A pilot study of evaluation of the antioxidative activity of resveratrol and its analogue in a 6-month feeding test in young adult mice.

Resveratrol, a polyphenolic phytoalexin, has free-radical scavenging activity and we found that it induces chromosomal aberrations, micronuclei, and sister chromatid exchanges in vitro. We synthesized its analogue 4-hydroxy-trans-stilbene (4-OH) and found that it has the same in vitro clastogenic activities as resveratrol, suggesting that the 4' hydroxy group of resveratrol is responsible for the effect. We fed resveratrol and 4-OH to young adult ICR mice at 0, 0.2, 2, or 20 ppm in their standard powder diet for 6 months and investigated the antioxidative effects. Half of each group was given 3000 ppm potassium bromate (KBrO(3)) in water for the last week to cause oxidative damage. Body weight gain tended to increase in males at 0.2 ppm resveratrol or 4-OH, and in females at 2 ppm 4-OH. Micronucleus (MN) analysis in bone marrow erythrocytes showed that the KBrO(3) tendency to induce MN was not prevented by the dietary resveratrol or 4-OH, which themselves did not induce MN under the present conditions. In this pilot study, resveratrol and 4-OH showed no obvious effect, either beneficial or adverse, at doses that are feasible in daily life for humans.

Usefulness of 11C-methionine-PET for predicting the efficacy of carbon ion radiation therapy for head and neck mucosal malignant melanoma.

The aim of this study was to determine whether l-methyl-[11C]-methionine (MET) positron emission tomography (PET) allows the prediction of outcomes in patients with head and neck mucosal malignant melanoma treated with carbon ion radiation therapy (CIRT). This was a retrospective cohort study involving 85 patients who underwent a MET-PET or MET-PET/computed tomography (CT) examination before and after CIRT. MET uptake in the tumour was evaluated semi-quantitatively using the tumour-to-normal tissue ratio (TNR). Local recurrence, metastasis, and outcome predictions were studied in terms of TNR before CIRT (TNRpre), TNR after CIRT (TNRpost), and the TNR change ratio. Kaplan-Meier curves revealed significant differences between patients with higher TNRpre values and those with lower TNRpre values in regard to local recurrence, metastasis, and outcome (log-rank test P<0.0001 for all three). There were also significant differences in metastasis rates and outcomes between patients with higher and lower TNRpost values (log-rank test P=0.0105 and P=0.027, respectively). The Cox proportional hazards model revealed TNRpre to be a factor significantly influencing the risk of local recurrence (hazard ratio (HR) 29.0, P<0.001), risk of metastasis (HR 2.67, P=0.024), and the outcome (HR 6.3, P<0.001). MET-PET or MET-PET/CT is useful for predicting the outcomes of patients with head and neck mucosal malignant melanoma treated with CIRT.

FlgB, FlgC, FlgF and FlgG. A family of structurally related proteins in the flagellar basal body of Salmonella typhimurium.

The flagellar basal body of Salmonella typhimurium consists of four rings surrounding a rod. The rod, which is believed to transmit motor rotation to the filament, is not well characterized in terms of its structure and composition. FlgG is known to lie within the distal portion of the rod, in the region where it is surrounded by the L and P rings, just before the rod-hook junction. The FlgC and FlgF proteins are also known to be flagellar basal-body components; by comparison of deduced and experimental N-terminal amino acid sequences we show here that FlgB is a basal-body protein. The flgB, flgC, flgF and flgG gene sequences and the deduced protein sequences are presented. The four proteins are clearly related to each other in primary sequence, especially toward the N and C termini, supporting the hypothesis (based on examination of basal-body subfractions) that FlgB, FlgC and FlgF are, like FlgG, rod proteins. From this and other information we suggest that the rod is the cell-proximal part of a segmented axial structure of the flagellum, with FlgB, FlgC and FlgF located (in unknown order) in successive segments of the proximal rod, followed by FlgG located in the distal rod; the axial structure then continues with the hook, HAPs and filament. Although the rod is external to the cell membrane, none of the four rod proteins contains a consensus signal sequence for the primary export pathway; comparison with the experimentally determined N-terminal amino acid sequence indicates that FlgB has had its N-terminal methionine removed, while the other three are not processed at all. This demonstrates that these proteins are not exported by the primary cellular pathway, and suggests that they are exported by the same flagellum-specific pathway as the flagellar filament protein flagellin. The observed sequence similarities among the rod proteins, especially a six-residue consensus motif about 30 residues in from the N terminus, may constitute a recognition signal for this pathway or they may reflect higher-order structural similarities within the rod.

