Impact of diabetes and glycaemic control on peripheral artery disease in Japanese patients with end-stage renal disease: long-term follow-up study from the beginning of haemodialysis.

AIMS/HYPOTHESIS: End-stage renal disease (ESRD) patients with diabetes have been regarded as being at the highest risk of cardiovascular disease. We therefore investigated the relationship between diabetes and the incidence of peripheral artery disease (PAD) in new haemodialysis patients. METHODS: We enrolled 1,513 ESRD patients who had just begun haemodialysis therapy. They were divided into two groups: those with (n = 739) and those without diabetes (n = 774). The endpoint was the development of PAD, defined as ankle brachial pressure index ≤ 0.9 or toe brachial pressure index <0.7 in patients with an ankle brachial pressure index >0.9. RESULTS: According to the Kaplan-Meier method, the 10 year event-free rate for development of PAD and lower limb amputation was significantly lower in the diabetes group than in the non-diabetes group (60.3% vs 82.8%, HR 2.99, 95% CI 2.27, 3.92, p<0.0001 and 93.9% vs 98.9%, HR 5.59, 95% CI 2.14, 14.7, p = .0005 for PAD and lower limb amputation, respectively). In patients with diabetes, quartile analysis of HbA1c levels showed that the highest quartile group (≥ 6.8% [51 mmol/mol]) had significant development of PAD and lower limb amputation compared with lower quartile groups (PAD HR 1.63, 95% CI 1.17, 2.28, p = .0038; lower limb amputation HR 2.99, 95% CI 1.17, 7.70, p = .023). CONCLUSIONS/INTERPRETATION: Diabetes was a strong predictor of PAD after initiation of haemodialysis therapy in patients with ESRD. In addition, higher HbA1c levels were associated with increased risk of developing PAD and requiring limb amputation in such diabetic populations.

Cloning of a streptomycin-production gene directing synthesis of N-methyl-L-glucosamine.

A Streptomyces bikiniensis DNA fragment complementing a deficiency in streptomycin (Sm) production was cloned on the plasmid vector pIJ385. Host strain S. bikiniensis SD 1 used as the recipient in cloning displayed deficiency in biosynthesis of N-methyl-L-glucosamine, one of the moieties of Sm. The cloned fragment on the multicopy plasmid pIJ385 conferred sevenfold increase in Sm production in comparison with the wild-type parental strain. By subcloning, the region complementing the Sm deficiency of SD 1 was narrowed to a 3.0-kb fragment.

Conversion of bialaphos to other oligopeptides containing phosphinothricin by Streptomyces hygroscopicus.

Two oligopeptides containing phosphinothricin were accumulated in the culture of bialaphos (BA) producer Streptomyces hygroscopicus SF1293 when large amount of BA was added to the culture at idio phase. One of the oligopeptides was a new substance, BA dimer (phosphinothricyl-alanyl-alanyl-phosphinothricyl-alanyl-alan ine), and the other was a known substance called If (phosphinothricyl-alanyl-alanyl-phosphinothricin). Though BA none producing mutants which were blocked at the steps 1, 10 and 13, respectively in BA biosynthesis also converted BA to BA dimer and If, the mutants blocked at the step 11 (alanylation) could not curry out the conversion. Antibiotic activities of BA dimer and If were lower than 1/100 of BA.

Biochemical activities of the derivatives of dehydrodicaffeic acid dilactone.

Activities of derivatives of dehydrodicaffeic acid dilactone (DDCAD) to inhibit catechol-O-methyltransferase (COMT), cyclic AMP phosphodiesterase (PDE) and DOPA decarboxylase (DDC) were examined. Among those tested, 2,6-bis-(5',6'-dibromo-4'-hydroxy-3'-methoxyphenyl)-3,7-dioxabicyclo-[3,3,0]-octane 4,8-dione was found to be the strongest inhibitor of both COMT and PDE. There were no derivatives which showed a stronger inhibition against DDC than the original compound, DDCAD.

Studies on inhibition of adenosine deaminase by isocoformycin in vitro and in vivo.

