Multi-platform metabolomics analyses of a broad collection of fragrant and non-fragrant rice varieties reveals the high complexity of grain quality characteristics.

The quality of rice in terms not only of its nutritional value but also in terms of its aroma and flavour is becoming increasingly important in modern rice breeding where global targets are focused on both yield stability and grain quality. In the present paper we have exploited advanced, multi-platform metabolomics approaches to determine the biochemical differences in 31 rice varieties from a diverse range of genetic backgrounds and origin. All were grown under the specific local conditions for which they have been bred and all aspects of varietal identification and sample purity have been guaranteed by local experts from each country. Metabolomics analyses using 6 platforms have revealed the extent of biochemical differences (and similarities) between the chosen rice genotypes. Comparison of fragrant rice varieties showed a difference in the metabolic profiles of jasmine and basmati varieties. However with no consistent separation of the germplasm class. Storage of grains had a significant effect on the metabolome of both basmati and jasmine rice varieties but changes were different for the two rice types. This shows how metabolic changes may help prove a causal relationship with developing good quality in basmati rice or incurring quality loss in jasmine rice in aged grains. Such metabolomics approaches are leading to hypotheses on the potential links between grain quality attributes, biochemical composition and genotype in the context of breeding for improvement. With this knowledge we shall establish a stronger, evidence-based foundation upon which to build targeted strategies to support breeders in their quest for improved rice varieties.

Frequent loss of p16 expression and its correlation with clinicopathological parameters in pancreatic carcinoma.

Expression of the p16 gene product in human primary pancreatic carcinoma (PC) was investigated in paraffin-embedded tissue using a monoclonal antibody against p16 protein, clone G175-405, by means of immunohistochemistry, and the correlation of results with various clinicopathological parameters was evaluated. All six cases of normal pancreas and all but 1 of 20 cases of chronic pancreatitis expressed p16 protein, whereas 37.5% (3 of 8) of cystadenomas and 41. 9% (26 of 62) of PCs lost p16 expression. There was a significant difference between chronic pancreatitis and PC for frequency of the loss of p16 expression (P < 0.01). Moreover, loss of p16 expression in pancreatic malignancy was significantly associated with histological grade (G1 versus G2 and G3, P < 0.01) but not with sex, age, clinical stage, tumor location, or resectability. The survival period was shorter and metastasis is more likely in those cases that did not show p16 expression than those that did.

Detection of mutations of p53 tumor suppressor gene in pancreatic juice and its application to diagnosis of patients with pancreatic cancer: comparison with K-ras mutation.

Because of the difficulty in obtaining biopsy specimens from pancreatic cancer patients, K-ras mutation analysis in pancreatic juice has been used for specific diagnosis. But recently, false positives have been obtained with this method. To improve the genetic diagnosis of pancreatic cancer, detection of p53 gene mutation in pancreatic juice was studied. Pancreatic juice was sampled endoscopically. Single-strand conformation polymorphism analysis was used for p53 mutation analysis. Furthermore, K-ras mutations at codon 12 were also studied in the same pancreatic cancer patients. Of 26 cases of pancreatic cancer, p53 mutations were detected in 11 (42.3%). No mutations were seen in the cases with mucin-producing adenoma nor with chronic pancreatitis. K-ras mutations were detected in 84.0% of cases by RFLP analysis, which has high sensitivity, and in 65.3% by hybridization protection assay, which has high specificity. Using a combination assay with both genes, genetic abnormalities were detected in 92.0% by RFLP and 73.1% by hybridization protection assay including two cases in which p53 alone was positive by both methods. The specificity of p53 mutation for pancreatic cancer is very high. Therefore, simultaneous analysis of p53 and K-ras mutation is suggested to enhance the genetic diagnosis of pancreatic cancer.

Infrequent expression of p21 is related to altered p53 protein in pancreatic carcinoma.

This study is designed to investigate the expression of p21 and its relation to altered p53 protein in pancreatic carcinoma (PC). Immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections of PC was performed using a monoclonal antibody against p21 (187), with a parallel examination of altered p53 protein. The expression of p21 was only found in 12 of 58 (20.7%) PCs and, moreover, was mainly restricted to the well-differentiated ductal epithelium. Sixty-four % (37 of 58) of PCs showed positive p53 staining, and this change was significantly related to the absence of p21 expression (P < 0.01). In a subgroup, the proportion of the undetectable p21 expression and the expression of p53 were increased with increasing tumor grade but decreased with advancing clinical stage. The results of the present study suggest that the absence of p21 expression is very common in PCs and appears to relate to altered p53 protein. Moreover, the abnormalities involving the expression of p21 and p53 may play a more important role in the development than in the progression of this malignancy.