Isolation of mutant lines with decreased numbers of chloroplasts per cell from a tagged mutant library of the moss Physcomitrella patens.

Eleven mutant lines exhibiting decreased numbers of chloroplasts per cell were isolated from 8 800 tagged mutant lines of Physcomitrella patens by microscopic observations. Chloronema subapical cells in wild-type plants had a mean of 48 chloroplasts, whereas chloroplast numbers in subapical cells in mutant lines 215 and 222 decreased to 75 % of that in the wild type. Seven mutant lines - 473, 122, 221, 129, 492, 207, and 138 - had about half as many chloroplasts as the wild type. Mutant line 11 had a few remarkably enlarged chloroplasts, and mutant line 347 had chloroplasts of various sizes. Whereas the cell volume was the same as in the wild type in mutant lines 222, 473, 221, 129, 492, and 207, the cell volume of the other mutants increased. The chloroplast number of leaf cells was the same as that of chloronema cells in each mutant line when gametophores could be formed. Treatment with ampicillin decreased the number of chloroplasts in all mutant lines. Southern hybridization using DNA in tags as probes showed that only one insertion occurred in mutant lines 473 and 221. To determine whether the tagged DNA inserted into the known genes for plastid division, we isolated the PpMinD1, PpMinD2, and PpMinE1 genes. Genomic polymerase chain reaction analysis showed that the PpFtsZ and PpMinD/E genes were not disrupted by the insertion of the tags in mutant lines 11 and 347, respectively.

Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesis.

The moss, Physcomitrella patens has been used as a useful material in many fields, because of its simple body plan, ease of gene targeting, and other reasons. Although many mutants have been reported, no method to isolate the corresponding genes was reported. We developed a gene tagging and gene-trap system in P. patens by using the shuttle mutagenesis technique, which has been used in the budding yeast. In 5264 tagged lines, 203 mutants with altered developmental or morphological phenotypes were obtained. In 129 of 4757 gene-trap lines, beta-glucuronidase (GUS) activity was detected in some tissue. Although multiple copies of a tag were detected in many tagged lines by Southern analyses, most copies are likely integrated at the same locus according to PCR analyses.

Genes for members of the GATA-binding protein family (GATA-GT1 and GATA-GT2) together with H+/K(+)-ATPase are specifically transcribed in gastric parietal cells.

mRNAs for novel DNA-binding proteins (GATA-GT1 and GATA-GT2) recognizing the (G/C)PuPu(G/C)NGAT(A/T)PuPy sequence and H+/K(+)-ATPase (proton pump) alpha subunit were detected in parietal cells of the rat gastric body mucosa by in situ hybridization. These results suggest that GATA-GT1 and GATA-GT2 together with H+/K(+)-ATPase are transcribed specifically in gastric parietal cells and that the two DNA-binding proteins may have important roles in cell specific gene regulation. Furthermore, we could detect parietal cells in different states of gene expression.

Inhibitory effects of natural carotenoids, alpha-carotene, beta-carotene, lycopene and lutein, on colonic aberrant crypt foci formation in rats.