Isocoformycin is a structural isomer of coformycin which has been demonstrated to be a potent inhibitor of adenosine deaminase. Isocoformycin showed a weaker inhibition of this enzyme than coformycin; the binding of coformycin to enzyme was irreversible, but isocoformycin inhibition was competitive with substrate. The Ki value of isocoformycin was 4.5 approximately 10 X 10(-8) M. Following intraperitoneal injection of isocoformycin in mice, the adenosine deaminase activity of homogenates of several organs was determined and the following ED50 values (50% inhibition doses) were observed: 29 mg/kg for thymus, 13 mg/kg for spleen, 80 mg/kg for liver and 20 mg/kg for kidney. The inhibition of adenosine deaminase in rabbit blood in vitro was also tested in comparison with coformycin.

New isoflavones, inhibiting catechol-O-methyltransferase, produced by Streptomyces.

In the screening of catechol-O-methyltransferase inhibitors in streptomyces culture filtrates, three new isoflavones were isolated. Their structures were shown to be 3',5,7-trihydroxy-4',6-dimethoxyisoflavone (I), 3',5,7-trihydroxy-4',8-dimethoxyisoflavone (II), 3',8-dihydroxy-4',6,7-trimethoxyisoflavone (III). I and II inhibited both catechol-O-methyltransferase and dopa decarboxylase, and showed hypotensive action. III was a specific inhibitor of catechol-O-methyltransferase, and showed no hypotensive action.

Dehydrodicaffeic acid dilactone, an inhibitor of catechol-O-methyl transferase.

In the screening of catechol-O-methyltransferase inhibitors, three compounds were isolated from the culture filtrate of a mushroom, Inonotus sp. One was 3,4-dihydroxycinnamic acid (caffeic acid) which had been reported as an inhibitor of this enzyme. The others were the dextrorotatory 2,6-bis-(3',4'-dihydroxyphenyl)-3,7-dioxabicyclo-[3,3,0]-octane 4,8-dione (dehydrodicaffeic acid dilactone) andits antipode. These new compounds inhibited both dopamine beta-hydroxylase and dopa decarboxylase and showed hypotensive activity in the SH rat.

The cloned Streptomyces bikiniensis A-factor determinant.

By cleavage with restriction endonucleases and cloning the resultant fragments, the A-factor determinant cloned from streptomycin-producing Streptomyces bikiniensis (Horinouchi et al., J. Bacteriol. 158: 481 approximately 487, 1984) was narrowed down to a 1.1-kilobase (kb) fragment. A hybrid multicopy plasmid (pAFB15) carrying the 1.1-kb fragment conferred A-factor production in a large quantity to A-factor-deficient mutants of S. bikiniensis and Streptomyces griseus as well as afsA mutants of Streptomyces coelicolor A3(2). A transcriptional control signal in the 1.1-kb fragment was identified by using a promoter-probe vector pARC 1. Plasmid pAFB15 also caused A-factor production with a marked gene dosage effect in four different Streptomyces strains which originally had no ability to produce A-factor and no DNA sequence homologous to the S. bikiniensis A-factor determinant, suggesting that precursors of A-factor are common metabolites in streptomycetes and that acquisition of only a single key enzyme encoded by the 1.1-kb fragment is sufficient for Streptomyces strains to synthesize A-factor.

Studies on auromomycin.

A new antitumor antibiotic, named auromomycin, was isolated from the culture broth of Streptomyces macromomyceticus, a macromomycin-producing strain. The antibiotic was recovered from the culture filtrate by salting out with ammonium sulfate and further purified by successive application of ion-exchange chromatography on Amberlite IRA-93 (Cl form) and DEAE-Sephadex (OH form), Gel filtration on Sephadex G-50 and hydrophobic chromatography on Octyl-Sepharose CL-4B. The antibiotic is an acidic polypeptide with a molecular weitht of 12,500 and an isoelectric point of pH 5.4 and consists of 16 different amino acids. It has characteristic absorption maxima at 273 nm and 357 nm in the ultraviolet spectrum and two minima at 280 nm and 350 nm in the optical rotatory dispersion spectrum. Auromomycin exhibits antibacterial activity not only against Gram-positive bacteria, but also Gram-negative bacteria. Antitumor activities of auromomycin were revealed against EHRLICH ascites carcinoma, ascites sarcoma 180, L1210 leukemia and LEWIS lung carcinoma. Auromomycin was found to be converted into macromomycin by adsorption chromatography on Amberlite XAD.

