MicroRNA-133a regulates the mRNAs of two invadopodia-related proteins, FSCN1 and MMP14, in esophageal cancer.

BACKGROUND: FSCN1 and matrix metalloproteinase 14 (MMP14) are both invadopodia-related proteins. We herein elucidate the tumourigenicity of these proteins and identify novel therapeutic agents in esophageal squamous cell carcinoma (ESCC). METHODS: FSCN1 and MMP14 were evaluated by immunohistochemistry and quantitative PCR, and microRNA (miR)-133a was also evaluated by PCR in surgical ESCC specimens. The roles of FSCN1, MMP14 and miR-133a were established in ESCC cells. RESULTS: The expression of FSCN1 or MMP14 was an independent poor prognostic factor according to a multivariate analysis of immunohistochemistry, and their co-expression correlated with the poorest overall survival (OS) out of all the examined factors. Additionally, their mRNAs significantly correlated and both inversely correlated with miR-133a in surgical specimens. Transfection of a miR-133a mimic decreased the mRNA and protein levels of both FSCN1 and MMP14 in ESCC cells. The knockdown of FSCN1 or MMP14 and transfection of a miR-133a mimic inhibited the proliferation and invasion of ESCC cells. Patients with a lower miR-133a expression have a significantly poorer OS than those with a higher expression. CONCLUSION: The combined expression of FSCN1 and MMP14 is associated with a poor prognosis, and miR-133a, which regulates their mRNAs, can serve as a strong tumour suppressor of ESCC.

Conformational dynamics monitored by His-179 and His-200 of isolated thermophilic F1-ATPase beta subunit which reside at the entrance of the 'conical tunnel' in holoenzyme.

When monitored by 1H NMR at various pH values, most of the C-2 proton signals from 12 His residues of the isolated beta subunit of thermophilic F1-ATPase (TF1) could be separately observed. Two of them were assigned to His-179 and His-200 which reside at the entrance of a 'conical tunnel' to reach catalytic site in the crystal structure of F1-ATPase. His-200 gave doublet, suggesting that this region is not a rigid alpha-helix in the isolated beta subunit. The binding of Mg.AMP-PNP changed the chemical shifts of His-179 and His-200 significantly. Although His-119 located at the opposite side of the conical tunnel was not affected by the nucleotide-binding, it contributed to the stability of beta subunit and the efficiency of the catalysis of the holoenzyme.

Prevention of diabetes by thymic hormone in alloxan-treated rats.

We investigated the effects of facteur thymique sérique (FTS), a thymic peptide hormone, on alloxan- and streptozotocin-induced diabetes in rats. Pretreatment with intravenous injection of FTS significantly suppressed both alloxan- and streptozotocin-induced hyperglycemia. The effects of FTS were time dependent. FTS suppressed hyperglycemia in a dose range of 1-6600 micrograms/kg. Alloxan-induced hyperglycemia was completely prevented when FTS was injected in doses of 40-50 micrograms/kg 1 min before injection of alloxan. Histological examination of islet areas showed that alloxan-induced destruction of beta-cells was inhibited by FTS. FTS had no significant effects on lymphocyte subsets and immunity-related cells or on plasma superoxide dismutase activity and total glutathione level. The blood half-life time of exogenously injected FTS was short (2-3 min), indicating acute internalization of FTS into pancreatic beta-cells. Our results suggested that FTS acutely and directly blocks some initial effect of alloxan, preventing the destruction of beta-cells.

Acute glucosuria after continuous glucocorticoid loading in the rat in vivo.

We investigated the effects of the continuous infusion of various steroids in rats on renal tubular reabsorption of glucose in vivo to elucidate the pathogenesis of steroid-induced glucosuria. Urinary glucose excretion increased 60 min after administration of dexamethasone (2.38 mM). By 120 min, urinary excretion of glucose was three times higher in the dexamethasone group than in the control group (24.1 +/- 4.6 versus 72.4 +/- 16.7 micromol); the plasma level of glucose did not increase. Dexamethasone had no effect on the resorption of 1,5-anhydro-D-glucitol, which is a glucose-resembling polyol that is actively absorbed by the renal tubules as glucose. Neither estradiol nor progesterone increased urinary excretion of glucose. These findings suggest that continuous administration of a high-dose glucocorticoid selectively influences the glucose reabsorption system in the kidney.

[Two cases of Campylobacter fetus subsp. fetus sepsis].