Inhibitory effect of four carotenoids prevalent in human blood and tissues against the formation of colonic aberrant crypt foci was examined in Sprague-Dawley rats. They received three intrarectal doses of N-methylnitrosourea in weak 1, and a daily gavage of de-escalated doses of carotenoids during weeks 2 and 5. Lycopene, lutein, alpha-carotene and palm carotenes (a mixture of alpha-carotene, beta-carotene and lycopene) inhibited the development of aberrant crypt foci quantitated at week 6, but beta-carotene did not. The results suggested that lycopene and lutein in small doses may potentially prevent colon carcinogenesis.

Transcriptional activation of H+/K+-ATPase genes by gastric GATA binding proteins.

H+/K+-ATPase (composed of alpha and beta subunits) and histamine H2 receptor are specifically expressed in gastric parietal cells. The GATA binding proteins (GATA-GT1 and GATA-GT2, also called GATA-6 and GATA-4, respectively) originally found in the gastric mucosa recognized a sequence motif [gastric motif, (G/C)PuPu(G/C)NGAT(A/T)PuPy] in the upstream regions of the ATPase genes [Tamura, S., Wang, X.-H., Maeda, M., and Futai, M. (1993) Proc. Natl. Acad. Sci. USA 90, 10876-10880]. These proteins activated the transcription of the reporter gene ligated downstream of the control region of the rat ATPase alpha or beta subunit gene but had no effect on the same reporter ligated downstream of the H2 receptor gene. Deletion analyses suggested that the upstream 249 (alpha gene) and 323 (beta gene) base pair sequences from the first letter of the initiation codon are sufficient for activation by the GATA proteins. Interestingly, two and three gastric motifs are located near the TATA-boxes of the alpha and beta genes, respectively. Mutagenesis studies demonstrated that the two motifs proximal to the TATA-box sequences of the ATPase alpha and beta subunit genes were essential for the activation. These results suggest that both the alpha and beta subunit genes are regulated similarly by the GATA binding proteins. The expression system established in this study is a useful system for analyzing the roles of GATA proteins in transcriptional regulation of the H+/K+-ATPase gene.

Effect of total parenteral nutrition enriched in branched-chain amino acids on metabolite levels in septic rats.

This study examined the effects of total parenteral nutrition (TPN) enriched with branched-chain amino acids (BCAAs) on metabolite levels of carbohydrate and protein metabolism in septic rats. Results also were obtained for standard amino acid hyperalimentation (conventional TPN). Septic peritonitis was induced in rats by cecal ligation and puncture. Two different experimental models were tested. In one, the two kinds of TPN were administered to the operated rats during the progress of sepsis (the septic phase). In the other, TPN was started immediately after surgical removal of the focal cecum (the recovery phase). The conventional and BCAA-enriched TPN solutions were isocaloric and isonitrogenous except that the percentage of BCAAs in the total amino acids by weight was 35.8% in BCAA-enriched TPN and 20.9% in conventional TPN. On the fifth postoperative day, TPN was discontinued, the animals were killed, and samples of arterial blood, liver, and rectus abdominis muscle were taken. BCAA-enriched TPN had a significant effect on nitrogen balance and survival rate in the septic phase model, and on muscle adenine nucleotide content in both models. Other metabolites showed similar changes in the two TPN groups. These results indicate that BCAA supplement in TPN improves nitrogen balance and peripheral cellular energy status and is thus clinically beneficial in preventive therapy for increased catabolism.

Alterations in metabolite levels in carbohydrate and energy metabolism of rat in hemorrhagic shock and sepsis.

