Electrolyte-free milk protein solution influences sodium and fluid retention in rats.

Milk is an effective post-exercise rehydration drink that maintains the net positive fluid balance. However, it is unclear which components are responsible for this effect. We assessed the effect of milk protein solution (MPS) obtained by dialysis on body fluid retention. Milk, MPS, milk electrolyte solution (MES), sports drink and water were administered to male Wistar rats at a dose of 6 ml/rat after treadmill exercise. Total body fluid retention was assessed by urine volume 4 h after administration of hydrating liquids. The rate of gastric emptying was evaluated by a tracer method using (13)C-labelled acetate. Plasma osmolality, Na and K levels, and urinary Na and K were measured by HPLC and osmometry, respectively. The gastric emptying rate was not delayed by MPS. During 4 h of rehydration, cumulative urine volumes differed significantly between treatment groups (P < 0·05) with 4·9, 2·2 and 3·4 ml from water-, milk- and MPS-fed rats, respectively. Thus, MPS elicited 50 % of the total body fluid retention of milk. Plasma aldosterone levels were significantly higher in MPS- and milk-fed rats compared with water-fed rats. Plasma osmolality was maintained at higher levels in MPS-fed rats than in water- and MES-fed rats (P < 0·05). Cumulative urine Na excretion was also suppressed in the milk- and MPS-fed groups compared with the MES-fed group. Our results demonstrate that MPS obtained by dialysis clearly affects net body water balance without affecting gastric emptying after exercise. This effect was attributed to retention of Na and water, and maintenance of plasma osmolality.

Happy experience of a medical doctor curing from pancreatic cancer: a 60-day diary.

Pancreatic cancer was found by an abdominal CT scan in a medical doctor, the author of this article, before the appearance of any symptoms. After considering all the imaging findings including the CT, MRI, and PET, a diagnosis was made. He was admitted in the University of Tokyo Hospital on the 14th day after the CT finding. On the 18th day, the operation was successfully performed, and no tumor invading to adjacent tissue was seen. On the 29th day, 11th day after the operation, he left the hospital with a drain still in place to excrete abdominal exudation. The remaining drain was finally removed on the 60th day, and the treatment by the surgeon was completed. The chronological events that occurred during these 60 days are described in diary form. There is nothing superior to early detection and early treatment in the fight against cancer. He recommends everybody to receive periodical medical examinations before praying for good luck.

Rapid rehydration and moderate plasma glucose elevation by fluid containing enzymatically synthesized glycogen.

Enzymatically synthesized glycogen (ESG) has high solubility and its solution has low osmotic pressure. Therefore ESG solution could be rapidly absorbed and could be adequate for water rehydration and carbohydrate supplementation during exercise. The object of this study was to evaluate the gastric emptying time and plasma glucose elevation after an administration of ESG solution in comparison with another carbohydrate solution by using a laboratory animal. Male BALB/c mice were administered 10% w/v solution of glucose, maltodextrin, starch, naturally synthesized glycogen (NSG) and ESG at a dose of 20 µL/g body weight for the measurement of gastric emptying rate (Experiment 1) and 10 µL/g body weight for the measurement of plasma glucose elevation (Experiment 2). The osmolarity of gastric content was lower in the ESG and maltodextrin group than the other carbohydrate group. Weight of gastric fluid was significantly lower in the ESG and water group than the glucose group (p<0.01). Plasma glucose level was significantly lower in the ESG group than the glucose group from 0 to 60 min after administration (p<0.01), whereas plasma glucose level was same from 60 to 120 min for the ESG and glucose group (p=0.948). In Experiment 3, BALB/c mice ran on a treadmill for 2 h and were administered 8% of ESG or glucose solution (1.75, 3.5 or 7.0 µL/g body weight) every 20 min during running. There was no difference in post-exercise muscle glycogen level. These data suggest that 1) ESG beverage does not disturb water absorption because of its short gastric emptying time and 2) ESG slowly elevates plasma glucose level and maintains it for a prolonged time compared to the glucose solution.

FGF-2 suppresses cellular senescence of human mesenchymal stem cells by down-regulation of TGF-beta2.