We encountered two relatively rare cases of sepsis due to Campylobacter fetus subsp. fetus (C. fetus). Case 1. A 54-year-old female with abdominal polysurgery developed a slight fever and vomiting in August 1984. Despite the administration of some digestive drugs by her family doctor, these symptoms continued. In mid-October, she was hospitalized with high fever with chill and rigor on the skin. On the third hospital day, C. fetus was detected in the blood culture. After combination chemotherapy of intravenous drip infusion of latamoxef (LMOX) (2 g/day) and oral administration of erythromycin (EM) (800 mg/day), her symptoms improved. Case 2. A 57-year-old male with diabetic retinopathy and nephropathy was hospitalized because of slight fever, general edema and pleural effusion. On the 6th hospital day, C. fetus was detected in the blood culture and he was diagnosed with sepsis. Under treatment with the intravenous drip of LMOX (2 g/day) and oral administration of EM (1200 mg/day), his condition improved. Both cases had common underlying diseases such as hypoproteinemia with edema and problems in the lower intestinal tract; the former had polysurgery and malabsorption syndrome, the latter had diffuse ulceration of the colon. Such underlying conditions may have permitted the invasion of C. fetus into the blood.

Development of a new haemostatic dissecting forceps utilizing controlled heat as an energy source.

We tested a prototype of a new thermal surgical system in animal experiments. This device utilizes controlled heat as an energy source and seals and divides small- to medium-size vessels. The forceps we used in the current study are shaped like dissecting forceps used in conventional open surgery, and their grippers can open bilaterally. Heating elements are built into a gripper. The temperature adjustment is controlled by monitoring the electric resistance. Since the new device utilizes no ultrasonic energy, unfavorable phenomena such as cavitations or mist production are not observed. In a preliminary experiment, 12 segments of animal arteries were sealed and cut by the prototype forceps. Five artery stumps did not burst at the maximum pressure of the manometer system (1471 mmHg). The other seven stumps showed burst pressure ranging from 525 mmHg to 1051 mmHg. It is feasible to utilize controlled heat as a new alternative energy source for haemostatic surgical dissection. The new thermal dissector we are in the process of developing showed safe and quick sealing and cutting of the vessels in the experimental settings.

[Effect of induction methods of pediatric anesthesia on serum myoglobin].

Two hundred and twenty nine children from infancy to middle teens divided into six groups were studied to determine the effect of induction methods of pediatric general anesthesia on serum myoglobin. After the slow induction with nitrous oxide-oxygen-halothane (GOF), the serum myoglobin increased significantly to 1774.7 +/- 4285.8 ng.ml-1 (mean +/- SD) by iv administration of succinylcholine 1 mg.kg-1. This significant increase of serum myoglobin was reduced by pretreatment with d-tubocurarine (dTc) 0.05 mg.kg-1 before administration of succinylcholine. However, it was not reduced by pretreatment with thiopental 3 mg.kg-1. After the rapid induction with thiopental 5 mg.kg-1 and succinylcholine 1 mg.kg-1, the serum myoglobin increased slightly to 387.6 +/- 596.5 ng.ml-1. But this increase in serum myoglobin was stopped by pretreatment with dTc 0.05 mg.kg-1 before the rapid induction. Serum myoglobin was not changed without succinylcholine after the slow induction with GOF. In each group, there was no relation with serum myoglobin and ages or fasciculation. Serum myoglobin was more influenced by the induction methods of pediatric general anesthesia than by ages.

IS1-encoded proteins, InsA and the InsA-B'-InsB transframe protein (transposase): functions deduced from their DNA-binding ability.

Insertion sequence IS1 encodes a transframe protein, InsA-B'-InsB, which is produced from two out-of-phase reading frames, insA and B'-insB, by translational frameshifting at a run of adenines. Unless the frameshifting event occurs, the InsA protein is produced from IS1. We found that cells harboring a plasmid carrying an IS1 mutant with a single adenine insertion in the run of adenines contained miniplasmids. Cloning and DNA sequencing analyses of the miniplasmids revealed that they had a deletion extending from an inverted repeat (IR) at the left end of IS1. This indicates that they were generated by IS1-mediated deletion due to efficient production of the InsA-B'-InsB transframe protein that is IS1 transposase. Both the InsA protein and transposase were partially purified as a fusion protein with collagen-LacZ by LacZ-specific affinity column chromatography. The InsA* and the collagenolyzed InsA* were found to bind specifically to a 24-bp region within each of the IRs at the ends of IS1. The transposase Tnp* and the collagenolyzed Tnp* were found to bind to the sequence with or without IR, but preferentially to that with IR. The nonspecific DNA-binding ability of transposase may be involved in recognition of the target DNA, an important process of transposition of IS1. Both InsA and transposase have the IR-specific DNA binding ability and a common polypeptide segment containing the alpha-helix-turn-alpha-helix motif, supporting the previous indication that InsA competes with transposase to bind to IRs and thus becomes a transposition inhibitor. Based on the observations described in this article, we speculate that transposase of IS1 consists of at least two domains, the N-terminal half, which almost entirely overlaps InsA, and the C-terminal half, which almost entirely overlaps B'-InsB. The frameshifting event adds the latter domain to the former to give the transposase activity recognizing IRs and the target sequence to initiate the transposition reaction.

