Feasibility of virtual surgical simulation in the head and neck region for soft tissue reconstruction using free flap: a comparison of preoperative and postoperative volume measurement.

In the head and neck region, preoperative evaluation of the free flap volume is challenging. The current study validated preoperative three-dimensional (3D) virtual surgical simulation for soft tissue reconstruction by assessing flap volume and evaluated fat and muscle volume changes at follow-up in 13 head and neck cancer patients undergoing anterolateral craniofacial resection. Patients received 3D virtual surgical simulation, and the volume of the planned defects was estimated by surgical simulation. Following en bloc resection of the tumor, the defect in the skull base was covered using a rectus abdominis myocutaneous flap. Following surgery, computed tomography scans were acquired at day 1 and at 6 and 12 months. Virtual planned defect was on average 227 ml (range, 154-315) and was 10% smaller than the actual flap volume in patients without skin involvement of the tumor. Between day 1 and 12 months post-surgery, the volume of fat and muscle tissue in the free flap dropped by 9% and 58%, respectively. Our results indicate that 3D virtual surgical simulation provides essential information in determining the accurate volume of the required free flap for surgical defect repair and may thus help improve surgical planning and functional and esthetic outcome.

Neural stem cell-based dual suicide gene delivery for metastatic brain tumors.

In our previous works, we demonstrated that human neural stem cells (NSCs) transduced with the cytosine deaminase (CD) gene showed remarkable 'bystander killer effect' on glioma and medulloblastoma cells after administration of the prodrug 5-fluorocytosine (5-FC). In addition, herpes simplex virus thymidine kinase (TK) is a widely studied enzyme used for suicide gene strategies, for which the prodrug is ganciclovir (GCV). To apply this strategy to brain metastasis treatment, we established here a human NSC line (F3.CD-TK) expressing the dual suicide genes CD and TK. We examined whether F3.CD-TK cells intensified the antitumor effect on lung cancer brain metastases. In vitro studies showed that F3.CD-TK cells exerted a marked bystander effect on human lung cancer cells after treatment with 5-FC and GCV. In a novel experimental brain metastases model, intravenously administered F3 cells migrated near lung cancer metastatic lesions, which were induced by the injection of lung cancer cells via the intracarotid artery. More importantly, F3.CD-TK cells in the presence of prodrugs 5-FC and GCV decreased tumor size and considerably prolonged animal survival. The results of the present study indicate that the dual suicide gene-engineered, NSC-based treatment strategy might offer a new promising therapeutic modality for brain metastases.

Bio rapid prototyping by extruding/aspirating/refilling thermoreversible hydrogel.

This paper reports a method for rapid prototyping of cell tissues, which is based on a system that extrudes, aspirates and refills a mixture of cells and thermoreversible hydrogel as a scaffold. In the extruding mode, a cell-mixed scaffold solution in the sol state is extruded from a cooled micronozzle into a temperature-controlled substrate, which keeps the scaffold in the gel state. In the aspiration mode, the opposite process is performed by Bernoulli suction. In the refilling mode, the solution is extruded into a groove created in the aspiration mode. The minimum width of extruded hydrogel pattern is 114 +/- 15 microm by employing a nozzle of diameter 100 microm, and that of aspirated groove was 355 +/- 10 microm using a 500 microm-diameter nozzle. Gum arabic is mixed with the scaffold solution to avoid peeling-off of the gel pattern from the substrate. Patterning of Sf-9 cell tissue is demonstrated, and the stability of the patterned cell is investigated. This system offers a procedure for rapid prototyping and local modification of cell scaffolds for tissue engineering.

Non-involvement of the K-ras mutation in colon carcinogenesis promoted by dietary deoxycholate in azoxymethane-treated rats.

Fisher-344 rats, whose ileum or jejunum had been surgically removed to change the influx of bile acids into the colon, were intraperitoneally administered with azoxymethane and fed on a diet containing deoxycholate for 39 weeks to induce colon cancer. Fecal bile acids in the ileum-resected group were 1.5-times and serum bile acids were about half of those in the jejunum-resected group. As a result, the incidence and number of tumors were higher in the ileum-resected group. In the total of 59 colon tumors (40 were in the ileum-resected group and 19 in the jejunum-resected group), 56 were carcinomas, including two well-differentiated invasive and two mucinous carcinomas found in the ileum-resected rats. However, only three carcinomas, two invasive and one non-invasive, had the K-ras mutation. These results demonstrate that the K-ras mutation was not essentially involved in deoxycholate-promoted colon carcinogenesis.

