Comprehensive comparison between locations of orthologous genes on archaeal and bacterial genomes.

MOTIVATION: Following an extensive search for orthologous genes between the complete genomes from archaea and bacteria, the spatial association of the orthologs has been investigated in terms of synteny, the conservation of the order of neighboring genes. However, the relationships between the relative locations of remote orthologs over entire genomes have not been shown. RESULTS: Comprehensive comparisons between the locations of orthologs on nineteen archaeal and bacterial genomes are presented by the location to location correspondence based on the gene-location distance. When the two genomes are rotated such that a pair of orthologs with the shortest distance is set in the same angle, a statistically significant number of orthologs maintain their relative locations between the genomes. Even by the short distances at the 5% significance level, the rotations are restricted within a narrow range, suggesting an intrinsic angle for realizing similar locations between the orthologs in each genome pair. Furthermore, the rotations in the restricted range agree with the replication origin and terminus sites for the analyzed genomes where such sites are known. The relationship between location-maintained orthologs and gene function is also discussed.

Protein surface amino acid compositions distinctively differ between thermophilic and mesophilic bacteria.

One of the well-known observations of proteins from thermophilic bacteria is the bias of the amino acid composition in which charged residues are present in large numbers, and polar residues are scarce. On the other hand, it has been reported that the molecular surfaces of proteins are adapted to their subcellular locations, in terms of the amino acid composition. Thus, it would be reasonable to expect that the differences in the amino acid compositions between proteins of thermophilic and mesophilic bacteria would be much greater on the protein surface than in the interior. We performed systematic comparisons between proteins from thermophilic bacteria and mesophilic bacteria, in terms of the amino acid composition of the protein surface and the interior, as well as the entire amino acid chains, by using sequence information from the genome projects. The biased amino acid composition of thermophilic proteins was confirmed, and the differences from those of mesophilic proteins were most obvious in the compositions of the protein surface. In contrast to the surface composition, the interior composition was not distinctive between the thermophilic and mesophilic proteins. The frequency of the amino acid pairs that are closely located in the space was also analyzed to show the same trend of the single amino acid compositions. Interestingly, extracellular proteins from mesophilic bacteria showed an inverse trend against thermophilic proteins (i.e. a reduced number of charged residues and rich in polar residues). Nuclear proteins from eukaryotes, which are known to be abundant in positive charges, showed different compositions as a whole from the thermophiles. These results suggest that the bias of the amino acid composition of thermophilic proteins is due to the residues on the protein surfaces, which may be constrained by the extreme environment.

Teaching a multidisciplinary approach to cancer treatment during surgical clerkship via an interactive board game.

BACKGROUND: Although educators agree that the approach to cancer management must be multidisciplinary, medical students usually observe cancer patients through the eyes of a single specialist at any given time. METHODS: In order to teach third-year medical students that cancer management is multidisciplinary, we developed the Oncology Game, an interactive, computer-assisted board game built on the principles of self-directed learning and student-student interaction. Eight "patients" with different histologic types of cancer are distributed randomly to 4 students, who play in teams of 2. The object is for the team to obtain the best treatment for its patients by advancing them via a roll of dice through surgical, medical, and radiation oncology clinics in the order most logical for the patient's particular cancer type. To test improvement in cognitive skills as a function of play, 16 students participated in a tournament taking parallel pretest and posttests before and after each round of play. RESULTS: Students demonstrated a statistically significant change in the total number of questions answered correctly each time they played the Oncology Game (F = 4.16, P = 0.018; Pretest Round 1: 8.88 +/- 0.58; Posttest Round 1: 9.63 +/- 0.42; Pretest Round 2: 10.75 +/- 0.62; Posttest Round 2: 11.5 +/- 0.85). Post hoc pairwise comparison revealed a significant improvement in student performance after playing two rounds of the Oncology Game. Based on the postgame survey, students felt they improved their understanding of oncologic principles (4.56 +/- 0.13), knowledge of malignancies (4.50 +/- 0.13), and appreciation for the multidisciplinary nature of cancer management (4.56 +/- 0.13). CONCLUSIONS: Improved test scores and postgame survey results demonstrate that third-year medical student students can learn about basic oncology principles and gain an appreciation for oncology as a multidisciplinary field of medicine through an interactive, computer-assisted board game.

