Protein crystallization in a 100 nl solution with new stirring equipment.

To investigate quantitatively the effects of stirring on protein crystallization, a new stirring system which can agitate a protein solution, approximately 100 nl, by providing Hagen-Poiseuille flow has been successfully developed. In addition, this new stirring system provides flow with a well defined pattern and velocity. Using this system, hen egg-white lysozyme was crystallized in 100-200 nl solutions while being stirred. The optimum stirring conditions for lysozyme crystals have been explored by evaluating the Reynolds (Re) number and the crystals obtained. Intermittent flow, as well as a low Re number, was found to contribute significantly to the growth of a smaller number of larger crystals.

Effect of rectal administration of rebamipide on dextran sulfate sodium-induced colitis: role of hepatocyte growth factor.

OBJECTIVE AND DESIGN: Since rebamipide is effective for the treatment of ulcerative colitis (UC), we examined the involvement of hepatocyte growth factor (HGF) in the action of rebamipide. MATERIALS: Fifty-five and forty female Balb/c mice, respectively, were used in Exp. 1 and 2. TREATMENT: 50 mg/kg/day rebamipide (Exp. 1) and 1 x 10(7) pfu pAxCAHGF (the CAG promoter-driving HGF gene in adenovirus vector) (Exp. 2) were intrarectally introduced after induction of colitis by 4 % dextran sulfate sodium (DSS). METHODS: Therapeutic effects were assessed by cell proliferation and apoptosis. RESULTS: Rebamipide caused proliferation of epithelial cells at 10 days after treatment, and decreased apoptosis at 10, 14 and 21 days, compared with controls. Expression of HGF was greatly increased in rebamipide-treated mice. pAxCAHGF caused cell proliferation and apoptosis, which showed the same pattern as with rebamipide treatment. CONCLUSIONS: Rectal administration of rebamipide is effective for DSS-induced colitis in association with induction of HGF.

Efficacy of imatinib mesylate (STI571) treatment for a patient with rheumatoid arthritis developing chronic myelogenous leukemia.

We report on an 80-year-old man with rheumatoid arthritis (RA) who presented with chronic myelogenous leukemia (CML). Five years after the onset of RA, the CML diagnosis was made. The patient was treated for CML with 300 mg of imatinib mesylate (STI; signal transduction inhibitor 571) for 8 weeks. Laboratory tests showed that the C-reactive protein level, percentage of cells exhibiting the Philadelphia chromosome (Ph1), WBC count, and Lansbury index for RA all dropped respectively from 7.5 mg/dl to 1.0 mg/dl, 74.9% to 1%, 25, 100/microl to 9900/microl, and 51% to 14%. Administration of imatinib mesylate is felt to be effective in treating not only CML but also RA in the active stage.

Diabetic neuropathies in brain are induced by deficiency of BDNF.

Diabetes is known to be one of the risk factors for dementia; however, neuropathic changes in the brain of patients with the disease have not been completely revealed. So in the present study, we investigated the brain function of rats with diabetes induced by streptozotocin (STZ), one of the most commonly used animal models for diabetes. In the diabetic rats, immediately working memory performance was impaired in the Y-maze task and neuronal cytoskeleton proteins such as calbindin, synaptophysin, and syntaxin were reduced. Furthermore, morphological observation by Golgi staining showed a decrease in the number of basal dendrites and abnormality of spine structure. Next, we measured the content of brain-derived neurotrophic factor (BDNF) in the diabetic brain, because BDNF is one of the essential proteins for the maintenance of neuronal functions including synapse function and neuronal transmissions. In the diabetic brains, both protein and mRNA levels of BDNF were severely reduced. These results suggest that, in diabetes, synapse dysfunction is, at least in part, caused by a failure of BDNF synthesis in the brain.

Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA.

Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.

[An electrophysiological study of ropivacaine on excised cervical vagus nerves of rabbit].

Ropivacaine, a new long acting local anaesthetic of amide type is structurally related to mepivacaine and bupivacaine. This study was designed to compare the in vitro potency and neurotoxicity of ropivacaine with those of other commercially available local anaesthetics using an isolated rabbit vagus nerve model. Ropivacaine dose-dependently suppressed the evoked compound action potentials of A beta nerve and C nerve components. Minimum concentration of ropivacaine for producing complete suppression of the compound action potentials of all components was 0.008%. Electron microscopic observation showed that ropivacaine did not destroy any peripheral nervous structures in concentrations up to 0.75%. When the neurotoxic effect of ropivacaine was compared, in terms of risk ratio (clinically used concentration/concentrations producing 2 hr irreversible block), with that of commercially available local anesthetics, the rank oder was dibucaine, tetracaine, lidocaine, bupivacaine and ropivacaine.