Soybean resistant proteins interrupt an enterohepatic circulation of bile acids and suppress liver tumorigenesis induced by azoxymethane and dietary deoxycholate in rats.

We found that azoxymethane and dietary deoxycholate induced liver tumors in rats. The incidence and the development of the tumor were closely related to the enterohepatic circulation of bile acids. The feeding of a high-molecular-weight fraction of soy protein digest (HMF) suppressed the tumorigenesis, probably due to the inhibitory effect of soybean resistant protein on reabsorption of bile acids in the intestine.

Cutting edge: naturally occurring soluble form of mouse Toll-like receptor 4 inhibits lipopolysaccharide signaling.

Toll-like receptors (TLRs) are a family of proteins playing important roles in host defense. Mice defective of functional TLR4 are hyporesponsive to LPS, suggesting that TLR4 is essential for LPS signaling. Here we report the cloning of an alternatively spliced mouse TLR4 (mTLR4) mRNA. The additional exon exists between the second and third exon of the reported mTLR4 gene and contains an in-frame stop codon. The alternatively spliced mRNA encodes 86 aa of the reported mTLR4 and an additional 36 aa. This alternatively spliced mTLR4 mRNA expressed a partially secretary 20-kDa protein, which we named soluble mTLR4 (smTLR4). In a mouse macrophage cell line, the exogenously expressed smTLR4 significantly inhibited LPS-mediated TNF-alpha production and NF-kappaB activation. Additionally, in mouse macrophages, LPS increased the mRNA for smTLR4. Taken together, our results indicate that smTLR4 may function as a feedback mechanism to inhibit the excessive LPS responses in mouse macrophages.

Antitumorigenic effects of several food proteins in a rat model with colon cancer and their reverse correlation with plasma bile acid concentration.

In order to obtain information on the preventive effects of various food proteins against colonic cancer, six groups of azoxymethane-initiated mature Fischer rats (n = 10) were fed respective diets different in protein sources such as bovine milk casein (casein), high-molecular-weight fraction from protolytic digest of soy protein isolate (soybean HMF), hen's yolk defatted protein (yolk protein), wheat gluten and codfish meat, which had been supplemented with sodium deoxycholate (hereinafter, DCA) as a cancer promoter except for an additional DCA-unfed casein group. All of the living rats at checkpoints during the feeding period were examined by the use of a bronchus fiberscope for colonic tumor incidence at 6 wk intervals between the 10th and 34th wk, from which both blood and feces samples were taken at times of endoscopy. Tumorigenesis in the colon was perceived by endoscopy at wk 22 in the group fed DCA casein only and at wk 28 in the other groups except the DCA-unfed casein group. At wk 34, both soybean HMF and yolk protein groups ranked inferior to the DCA-unfed group in tumor incidence. When plasma steroid or lipid concentration was plotted against tumor incidence at wk 28 or 34, positive correlations were found between plasma bile acid concentration and tumor incidence at both weeks. With the exception of the DCA-unfed casein group, plasma bile acid concentration was reversely correlated to fecal bile acid excretion. Taken altogether, these results suggest that bile acids at higher concentrations in the plasma may serve as risk factors of colon tumor incidence.

Preventive effect of soybean resistant proteins against experimental tumorigenesis in rat colon.

The insoluble 'high-molecular-weight' fraction (HMF) centrifugally separable after digestion of soy protein isolate with a microbial protease of the exo-type, of which about a quarter is regarded as an indigestible 'resistant protein,' was examined for its preventive effect against colonic tumorigenesis in a model system with male F-344 rats. The rats were intraperitoneally injected with azoxymethane (15 mg/kg BW) once a week for 3 wk and were fed a 20.6% HMF diet (+0.4% DL-Met) or 14.7% casein diet (+0.3% DL-Met) supplemented with 0.2% sodium deoxycholate (DCA) or without supplementation. Twelve wk later, 5 rats of each group were inspected for formation of tumors but no tumors were visible to the naked eye. The DCA-fed casein group was conspicuous for a low count of aberrant crypt foci. At 39 wk, 6 rats of the DCA-fed casein group (n = 10) and 3 rats of the DCA-fed HMF group (n = 9) had a total of 18 tumors with a major axis of 4.0 +/- 0.4 mm and 3 tumors with an axis of 2.0 +/- 0.1 mm, respectively, in contrast to only a single tumor for the DCA-unfed casein group (nil for the DCA-unfed HMF group). The difference in tumor number and size was considered significant between these DCA-fed casein and HMF groups; that is to say, HMF feeding retarded tumor development despite the frequent occurrence of pre-neoplastic lesions. In addition, fecal bile acid excretion was much more elevated by HMF feeding than by casein feeding. It can be assumed from these observations that the antitumorigenicity of HMF is due to the inhibitory effect of soybean resistant proteins on reabsorption as well as the mucosal contact of bile acids in the intestine.