Overexpression of p8 is inversely correlated with apoptosis in pancreatic cancer.

A recently identified gene, p8, has cell growth-promoting activity and is strongly induced in acute pancreatitis. In this study, we detected p8 and single-stranded DNA (ssDNA) for apoptosis by immunohistochemistry in human pancreatic cancer. The p8 was overexpressed (>30% per 1000 cancer cells) in 26 of 44 (59%) pancreatic cancers, and apoptosis (ssDNA-positive cells >10% per 1000 cancer cells) was recognized in 18 of 44 (41%) pancreatic cancers. There was a significant inverse correlation between the p8 overexpression and apoptosis (P < 0.05). Moreover, the expression pattern of high p8 and low ssDNA was seen significantly more often in lower age (<65 years), in moderately or poorly differentiated cancers, and in node-positive cases (P < 0.05). The p8 expression and apoptosis were not significantly correlated with survival. These results suggest that p8 overexpression is involved in antiapoptotic activity and the biological characteristics of pancreatic cancer.

Expression of p8 in human pancreatic cancer.

The p8 gene is a recently identified gene with mitogenic activity. p8 expression is induced in acute pancreatitis, pancreatic development, and regeneration. However, the expression of p8 in pancreatic cancer is not reported. We investigated p8 expression in 72 human pancreatic tissues, including 38 pancreatic cancers (PCs), by immunohistochemistry. p8 was overexpressed (positive cells >25% in 1,000 cells) in 71% (27 of 38) of PCs, but in only 17% (3 of 18) of chronic pancreatitis cases. There was no overexpression in mucinous cystadenoma or in normal pancreas. The p8 overexpression rate in PC was significantly higher than that in other conditions (P < 0.05). Reverse transcription-PCR analysis confirmed p8 mRNA overexpression (tumor/nontumor ratio >2) in 75% (3 of 4) of PCs. p8 was overexpressed also in human pancreatic cancer cell lines (MIA PaCa-2 and PANC-1). These results suggest that p8 is involved in the development of pancreatic cancer, reflecting its mitogenic activity.

Colocalization of vascular endothelial growth factor (vascular permeability factor) and insulin in pancreatic islet cells.

Previously, we found that the islet of pancreas stained with a antibody against the vascular permeability factor (VPF; also known as vascular endothelial growth factor, VEGF) protein. To determine how common this reaction was and whether it was a specific reaction for the islet, we examined its expression and specific cellular localization. Two different antibodies directed against VPF/VEGF peptide revealed an intense reaction for beta-cells in the human islets of Langerhans, and several human beta-cell tumors (insulinomas), but no reaction, were detectable in the vascular endothelium. In the fetal pancreas (second and third trimesters), the VPF/VEGF peptide was detected in immature islets. Northern blot analysis of cell lines derived from rodent insulinomas revealed expression of VPF/VEGF messenger ribonucleic acid. Western blot analysis of conditioned medium from one of these cell lines showed the presence of the released VPF/VEGF protein. These findings indicate that beta-cells have a specific role other than endocrine function in the pancreas. VPF/VEGF in beta-cells may be involved in the maintenance and control of permeability within the islet capillary system.

The amino acid sequence of a 20 kDa bifunctional subtilisin/alpha-amylase inhibitor from bran [correction of brain] of rice (Oryza sativa L.) seeds.

A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the alpha-amylase from the larvae of the red flour beetle (Tribolium castaneum) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion-exchange chromatography on DEAE-Cellulose and Toyopearl CM-650, and preparative HPLC on Vydac C18. The complete primary structure was determined by automatic degradation of the intact, reduced and S-alkylated protein, and by manual DABITC/PITC micro-sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, elastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds.

A flow cytofluorometric study on age-dependent ploidy class changes in mouse hepatocyte nuclei.