Ultrasonographic assessment of the risk of injury to branches of the middle hepatic vein during laparoscopic cholecystectomy.

BACKGROUND: Although hemorrhage from the gallbladder bed during laparoscopic cholecystectomy is one of main reasons for conversion to open cholecystectomy, the cause of this life-threatening complication is unclear. PATIENTS AND METHODS: Color Doppler ultrasound was used to examine the cause of venous hemorrhage from the gallbladder bed during laparoscopic cholecystectomy in 4 patients postoperatively and to examine the anatomic relationship between the gallbladder bed and branches of the middle hepatic vein in 50 healthy volunteers. RESULTS: Injury to a large branch of the middle hepatic vein adjacent to the gallbladder bed was diagnosed in all 4 patients. One patient required conversion to open cholecystectomy while the bleeding in 2 patients was immediately controlled by direct pressure with the gallbladder. The branch of the middle hepatic vein was completely adherent to the gallbladder bed in 5 of the 50 volunteers, and in 1 the diameter of the branch was as large as 3.5 mm. In 3 volunteers branches 3.0 to 3.8 mm in diameter traversed as close as 1.0 mm from the gallbladder bed. CONCLUSIONS: Patients with large branches of the middle hepatic vein close to the gallbladder bed are at risk of hemorrhage during laparoscopic cholecystectomy and should be identified preoperatively with ultrasound.

Genetic variation in the promoter region of the beta-fibrinogen gene is associated with ischemic stroke in a Japanese population.

Evidence suggests that an allelic variation in the beta fibrinogen gene may confer an increased risk of coronary artery disease and stroke. The role of the beta fibrinogen gene polymorphism and fibrinogen levels in ischemic stroke has not been determined in Japanese, who are more prone to stroke than to coronary artery disease compared with Caucasians. We investigated the associations between ischemic stroke, plasma fibrinogen level, and a HaeIII restriction fragment length polymorphism (G/A(-455)) located at -455 bp from the start of transcription of the beta fibrinogen gene in 85 hypertensive patients with ischemic stroke (stroke group), 85 hypertensive patients without ischemic stroke (nonstroke group) and in 84 normotensive subjects matched for age, sex, and smoking status recruited at an annual health examination (normotensive group). The frequency of non-cutting allele (designated A(-455) allele) in the control group was 0.07 [95% CI: 0.03-0.11]; this value was significantly lower than that previously reported in Caucasians (0.19-0.26). The A(-455) allele frequency of the nonstroke group and stroke group were 0.08 [95% CI: 0.04-0.12] and 0.15 [95% CI: 0.10-0.21]. A(-455) allele frequency of the stroke group was significantly higher than that of the control group chi2 = 5.63, P= 0.018) and the nonstroke group chi2 = 4.00, P= 0.043). The mean +/- SD fibrinogen level was significantly higher in the stroke group than that in the normotensive group (277 +9/- 64 mg/dl versus 257 +/- 52 mg/dl, P < 0.03), but that of the nonstroke group was not significantly different compared with both normotensive and stroke groups. In conclusion, the positive association between the fibrinogen genotype G/A(-455) and ischemic stroke in hypertensive patients was independent of other risk factors. These results suggest that fibrinogen A(-455) allele may be an independent risk factor for ischemic stroke in the Japanese population.

Wheat MADS box genes, a multigene family dispersed throughout the genome.

Polymerase chain reaction (PCR) with degenerate primers was utilized for partial cloning of the MADS box gene family from wheat (Triticum aestivum L.). PCR products corresponding to a part of the MADS box region were cloned and sequenced. Eleven individual clones sequenced were classified into seven types on the basis of the nucleotide sequence and five types on the deduced amino acid sequence, which included two wheat-specific MADS box protein sequences. RT-PCR analysis with degenerate primers revealed preferential expression of the MADS box genes in young spikes. Furthermore, genomic Southern blot analysis with degenerate PCR products as probes indicated that wheat MADS box genes constitute a multigene family and are dispersed throughout the genome.

Laparoscopic cholecystectomy with an ultrasound surgical aspirator.

Laparoscopic cholecystectomy using an ultrasound surgical aspirator has been performed in our department since March 1991. The horn cover was altered in order to be inserted through a trocar 10 mm in diameter. The main purpose of this device is to explore Calot's triangle by fragmentation and aspiration of the fatty tissue without damaging the nerves, vessels, and cystic duct. First the serosa of the Calot's triangle is cut via electrocautery with the sharp-angle hook dissector we designed. Then the cystic duct and cystic artery are efficiently exposed by the ultrasound surgical aspirator. This procedure is perfectly adapted for laparoscopic cholecystectomy. We obtained favorable results with the ultrasound surgical aspirator in 135 cases including 40 cases with a negative gallbladder, as evaluated by endoscopic retrograde cholangiography. In conclusion, the ultrasound surgical aspirator is suitable for skeletonizing the cystic duct and cystic artery, and the procedure is perfectly safe.