Human mesenchymal stem cells (hMSCs) are able to both self-replicate and differentiate into a variety of cell types. Fibroblast growth factor-2 (FGF-2) stimulates the growth of hMSCs in vitro, but its mechanisms have not been clarified yet. In this study, we investigated whether cellular senescence was involved in the stimulation of hMSCs growth by FGF-2 and the expression levels of transforming growth factor-beta1 and -beta2 (TGF-betas). Because hMSCs were induced cellular senescence due to long-term culture, FGF-2 decreased the percentage of senescent cells and suppressed G1 cell growth arrest through the suppression of p21(Cip1), p53, and p16(INK4a) mRNA expression levels. Furthermore, the levels of TGF-betas mRNA expression in hMSCs were increased by long-term culture, but FGF-2 suppressed the increase of TGF-beta2 mRNA expression due to long-term culture. These results suggest that FGF-2 suppresses the hMSCs cellular senescence dependent on the length of culture through down-regulation of TGF-beta2 expression.

Single-injection ornithine decarboxylase-directed antisense therapy using atelocollagen to suppress human cancer growth.

BACKGROUND: Substantial evidence supports a direct role of ornithine decarboxylase (ODC) in the development and maintenance of human tumors. Although antisense oligonucleotide therapy targeting various genes are useful for cancer treatment, 1 of the major limitations is the problem of delivery. A novel antisense oligonucleotide delivery method is described that allows prolonged sustainment and release of ODC antisense oligonucleotides in vivo using atelocollagen. METHODS: The effect of ODC antisense oligonucleotides in the atelocollagen on cell growth of gastrointestinal cancer (MKN 45 and COLO201) and rhabdomyosarcoma (RD) was studied in vitro using a cell-counting method with a hemocytometer. In vivo, the effect of intratumoral, intramuscular, and intraperitoneal single administration of ODC antisense oligonucleotides in the atelocollagen on tumor growth of MKN45, COLO201, and RD cells was studied. ODC activity and polyamine contents were measured. RESULTS: In vitro, ODC antisense oligonucleotides in the atelocollagen remarkably suppressed MKN45, COLO201, and RD cell growth. A single administration of antisense oligonucleotides in the atelocollagen via 3 routes remarkably suppressed the growth of MKN45, COLO201, and RD tumor over a period of 35-42 days. CONCLUSIONS: As various human cancers significantly express ODC, the results strongly suggest that this new antisense method may be of considerable value for treatment of human cancers.

Novel function of lipids as a pheromone from the Harderian gland of golden hamster.

Sexual diversity of ADG in Harderian gland of golden hamster was demonstrated on TLC. Female ADG contained iso- and anteiso-branched acyl and alkyl components, but male ADG contained only straight chain ones, which suggested the hormonal control of the expression of acyl-CoA dehydrogenases in the catabolism of BCAA. Acyl-CoA dehydrogenases were not expressed in the absence of testosterone, and then isovaleryl-CoA, 2-methylbutyryl-CoA, and isobutyryl-CoA accumulated, and acted as primers for the synthesis of iso- and anteiso-branched fatty acids. The incorporation of [U-(14)C] leucine into lipids was monitored by TLC. The cholesterol fraction was labeled in males but not in female, which means that cholesterol was not produced from BCAA in female gland due to the lack of expression of acyl-CoA dehydrogenases. We monitored the behavior of male hamsters toward female gland lipids, and found slightly greater attractiveness in female ones than that in male ones although the difference was not significant. Considering the lifestyle of golden hamster in nature, we propose a hypothesis that the lipids from the Harderian gland of golden hamster serve as a pheromone to declare their territory and to seek the mate with good congeniality.

Distribution and metabolism of dihomo-gamma-linolenic acid (DGLA, 20:3n-6) by oral supplementation in rats.

We compared the dietary effects of dihomo-gamma-linolenic acid (DGLA) contained in the DGLA oil produced by a fungus with gamma-linolenic acid (GLA) on the fatty acid composition. Wistar rats were fed with three kinds of oil for two weeks as follows: (i) control group: corn oil; (ii) GLA group: borage oil; (iii) DGLA group: DGLA oil/safflower oil = 55:45. The DGLA concentrations in the liver, serum, and brain of the DGLA group were higher than those of the GLA oil group. We also examined the dose effect of DGLA. The DGLA levels in the liver, serum, and brain significantly increased with increasing dosage of DGLA in the diet. DGLA administration significantly increased the ratio of PGE1/PGE2 in the rat plasma. The mechanism for GLA administration to improve atopic eczema is thought to involve an increase in the concentration of DGLA metabolized from GLA, so these results suggest that the dietary effect of DGLA would be more dominant than GLA.