To quantitate the age-dependent hyperploidization of mouse hepatic cell nuclei, a cytophotometric study using FACS III was performed. The isolated nuclei from the liver of C57BL/6CrSlc mice at various ages were stained with chromomycin A3. The fraction of polyploid nuclei was increased with age and invariably greater in male than in female animals of corresponding ages. The highest ploidy class observed was 16C, but from the analysis of polyploidization ratio, increase in 4C and 8C nuclei was considered to be the characteristic of the age-related change.

Immunocytochemical demonstration of prolactin interaction with choroid plexus in aging and acute hyperprolactinemia.

Prolactin (PRL) exerts a direct effect on the central nervous system, reaching the PRL-responsive brain regions via cerebro-spinal fluid (CSF). The hormone enters the CSF by a specific receptor-mediated transport mechanism that is localized on the epithelium of the choroid plexus (CP) of brain ventricles. PRL interactions with the CP in aging were examined in young (3-month) and old (27-month) female Wistar rats using immunocytochemistry (immunogold technique). The enhancement of PRL uptake by the CP in animals at both ages was achieved by the modelling of acute hyperprolactinemia. A great age-related difference was found in the intensity of immunocytochemical reaction under activated conditions, the uptake of PRL by CP being significantly higher in young animals than in old. The character of the colloidal gold particle distribution in different components of CP epithelial cells appeared to be the same in both age groups. The weakening of PRL-transporting capacity in the CP of old animals may constitute one aspect of the alteration of neuroendocrine regulation in the CP-CSF system that occurs during aging.

Age-associated ultrastructural changes in the aortic intima of rats with diet-induced hypercholesterolemia.

The ultrastructure of the aortic intima and serum lipid levels in Fischer 344 rats were examined at the ages of 12, 18 and 24 months after feeding the animals an atherogenic diet (2% cholesterol, 0.25% sodium cholate, 5% beef fat) for 6 months. Structural changes in the intima were noticeable only at the age of 24 months. In control rats, the endothelial cells were irregular in shape and each had a well-developed Golgi complex and a few lipid droplets. Simultaneously, reticular, basal lamina-like material and electron-dense granules of extracellular liposomes accumulated in the subendothelium. In fat-fed rats, these structural changes were more conspicuous in association with hypercholesterolemia, and numerous monocytes with lipid droplets were attached to the endothelium, occasionally invading into the subendothelium. Slight foam cell lesions were evident in the intima. The finding that older rats were more susceptible to the atherogenic diet suggests that atherogenesis in the rat is promoted by intrinsic age-associated changes in the aortic intima.

Studies on the structure-function relationship of the HNK-1 associated glucuronyltransferase, GlcAT-P, by computer modeling and site-directed mutagenesis.

All members of a glucuronyltransferase (GlcAT) gene family cloned to date contain four conserved regions (modules I-IV), which are widely located in the catalytic domain. In order to understand the biological significance of these modules, we investigated the structure-function relationship of GlcAT-P by means of the combination of site-directed mutagenesis and computer aided three-dimensional modeling. The wild-type and mutant GlcAT-Ps were expressed in Escherichia coli as glutathione-S-transferase (GST)-fused soluble proteins. Most of the mutants in which a polar amino acid within the modules was replaced with alanine lost their transferase activity almost completely, while all of the mutants in which the replacement was outside these modules retained the original catalytic activity. A three-dimensional (3-D) model of GlcAT-P was constructed by computer simulation with the three-dimensional structure of adenylate kinase (1AKE) as a template. This model predicted that the large catalytic domain of GlcAT-P forms a globular shape with a Rossmann-fold motif consisting of five alpha-helix and beta-sheet repeats. The putative catalytic pocket consisting mainly of modules I-III is surrounded by a cluster of polar amino acids, which are essential for the transferase activity and also for the binding to the acceptor substrate (essential amino acids), asialo-orosomucoid. There is the second cluster of essential amino acids almost on the opposite surface of the molecule, in which an aspartic acid repeat (DDD) is located. The biological significance of the second cluster is currently not clear but it may be associated with the interaction of the enzyme with modulation molecules, manganese and membrane phospholipids.

Pineal gland in old age; quantitative and qualitative morphological study of 168 human autopsy cases.