Elimination of Na+-dependent bile acid transporter from small intestine by ileum resection increases [correction of increase] colonic tumorigenesis in the rat fed deoxycholic acid.

Ileal Na+-dependent bile acid transporter (ISBT) constituting a gateway to enterohepatic circulation of bile acids occurs exclusively at the distal site of the small intestine. In the present study, we examined colonic tumorigenesis promoted by deoxycholic acid in relation to the expression of the ISBT. For this purpose, the small intestine of a Fischer-344 rat was resected a length of 20 cm above the ileo-cecal valve (ileal resection) or below the duodenum (jejunal resection). Then, rats were treated with an intraperitoneal injection of azoxymethane (15 mg/kg body wt.) once a week for 3 weeks and fed a 20% casein diet supplemented with 0.2% deoxycholate for 39 weeks. Northern blot analysis demonstrated that the ISBT mRNA was hardly detectable in ileum-resected rats. The excretion of fecal bile acids was 1.5-fold higher in the ileum-resected group than in the jejunum-resected group (P < 0.05). On the contrary, the serum bile acids concentration of ileal-resected rats was about one-half of that of jejunum-resected animals (P < 0.05). The tumor incidence and the total tumor number were significantly higher in the ileum-resected group than in the jejunum-resected one (P < 0.05). Interestingly, no tumor was found at the proximal colon in the jejunum-resected group while tumors developed frequently at the proximal site as well as mid and distal colon in the ileum-resected group. These observations demonstrate that malabsorption of bile acids owing to the lack of ISBT enhanced colon tumorigenesis.

Feeding soybean resistant protein to rats raises fecal bile acid excretion but counteracts a deoxycholate-caused decrease in colonic aberrant crypt foci.

A high-molecular-weight fraction after removal of water-soluble peptides from proteinase-treated soybean protein isolate (referred to as HMF) was examined for its effect on preneoplastic lesions in the rat colon. For this purpose, male Fisher-344 rats 7 wk old were divided into 8 groups (n = 5), of which 6 groups received 3 injections of azoxymethane (AOM, 15 mg/kg of body weight) for 3 wk once a week, while all were fed HMF or casein diets supplemented with or without deoxycholic acid (DCA) over a period of 4 wk. Two groups of AOM-treated rats were allowed free access to HMF or casein diets without supplemental DCA, respectively, while the others were pair-fed so as to be well matched in their food intake. There were no significant differences in growth parameters among the pair-fed groups. Feeding HMF diets raised fecal lipid and acidic steroid excretions to a greater extent than feeding casein diets, secondary bile acids being conspicuous among acidic steroids in the excreta irrespective of the presence or absence of DCA supplementation. As a result of observation for colonic aberrant crypt foci (ACF), the intake of HMF proved to reverse the reduction of ACF appearance by DCA. This result implies that secondary bile acids are caught and brought out by HMF, or rather its derivative "resistant protein," so as not to keep contact with colonic mucosae.

Characterization, cDNA cloning, and functional expression of mouse ileal sodium-dependent bile acid transporter.

Mouse ileal sodium dependent bile acid transporter (ISBT) was characterized using isolated enterocytes. Only enterocytes from the most distal portion showed Na+-dependent [3H]taurocholate uptake. Northern blot analysis using a probe against mouse ISBT revealed the expression of mouse ISBT mRNA to be restricted to the distal ileum. The Km and Vmax for Na+-dependent [3H]taurocholate transport into isolated ileocytes were calculated as 27 microM and 360 pmol/mg protein/min, respectively. Uptake of [3H]taurocholate was inhibited by N-ethylmaleimide. We have cloned ISBT cDNA from mouse ileum. The cDNA included the entire open reading frame coding 348 amino acid protein with seven hydrophobic segments and two N-glycosylation sites. COS-7 cells transfected with the expression vector containing this cDNA expressed Na+-dependent [3H]taurocholate uptake activity with a Km of 34 microM.

Soybean curd refuse alleviates experimental tumorigenesis in rat colon.