A comprehensive classification system for lipids.

Lipids are produced, transported, and recognized by the concerted actions of numerous enzymes, binding proteins, and receptors. A comprehensive analysis of lipid molecules, "lipidomics," in the context of genomics and proteomics is crucial to understanding cellular physiology and pathology; consequently, lipid biology has become a major research target of the postgenomic revolution and systems biology. To facilitate international communication about lipids, a comprehensive classification of lipids with a common platform that is compatible with informatics requirements has been developed to deal with the massive amounts of data that will be generated by our lipid community. As an initial step in this development, we divide lipids into eight categories (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides) containing distinct classes and subclasses of molecules, devise a common manner of representing the chemical structures of individual lipids and their derivatives, and provide a 12 digit identifier for each unique lipid molecule. The lipid classification scheme is chemically based and driven by the distinct hydrophobic and hydrophilic elements that compose the lipid. This structured vocabulary will facilitate the systematization of lipid biology and enable the cataloging of lipids and their properties in a way that is compatible with other macromolecular databases.

Mapping Japanese medical terms to UMLS Metathesaurus.

This paper introduces and reports the results for a project to map Japanese medical terms to the UMLS Metathesaurus. The "Thesaurus for Medical and Health related Terms version 5" published in 2003 by the Japan Medical Abstracts Society and UMLS version 2002AC provided by NLM were used in this study. The goal was to judge the validity of the correlation between the Japanese and English terms that belong to the same MeSH concept. Fifteen medicine, nursing, and library science professionals, excluding JAMAS, used a custom designed Web interface to perform this task. About 10% of the concepts were judged as invalid, and the reasoning behind these failures were analyzed. Experience from this project can be used to estimate the manpower required to revise the Japanese thesaurus after future revisions to UMLS or MeSH.

Effect of simultaneous administration of vitamin C, L-cysteine and vitamin E on the melanogenesis.

The effect of simultaneous administration of vitamin C (ascorbic acid), L-cystein (Cys) and vitamin E (tocopherol) on the melanogenesis in vivo and in vitro was studied. Forty-eight brownish guinea pigs were divided into 4 groups as follows: VC group, VC+Cys group, VC+Cys+VE group and control group. They were given these vitamins by oral administration every day. UV-B exposure (0.384 J/cm2) on their depleted back skin was done at the day 8, 10, 12, 15 17 and 19. After UV-B irradiation, vitamins were administrated further 3 weeks. The luminosity score was measured using a Color Reader CR-11 (Minolta, Co) and the numbers of DOPA-positive melanocytes of their back skin were counted. B16 melanoma cells were incubated with VC, N-acetyl cystein (NAC) and VE. After 4 days of incubation, cells were harvested. The melanin contents and the tyrosinase activities in cells were measured. The luminosity score in the VC+VE+Cys group was higher than those in the other groups. The numbers of DOPA-positive melanocytes of guinea pigs treated with VC, VE and Cys were significantly decreased compared with those in VC group. In B16 melanoma cells, simultaneous treatment of VC, VE and NAC was the most effective to decrease the melanin contents and to inhibit tyrosinase activity.

Cholestanol metabolism, molecular pathology, and nutritional implications.

Cholestanol, not cholesterol, is a minor component in the human body and in foods, but an increase in cholestanol concentration in serum induces a pathological condition named cerebrotendinous xanthomatosis (CTX). In our investigation of this disease for more than 25 years, a procedure for quantification of cholestanol by high-performance liquid chromatography and an assay method for sterol 27-hydroxylase were established, and several mutations of the CYP 27 gene in 10 CTX families were identified. We also established experimental animal models with symptoms of CTX by feeding a high cholestanol diet. Corneal dystrophy and gallstones were produced in mice, and an apoptosis of cerebellar neuronal cells was observed in rats. We propose the following underlying mechanism of CTX pathogenesis: When cholesterol in the plasma membrane is replaced by cholestanol to some extent, the membrane fluidity is reduced, and the calcium channel fails to open, inducing cell death. CTX patients are treated with oral administration of chenodeoxycholic acid, which reduces the cholestanol concentration in serum. Cholestanol has a toxic effect, and an imbalance of the cholesterol/cholestanol ratio in plasma membrane is suspected to cause the disturbance of calcium channel function of the membrane.

Analysis of the comprehensive effects of polyunsaturated fatty acid on mRNA expression using a gene chip.