Prorenin-renin axis in synovial fluid in patients with rheumatoid arthritis and osteoarthritis.

This study was undertaken 1) to determine whether or not renin is present in synovial fluid in patients with rheumatoid arthritis and osteoarthritis, and, if present, 2) to investigate whether it is synthesized in synovial fluid, or it is only transported from the circulation into the synovial cavity. The active renin concentration (indirect) was measured with angiotensin I radioimmunoassay kits. Inactive renin was converted into active renin with Sepharose-bound trypsin. Both active and inactive forms of renin were found in synovial fluid. They were significantly higher in patients with rheumatoid arthritis (n = 9) than in those with osteoarthritis (n = 16). In plasma, the concentration of inactive renin was significantly higher (P less than 0.001) in the former. Albumin, transferrin, alpha 2-macroglobulin, ceruloplasmin and immunoglobulins G and M were also found in synovial fluid. In each disease, a plot of the log ratio of synovial fluid to the serum concentration against the log molecular weight of each protein gave an approximately straight line curve, suggesting that these proteins are derived from the circulation and are transported into the synovial cavity. In contrast, the ratio of synovial fluid to plasma concentrations of active renin was significantly higher than that predicted on the basis of the above-mentioned interrelationships in both diseases, whereas the ratio of inactive renin was significantly lower. These findings suggest that 1) inactive and active renin are filtered into the synovial fluid from the circulation, and that 2) inactive renin is converted into the active form in the fluid.

Inactive renin release from the human arm vascular wall during upper arm occlusion: relation to beta-adrenoceptors.

To investigate the extrarenal release of renin, plasma active renin (PAR) and plasma trypsin activatable or inactive renin (PIR) in blood taken simultaneously from bilateral median cubital veins were measured in seven normal men before and after occlusion of the right upper arm with a standard cuff inflated halfway between systolic and diastolic blood pressures for 15 min. After 15 min, both PAR (P less than 0.05) with PIR (P less than 0.01) increased significantly in occluded arms compared with 0 min. PIR rose significantly (P less than 0.01) in occluded arms compared with controls at 15 min, but PAR was unchanged. The same studies were repeated after pretreatment with oral doses of either 1 mg prazosin or 10 mg propranolol at 1600 h and 2400 h on the previous day and 1 hour before the experiment. After prazosin, both PAR (P less than 0.05) and PIR (P less than 0.01) in occluded arms increased significantly at 15 min compared with those at 0 min and PIR rose significantly (P less than 0.01) in occluded arms compared with controls at 15 min, as observed in the control studies. On the other hand, after propranolol, the significant increment of PAR in the occluded arms was abolished. Moreover, the significant difference of PIR between the occluded and the control arms at 15 min was also abolished. These results indicate that inactive renin may be present in the vascular wall of the arm and released into circulation by a stimulus such as arm occlusion by mechanisms related to beta-adrenoceptors.

Percutaneous stenting for malignant biliary stenosis.

Percutaneous stenting for malignant biliary stenosis is quite beneficial to patients with unresectable or recurrent disease, tremendously improving the quality of their lives. Percutaneous transhepatic biliary drainage (PTBD) was attempted in 92 patients with obstructive jaundice during the period between January 1986 and July 1989. Implantation of an endoprosthesis was performed in 14 cases (15.2%) and succeeded in 12 (85.7%). When a guide wire could not be passed distally across the stricture site, percutaneous transhepatic cholangioscopy (PTCS) through the dilated PTBD fistula was carried out to enable its passage. PTCS is also valuable in the preoperative diagnosis of obstructive jaundice. The patients who are not candidates for surgery are suitable for this procedure. A Miller double-mushroom stent is used as the endoprosthesis in the majority of cases. One patient with recurrent hepatoma has lived at home with this stent for greater than 3 years due to repeated transarterial embolization and chemotherapy and does not need to wash or change the stent.

Beneficial effect of amosulalol and phentolamine on post-hypoxic recovery of contractile force and energy metabolism in rabbit hearts.