7-Hydroxyguanine, a novel antimetabolite from a strain of Streptomyces purpurascens. I. Taxonomy of producing organism, fermentation, isolation and biological activity.

Strain A-347, an actinomycete isolated from a soil sample, was found to produce a new antimetabolite, 7-hydroxyguanine. The aerial mycelium formed spiral spore chains with spiny spore surface. The chemical composition of strain A-347 indicated that it was an actinomycete of cell wall Type I. From its morphological, cultural, physiological characteristics and direct comparison with the type culture, this strain was classified as Streptomyces purpurascens. 7-Hydroxyguanine was purified from the broth filtrate by ion exchange chromatography and crystallized from hot 2 M NH4OH. 7-Hydroxyguanine inhibited the growth of experimental tumors such as L1210 leukemia.

The bialaphos resistance gene (bar) plays a role in both self-defense and bialaphos biosynthesis in Streptomyces hygroscopicus.

We inactivated the bialaphos (BA) resistance gene (bar) of a BA producer, Streptomyces hygroscopicus, by the gene replacement technique. The resulting BA-sensitive mutant (Bar-) was able to produce little BA but considerable amount of an intermediate demethylphosphinothricin (DMPT). The Bar- mutant was still able to convert the N-acetyl derivative (AcDMPT) of DMPT to BA. Introduction of normal bar containing plasmid restored both BA resistance and BA biosynthesis to levels as high as the parental BA producer. By contrast, introducing a multi copy glutamine synthetase gene (glnA) into the Bar- mutant restored BA resistance but not BA production. Thus, the bar gene plays a crucial role in both self-defense and a step of BA biosynthesis in the BA-producing S. hygroscopicus.

Replacement of Streptomyces hygroscopicus genomic segments with in vitro altered DNA sequences.

We have developed a method for gene replacement in Streptomyces hygroscopicus which permits introduction of an in vitro derived mutation carried on a plasmid into the chromosome. We constructed the plasmid pMSB212 which can replicate in S. hygroscopicus and contains the step5 gene of the bialaphos biosynthetic pathway which was inactivated by a frame-shift mutation caused by filling in the cohesive ends of the EcoR I site in the structural gene. pMSB212 was introduced into a bialaphos producer strain and by protoplast regeneration of the primary thiostrepton-resistant transformants, non-producing mutants, were obtained. Biochemical and genetical analyses indicated that these mutants were specifically blocked by introduction of the frame-shift mutation in the step5 gene on the chromosome. This method will enable us to obtain isogenic mutants of known genes and to identify new genes encoded on a cloned fragment.

The bialaphos biosynthetic genes of Streptomyces hygroscopicus: cloning and analysis of the genes involved in the alanylation step.

We have isolated and studied the genes involved in the alanylation step in the biosynthesis of a herbicide, bialaphos which is produced by Streptomyces hygroscopicus. Three bialaphos-nonproducing mutants, NP60, NP61 and NP62, isolated from S. hygroscopicus by treatment with N-methyl-N'-nitro-N-nitrosoguanidine were defective for the alanylation step and were not restored to productivity by any locus of the gene cluster previously cloned. Three plasmids were isolated using NP60, NP61 and NP62 as recipients. The genes which restored productivity to NP61 and NP62 hybridized to the contiguous region of the bialaphos biosynthetic gene cluster. The gene cluster involved in the bialaphos production was about 35 kb long. The gene which restored productivity to NP60 did not hybridize to the bialaphos biosynthetic gene cluster. VM3 and VM4, putative alanylation blocked mutants, were derived from a bialaphos producer by gene replacement of an unidentified region of the biosynthetic gene cluster with an in vitro altered DNA sequence. The genes which restored productivity to VM3 and VM4 were located between the genes which code for phosphinomethylmalic acid synthase and demethylphosphinothricin acetyltransferase in the cluster. These results suggest that multiple genes are involved in the alanylation step.