Multiple arterial thromboembolisms in a patient with the 20210 A prothrombin gene mutation.

The most recently characterized genetic defect contributing to venous thrombophilia is the 20210 A prothrombin gene mutation. We describe a patient with this defect who had arterial thrombosis resulting in considerable mesenteric ischemia. Several environmental factors, which might otherwise be considered of low thrombotic risk, may also have contributed to her condition. The recognition of the potential for novel presentations of hypercoagulable states may contribute to a reduction in the morbidity associated with acute mesenteric ischemia.

Simultaneous colonisation of Helicobacter pylori with and without mutations in the 23S rRNA gene in patients with no history of clarithromycin exposure.

BACKGROUND: It was recently reported that A to G transition mutations at positions 2143 and 2144 in the 23S rRNA gene are associated with clarithromycin resistance in Helicobacter pylori. AIMS: To study the incidence and mechanism of development of clarithromycin resistance by analysing these mutations. SUBJECTS: Eighty two H pylori positive patients who had an endoscopic examination and no history of treatment with macrolide antibiotics. METHODS: Clarithromycin resistance was screened for by polymerase chain reaction-restriction fragment length polymorphism of the 23S rRNA gene coupled with antibiotic susceptibility testing. In clinical isolates with mutations or resistance, mutations in individual colonies were analysed by direct sequencing. RESULTS: Of the 79 amplicons (DNA fragments amplified by polymerase chain reaction), Alw26I and MboII digestion disclosed the mutation in four (5%) and one (1%) respectively. However, the Alw26I cleavage was incomplete in two of the four amplicons, as was the MboII cleavage. Individual colony analysis of the isolates with incomplete cleavage patterns showed the presence of both wild type and mutated strains in the 23S rRNA genes. CONCLUSIONS: Both clarithromycin sensitive and resistant strains colonised in some patients with no history of exposure to macrolides. The results suggest that resistant strains may not be formed but selected by clarithromycin administration.

Influence of 5-fluorouracil on colonic healing and expression of transforming growth factor-beta 1.

BACKGROUND: Administration of chemotherapeutic agents in the immediate postoperative period may have beneficial effects by decreasing local cancer recurrence rates, but this must be weighed against possible impairment of wound healing. Since local expression of transforming growth factor-beta1 (TGF-beta1) is normally upreglated following creation of experimental colonic anastomoses, this study examines the effects of 5-fluorouracil (5-FU) on colonic healing and on the local expression of TGF-beta1. MATERIALS AND METHODS: Forty-eight male Sprague-Dawley rats underwent transection of the descending colon with primary anastomosis and were then randomly assigned to receive either intraperitoneal 5-FU (20 mg/kg/day) or saline (SAL). On Postoperative Days (PODs) 3, 5, and 7, bursting pressure (BP, mm Hg) and bursting energy (BE, mm Hg xs) were determined in situ. Anastomotic and nonoperated segments of colon were harvested and analyzed using the semiquantitative reverse transcriptase-polymerase chain reaction to determine the relative expression of TGF-beta1 normalized to that of a constitutive gene. RESULTS: Progressive increases in BP and BE were observed in both the 5-FU and the SAL groups, across the time course examined. Overall, these measures were decreased in the 5-FU groups compared to SAL, significantly so on PODs 5 and 7; BP, 127.8 +/- 7.6 vs 161.1 +/- 7.2 and 139.9 +/- 10.9 vs 186.0 +/- 8.6; BE, 1093.6 +/- 190.0 vs 2207.9 +/- 308.2, and 1518.5 +/- 326.5 vs 3279.3 +/- 225.7, respectively. Anastomotic TGF-beta1 expression also increased progressively in both groups over the postoperative time course. Expression in the 5-FU group, however, was significantly decreased compared to that in the SAL group on POD 3; 0.42 +/- 0.05 vs 0.84 +/- 0.04. Interestingly, this preceded the reduction in BP and BE in the 5-FU group on PODs 5 and 7. TGF-beta1 expression in nonoperated colonic segments did not change during the time points studied or in response to 5-FU administration. CONCLUSIONS: Wound healing following a colonic anastomosis is associated with local increases in TGF-beta1 expression, which in turn is diminished by the administration of 5-FU. If this deleterious effect on wound healing could be counteracted, then chemotherapy administration in the immediate postoperative period may become safer.

