Structural change and catalytic activity of horseradish peroxidase in oxidative polymerization of phenol.

The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.

Measurement of protein C inhibitor in seminal plasma is useful for detecting agenesis of seminal vesicles or the vas deferens.

Protein C inhibitor (PCI), a plasma serine protease inhibitor of activated protein C, is present at high concentrations in the seminal plasma of normal subjects and is decreased in some infertile patients. We measured the concentrations of PCI, prostate-specific antigen, and fructose in the seminal plasma of infertile patients (n = 125) and of normal subjects (n = 13). We also measured time-dependent changes in the concentrations of PCI and fructose in seminal plasma after ejaculation. A weak correlation was found between the levels of PCI and fructose (r = 0.268, P = 0.016). The PCI level in seminal plasma of patients with seminal vesicle and/or vasal agenesis was significantly lower (P < .01) than in normal subjects. The level of fructose in seminal plasma decreased in vitro in a time-dependent manner after ejaculation, whereas the concentration of PCI was stable at 48 hours after ejaculation. These data suggest that PCI in seminal plasma, as well as fructose, may become one of the markers for agenesis of seminal vesicles and/or the vas deferens.

The primary electron acceptor of green sulfur bacteria, bacteriochlorophyll 663, is chlorophyll a esterified with Delta2,6-phytadienol.

The primary electron acceptor of green sulfur bacteria, bacteriochlorophyll (BChl) 663, was isolated at high purity by an improved purification procedure from a crude reaction center complex, and the molecular structure was determined by fast atom bombardment mass spectroscopy (FAB-mass), (1)H- and (13)C-NMR spectrometry, double quantum filtered correlation spectroscopy (DQF-COSY), heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectral measurements. BChl 663 was 2.0 mass units smaller than plant Chl a. The NMR spectra showed that the macrocycle was identical to that of Chl a. In the esterifying alcohol, a singlet P7(1) signal was observed at the high-field side of the singlet P3(1) signal in BChl 663, while a doublet peak of P7(1) overlapped that of P11(1) in Chl a. A signal of P7-proton, seen in Chl a, was lacking, and the P6-proton appeared as a triplet signal near the triplet P2-proton signal in BChl 663. These results indicate the presence in BChl 663 of a C=C double bond between P6 and P7 in addition to that between P2 and P3. The structure of BChl 663 was hence concluded to be Chl a esterified with 2,6-phytadienol instead of phytol. In addition to BChl 663, two molecules of the 13(2)-epimer of BChl a, BChl a', were found to be present per reaction center, which may constitute the primary electron donor.

Time-dependent structure and activity changes of alpha-chymotrypsin in water/alcohol mixed solvents.

Secondary structure of alpha-chymotrypsin in water/ethanol was investigated by circular dichroic (CD) spectroscopy. The changes in catalytic activity were discussed in terms of structural changes of the enzyme. Alpha-chymotrypsin formed beta-sheet structure in water/ethanol (50/50 by volume), but it was substantially less active as compared to that in water. At water/ethanol 10/90, alpha-chymotrypsin took on a native-like structure, which gradually changed to beta conformation with concomitant loss of activity. Change of solvent composition from water/ethanol 50/50 to 90/10 or 10/90 by dilution with water or ethanol, respectively, led to partial recovery of native or native-like structure and activity. In water/methanol, alpha-chymotrypsin tended to form stable beta-sheet structure at water/methanol ratios lower than 50/50, but the catalytic activity decreased with time. Change to alpha-helix structure with substantial loss in catalytic activity was observed when alpha-chymotrypsin was dissolved in water/2,2,2-trifluoroethanol with water contents lower than 50%. In water/2,2,2-trifluoroethanol 90/10, alpha-chymotrypsin initially had the CD spectrum of native structure, but it changed with time to that characteristic of beta-sheet structure.

Paravesical granuloma after inguinal herniorrhaphy. Case report and review of the literature.