Primary cardiac leiomyosarcoma growing rapidly and causing right ventricular outflow obstruction.

Leiomyosarcomas are extremely rare primary cardiac tumors. We report a rapidly growing primary leiomyosarcoma of the right ventricle, which obstructed the right ventricular outflow tract within one month after symptom onset in a 68-year-old man. Two-dimensional echocardiography was useful in diagnosing the extent and progression of the tumor. The tumor was surgically resected on an emergency basis, and the right ventricle and pulmonary artery were successfully reconstructed. Recurrence of the tumor on the right ventricle was observed, and the patient was overcome by sudden dyspnea and died three months after surgery.

Effect of artificial carbon dioxide foot bathing on critical limb ischemia (Fontaine IV) in peripheral arterial disease patients.

BACKGROUND: It has been reported that artificial carbon dioxide (CO(2)) foot bathing improves subcutaneous microcirculation in peripheral arterial disease (PAD) patients. However, the effect for critical limb ischemia (CLI) with ulceration or gangrene (Fontaine stage IV) is not identified. The physiological effects of CO(2) bathing and the outcome of limb salvage in such patients were studied. METHODS: In 18 healthy volunteers (Study I), the dorsal pedis peripheral blood flow was measured by a laser Doppler flow-meter during CO(2) foot bathing (1000 ppm, 37 degrees C) for 10 min. A Holter electrocardiogram was also recording in the same period. Blood flow and cell volume significantly increased during bathing. Eighty-three CLI limbs (Fontaine IV) in 68 PAD patients (Study II) underwent artificial CO(2) foot bathing (for 10 minutes twice daily >2 months) were followed up >6 months. RESULTS: In Study I, analysis of heart rate variability showed that high frequency amplitude (HFA) considerably increased and the ratio of low frequency amplitude to HFA (LF/HF) noticeably decreased during bathing. In Study II, 69 limbs (83.1%) could be salvaged. Twenty-seven of 28 limbs (96.4%) which have ulcer and gangrene in only one toe, 13/16 limbs (81.2%) in multiple toes and 29/39 limbs (74.4%) in all toes and/or heel respectively were saved. CONCLUSIONS: The effect of CO(2) enriched water on the subcutaneous microcirculation might be brought about by peripheral vasodilation reflected by increased parasympathetic and decreased sympathetic activity, and the artificial CO(2) foot bathing is clinically effective on salvage of CLI (Fontaine stage IV) limbs.

[Reoperation for diffuse supravalvular aortic stenosis with Williams syndrome--extended patch aortoplasty and extra-anatomic bypass from the ascending aorta to the descending aorta in a median sternotomy].

A case of diffuse supravalvular aortic stenosis (SVAS) with Williams syndrome is reported. In this case of severe diffuse SVAS, we performed the diamond-patch aortoplasty in a child. However he has been suffering from residual SVAS. At 9-years old, the myocardial injury was noted by myocardial scintigraphy. Preoperative cardiac catheterization and angiography revealed the hypoplastic ascending aorta and arch with a pressure gradient of 89 mmHg at the distal site from the left subclavian artery. Through only a median stenotomy, an extended patch aortoplasty between the valsalva sinus and distal arch was performed and an extraanatomic bypass from the ascending aorta to the descending aorta was employed using a 10 mm tube graft. We realize this technique is available because this method can relieve the left ventriculus of the pressure load and operate via only median sternotomy.

[Decreasing sarcoplasmic reticular calcium gives rise to myocardial protection--the effect of thapsigargin for myocardial protection under conditions of normothermia].

