Major groove recognition by three-stranded beta-sheets: affinity determinants and conserved structural features.

We present the results of a rational mutagenesis and binding-affinity study of the three-stranded beta-sheet-DNA interface in the complex formed by the amino-terminal DNA-binding domain of the Tn916 integrase protein and its cognate binding site. The relative importance of interfacial contacts present in its NMR-derived solution structure have been tested through mutagenesis, fluorescence anisotropy, and intrinsic quenching DNA-binding assays. We find that seven protein-DNA hydrogen bonds (two base-specific and five to phosphate groups) significantly contribute to the level of affinity. These interactions span the entire DNA-binding surface on the protein, but primarily originate from residues in only two strands of the sheet and loop L2. Interestingly, we show that highly populated, precisely defined intermolecular hydrogen bonds in the ensemble of conformers are invariably important for DNA-binding, implying that NMR-derived solution structures provide direct insight into the energetics of recognition. Unusual three-stranded beta-sheet-DNA interfaces have recently been discovered in three unrelated protein-DNA complexes. A comparative analysis of these structures reveals similar sheet positioning, the presence of two invariant interfacial contacts to the phosphodiester backbone, and two semi-conserved base-specific hydrogen bonds. Two of these conserved contacts significantly contribute to the affinity of the integrase-DNA complex, suggesting that the three-stranded beta-sheet DNA-binding motif exhibits conserved principles of recognition.

Remarkably enhanced inhibitory effects of hybrid liposomes on the growth of specific tumor cells.

The highly specific inhibitory effects of the hybrid liposomes composed of 90 mol% L-alpha-dimyristoylphosphatidylglycerol and 10 mol% polyoxyethylene (10) dodecyl ether on the growth of lung adenocarcinoma and stomach tumor cells in vitro were obtained for the first time.

Isolation and characterization of a cytokinin up-regulated gene from tobacco mesophyll protoplasts.

We have isolated a cytokinin up-regulated cDNA clone, H13, from an early stage of cultured tobacco mesophyll protoplasts by a differential display method. The expression of this gene was specifically induced by natural and synthetic cytokinins including N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30), a diphenylurea-type cytokinin, although the simultaneous presence of auxin was also required. It seems that the preceding treatment of the tobacco mesophyll protoplasts by auxin is necessary for the gene to respond to cytokinin. The addition of a cytokinin antagonist, compound 182, which suppressed the induction of cell division in tobacco mesophyll protoplasts, completely abolished the expression of this gene. Though the predicted gene product of H13 did not suggest us any sequences of defined functions, two domains of the predicted sequence had significant homology to several reported sequences in the data base. The gene product of H13 is proposed to have a role in regenerating cell wall in cultured protoplasts, since a cDNA clone E6, from cotton fiber cells, which has the most closely related structure to H13, has been isolated from cells which showed active cellulose synthesis. This supposition is supported by the evidence that in the absence of cytokinin, cell wall regeneration was significantly suppressed, resulting in failure of the induction of cell division. Thus, the gene product of H13 is supposed to have a role in regenerating cell walls and facilitating the progression of the cell cycle, resulting in the sustained cell division of tobacco mesophyll protoplasts.

Spring coil retraction in coil occlusion of persistent ductus arteriosus.

AIMS: To present the short and intermediate term results of coil occlusion of persistent ductus arteriosus and the results of radiographic measurements of spring coils implanted to treat patent ducts. PATIENTS: 22 children underwent coil occlusion. Their ages ranged from 2 years 9 months to 12 years 10 months (mean (SD) age, 6.5 (3.6) years). The duct diameter ranged from 1.0 to 3.5 mm at the narrowest point (mean 2.6 (0.7) mm). In 11 of the children regular coils were implanted using the non-attached system, while in the other 11 the detachable coil embolisation system was used. RESULTS: 12 children (55%) had no significant residual leaks immediately after procedures involving a single coil delivery. The remaining 10 (45%) had residual leaks immediately after the procedure, although no patient with a large duct showed residual leakage 18 months after the procedure. Radiographic measurement of the coils showed that all implanted coils retracted to 65-85% of their original size immediately after occlusion. This retraction was more evident in patients showing spontaneous closure of the residual shunt or having a coil 8 mm in diameter. CONCLUSIONS: Coil embolisation is an acceptable method for occluding persistent ductus arteriosus. Retraction of implanted coils is common in the follow up period. Such retraction may be related to spontaneous closure of residual shunt after embolisation.