Streptozotocin treatment upregulates uncoupling protein 3 expression in the rat heart.

Diabetic rats have a deficiency in their heart ATP concentrations, and although the mechanism remains to be elucidated, this deficiency may involve increased uncoupling of oxidative phosphorylation. To investigate whether heart uncoupling proteins (UCPs) are subject to transcriptional regulation in diabetes, we examined changes in UCP mRNA expression in the heart of streptozotocin-induced diabetic (STZ-DM) rats. Heart UCP3 mRNA expression significantly increased by 9.4-fold in STZ-DM rats, while levels of UCP2 mRNA expression were not significantly altered. Insulin supplementation in STZ-DM rats returned UCP3 mRNA concentrations to control levels. The expression of UCP3 mRNA was similarly elevated in the heart of fasted rats, which also have hypoinsulinemia and hyper-free fatty acidemia but, unlike the STZ-DM rats, are hypoglycemic. Since hyperinsulinemia alone was previously reported to not affect UCP3 gene expression in the muscle, these results indicate that hyper-free fatty acidemia is a potent enhancer of UCP3 gene expression in the diabetic rat heart. Interestingly, we found no changes in UCP3 mRNA levels in Zucker fatty (fa/fa) rats with excessive chronic hyper-free fatty acidemia, which suggests that upregulation of heart UCP3 mRNA may depend on an acute change in free fatty acid concentrations rather than on their sustained elevation. High-energy ATP deficiencies in the diabetic rat heart may primarily result from proton leakage due to the upregulation of UCP3 expression.

Effect of bowel preparation and a fiber-free liquid diet on expression of transforming growth factor and procollagen in colonic tissue preoperatively and postoperatively.

PURPOSE: Dehiscence of colonic anastomoses is prevalent and potentially fatal. In an attempt to reduce the likelihood of anastomotic dehiscence, the colon is cleansed before surgery and fiber-free diets are prescribed postoperatively. However, fiber-free diets induce colonic atrophy and impair healing. This study was designed to investigate the effect of bowel preparation and postoperative fiber-free diet on the local gene expression of transforming growth factor-beta 1 and procollagen type I. METHODS: Four Sprague-Dawley rats underwent bowel preparation with a fiber-free liquid diet and polyethylene glycol in a balanced electrolyte solution for two days (fiber-free preoperative diet group), whereas four rats received standard chow with fiber (preoperative diet with fiber group). On the third day tissue was obtained from the descending colon of each rat to assess the effect of bowel preparation. Forty additional rats had their bowels prepared and underwent transection of the descending colon and anastomosis. These rats were then randomly assigned to continue on the liquid diet (fiber-free postoperative diet group) or rat chow (postoperative diet with fiber group). On postoperative days 3, 5, 6, 7, and 14, colonic tissue was obtained from the anastomosis and analyzed with the use of semiquantitative reverse transcriptase-polymerase chain reaction to examine the relative expression of transforming growth factor-beta 1 and procollagen type I genes normalized to that of a constitutive gene. RESULTS: There was a decrease in the expression of the transforming growth factor-beta 1 and the procollagen type I genes in the fiber-free preoperative diet group compared with the preoperative diet with fiber group; however, this difference only reached statistical significance for procollagen type I. Postoperatively, significant increases in the expression of the transforming growth factor-beta 1 and procollagen type I genes over baseline levels were observed around postoperative day 7 in both groups, which temporally correlates with active phases of collagen deposition in the wounded colon. Expression of the procollagen type I gene, however, was significantly decreased at this time in the fiber-free postoperative diet group compared with the postoperative diet with fiber group. CONCLUSION: Although necessary to reduce septic complications, preoperative bowel preparation has a detrimental effect on the expression of transforming growth factor-beta 1 and procollagen type I. A postoperative fiber-free liquid diet also may be detrimental to the expression of these transcripts in the bowel. Alternative methods for delivery of colonic fuels are needed to create a better environment for colonic healing while eliminating bacteria and bulk.

Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae.

Open reading frames in the genome of Saccharomyces cerevisiae were screened for potential glycosylphosphatidylinositol (GPI)-attached proteins. The identification of putative GPI-attached proteins was based on three criteria: the presence of a GPI-attachment signal sequence, a signal sequence for secretion and a serine- or threonine-rich sequence. In all, 53 ORFs met these three criteria and 38 were further analyzed as follows. The sequence encoding the 40 C-terminal amino acids of each was fused with the structural gene for a reporter protein consisting of a secretion signal, alpha-galactosidase and a hemagglutinin (HA) epitope, and examined for the ability to become incorporated into the cell wall. On this basis, 14 of fusion proteins were classified as GPI-dependent cell wall proteins because cells expressing these fusion proteins: (i) had high levels of alpha-galactosidase activity on their surface; (ii) released significant amounts of the fusion proteins from the membrane on treatment with phosphatidylinositol-specific phospholipase C (PI-PLC); and (iii) released fusion proteins from the cell wall following treatment with laminarinase. Of the 14 identified putative GPI-dependent cell wall proteins, 12 had novel ORFs adjacent to their GPI-attachment signal sequence. Amino acid sequence alignment of the C-terminal sequences of the 12 ORFs, together with those of known cell wall proteins, reveals some sequence similarities among them.

Antagonistic regulation of cell migration by epidermal growth factor and glucocorticoid in human gastric carcinoma cells.

Epidermal growth factor (EGF) induced the disruption and scattering of colonies of TMK-1, a cell line derived from a human gastric carcinoma. A stimulatory action of EGF on cell migration was also observed as determined by a wound assay. However, these actions of EGF were inhibited if the cells were pretreated with dexamethasone, a synthetic glucocorticoid. Dexamethasone increased cell adhesion to collagen type IV and laminin, but not to poly-L-lysine and fibronectin. In contrast, EGF did not affect cell adhesion to these extracellular matrices whether dexamethasone was present or not. Dexamethasone enhanced the protein levels of both alpha1 and beta1 integrin subunits, and that of the alpha1 beta1 heterodimer. Further, flow cytometric analysis revealed that dexamethasone increased the expression of beta1 and alpha1 integrin subunits at the cell surface, whereas EGF increased expression of beta1 and alpha2 subunits at the cell surface. Antibodies against alpha1 and beta1 integrin subunits inhibited the increased cell adhesion seen in the presence of dexamethasone. An immunofluorescence study indicated that dexamethasone increased the formation of focal adhesions along the entire edges of cell colonies. In contrast, EGF led to the formation of focal adhesions preferentially at the cell front, and this EGF-induced preferential formation was not observed if the cells were pretreated with dexamethasone. These results suggest that glucocorticoid increased cell adhesion to the extracellular matrix via alpha1 beta1 integrin, and thereby antagonized EGF-induced cell migration.

Gastrojejunal fistula caused by gastric ulcer.

We report a case of gastrojejunal fistula caused by benign gastric ulcer, a very rare condition. The patient was an 81-year-old-woman who had had multiple recurrences of gastric ulcer. She also had diabetes mellitus. She was admitted to our hospital because of a left femoral head fracture, necessitating a mechanical bone head exchange operation. She had severe abdominal pain and anemia on the 48th postoperative day. Gastroendoscopic examination revealed a giant ulcer with a long-axis diameter of more than 5cm on the lesser curvature of the gastric body. She was treated with intravenous famotidine and all oral intake was restricted; her symptoms were alleviated. Two weeks later, a fistula had formed between the stomach and the jejunum just anal to the duodeno-jejunal flexure. She was placed on an ulcer diet, and was discharged with no symptoms on the 151st postoperative day. She has remained asymptomatic for 1 1/2, years to date. Lack of H2-antagonist administration, operative stress, and administration of ipriflavone appeared to have induced gastric ulcer recurrence, and formation of the fistula between the stomach and the jejunum seemed to have been facilitated by the patient being very lean and having minimal mesenteric adipose tissue.

A theoretical method for evaluating the relative importance of positive selection and neutral drift from observed base changes.