Deceasing sarcoplasmic reticular (SR) calcium may contribute to the myocardiac protection against ischemia and reperfusion-induced injury. Therefore, using the isolated working rat heart model, we investigated the effect of Thapsigargin (TH)-induced SR calcium diminution on the myocardial protection when added either before onset of ischemia or at time of reperfusion under conditions of normothermic ischemia. Hearts (n = 6/group) from male Wistar rats were aerobically (37 degrees C) perfused (20 min) with bicarbonate buffer. In the experimental protocol A, this was followed by a 3 min infusion of St. Thomas' Hospital cardioplegic solution No. 2 (STS) containing various concentrations of TH. Hearts were then subjected to 34 min of normothermic (37 degrees C) global ischemia and 35 min of reperfusion (15 min Langendorff, 20 min working). Reperfusion cardiac functions at 20 min of working perfusion was measured and compared with the preischemia values. STS added to 0.1 and 0.25 mumol/L TH improved recovery of aortic flow after 20 min reperfusion from 47 +/- 3% in the TH free controls to 62 +/- 3, 63 +/- 2% (n = 6) (p < 0.05). There was no difference in creatine kinase (CK) leakage during Langendorff reperfusion between the TH treated groups and the control group. In the experimental protocol B, 3 min of cardioplegia without TH and 34 min of ischemia (37 degrees C) were followed by a 10 min Langendorff reperfusion with various concentrations of TH, then 10 min Langendroff reperfusion for washing out, and 20 min working reperfusion. When TH was added to reperfusate the recovery of aortic flow did not change, 0.5 mumol/L TH group had the detelious effect. Thus, TH, when added to the cardioplegia, enhanced myocardial protection. We conclude that lessened uptake of Ca2+ into sarcoplasmic reticulum by inhibitors of the Ca(2+)-ATPase pump can decrease ischemia and reperfusion-induced injury.

[Use of ketamine combined with local anesthetics in epidural anesthesia].

Postoperative pain relief and sedation with epidural ketamine were studied. Twenty-four patients for elective upper abdominal surgery were divided into 4 groups. Epidural catheter was inserted into thoracic epidural space before induction of general anesthesia. In each group, either 0.25% bupivacaine 5 ml only, ketamine 0.1 mg.kg-1 + bupivacaine 5 ml, or ketamine 0.3 mg.kg-1 + bupivacaine 5 ml, or ketamine 0.5 mg.kg-1 + bupivacaine 5 ml was injected into epidural catheter for complaint of pain in recovery room. In ketamine injected groups, blood pressure and heart were unchanged, but respiration rate increased significantly. Patients in ketamine 0.3 or 0.5 mg.kg-1 injected groups, pain relief and sedation score were significantly intensified, but patients in ketamine 0.5 mg.kg-1 injected group, incidence of pain in the back during injection and headache was high. We conclude that epidural ketamine is useful for postoperative pain relief, and the superior dose of epidural ketamine is 0.3 mg.kg-1.

[Changes in analgesic levels, plasma concentrations and epidurogram during long-term continuous epidural block].

The change of analgesic levels, plasma concentrations and the radiographical changes of epidural space due to continuous epidural block were studied in 54 patients receiving pain relief. All the patients received 2 ml per hour of 2% lidocaine or 0.5% bupivacaine via the catheter over two weeks. The effect of epidural block was determined on 1, 2, 4, 7, 10 and 14 days, the epidurogram was performed on 1, 7, 14 days, and plasma concentration of lidocaine or bupivacaine was measured on 1, 3, 7, 14 days. We removed catheters in 8 patients because of infection around the catheter, pain in the back during injection, the leakage of anesthetics, and spontaneous removal. The effects and plasma concentrations of epidural block decreased significantly after a week, and the mean segmental number of radiographic spread decreased significantly after two weeks. The decreasing effect or duration of epidural block was due to decrease in spread of anesthetics because of adhesion around the catheter in the epidural space. We conclude that the safety period of continuous epidural block is within 2 weeks.

[An elderly case of ruptured aortic arch aneurysm with hemorrhagic cardiac tamponade].

We reported a case of successful aortic arch replacement using selective cerebral perfusion for ruptured distal aortic arch aneurysm (DAAA) with cardiac tamponade. A 80-year-old man who had preoperative episode of severe chest pain. Computed tomography showed saccular DAAA and pericardial effusion. He was diagnosed as ruptured DAAA with hemorrhagic cardiac tamponade. We performed urgent graft replacement of the aortic arch using selective cerebral perfusion. Postoperatively he had no complication. Thirty days after the operation he was discharged from the hospital and he is now leading a normal life.