Volumegraph (overlaid three-dimensional image-guided navigation). Clinical application of augmented reality in neurosurgery.

OBJECTIVE: We have developed an overlaid three-dimensional image (Volumegraph)-guided navigation system that allows navigation during operative procedures. The three-dimensional image is superimposed on the patient's head and body via a semi-transparent mirror. The Volumegraph can display three-dimensional images in the air by a light beam which is based on CT/MRI. METHOD: The system consists of a Volumegraph (thin plate of three-dimensional recorded medium), a Volumegraphscope and an original designed triangular-shaped marker system for registration. The three-dimensional data obtained from CT and MRI before the operation were processed by a computer. Such image data are applied for preoperative investigation to recognize the three-dimensional structure of organs and tumor. These reconstructed three-dimensional images were superimposed and registered at the patient's head according to a fiducial marker (registration). Then the operator can operate with this three-dimensional-image-guided navigation system. RESULTS: Based on clinical application in 7 cases, the system was found to be advantageous because the surgical procedures could be navigated easily by augmented reality in the surgical field. Invisible parts of the surgical field were supplemented with the overlaid three-dimensional images (Volumegraph) as if it were the virtual operative field. At another time, spatial positioning and overlaid visualization by the Volumegraph was useful for identifying anatomical structures and functional location in the image. CONCLUSION: This preliminary study of overlaid three-dimensional-image-guided navigation demonstrated its clinical usefulness. The application of augmented reality in the surgical field makes it possible to do a neurosurgical intervention easily and accurately.

[Specific inhibitory effect of hybrid liposomes on the growth of hybridoma cells in vitro].

Remarkably high inhibitory effects of the hybrid liposomes composed of L-alpha-dimyristoyl-phosphatidylcholine (DMPC) and polyoxyethylenealkyl ether (C14(EO)n, n = 6-8 and C12(EO)n, n = 8-12)) on the growth of human lymphoma-human B-lymphocyte hybridoma (HF) cells in vitro were obtained. The hybrid liposomes composed of 90 mol% DMPC/10 mol% C14(EO)n (n = 6-8) or C12(EO)n (n = 8-12) were more fluid as compared with 90 mol% DMPC/10 mol% C14(EO)4 or C12(EO)n (n = 4, 23) hybrid liposomes on the basis of fluorescence polarization measurements. These results suggest that the inhibitory effects of the hybrid liposomes on the growth of HF cells should be related to the membrane fluidity. No toxicity to normal rats in vivo was observed in the experiment using 90 mol% DMPC/10 mol% C14(EO)7 or 90 mol% DMPC/10 mol% C12(EO)12 hybrid liposomes.

High affinity of hybrid liposomes for normal human epidermal keratinocytes in vitro.

A high affinity of hybrid liposomes towards normal human epidermal keratinocytes was observed. The fluorescence micrograph showed that hybrid liposomes may be incorporated into the keratinocytes by fusion.

Production of acetic acid by Clostridium thermoaceticum in electrodialysis culture using a fermenter equipped with an electrodialyser.

Electrodialysis culture of Clostridium thermoaceticum increased the yield of acetate by its continuous removal. In normal batch cultures without pH control the yield was 4.2 g acetic acid/800 ml, while in pH-controlled culture it was 16.8 g/800 ml. Although electrodialysis cultures gave almost the same yield (15.4 g/800 ml) as that in pH-controlled cultures, sparging CO2 into the broth in electrodialysis culture increased the amount of acetic acid to 22.3 g/800 ml. CO2 sparging into normal cultures with or without pH control did not significantly increase the amount of acetate produced but yields, in terms of amounts of glucose consumed, were higher than without sparging. The theoretical yield was almost obtained in pH-controlled, electrodialysis cultures with CO2 sparging.

Successful balloon mitral commissurotomy in a small child: use of small Inoue balloon catheter.