To evaluate the relative importance of positive selection and neutral drift from the nucleotide base changes observed in the homologous alignment of genes, a theoretical equation of base changes is formulated by including both the influence of selection and the base substitutions due to mutations. Under the assumption that the average rate of base substitutions estimated from synonymous changes is the "true" mutation rate applicable at all positions, this method is applied to the vertebrate globin gene family, and evaluates the departures of base change rates from the "true" mutation rate at the first and second codon positions as a consequence of preferential selection for the conservation of important function. In addition to the strong effect of selection on the amino acid residues in the internal region mostly common to myoglobin and hemoglobin chains, the distinctive directions of selective parameter values are seen at sites on the globin surface, distinguishing the subunit contact residues of hemoglobins from the polar residues on the surface of myoglobins. Moreover, this effect of selection distinguishing between the myoglobin and hemoglobin chain genes becomes weaker in cold-blooded vertebrates, especially in fish, strongly suggesting the possibility that the clear distinction between these globins is a result of selection out of the changes regarded as neutral ones in an ancestor of vertebrates. Thus, the present method may also serve to investigate the homology of many other proteins from the aspect of molecular evolution, mainly focusing on the evolution of their biological functions.

Evolution of the self-reproducing system to the biosynthesis of the membrane: an approach from the amino acid sequence similarity in proteins.

In order to study the problem of how the biomembrane synthesis started in the evolutionary process of the self-reproducing system, we carry out an extensive similarity search of the sequence data stored in databases, using the acetyl-CoA carboxylase, fatty acid synthase and the enzyme proteins leading to the combination of sn-glycerol 3-phosphate and fatty acid as the query sequences. With the use of the FASTA program (Pearson & Lipman, 1988), the proteins that carry an amino acid sequence showing similarity to any of the query sequences are picked up under the criterion of statistical significance of more than 6.0 for the homology, then classified according to the functional blocks where they operate. Finally they are filtered to the enzyme proteins in the metabolic pathways and to the DNA- or RNA-interacting proteins in the translation, transcription and replication apparatuses by eliminating proteins such as membrane proteins, lipase etc. which seem to have been generated after the appearance of the biomembrane. The distribution of the proteins thus selected shows a clear pattern that the amino acid sequences showing considerable similarity to the biomembrane synthetic proteins are concentrically found in the enzyme proteins in and around the section of glycolytic pathway from glyceraldehyde 3-phosphate to pyruvate while the DNA- or RNA-interacting proteins similar to the query sequences are distributed sparsely over the translation, transcription and replication systems. The assignment of similarity regions ascertains that considerable regions of most biomembrane synthetic proteins are covered by the enzyme proteins in and around the glycolytic pathway. Although acetyl-CoA carboxylase and fatty acid synthase are full of variety in the constitution of active domains depending on species, the above-mentioned pattern is also obtained by using either the monofunctional or the multifunctional type of proteins as the query sequences. Thus, the evolution towards biomembrane synthesis may be positioned as an event following the establishment of a section of glycolytic pathway from glyceraldehyde 3-phosphate to pyruvate. The causality of this evolution from the glycolytic pathway to the biomembrane synthesis is also discussed in connection with the absorption of protons released in the glycolytic process.

[Successful repair of distal type of aortopulmonary window by deep hypothermia with circulatory arrest].

A one year old boy with the distal type of aortopulmonary window was successfully repaired by the transaortic approach and discharged two weeks after operation. Exposure of the defect was enhanced by using deep hypothermia with circulatory arrest so that aortic cannula and cross-clamp could be removed. 2 months before the operation, he needed the respiratory support during a month because of respiratory syncytial viral infection, cardiac failure and equivalent pulmonary hypertension. Postoperative cardiac catheterization after 4 months revealed that PA pressure decreased to 25/5 mmHg without any medication. Aortogram showed the left single coronary artery.

Bone metastasis from colorectal cancer in autopsy cases.