For comparison of the extent of metabolite content alteration caused by etiologically different types of shock, septic peritonitis and hemorrhagic shock (mean arterial blood pressure at 40 mm Hg for 1 h or 2 h) were produced in rats. Contents of metabolites were determined in the liver and the muscle. Characteristic differences were found in the alteration modes of hepatic lactate level, muscle adenine nucleotide concentrations, and muscle protein content between these shock models. Rapid and significant alterations were observed in the levels of adenine nucleotides, glucose-6-phosphate and lactate in the liver in both types of shock. Hepatic energy charge and contents of glycogen and protein also significantly decreased. On the other hand, noticeable changes in the muscles were elevation of lactate level and the decrease of phosphocreatine and protein concentrations. Another distinct change was the decrease of total adenine nucleotide content in the muscle of septic rats, whereas it remained unchanged in the muscle of hemorrhagic shock rats. Thus, the changes of metabolite levels did not occur simultaneously in different tissues, and their rate and magnitude varied between different types of shock. The difference in adaptive response of metabolism may result in pathophysiologic diversity in shock.

Alterations in levels of plasma phenylalanine and its catabolism in the liver of stressed rats.

This study examined the relationship between elevation of blood phenylalanine (Phe) concentrations often observed in trauma or infected patients without hepatic dysfunction and alterations of liver Phe catabolism. Rats underwent pathophysiologically different stresses, either sepsis or scald injury. The catalytic activity of hepatic Phe hydroxylase (PH) in the septic rats, as measured after preincubation with Phe, decreased to 60% of the control values; this in vitro result suggests a reduction of enzyme species activated by its substrate. Phe was degraded in the septic rats to a similar extent to that in controls, when measured by pulse administration of [1-14C]-Phe. In the scalded rats whose plasma Phe level showed a comparable but transient increase, no significant alterations occurred in Phe catabolism and enzyme activities. The changes in plasma glucagon and catecholamine levels were consistent with those of the enzyme activities involved in Phe and tyrosine (Tyr) catabolism in the stressed groups. These results indicate that inadequate activation of native PH by regulatory mechanisms involving Phe in vivo was also associated with the accumulation of plasma Phe in infected rats during massive mobilization of amino acids from muscles under conditions of enhanced and sustained catabolism.

Biosystematic studies on the family Tofieldiaceae I. Phylogeny and circumscription of the family inferred from DNA sequences of matK and rbcL.

In order to specify phylogenetic positions of the genera of Tofieldiaceae (Tofieldia, Triantha, Pleea, Harperocallis, Isidrogalvia), and to suggest reasonable circumscription of the family and genera of Tofieldiaceae, we determined DNA sequences of matK and rbcL for each genus of the family, and analyzed them phylogenetically with the 45 families and 113 genera of the monocots other than Tofieldiaceae, whose matK and rbcL sequences have already been reported. We found that Tofieldia, Triantha, Pleea, and Harperocallis form the same clade, which receives 100% bootstrap support. This clade can be regarded as corresponding to Tofieldiaceae, and is embedded in the clade of Alismatales (98%). On the other hand, Isidrogalvia is not included in this Tofieldiaceae clade, and positioned as sister to Narthecium (100%), embedded in the clade of Nartheciaceae (Dioscoreales) (100%). In the Tofieldiaceae, Pleea first diverges from the remaining three genera (100%), and then, Harperocallis diverges from the Tofieldia- Triantha complex (100%). In the Tofieldia- Triantha complex, five Tofieldia species form the same clade (100%), and two Triantha species form another clade (100%). Thus, Isidrogalvia should be transferred from Tofieldiaceae to Nartheciaceae. As Isidrogalvia, as well as the Nartheciaceae, have the carpels that are fully connate into a single style, Isidrogalvia fits the Nartheciaceae well with respect to carpel connation. After this transfer, the Tofieldiaceae correspond mainly to plants with almost free carpels and three styles. Pleea is better treated as an independent genus than included in Tofieldia. Triantha can be treated either as an independent genus or as congeneric with Tofieldia.

Metabolic effect of short-term total parenteral nutrition highly enriched with leucine or valine in rats recovering from severe trauma.