A 15-month-old girl weighing 5.8 kg, in whom congestive heart failure developed due to congenital mitral stenosis, was successfully treated by percutaneous transluminal mitral commissurotomy using a small Inoue balloon catheter. Percutaneous transluminal mitral commissurotomy using a small Inoue balloon catheter may be a first-choice treatment for small children with congenital mitral stenosis.

Development and evaluation of a novel sterilizer with rotary vibrators.

A novel steam-air sterilizer with rotary vibrators was developed with the aim of promoting heat transfer and the effective sterilization of foods. To evaluate the effects of the sterilizer, experiments to examine heat transfer and sterilization tests were carried out. In the experiments to examine heat transfer, heat penetration factors of sterilizer with vibration, j and fh values, were 1.70 and 1.50, respectively, while those of sterilizer without vibration were 1.83 and 2.32, respectively. In the sterilization with vibration, no surviving cells were detected after 3 min, whereas 7 min were required for sterilization without vibration. The rate of the amino carbonyl reaction was repressed by heat treatment with vibration.

Intermediate-term results of balloon valvuloplasty for isolated and complicated pulmonary valve stenosis.

UNLABELLED: The purpose of this study is to report our experience regarding the acute and intermediate-term results of balloon pulmonary valvuloplasty (BPV) in various types of congenital pulmonary valve stenosis. METHODS AND RESULTS: Twenty-four consecutive patients with a median age of 6.6 years (ranging from 1 month to 24 years old) underwent BPV between January 1988 and September 1991. These patients were divided into 2 groups; Group 1 consisting of 13 patients with isolated pulmonary valve stenosis, and Group 2 consisting of 11 patients with complicated pulmonary valve stenosis (supravalvular, subvalvular, valved conduit and post-right ventricular outflow reconstruction). Mean peak systolic pressure gradients from the right ventricle to the pulmonary artery were as follows: In group 1, 48 +/- 21 (mean +/- SD) mmHg before BPV, 18 +/- 8 mmHg immediately after BPV and 13 +/- 5 mmHg at the longest follow-up based on catheterization or Doppler echocardiographic studies. The gradients in group 2 were 65 +/- 28 mmHg before BPV, 46 +/- 25 mmHg immediately after BPV and 47 +/- 21 mmHg at the longest follow-up. CONCLUSIONS: BPV provides both acute and intermediate-term gradient relief in patients with isolated pulmonary valve stenosis. In complicated pulmonary valve stenosis, on the other hand, the effect of BPV was unsatisfactory and appears to depend on the mechanism of associated obstruction. Therefore accurate evaluation of the anatomy of associated obstruction in the pulmonary valve region is needed to determine that BPV is indicated.

Plasma endothelin concentration: relation with vascular resistance and comparison before and after balloon dilatation procedures.

Endothelin (ET) is a potent vasoconstrictor peptide with an as yet uncertain physiological role in cardiovascular disease. We measured blood plasma ET concentrations using a recently developed radioimmunoassay and analysed the relations between ET concentration, systemic arterial pressure and systemic vascular resistance. In addition, ET levels before and after percutaneous balloon valvuloplasty and angioplasty were measured. Fifty-one patients were studied: (1) 13 patients with small left-to-right shunting or Kawasaki heart disease (age ranged from 4 to 144 months); (2) 10 patients who had undergone balloon valvuloplasty or angioplasty (age ranged from 1 to 233 months) and (3) 28 healthy infants and children (age ranged from 3 to 152 months). Systemic vascular resistance was calculated by the formula (mean aortic pressure--mean right atrial pressure) X 80/cardiac output (dyne.sec.cm-5). Plasma ET concentrations in healthy children less than 2 years were significantly higher than those over 2 years (2.48 +/- 0.62 vs 1.31 +/- 0.53 pg/ml). In eight patients in groups 1 and 2, plasma ET concentration in the pulmonary artery (2.00 +/- 0.43 pg/ml) was significantly lower than that in the femoral vein (2.39 +/- 0.69 pg/ml) and aorta (2.23 +/- 0.59 pg/ml), suggesting ET secretion derived from endothelial cells in peripheral pulmonary vessels. There was a significant positive correlation between ET concentrations in the femoral vein and systemic vascular resistance (r = 0.55, p less than 0.05). After balloon dilatation ET concentration rose from 2.15 +/- 0.82 pg/ml to 2.61 +/- 1.38 pg/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and some properties of a thermostable metal proteinase produced by Thermomicrobium sp. KN-22 strain.