The incidence of bone metastasis from colorectal cancer is reported to be 10.7% in autopsy cases. However, the characteristics of the primary cancers, as well as the patterns of bone metastasis, remain unclear. We analyzed the clinical and autopsy records of 118 patients with primary colorectal cancer treated either surgically or conservatively and eventually autopsied between 1970 and 1987 at Toranomon Hospital in Tokyo. Bone metastasis was detected in 23.7% (28/118). The average age of patients with bone metastasis was lower than that in patients without bone metastasis (P < 0.02). Cancers to the rectum and cecum were accompanied by bone metastasis more frequently than cancers of other portions of the colon. Signet-ring cell carcinoma showed a high incidence of bone metastasis (P = 0.041). Bone metastasis from colorectal cancer was associated with liver or lung metastases (P < 0.0001). These results indicated that bone metastasis from colorectal cancer is not as infrequent as previously described.

Dopaminergic activity and endorenal dopamine synthesis in non-insulin dependent diabetes mellitus.

In the present study we tried to clarify the differences of the cardiovascular and renal responses to feeding in relation to the peripheral dopamine system. In control subjects (C), ingestion of protein (100 g) induced an increase in Ccr accompanied by an increase in tubular sodium excretion (FENa+). Patients with non-insulin dependent diabetic (NIDDM), a protein-induced increase in Ccr was comparable to that in C, while FEN+ did not change following protein. Since an increase in urinary 3,4-dihydroxyphenylacetic acid was blunted in NIDDM, an impaired natriuretic response to high protein may be results from an insufficient synthesis of renal dopamine. Plasma dopamine and its metabolites in NIDDM following protein tended to be greater than in C. Protein induced a greater decrease in blood pressure (BP) in NIDDM, but no increase in pulse rate was observed. An ordinary diet containing 10 g of protein also induced a decrease in BP. A reflex tachycardia was observed in C and normotensive NIDDM but not in hypertensive one. In normotensive NIDDM, plasma dopamine and norepinephrine increased after the diet, while in hypertensive NIDDM there were no increases in catecholamines. From these results it is suggested that the relatively elevated peripheral dopaminergic activity and the blunted dopamine synthesis in the kidney may be responsible for the abnormal cardiovascular and renal responses to feeding in patients with NIDDM.

Gene expression of aromatic L-amino acid decarboxylase mRNA in the kidney of normotensive and hypertensive rats.

Peripheral dopamine (DA) synthesis and release increase during hypertensive stage of spontaneously hypertensive rats (SHR). DA is generated from 3,4-dihydroxyphenylalanine by L-amino acid decarboxylase (AADC). We have studies urinary DA and DA metabolites and the gene expression of neuron and non-neuron specific AADC mRNA in the kidney of SHR. Compared to Wister-Kyoto rats (WKY), there was an increased urinary free DA and DOPAC excretions in 8 and 12 week-old SHR. At the age of 16 weeks, the difference in free DA excretion between SHR and WKY rats disappeared, although the urinary DOPAC excretion remained significantly higher in SHR, but urinary HVA excretion did not differ from WKY rats. The expression of the neuron specific AADC mRNA in the kidney of SHR and WKY rats was not detected, but the non-neuron specific AADC mRNA in the kidney of SHR and WKY rats was detected. The gene expression of the non-neuron specific AADC mRNA tended to decrease with age in SHR. The results suggest that a decrease in renal DA production with age may be caused by diminished expression of non-neuron specific AADC mRNA in kidney.

Postprandial changes in noradrenergic and dopaminergic activity in patients with essential hypertension.

In order to elucidate the role of noradrenergic and dopaminergic activity in the pathogenesis of postprandial hypotension, the effect of feeding of ordinary diet on blood pressure, pulse rate, plasma catecholamine and other circulating vasoactive substances such as insulin were examined in mild essential hypertensive patients (EH) and their age-matched control subjects (N). Mean blood pressure significantly decreased in both N and EH after feeding, and the decrease tended to be greater in EH than in N. Feeding induced a marked increase in plasma norepinephrine in both N and EH. Plasma dopamine significantly increased following feeding was observed in N, while the increase in plasma dopamine following feeding was blunted in EH. The ratio of norepinephrine to dopamine following in EH was significantly greater than that in N. From these results, it is suggested that the feeding-induced stimulation of noradrenergic activity may be a result from the decrease in blood pressure, and that the blunted response of dopaminergic activity in EH may reflect the enhanced conversion of dopamine to norepinephrine probably due to the enhanced activity of dopamine beta-hydroxylase in the sympathetic nerves.