Paravesical granuloma after herniorrhaphy is an unusual complication due to infected suture material and often mimics bladder or urachal malignancy. We present 3 cases of this disease and reviewed 21 previously reported cases. Our patients underwent hernial repair 2-7 years before examination and presented urinary symptoms. They were treated with antibiotics and underwent exploration of the inguinal wound infection but the symptoms did not resolve. En bloc excision and partial cystectomy were performed and resulted in complete resolution of the symptoms. It is important to consider paravesical granuloma in patients who had undergone herniorrhaphy in the differential diagnosis of bladder or urachal tumors.

[Extracorporeal shock wave lithotripsy in patients with coagulopathies: report of three cases].

We describe our experience with extracorporeal shock wave lithotripsy (ESWL) in three patients with coagulation disorders (one case of hemophilia A, and two cases of thromboasthenia). We successfully performed ESWL using factor VIII or transfusion of platelets without any severe hemorrhagic complications, such as perirenal and subcapsural hematomas. We consider that adequate supplement of coagulation factor or platelets may lower the risk of hemorrhagic complications in coagulopathic patients who undergo ESWL.

[Laparoscopic fenestration of lymphocele after renal transplantation: report of two cases].

Lymphocele after renal transplantation is a complication that often requires surgical treatment. We performed laparoscopic drainage for the postoperative lymphocele in two renal transplantation patients. The patients were suffering from perineal discomfort and urinary frequency due to the lymphocele after the renal transplantation. Percutaneous catheter drainages and injections of tissue sclerosing agents such as povidone-iodine solution and tetracycline were not effective. Then fenestration of the lymphocele by laparoscopic technique was done. This procedure made the lymphatic fluid free from the lymphocele in the retroperitoneal space to the peritoneal cavity following creation of a peritoneal window. There were no apparent complications. The patients were free from symptoms and had no relapse 10 months after the operation. We report the procedure and the results of the laparoscopic fenestration.

Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli.

If ribozymes are to be exploited in vivo, it is necessary to select ribozymes that are functional in the intracellular environment. Ribozymes selected in the intracellular environment should retain their function in vivo as well as in vitro. We have devised a novel system for selection of active ribozymes from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our first attempt, a sequence encoding either an active or an inactive ribozyme was connected upstream of the gene for DHFR. Each plasmid was designed such that, when the ribozyme was active, the ribozyme would cleave the target site and, as a result, the rate of production of DHFR would be high enough to endow resistance to trimethoprim (TMP). However, a critical defect may be associated with introduction of a ribozyme upstream of the DHFR gene because, during actual screening for active ribozymes on the 5' side from a pool of random sequences, there is the danger of selecting sequences that are not related to the activity of ribozymes. Indeed, some upstream linker sequences affected the level of expression of the DHFR protein and, as a result, the resistance of Escherichia coli to TMP. Therefore, we newly constructed a 3'-connected ribozyme system, and activities in vivo of 5'-connected and 3'-connected ribozymes were compared. We found that the cleavage efficiencies in vivo were nearly identical for the two types of ribozyme, 24% for the 5'-side ribozyme and 23% for the 3'-side ribozyme, indicating that polysomes did not seem to inhibit the action of the 3'-connected ribozyme. In both cases, when cells were transformed with a 1 : 1 mixture of active and inactive ribozyme-coding plasmids, it was mainly the cells that harbored the active ribozyme that survived in the presence of TMP.

[Selective transcatheter embolization for renal artery aneurysms: report of three cases].