Age-related changes of human pineal gland were studied morphologically on 168 autopsy cases. Pineal weight in the formalin-fixed condition was 99 +/- 56 mg in males (n = 88, mean age 78 years) and 91 +/- 41 mg in females (n = 80, mean age 79 years), which showed no sexual difference (P = 0.290). There was no correlation between pineal weight and age of the patient (r = 0.0678, P = 0.191, n = 168). The pineal volume calculated as a spheroid was directly proportional to the weight (r = 0.904, n = 167, P = 0.000). A significant correlation existed between pineal weight and the degree of calcification (r = 0.231, P = 0.002, n = 156). The density of the pineals decreased with age (r = -0.164, P = 0.017, n = 167). The degree of calcification and cyst formation did not increase with age, but the grade of cyst formation was related to the pineal volume (P = 0.0002). Some pineals of the patients over 90 years old showed no calcification and appeared indistinguishable from the ones of the younger subjects. The weight and volume of the pineals of the patients with hypertension was appreciably greater than those of the non-hypertensive patients. These results indicate that human pineals do not necessarily degenerate progressively after involution.

An immunohistochemical analysis of p27 expression in human pancreatic carcinomas.

p27 is known as one of the candidate tumor suppressor genes. Although abnormalities including deletion and mutation in this gene are rarely detected, loss of p27 expression has been reported to be correlated with the high degree of malignancy in many human cancers. In the present study, we investigated the status of p27 expression in human pancreatic carcinoma (PC) and assessed the clinicopathologic significance of loss of p27 expression in the development and progression of this malignancy using immunohistochemistry. No nuclear staining for p27 protein existed in 65 (65.7%) of the 99 PC tumors examined. Furthermore, loss of p27 expression was correlated with tumor grade (G1 versus G2 or G3, p < 0.05) or clinical stage (I and II versus III and IV, p < 0.01). The above data suggest that loss of p27 expression is a very common event in PC, and moreover, this alteration might contribute to the progression of this malignant disease. Key Words: Pancreatic carcinoma-p27 protein expression-Immunohistochemistry.

Usefulness of supernatant of pancreatic juice for genetic analysis of K-ras in diagnosis of pancreatic carcinoma.

AIMS: To ascertain whether analysis of K-ras mutations at codon 12 (KRM) in the supernatant of pure pancreatic juice (PPJ) is more useful for the diagnosis of pancreatic carcinoma (PCa) than that in sediment, the authors analyzed KRM in DNA extract from not only the sediment but also the supernatant of PPJ and compared the results. METHODOLOGY: PPJ was collected endoscopically from 19 patients with PCa and 25 patients with chronic pancreatitis (CP). DNA was extracted from the supernatant and the sediment of PPJ. Mutant allele-specific amplification (MASA) was performed for KRM analysis with the DNA extracts from these samples. RESULTS: The incidence of KRM in the supernatant of PPJ was 89% (17 of 19) in patients with PCa and 28% (7 of 25) in patients with CP, whereas that in the sediment was 79% (15 of 19) in patients with PCa and 20% (5 of 25) in patients with CP. Although there was no significant difference in KRM incidence between supernatant and sediment, the positive rate of KRM was higher in the former. Additionally, with regard to the PCa cases, KRM were found in the supernatant alone in four cases and in the sediment alone in two cases. Consequently, by a combination assay, all of the patients with PCa showed KRM in either the supernatant or sediment of PPJ. Although there was no relation between the incidence of KRM in PPJ and the location and size of tumor, and clinical stage of carcinoma in the patients with PCa, two patients with clinical stage I disease showed KRM in the supernatant. CONCLUSION: These results suggest that the positive rate of KRM in the supernatant is not lower than that in the sediment, and simultaneous analysis of KRM in the supernatant and sediment of PPJ enhances the genetic diagnosis of PCa.

Complete regression of colonic adenomas after treatment with sulindac in Gardner's syndrome: a 4-year follow-up.

A 22-year-old woman with Gardner's syndrome in whom long-term sulindac therapy, without surgical treatment, was effective in inducing complete regression of colonic adenomas is reported. One hundred milligrams of sulindac was administered twice daily after endoscopic polypectomy. Follow-up colonoscopy 6 months later revealed an encouraging regression of colonic adenomas. The tumors had disappeared after 40 months of sulindac treatment. A sustained effect was identified even after 51 months. Ten milligrams of famotidine was coadministered to prevent side effects of sulindac. Although the effect of sulindac on colorectal adenomas may be transient, this therapy may be useful for postponing prophylactic colectomy, especially for the sparse type of familial adenomatous polyposis.