An extreme thermophile that produces a heat-stable proteinase was isolated from hot-spring water and classified as Thermomicrobium sp. KN-22 (growth temperature, 50-83 degrees C; and optimum growth temperature, 70 degrees C). The proteinase was purified from the culture broth of this strain by fractionation with ammonium sulfate, chromatography on columns of DEAE-cellulose and CM-Sepharose CL-6B, and HPLC on TSKgel CM-5PW. The purified enzyme gave a single band on SDS-polyacrylamide gel electrophoresis and a single peak after HPLC (yield 8.8%). The enzyme had maximum activity at pH 8.5 and at 75 degrees C and it was stable up to 60 degrees C. The molecular weight of the enzyme was 35,000 by SDS-PAGE. Since the enzymatic activity was completely inhibited by EDTA, o-phenanthroline, and phosphoramidon, it appears that the enzyme is a metal proteinase.

Immunosuppressive therapy in the management of acute myocarditis in children: a clinical trial.

To assess whether steroid therapy influenced the clinical course of myocarditis in a pediatric population, findings in 13 consecutive infants and children (8 female, 5 male) with biopsy-proved myocarditis were reviewed. The mean age was 5.7 +/- 4.8 years (range 1.1 to 14.8). Congestive heart failure was present in all as were ST-T wave changes, cardiomegaly and pulmonary edema on chest roentgenogram. Echocardiography demonstrated pericardial effusion in five patients and mitral regurgitation in eight. Mean left ventricular ejection fraction was 34 +/- 12%. Prednisone was administered to all patients; one patient also received azathioprine. There was one death. All survivors showed clinical improvement with normalization of ECG changes, heart size and systolic function. No significant side effects occurred. Repeat myocardial biopsy in eight patients demonstrated improvement in all eight and elimination of the inflammatory infiltrate in six. Immunosuppressive therapy in this pediatric population appeared useful in improving the clinical course and cardiac function in acute myocarditis with no adverse side effects.

Balloon occlusion aortography.

We review the validity of balloon occlusion aortography (BOA) on the basis of our personal experience with 18 patients with congenital heart disease (mean weight 4.55 g, including 8 neonates). Four of the 18 patients underwent aortic arch angiography using balloon occlusion of the descending aorta. Pulmonary angiography was also performed in 9 patients via a patent ductus arteriosus and in 3 patients via a Blalock-Taussig shunt. The remaining 2 patients underwent coronary arteriography by balloon occlusion of the ascending aorta. The information obtained was satisfactory in 17 of the 18 patients. However, in one patient with a double-outlet right ventricle and pulmonary stenosis, the pulmonary arteries were not clearly visualized because of dominant antegrade flow from the right ventricle. BOA is a safe and useful procedure which can be used to image the aortic arch, pulmonary artery, and coronary arteries in infants with congenital heart diseases. In children over 3 years of age, however, the balloon may not be able to occlude the appropriate site of the aorta, so selective angiography is required to obtain precise information.

Clinical features of aortic arch anomaly with malalignment ventricular septal defect.

The clinical features and outcome after various surgical procedures on 9 patients with coarctation or interruption of the aortic arch and malalignment ventricular septal defect (group 1) were compared with those of 9 patients with the arch anomaly without malalignment ventricular septal defect (group 2). Cardiomegaly and metabolic acidosis were prominent in group 1. Five of the 9 patients in group 1 died in the immediate postoperative period (56% mortality), but no operative deaths occurred among 8 patients in group 2 (p less than 0.01). The ratio of left ventricular outflow tract to ascending aortic diameter was 0.59 +/- 0.09 in group 1 and 1.03 +/- 0.11 in group 2 (p less than 0.01). Three of 4 patients with a ratio of less than 0.6 died, but no operative deaths occurred among the 6 patients who had a palliative operation and in whom the ratio was more than 0.6. These data suggest that left ventricular outflow tract obstruction is critical when the ratio of left ventricular outflow tract to ascending aortic diameter is 0.6 or less. The presence of severe left ventricular outflow tract obstruction necessitates modification of the present surgical strategy.