO P IPER and T RACY. The favourable conditions for DOPA accumulation were found to be suboptimal conditions for growth, i.e., low concentration of phosphate, low concentration of 2,4-D, high concentration of kinetin. Light promoted DOPA accumulation. The best condition for DOPA accumulation was discussed in relation to protein synthesis.

Evaluation of pericardial effusion with computed tomography.

Evaluation of pericardial effusion was attempted with computed tomography in 11 patients. The volume and distribution of pericardial fluid were assessed with satisfactory resolution and the nature of the fluid was estimated by the difference in x-ray transparency (CT numbers). The volume of pericardial fluid calculated by tomographic methods ranged from 25 ml. to 585 ml. and agreed well with the surgically drained fluid volume. The CT numbers of the pericardial effusion due to renal or heart failure, acute viral pericarditis, hypothyroidism, and hemopericardium were +12 to +13, +20, +28 to +30, and +26 to +40, respectively. Therefore the volume and gross nature of the pericardial fluid could be estimated noninvasively with computed tomography.