Selective transcatheter embolization using an interlocking detachable coil (IDC) or detachable balloon was performed in three patients with renal artery aneurysms. All aneurysms were of the saccular type, located on segmental branches of the renal artery. After the embolization procedures, the levels of LDH, GOT, WBC and the body temperature were transitionally elevated in all patients. Complete occlusion of the aneurysms were achieved in all cases. Good clinical results were achieved in two of these patients without any renal dysfunction. However, local infarction of ipsilateral renal parenchyma occurred in one patient immediately after the embolization and a Tc-99m-DMSA renal scintigraphy study suggested renal dysfunction after infarction, and the level of serum renin activity was slightly elevated. Selective transcatheter embolization may constitute an acceptable therapeutic approach for renal artery aneurysms because beside avoiding surgery, it has a low risk of complications.

[A case of xanthogranuloma of the urinary bladder following herniorrhaphy].

We report a case of xanthogranuloma of the bladder following herniorrhaphy. The patient complained of urinary frequency, residual feeling, and presented with a solid mass located on the dome of the bladder. He had undergone previous inguinal herniorrhaphy. Transurethral biopsy revealed an inflammatory change. Partial cystectomy was carried out for en bloc removal of the tumor. The histological diagnosis was xanthogranuloma of the bladder. Postoperatively, the symptoms disappeared. This is the sixth case report of xanthogranuloma of the urinary bladder and the first case following herniorrhaphy in the Japanese literature.

Increase of catalytic activity of alpha-chymotrypsin by metal salts for transesterification of an amino acid ester in ethanol.

alpha-Chymotrypsin-catalyzed transesterification of N-acetyl-L-tyrosine methyl ester in ethanol was markedly accelerated by addition of small amounts of divalent metal salts. The reaction rate dependent not only on the nature of metal ions but also on the nature of anionic counter ions. Calcium acetate was the most effective among the metal salts used. The reaction followed Michaelis-Menten kinetics, and it was found that the reaction increase is due to the increase in kcat.

Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli.

For use of ribozymes in vivo, it is desirable to select functional ribozymes in the cellular environment (in the presence of inhibitory factors and limited concentrations of mandatory Mg2+ ions, etc.). As a first step toward this goal, we developed a new screening system for detection in vivo of an active ribozyme from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our DHFR expression vector, the sequence encoding either the active or the inactive ribozyme was connected to the DHFR gene. The plasmid was designed such that, when the ribozyme was active, the rate of production of DHFR was high enough to endow resistance to trimethoprim (TMP). We demonstrated that the active ribozyme did indeed cleave the primary transcript in vivo, whereas the inactive ribozyme had no cleavage activity. Cells that harbored the active-ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored the inactive-ribozyme-coding plasmid. Consequently, when cells were transformed by a mixture that consisted of active- and inactive-ribozyme-coding plasmids at a ratio of 1:1, (i) mainly those cells that harbored active ribozymes survived in the presence of TMP and (ii) both active- and inactive-ribozyme-harboring cells grew at an identical rate in the absence of TMP, a demonstration of a positive selection system in vivo. If the background "noise" can be removed completely in the future, the selection system might usefully complement existing selection systems in vitro.

[Effect of pamidronate and interferon-alpha on bone and lung metastases and hypercalcemia in a patient with renal cell carcinoma].

Pamidronate is a second generation bisphosphonate used for treating tumor-induced hypercalcemia and for preventing the development of new bone metastasis. A 47-year-old man with renal cell carcinoma was admitted in our institution because of hypercalcemia with multiple metastasis in bone, lung and lymph nodes. After embolization of the right renal artery, the patient was treated with pamidronate and interferon-alpha. Intravenous pamidronate significantly reduced bone pain and normalized the serum calcium level. The pulmonary metastasis responded to interferon therapy after 2 months of therapy. Radical nephrectomy was then carried out. Paraaortic lymph nodes were found to be necrosed completely. Ossification of osteolytic lesions was observed after two months of therapy and metastatic lesions in the lung showed complete remission (CR) after six months of therapy.

Characterization of semenogelin II and its molecular interaction with prostate-specific antigen and protein C inhibitor.