Pneumatosis intestinalis and hepatic portal venous gas caused by mesenteric ischemia in an aged person.

An 82-year-old woman complaining of abdominal pain and vomiting was admitted to our emergency department. Abdominal X-ray, ultrasonography, and computed tomography showed hepatic portal venous gas, as well as pneumatosis intestinalis. We first suspected superior mesenteric arterial thrombosis. However, her physical findings, including computed tomography scanning and laboratory data, did not support the presence of bowel necrosis. The gas disappeared after 1 day. After the 12th day, she had recovered with conservative therapy, and she was discharged on the 41st day. Many reports indicate that hepatic portal venous gas is often associated with bowel necrosis, and urgent operation is recommended in such instances. In this patient, total colonoscopy on the 7th day revealed longitudinal redness, suggesting mesenteric ischemia. Thus, we speculate that this is a rare case of mesenteric ischemia without bowel necrosis associated with both pneumatosis intestinalis and hepatic portal venous gas.

Mucin-producing biliary papillomatosis associated with gastrobiliary fistula.

We report a case of mucin-producing biliary papillomatosis in a 78-year-old woman. Abdominal ultrasound (US) and computed tomography (CT) showed wall thickening and dilatation of the intrahepatic bile duct (IHBD), as well as a nodular lesion, 1.2 cm in diameter, in the left branch of the IHBD. Gastric endoscopy revealed excretion of bile-containing mucin on the anterior wall of the body of the stomach. Endoscopic ultrasonography (EUS) showed gastrobiliary fistula and discharge of mucin into the stomach. Needle biopsy of the biliary tumor revealed papillary proliferation, but no malignant cells were recognized histologically. Therefore this patient was diagnosed as having mucin-producing biliary papillomatosis forming gastrobiliary fistula. She did not present with obstructive jaundice, probably because of the fistula. She is alive, without obstructive jaundice, 16 months after the diagnosis without having had surgery. This is, to our knowledge, the first reported case of biliary papillomatosis forming gastrobiliary fistula and with the patient free of obstructive jaundice.

Drug interactions of zonisamide with phenytoin and sodium valproate: serum concentrations and protein binding.

The influence of co-medication with zonisamide (ZNS) on the serum concentration and protein binding of phenytoin (PHT) and sodium valproate (VPA) was studied in 21 pediatric patients. No significant correlation between the daily ZNS dose, and total serum concentrations, free concentrations or free fractions (FF) of PHT or VPA was observed. The patient study showed that changes in the FF of PHT and VPA were correlated more closely with the serum protein and bilirubin levels than changes in the ZNS dosage. An in vitro study revealed that the addition of ZNS caused decreases in the FF of PHT and VPA. However, these decreases were within the range of measurement error and were negligible. In conclusion, no significant effect of ZNS on the serum concentration or protein binding of PHT or VPA was demonstrated.

Thermal and physicochemical properties of rice grain, flour and starch.

Three types of rices, namely, Thailand rice (Indica), Nipponbare (Japonica), and Himenomochi (Japonica waxy), in grain, flour, and starch forms have been studied for their thermal and physicochemical properties. In grain form, Indica was slender and Japonica rices were bold and thick. Indica had the highest protein and amylose equivalent. Protein contents in isolated starches varied from 0.2 to 0.9%. Cooked Indica grain was hardest and waxy rice was softest; stickiness was highest in Japonica rice. Glass transition temperature (T(g)) was highest in Indica rice flour (approximately 222 degrees C) and almost the same in Japonica rice flours. Melting point was highest for Japonica (approximately 264 degrees C) and almost the same for Japonica waxy and Indica rice flours. T(g) values of starches were almost the same in Indica and Japonica waxy (approximately 237 degrees C); defatting caused reduction in this property in all of the starches. Highest melting point was shown by Indica starch (approximately 276 degrees C) and was almost the same for the other two starches. Protein and fats play a critical role in glass transition and melting points of rice flours and their respective starches. Viscosities of the cooked pastes of flour and starch during cooking in an RVA instrument and their gel and other properties have been discussed.