To investigate the comprehensive effects of polyunsaturated fatty acids (PUFA) on gene expression, we analyzed changes of mRNA expression in PUFA-treated HepG2 cells using a DNA micro array. We incubated HepG2 cells for 24 h with or without 0.25 mM oleic acid (OA), arachidonic acid (AA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), and then compared the expression profiles of thousands of genes using a GeneChip. PUFA influenced the expression of various genes related to cell proliferation, growth and adhesion, as well as for many transcription factors including sterol regulatory element binding proteins (SREBP). Treatments with AA, EPA, and DHA repressed the expression of genes related to cholesterol and lipid metabolism. Moreover, data from gene chip analysis proved that PUPA reduced the expression ofprostasin, which is a serine protease. By measuring the mRNA levels of SREBPs, mevalonate pyrophosphatase and prostasin using quantitative RT-PCR, we confirmed the effect of PUFA revealed by gene chip analysis. These data might provide useful clues with which to explore novel functions of PUPA.

Effects of sesamin and capsaicin on the mRNA expressions of delta6 and delta5 desaturases in rat primary cultured hepatocytes.

Sesamin, a lignan in sesame seeds and sesame seed oil, and capsaicin, the pungent principle of hot red pepper, affect lipid metabolism. Sesamin specifically inhibits delta5 desaturase activity in the Mortierella alpina fungus and rat liver microsomes, however, the effects of sesamin and capsaicin on mRNA expressions of delta6 and delta5 desaturases are not still clear. In this study, we investigated the effects of sesamin and capsaicin on the desaturation indexes of delta6 [(gamma-linolenic acid+dihomo-gamma-linolenic acid)/linolenic acid, (GLA+DGLA)/LA] and delta5 (arachidonic acid/DGLA, AA/DGLA) and mRNA expressions of delta6 and delta5 desaturases in rat primary cultured hepatocytes. To measure the mRNA expressions of delta6 and delta5 desaturase, hepatocytes were cultured in the presence of sesamin or capsaicin for 24 h. To investigate the delta6 or delta5 desaturation index, hepatocytes were cultured in the presence of LA or DGLA, respectively, with sesamin or capsaicin for 24 h. The fatty acid composition of the cells was measured by GLC. The mRNA expressions of delta6 and delta5 desaturases were detected by real time quantitative RT-PCR. Sesamin and capsaicin had no effect on the mRNA expressions of delta6 and delta5 desaturases in rat hepatocytes. Capsaicin had no effect on both delta6 and delta5 desaturation indexes, either. On the other hand, sesamin significantly reduced the index of delta5 desaturation but not delta6 desaturation. These results suggested that sesamin reduced the delta5 desaturation index without the changing of the delta5 desaturase mRNA level.

In vivo role of caspases in excitotoxic neuronal death: generation and analysis of transgenic mice expressing baculoviral caspase inhibitor, p35, in postnatal neurons.

Caspases, a family of cysteine proteases, are thought to be critical mediators of apoptosis. To examine the role of neuronal caspases in excitotoxic neurodegeneration in vivo, we have generated transgenic mice expressing the baculovirus protein p35, a potent viral caspase inhibitor, using the neuron-specific calmodulin dependent kinase-II alpha (CaMKII-alpha) promoter. The expression of p35 was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. We analyzed caspase activation and cell death by employing an experimental paradigm, in which the excitotoxin kainate (KA) was injected into CA1 of hippocampus and the distribution of the caspase-generated actin fragment was detected immunohistochemically. While kainate treatment led to selective neuronal death in the CA1, CA3 and CA4 of non-transgenic control mice, we observed restricted caspase activation only in the CA3 sector. The transgenic expression of p35 consistently inhibited the kainate-induced caspase activation, but failed to influence the death of neurons to any extent. In addition, we observed concomitant early calpain activation in the specific areas where neurons underwent degeneration in both the transgenic and non-transgenic mice. These results indicate that p35-inhibitable caspases play rather minor roles in the kainate-induced excitotoxicity and that the relative contribution of calpain is likely to be greater than that of caspases.

A possible intermediate step during apoptotic execution.