Interaction of theta-toxin (perfringolysin O), a cholesterol-binding cytolysin, with liposomal membranes: change in the aromatic side chains upon binding and insertion.

To understand the mechanism of membrane lysis by theta-toxin (perfringolysin O) from Clostridium perfringens, a cholesterol-binding, pore-forming cytolysin, we undertook a spectroscopic analysis of the structural changes that occur during the lytic process using lipid vesicles. In particular, the spectra were compared with those obtained using a modified theta-toxin, MC theta, that binds membrane cholesterol without forming oligomeric pores, thus bypassing the oligomerization step. The interaction of theta-toxin with liposomes composed of cholesterol and phosphatidylcholine but not with cholesterol-free liposomes caused a remarkable increase in the intensity of the tryptophan fluorescence emission spectra and ellipticity changes in the near- and far-UV CD peaks. A CD peak shift from 292 to 300 nm was specific for theta-toxin, suggesting oligomerization-specific changes occurring around tryptophan residues. Structural changes in the aromatic side chains were detected in the near-UV CD and fluorescence spectra upon MC theta-liposome interaction, although the far-UV CD spectra indicate that the beta-rich secondary structure of MC theta is well-conserved after membrane binding. Quenching of the intrinsic tryptophan fluorescence of MC theta by brominated lecithin/cholesterol liposomes suggests that theta-toxin inserts at least partly into membranes in the absence of oligomerization. These results indicate that regardless of oligomerization, the binding of theta-toxin to cholesterol induces partial membrane insertion and triggers conformational changes accompanied by aromatic side chain rearrangement with retention of secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonstereospecific actions of ketamine isomers on the force of contraction, spontaneous beating rate, and Ca2+ current in the guinea pig heart.

Ketamine possesses stereospecific actions of anesthesia with the S(+)-isomer being three to four times as potent an anesthetic as the R(-)-isomer. We investigated the mechanical and electrophysiologic effects of ketamine isomers in guinea pig cardiac preparations. Both isomers decreased the contractile force of electrically driven papillary muscles and the spontaneously beating rate of the right atria in a concentration-dependent manner. There were no significant differences between the S(+)- and R(-)-isomers for these measured variables. Consistent with the results from mechanical experiments, electrophysiologic experiments using whole cell voltage clamp techniques revealed that both isomers suppressed identically the transsarcolemmal Ca2+ current (ICa), which plays a role in the generation of the force of contraction and the spontaneous firing of sinoatrial node cells. In conclusion, the optical isomers of ketamine have equipotent cardiodepressant effects in the guinea pig. Inasmuch as the S(+)-isomer is the more potent anesthetic, it could offer significant clinical advantage over the R(-)-isomer or the racemate, in terms of impairment of cardiac functions, if the present results could be extrapolated to the clinical setting.

Transport and accumulation of 1,5-anhydro-D-glucitol in the human erythroleukemia cell line K-562.

The transport and intracellular accumulation of 1,5-anhydro-D-glucitol (AG) was studied in the human erythroleukemia cell line K-562 by gas chromatography-mass spectrometry in conjunction with liquid scintillation spectrometry. K-562 cells contained 106 +/- 6 nM/10(6) cells of free AG, primarily in the cytosol. Addition of physiologic amounts of AG to the extracellular medium resulted in rapid intracellular incorporation of AG, with a half-saturation time of 5 s. Intracellular accumulation was linear for 2 h and subsequently reached saturation. AG uptake was temperature and concentration dependent with an apparent Km of 127 mM. AG uptake and accumulation was not inhibited by fructose, fucose, galactose, mannose, glucose, or 3-O-methyl-D-glucose and was less affected by cytochalasin B or phloretin than that of 2-deoxyglucose. Phloridzin did not affect AG uptake but did inhibit 2-deoxyglucose uptake. Efflux of AG from K-562 cells depended on external AG concentration alone and was not affected by extracellular glucose concentration. Intracellular AG concentration decreased rapidly and reached zero within 10 min following removal of AG from the external medium. We therefore propose that both transport and countertransport of AG in K-562 cells are mediated by a specific carrier system.