Adult Fischer-344 rats which underwent administration of azoxymethane were fed diets containing soybean curd refuse (SCR) or a high-molecular-weight fraction of soy protein digest (HMF), or Hammarsten casein (CAS) as a protein source over a period of 34 weeks. All the living rats of each group at 22, 28 or 34 weeks were endoscopically inspected for tumor incidence in the colon. SCR turned out to be comparable to HMF in anti-tumorigenicity, or rather better than HMF.

Protein-nutritive assessment of sake lees obtained by brewing from liquefied rice.

Sake lees obtained by brewing from liquefied rice were deprived of water and alcohol by lyophilization, and then examined for nutritional availability with the aid of proximate food analysis, amino acid analysis and animal experiment. Freeze-dried sake lees powder was comprised of 44.6% protein, 37.4% carbohydrate, 2.5% fat, 6.7% fiber, 1.8% ash and 7.2% moisture (alcohol < 0.1%), of which the nutritive value (amino acid score) was estimated as 89.6 when compared with the amino acid requirements for preschool children (FAO/WHO/UNU, 1985). Sake lees protein had been, however, appreciably improved in the limiting amino acid "lysine" relative to polished rice protein. As a result of an animal experiment, the rats fed a 50% sake lees powder diet proved to be equal in growth to those fed a 20% casein (control) diet, although the former diet had to be supplemented with vitamins and minerals, which were in shortage as compared to the control diet. On the other hand, the feeding of sake lees powder was effective in lowering the serum triacylglycerol concentration. Accordingly, sake lees powder can be assessed as a favorable candidate for not only protein-rich but also hypolipidemic provisions.

Antioxidative activity of barley hordein and its loss by deamidation.

Barley hordein was comparable to maize zein in antioxidation under a powder model system. Various deamidated "hordein" preparations were obtained and examined for their molecular-size distribution (by Sephacryl S-100 gel filtration), hydrophobicity (by fluorescence measurement using fluorescent probes) and antioxidative activity (by the ferric thiocyanate method). Deamidation caused fragmentation of the hordein molecule and simultaneously lowered its fatty acid-binding capacity rather than its surface hydrophobicity. Then, the antioxidative activity diminished with increasing deamidation. When the fatty acid-binding capacity was plotted against the antioxidative activity, a high correlation (r2 = 0.92) was observed between these two events.

Comparison of ANG II with other growth factors on Egr-1 and matrix gene expression in cardiac fibroblasts.

The purpose of the present investigation was to compare the effects of angiotensin II (ANG II) other growth factors implicated to play a role in ventricular hypertrophy on cardiac fibroblast changes associated with cardiac remodeling. These changes included induction of early growth response (Egr-1) gene and increases in message levels of extracellular matrix proteins. ANG II treatment (10(-10)-10(-6) M) of rat cardiac fibroblasts induced 1) Egr-1 and 2) a fourfold (P < 0.02) increase in fibronectin and a twofold (P = 0.05) increase in laminin mRNA levels but no increases in that of collagens I, III, or IV at 24-48 h, and 3) a decrease in AT1-receptor mRNA levels to 26% (P < 0.001) of basal at 4-6 h. These effects were all inhibited by the AT1-receptor blocker, losartan, but not AT2-receptor blockers. Immunostaining of cultured cells with antibody against rat fibronectin demonstrated positive staining of cells in serum-free medium; staining was more intense in cells treated with ANG II (10(-6) M, 48 h). Fluorescent-activated cell sorting using an antibody against rat AT1 receptor demonstrated a receptor signal in cells maintained in serum-free medium; however, the receptor signal was not detectable in ANG II-treated cells. Serum and epidermal growth factor (EGF) also induced Egr-1, but norepinephrine (NE) and endothelin (ET) had no effect. Serum increased fibronectin mRNA levels by twofold (P < 0.05). EGF, NE, and ET had no effect on matrix gene expression. Serum, EGF, and NE also transiently downregulated AT1-receptor mRNA levels at 4-6 h of treatment. These results demonstrate that 1) ANG II both induces protooncogene expression and enhances fibronectin mRNA levels in cultured cardiac fibroblasts, whereas EGF only induces Egr-1, and NE and ET have no effects on either function; 2) ANG II effects are primarily mediated by the AT1 receptor; and 3) growth factors can regulate AT1-receptor mRNA levels. Thus ANG II, relative to NE, ET, and EGF, appears to play a prominent and direct role in fibroblast changes associated with cardiac hypertrophy.

Decreased Antioxidative Activity of Maize Zein in Response to Deamidation Rate.