1. The effects of phentolamine, an alpha-adrenoceptor blocking agent and amousulalol, an alpha 1 and beta-adrenoceptor antagonist on hypoxia-induced impairment in cardiac function and metabolism were examined using the isolated heart Langendorff preparation of the rabbit. 2. Hypoxia induced cessation of cardiac contractile force, a rise in resting tension, a decrease in myocardial high-energy phosphates, an increase in tissue calcium content and the release of ATP metabolites from the heart. Subsequent reoxygenation resulted in little recovery of cardiac contractile force, and there were further increases in tissue calcium content and in the release of creatine kinase from the heart. 3. Treatment of hypoxic hearts with either 83 microM phentolamine or 45 microM amosulalol resulted in a suppression of the rise in resting tension, the tissue calcium accumulation and the release of creatine kinase and ATP metabolites during hypoxia. This treatment also elicited significant recovery of cardiac contractile force, restoration of myocardial high-energy phosphates, suppression of the release of creatine kinase and the accumulation of tissue calcium during reoxygenation. Both 83 microM phentolamine and 45 microM amosulalol a significant prolongation of the effective refractory period of rabbit isolated atria. 4. Lower concentrations of phentolamine (16 microM) and amosulalol) (9 microM), which are sufficient to exert an alpha-adrenoceptor blocking action, did not elicit an appreciable effect on the post-hypoxic recovery of cardiac contractile force. 5. These results suggest that phentolamine and amosulalol are capable of protecting the myocardium from hypoxia-induced derangements in cardiac function and metabolism. This effect is probably attributable to their membrane stabilizing effect, rather than to their alpha-adrenoceptor blocking action.

Possible mechanism by which coenzyme Q10 improves reoxygenation-induced recovery of cardiac contractile force after hypoxia.

To elucidate possible mechanisms by which coenzyme Q10 enhances reoxygenation-induced recovery of cardiac contractile force after hypoxia, rabbit hearts were subjected to hypoxic perfusion for 20 min, followed by 45 min-reoxygenation with or without pretreatment with coenzyme Q10. Hypoxia induced a decline in cardiac contractile force, a decrease in myocardial high-energy phosphates and a release of ATP metabolites and creatine phosphokinase from the perfused heart. Upon reoxygenation the rate of release of ATP metabolites subsided, but no appreciable recovery of the loss of contractile force and the reduction of myocardial ATP content was seen, and the release of creatine phosphokinase was increased further. Pretreatment of rabbits with coenzyme Q10 resulted in an appreciable recovery of cardiac contractile force and of myocardial ATP content upon reoxygenation. The release of creatine phosphokinase from hearts during hypoxia and reoxygenation was inhibited completely by the pretreatment. Changes in the UV absorbance of the perfusate suggested that coenzyme Q10 reduced the loss of ATP metabolites from hypoxic hearts. Furthermore, high-performance liquid chromatographic analysis indicated that coenzyme Q10 attenuated the release of inosine and hypoxanthine from the hearts and decreased myocardial inosine and adenosine content of the hypoxic heart, suggesting that coenzyme Q10 retards the breakdown of ATP metabolites which are possible substrates for a salvage synthesis of ATP, when oxygen is replenished. This could account for an appreciable restoration of ATP, and eventually provide a significant recovery of cardiac contractile force upon reoxygenation.

[A case of remission of metastatic gastric cancer lesions in the chest and abdominal wall by 5'-DFUR administration].

52-year-old female was weakened and bed-ridden by metastasis of gastric cancer to the chest and abdominal wall, and peritonitis carcinomatosa 2 years after gastrectomy. One week after administration of 5'-DFUR, 1200 mg/d, p.o., a clinical effect was seen. In 3 weeks metastatic lesions had diminished in size, ascites had decreased and the patient had regained her appetite and put on weight. She recovered sufficiently to be able to do some daily work. Pathological degeneration and necrosis of cancer cells were observed. Although she died 8 months after remission, 5'-DFUR was effective in improving her condition and prolonging her life.

[Fundamental and clinical studies on cefoperazone in pediatrics, with special reference to renal toxicity].

Fundamental, clinical studies on cefoperazone (CPZ), a new synthetic antibiotic of cephalosporins was conducted to obtain results as follows. This preparation, 11.4-50 mg/kg was administered to 11 cases of children by intravenous drip infusion for 30 minutes, and serum levels were studied. The highest serum level at the completion of infusion was 40.0-138.0 mcg/ml. A dose response was observed. The half-life in serum averaged 1.55 hours except 2.4 hours observed with 1 case of liver dysfunction. When the urinary excretion during 30 minutes drip infusion was examined, the urinary recovery rate at 0-6 hours in 2 cases of children averaged 17.2% and was low at 5.3% in 1 case of membranoproliferative glomerulonephritis. In terms of the clinical effect on bacterial infections in pediatric field, CPZ proved effective in all of the 21 cases in which it was used alone. And it showed an excellent antibacterial activity against all the bacteria isolated from cases used as the subjects. When side effects were studied, diarrhea and slight impairment of the liver were observed, but all were transient. As a result of a study on the renal toxicity of CPZ, CPZ was deemed as being a drug which hardly causes disturbance of renal tubules judging from the aspect of the beta-D-N-acetylglucosaminidase activity in urine and variations in the urine beta 2-microglobulin levels.