Functional conformation changes in the TF(1)-ATPase beta subunit probed by 12 tyrosine residues.

The effect of nucleotide binding on the structure of the F(1)-ATPase beta subunit from thermophilic bacillus PS-3 (TF(1)beta) was investigated by monitoring the NMR signals of the 12 tyrosine residues. The 3,5-proton resonances of 12 tyrosine residues could be observed for the specifically deuterated beta subunit. The assignment of 3,5-proton resonances of all of the tyrosine residues was accomplished using 14 mutant proteins, in each of which one or two tyrosine residues were replaced by phenylalanine. Binding of Mg. ATP induced an upfield shift of Tyr(341) resonance, suggesting that their aromatic rings are stacked to each other. Besides Tyr(341), the signal shift observed on Mg.ATP binding was restricted to the resonances of Tyr(148), Tyr(199), Tyr(238), and Tyr(307), suggesting that Mg.ATP induces a conformational change in the hinge region. This can be correlated to the change from the open to closed conformations as implicated in the crystal structure. Mg.ADP induced a similar but distinctly different conformational change. Therefore, the intrinsic conformational change in the beta subunit induced by the nucleotide binding is proposed to be one of the essential driving forces for the F(1) rotation. Reconstitution experiments showed that Tyr(277), one of the four conserved tyrosines, is essential to the formation of the alpha(3)beta(3)gamma complex.

Usefulness of troglitazone administration to obese hyperglycaemic patients with near-normoglycaemia.

AIMS: We assessed the effectiveness of 400 mg/day of troglitazone administered to hyperglycaemic patients with near-normoglycaemia who were obese and who had hyperinsulinaemia. RESULTS: The area under the plasma glucose curve in oral glucose tolerance tests (OGTT) significantly decreased from 39.8 +/- 19.4-20.5 +/- 10.2 mg/dL. h and the area under the insulin-response curve from 31.8 +/- 22.5-12.2 +/- 5.7 microU/ml x h 4 months after the start of treatment. The level of HbA1c significantly improved from 6.6 +/- 0.2 to 6.3 +/- 0.2% (p < 0.05) by 1 month after administration, and that of serum 1,5-anhydroglucitol (1,5-AG) from 12.6 +/- 1.1-18.3 +/- 2.5 micro/ml (p < 0.05). In some cases, recovery of the first-phase insulin secretion was observed. CONCLUSIONS: These findings suggest that the administration of this insulin sensitizer is useful in the treatment of obese Japanese subjects with borderline or mild diabetics accompanied by hyperinsulinaemia.

Prolonged hyperalimentation as a possible cause of renal tubular dysfunction: evaluation of 1,5-anhydro-D-glucitol resorption and N-acetylglucosaminidase excretion in humans.

1. A major polyol found in the sera and other tissues of humans, 1,5-anhydro-D-glucitol, is mainly ingested in the diet and is excreted in urine. We compared the influence of the long-term administration of total parenteral nutrition free of 1,5-anhydro-D-glucitol with that of total enteral nutrition on the serum level of 1,5-anhydro-D-glucitol in 46 patients who could not take food by mouth. 2. The serum concentration of 1,5-anhydro-D-glucitol and its kinetics remained unchanged in the group receiving total enteral nutrition (n = 21) over a period of 12 months. However, after 1 month on total parenteral nutrition (n = 25), the serum level of 1,5-anhydro-D-glucitol decreased, falling to about one-sixth the pretreatment level in the 12th month. Because the serum level of 1,5-anhydro-D-glucitol continued to decline, falling below the limit at which its renal reabsorption is normally activated, this decrease did not seem to be caused directly by a nutritional deficiency of this substance. 3. The urinary excretion of 1,5-anhydro-D-glucitol was closely correlated (r = 0.792) with that of N-acetyl-beta-glucosaminidase; but not with the serum creatinine level or of the urinary excretion of microalbumin or of urinary beta 2-microglobulin. We observed no glucosuria, hyperuricuria or changes in serum electrolytes during total parenteral nutrition. 4. The reduction in the serum level of 1,5-anhydro-D-glucitol and the urinary excretion of N-acetyl-beta-glucosaminidase were correlated with the duration of total parenteral nutrition administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Post-load glucose measurements in oral glucose tolerance tests correlate well with 1,5-anhydroglucitol, an indicator of overall glycaemic state, in subjects with impaired glucose tolerance.