Many proteases are known to be involved in apoptosis. Among them, interleukin-1beta converting enzyme (ICE) and its family proteases, which are called caspases, play critical roles in the execution stage of apoptosis. We previously reported that a proteasome-inhibitor, benzyloxycarbonyl Leu-Leu-leucinal (ZLLLal), induced apoptosis in MOLT-4 cells. In the present study, in order to analyze the detailed mechanism of ZLLLal-induced apoptosis, we examined the effect of a caspase-inhibitor, acetyl(Ac)-Tyr-Val-Ala-Asp-chloromethyl ketone (AcYVADcmk), on ZLLLal-induced apoptosis in the cells. Agarose gel electrophoresis revealed that low concentrations of AcYVADcmk efficiently suppressed apoptotic DNA fragmentation. However, the cells presented morphology different from normal, apoptotic or necrotic cells, although DNA fragmentation was suppressed. The same examination was performed on the cells with anti-Fas antibody-induced apoptosis, and the same results were obtained. Some cells with a similar morphology were found even without the caspase-inhibitor in the early stage of anti-Fas antibody-induced physiological apoptosis. In addition, apoptotic cascade was reactivated by washing out the caspase inhibitor from the DNA degradation-suppressed cells. Therefore, this newly found morphological feature shows the presence of a step prior to caspase activation in the cells, and this is the first report presenting the pre-caspase-activated step in the apoptotic cascade.

Coexpression of glucose transporter 1 and matrix metalloproteinase-2 in human cancers.

BACKGROUND: Cancer cells express higher levels of glucose transporter proteins (Gluts) than do normal cells. Glut-1 overexpression is associated with invasiveness. Because matrix metalloproteinase-2 (MMP-2) is also overexpressed in cancer cells and is associated with invasiveness, we tested the hypothesis that Glut-1 may regulate MMP-2 expression. METHODS: We transiently transfected Glut-1 complementary DNA (cDNA) or Glut-1 antisense oligonucleotides in the human rhabdomyosarcoma cell line RD and analyzed MMP-2 mRNA expression and cell invasiveness. Empty vector and sense oligonucleotides were used for controls. We analyzed MMP-2 promoter activity in transfectants with a luciferase reporter assay and with p53 and Ets-1 gel mobility shift assays. Eight human cancer cell lines and 80 human cancer specimens were analyzed for coexpression of Glut-1 and MMP-2 by western blot and immunohistochemical analyses, respectively. RESULTS: Overexpression of Glut-1 in RD cells increased MMP-2 expression 4.3-fold (95% confidence interval [CI] = 3.7-fold to 4.9-fold) and invasiveness 3.2-fold (95% CI = 2.6-fold to 3.8-fold) relative to control transfected cells. Conversely, suppression of Glut-1 expression by antisense oligonucleotides decreased MMP-2 expression by 71.5% (95% CI = 71.1% to 71.9%) and invasiveness by 53.0% (95% CI = 47.5% to 58.5%). Glut-1-mediated MMP-2 expression involved the binding of the transcription factor p53 but not Ets-1. All eight human cancer cell lines coexpressed Glut-1 and MMP-2 by western blotting, and 45 of 80 human tumor tissues coexpressed Glut-1 and MMP-2 by immunohistochemistry. CONCLUSIONS: MMP-2 expression and cell invasiveness are tightly associated with Glut-1 expression in human cancer cell lines. Because suppression of Glut-1 decreased MMP-2 expression and cancer cell invasion, Glut-1 could be a target for therapy of various cancers that overexpress Glut-1.

Overexpression of ornithine decarboxylase enhances endothelial proliferation by suppressing endostatin expression.

Angiogenesis, an essential process for tumor growth, is regulated by endothelial proliferation factors and their inhibitors such as endostatin. Endostatin, a carboxyl-terminal fragment of type XVIII collagen, inhibits endothelial proliferation, angiogenesis, and tumor growth. Ornithine decarboxylase (ODC), a molecule that is overexpressed in various cancers, is associated with promoting tumor growth and angiogenesis. We found that ODC-overexpressing human cancer cells and breast cancer specimens showed suppressed expression of type XVIII collagen and endostatin. We hypothesized that ODC overexpression may facilitate angiogenesis in tumors by suppressing endostatin expression. ODC-overexpressing COS cells, which showed suppressed type XVIII collagen and endostatin expression, were established. Conditioned media derived from these cells, containing decreased levels of endostatin, induced significant endothelial proliferation. ODC-overexpressing cells, when transplanted into nude mice, suppressed type XVIII collagen expression and promoted neovascularization in vivo. Thus, overexpression of ODC facilitates endothelial proliferation by suppressing endostatin expression.