Ten samples with different deamidation rates were prepared from commercially available maize zein by heat treating with 0.05 n HCl in 70% ethanol, and were examined for their antioxidative activity in simply mixed protein-fatty acid (10:1) model systems at moderate humidity. A regression line with a correlation coefficient of r = - 0.946 was obtained, when the linoleate/palmitate ratio on day 3 was plotted against the percentage of deamidation.

Effects of short chain fatty acid, sodium butyrate, on osteoblastic cells and osteoclastic cells.

1. Sodium butyrate increased alkaline phosphatase (ALP) activity of cloned osteoblastic cell line MC3T3-E1 by the stimulation of de novo enzyme synthesis. 2. Sodium butyrate did not affect mature osteoblastic cells but affected preosteoblastic cells. 3. Sodium butyrate decreased tartrate resistant acid phosphatase (TRACP)-positive multinucleated cells (MNC) formation from bone marrow cells. This related to the cytotoxicity of sodium butyrate on bone marrow cells.

Comparative effect of cadmium on osteoblastic cells and osteoclastic cells.

Cadmium(Cd) has been thought to disturb the bone metabolism directly. The mechanism for the bone lesion is unknown, however. To examine the effects of cadmium on bone metabolism, we compared its effects on osteoblasts and osteoclasts in vitro. We used an established cell line, MC3T3-E1, as osteoblasts and tartrate resistant acid phosphatase (TRACP)-positive multi-nucleated cells (MNC) formed by a bone marrow culture system as osteoclasts. Alkaline phosphatase (ALP) activity was decreased by 10(-7) M Cd and DNA content and hydroxyproline content of osteoblastic cells were decreased by 10(-5) M Cd. Cadmium at 10(-7) M inhibited the osteoclastic cell formation from mouse bone marrow in the presence of 10(-8) M 1 alpha, 25(OH)2 vitamin D3. A 100-fold higher concentration of zinc(Zn) simultaneously added to the cadmium-containing medium prevented the toxicity of cadmium to osteoclastic cells as observed in the culture of osteoblastic cells. These results indicate that both bone formation and bone resorption are inhibited by cadmium. The responses of osteoclasts and osteoblasts to cadmium in this culture system were the same and the responses of cadmium-damaged osteoblasts and osteoclasts to zinc were also similar. These results suggest that another mechanism by which cadmium could cause bone damage should be considered in addition to the specific induction of osteoclastic cells by Cd.

Postprandial Changes in the Function of Digestive Organs in Rats Meal-fed Twice a Day with 40% Casein-based and Protein-free Diets.

Growing rats were meal-fed twice a day in the morning (9:00-11:00) and night (20:0-22:00) with 40% casein-based and protein-free diets in turn, or vice versa. Twenty-five days later, the animals were killed by fours in each group at 9:00, 11:00, 14:00, and 18:00, to excise their small intestines and pancreas. Immediately the jejunal mucosa was scraped together and assayed for [(3)H]leucine incorporation into protein as well as [(3)H]leucine absorption in epithelial cells. Concurrently with the assay, tissue-specific hydrolase activities were measured. Although small intestinal intrinsic hydrolases did not fluctuate much in activity, the activities of digestive enzymes in the pancreas increased after the intake of the 40% casein-based diet and decreased after the intake of the protein-free diet. This can be accounted for by the supply of synthesis materials for these digestive enzymes. Interestingly a reverse tendency was observed for both the in vitro 'protein synthesis' and 'amino acid transport' capacities of jejunal mucosal scrapings. Such functional changes are probably under the control of circadian rhythm cued by a feeding schedule.

Two-dimensional Analysis of Voluntary Locomotor Activity in Rats; Compact Apparatus and Its Application from Nutritional Aspects.

A convenient apparatus for measuring the locomotor activity of caged rats was produced from thin metallic chest, video camera, personal computers, fluorescent lamp, infrared lamp, etc. at a cost not exceeding 200,000 yen. This apparatus was large enough for a growing rat to move about at will, whose location and locomotion were memorized at intervals of a second with the connected personal computer. For this reason, the apparatus is more suitable for monitoring 'voluntary' or 'spontaneous' activity than a running wheel or so-called 'Animex' apparatus. The behavior of rats under nutritionally different conditions, as well as those accustomed to meal-feeding either with high-protein and protein-free diets or with diets containing 10% perilla or safflower oil, was successively measured. As a result, it was assumed that the relative locomotor activity would be affected by body mass rather than by the preference for a particular diet and that the time to search for food would not necessarily be shortened by ingesting perilla oil.