Using both cross-sectional and longitudinal methods, we investigated the relationship between post-load serum glucose concentration in a 75 g oral glucose tolerance test (OGTT) and overall glycaemic state in subjects with impaired glucose tolerance (IGT). Glycaemic state was assessed by measuring glycated haemoglobin (HbA1c) and the serum concentration of 1,5-anhydroglucitol (1,5-AG). In the cross-sectional study, the concentration of 1,5-AG, while remaining within a normal range, was reduced to a degree proportional to the post-load glycaemic level. Although the correlation between HbA1c and post-load plasma glucose was relatively weak (r=0.281, P<0.001), a significant inverse correlation (r=-0.824, P<0.0001) was found between 1,5-AG and mean post-load plasma glucose concentration in 211 subjects with IGT. Fasting plasma glucose (r=-0.539, P<0.0001) and 2 h plasma glucose (r=-0.621, P<0.0001) were correlated with 1,5-AG less strongly than was post-load glycaemia. Both 1,5-AG and HbA1c were correlated weakly but significantly with the fasting insulin concentration. In the longitudinal study we measured 1,5-AG and mean post-load plasma glucose with an OGTT once yearly for 10 years in 15 subjects with IGT. Strong inverse correlations were seen between 1,5-AG and mean post-load plasma glucose in each subject (range of r values among subjects of -0.584 to -0.978). These findings suggest a close relationship between post-load plasma glucose concentration measured by OGTT and overall glycaemic state in subjects with IGT.

Clinical usefulness of serum 1,5-anhydroglucitol in monitoring glycaemic control.

BACKGROUND: To evaluate prospectively the clinical value of measuring serum concentrations of 1,5-anhydroglucitol (1,5AG) in monitoring glycaemia in patients with newly diagnosed non-insulin-dependent diabetes mellitus (NIDDM), we measured serum 1,5AG in 56 such patients. METHODS: 28 patients (group A) were started on, and continuously received, an oral hypoglycaemic agent for at least 6 weeks. The other 28 patients (group B) were given such agents for 4 weeks, and then stopped taking them for at least 2 weeks. All patients were then followed for an additional 10 weeks. Serum 1,5AG, fructosamine, glycated haemoglobin (HbA1c), and self-monitoring of blood glucose were monitored every 14 days for 16 weeks. FINDINGS: When sudden worsening of glycaemia occurred within 2 weeks, entailing withdrawal of oral treatment, 1,5AG accurately detected the slight change in glycaemia whereas HbA1c and fructosamine both failed to detect it. Although the change was detected by measurement of fasting plasma glucose (FPG) concentrations, FPG was less sensitive than 1,5AG. In patients with "near-normoglycaemia" (HbA1c about 6.5%) in the preceding 8 weeks, those who showed a lower concentration of 1,5AG (<10.0 micrograms/mL) manifested a higher mean daily plasma glucose concentration even though HbA1c measurement suggested good control of glycaemia. Results of 1,5AG were correlated more strongly with the FPG (r=0.790) and mean daily plasma glucose (r=-0.835) estimated on the same day than those estimaoffted in the preceding 2, 4 and 8 weeks, and with a fall in the Spearman correlation coefficient at any preceding time interval. INTERPRETATION: Because 1,5AG accurately detected a slight change in glycaemia without delay, it is suitable for use in monitoring for strict control of glycaemia, an important clinical goal.

Nematocidal activity of long alkyl chain amides, amines, and their derivatives on dog roundworm larvae.

The nematocidal activity of amides and amines having a long alkyl chain against the second-stage larva of dog roundworm, Toxocara canis, was examined. Long chain acyl amides with smaller substituents on the nitrogen showed stronger activity and the activity of cyclic amine amides was stronger than that of acyclic ones. In a series of homologous amides, the activity was dependent on the alkyl chain length: it reached a maximum at an optimal chain length and decreased in both shorter and longer homologues. The relationship between the activity and hydrophobicity of the homologues was analysed by the use of the bilinear model. The hydrophobicity of a compound, which gives a maximal activity, was similar for all neutral amides, but amides which have an additional amine group in the molecule had different values. Tertiary amines and their salts having a long alkyl chain also showed nematocidal activities comparable to those of the corresponding amides. The salts killed the larva at concentrations lower than their critical micell concentration, suggesting that they behave as a single molecule for the nematocidal action.