The metabolic impact of infusing a large amount of leucine (Leu) or valine (Val) was examined with regard to the corrective effect of total parenteral nutrition (TPN). Rats recovering from severe sepsis received either Leu- or Val-enriched TPN solution for 30 hours. The in vivo behavior of the amino acids administered was explored by a pulse injection of 14C-labeled Leu or Val. The recovery of 14CO2 from Leu increased by 64% in the septic rats of Leu-TPN group (41% of dose; p less than .01), as compared with control rats receiving the same TPN solution, whereas no significant rise in the 14CO2 recovery from Val occurred in the septic rats given Val-TPN (45% of dose) in comparison with the corresponding controls. The enhancement of Leu catabolism to CO2 in the Leu-TPN group was compatible with the alterations of urinary nitrogen excretion, plasma Leu level, and metabolite contents of liver and muscle. The only difference in metabolite levels observed between the two TPN groups was in hepatic total adenine nucleotides. Plasma amino acid levels were largely unaffected by infusion of these TPN solutions highly enriched with branched-chain amino acids (45%), except for an approximately threefold elevation of the Val level in Val-TPN rats. Thus, when administered in a large quantity during such short-term TPN, Leu can exert its metabolic effect without causing an imbalance in plasma amino acids under severe catabolic conditions.

Immediate stimulation of protein metabolism in burned rats by total parenteral nutrition enriched in branched-chain amino acids.

The effects of two kinds of total parenteral nutrition (TPN) on energy and protein metabolism were examined in rats subjected to 15% full-thickness scald burns in the absence of septic complications. One type of TPN was enriched in branched-chain amino acids (BCAAs), especially leucine (45% BCAA content), and the other was conventional TPN (21% BCAA content). Burned rats received isocaloric and isonitrogeneous TPN solutions for 48 hr after resuscitation by saline infusion for 24 hr. Liver and rectus abdominis muscle were removed from the rats at 7, 24, 48, and 72 hr. The concentrations of adenine nucleotides, RNA, protein, glucose-6-phosphate, hepatic glycogen, muscle phosphocreatine, and 3-methylhistidine were determined. Metabolic alterations occurred during the period of saline resuscitation (0-24 hr). At 48 hr the RNA and protein levels were significantly more improved in the BCAA-TPN group than in the conventional TPN group. At 72 hr, however, the results for the two groups were similar in most metabolite levels. Thus, BCAA-TPN enriched in leucine rapidly stimulated protein synthesis in the liver and muscle. This rapid effect may make it useful during the initial nutritional management of severe trauma patients.

Glutamate in enteral nutrition: can glutamate replace glutamine in supplementation to enteral nutrition in burned rats?

BACKGROUND: Glutamine (GLN) plays many important roles for the enterocytes in health and disease, but no liquid enteral products contain GLN because of its instability. We hypothesized that glutamate (GLU) may replace GLN in supplementation to an enteral diet, and compared the metabolic effect of GLU and GLN on the gut to each other. METHODS: Rats suffering from a 30% burn received an enteral diet containing 30% GLU (m/w to total amino acids; GLU group), 30% GLN (GLN group), or a standard amino acid formula (CTR group). After a 64-hour feeding period, the small intestine and the portal and arterial blood were harvested to observe portal and arterial amino acid levels, and glutaminase activity and glutathione in the jejunal mucosa. In another study, 3H uptake into the mucosal protein was examined after a massive dose injection of 3H-phenylalanine. RESULTS: Alanine, a product of GLN or GLU catabolism, significantly increased in the portal blood of the GLU group compared with the GLN group. In the gut mucosa of the GLU group, 3H uptake into protein and total glutathione were higher than those of other two groups. GLN did not elevate the glutaminase activity. Arterial GLU levels increased in the GLU group, however remained within safety limits. CONCLUSIONS: Enterally delivered GLU may be a preferable fuel for the enterocytes and enhance the mucosal protein synthesis. GLU probably can substitute for GLN in supplementation to an enteral diet regarding many roles GLN plays in the intestinal mucosa under stress situations.