The serine protease, prostate-specific antigen (PSA), its protein substrates, semenogelin (Sg) I and II, and protein C inhibitor (PCI) have been described as components of human seminal plasma. PCI was found to inhibit the PSA-catalyzed degradation of insoluble coagula Sg I + II by forming a PSA-PCI complex. Digestion of seminal coagula with PSA released PCI and PSA-PCI complex from the coagula into a soluble phase, suggesting the presence of active PCI binding to the coagula. To investigate the molecular interaction of Sg with PSA and PCI, we purified Sg II from seminal coagula as a soluble form and found that Sg II is glycosylated with heterogeneous carbohydrate moieties. Sg II bound to the solid-phase complex of diisopropylfluorophosphate (iPr2FP) and PSA with an apparent dissociation constant (kd) of 41 nM and to PCI with a Kd of 28 nM. The binding of Sg II to iPr2P-PSA was not affected by PCI and that of Sg II to PCI was not affected by iPr2P-PSA, suggesting that Sg II forms a ternary complex with PSA and PCI. The bindings of Sg II to both iPr2P-PSA and PCI were influenced by pH, ionic strength, heparin, dextran sulfate, and divalent cations, particularly by Zn2+. Treatment of Sg II with heparinase, heparitinase, N-glycanase, or with O-glycanase following sialidase did not affect the binding of Sg II to iPr2P-PSA and PCI. These findings suggested that PCI bound to Sg in seminal vesicles regulates the PSA-catalyzed degradation of Sg in seminal plasma, and that the binding of PCI and PSA to Sg is modulated by several factors such as pH, ionic strength, divalent cations, and heparin-like substances in seminal plasma.

[Asymptomatic unilateral adrenal medullary hyperplasia with a cyst: case report].

A 45-year-old man was admitted with asymptomatic adrenal tumor. He had normal plasma and urinary catecholamine levels. The swelling of right gland was detected by CT scan and MRI. Selective venous samplings were performed and the level of catecholamine into the right adrenal vein were much higher than that into the left one. On July 15th, 1993, a right adrenalectomy was carried out and the right adrenal gland with a large cyst could be found. The ratio of cortical area to medullary area was about 4:1. Therefore, it was pathologically diagnosed as adrenal medullary hyperplasia with a cyst. There are relatively few reports of adrenal medullary hyperplasia. Before the operation, the patients were mostly diagnosed as pheochromocytoma by the results of laboratory studies and their symptoms. The condition of this disease is usually bilateral and often associated with type II multiple endocrine neoplasia (MEN II). We describe a case of asymptomatic unilateral adrenal medullary hyperplasia.

[Renal angiomyolipoma: diagnosis and treatment].

In 10 years the diagnosis of renal angiomyolipoma (RAML) was made in 14 patients (male-to female ratio 1:3.7) at our institution; 1 case was associated with tuberous sclerosis (TS) and 1 case had regional lymph node involvement. A statistical study was done on data taken from 739 cases of RAML in the Japanese literature, including our cases. The male to female ratio was 1 to 3. Twenty eight percent of the cases were associated with TS. The ratio of bilateral cases to the unilateral one was 1 to 3. The main clinical signs were flank pain, abdominal mass, hematuria and fever elevation. Recently the ratio of nephrectomy has decreased to 30%. The percentage of detecting the fat component by ultrasonography (US), computed tomography (CT) and magnetic resonance imaging were 88.1%, 86.5% and 80.8% respectively. The percentages of visualizing hypervascularity, aneurysms, absence of arterio-venous shunt and onion peel appearance by selective renal angiography were 77.3%, 71.4%, 48.1% and 4.9% respectively. Small (less than 3 cm), asymptomatic, simple lesions with adipose component may be observed annually by CT and US until more experiences is gained with surveillance of these patients. Embolization was useful for emergency cases or pre-treatment of nephron sparing surgery, but insufficient by itself. As there still remain problems in the diagnosis of RAML, especially in the case of very small tumors, in the case with almost no adipose component and in the case associated with renal cell carcinoma, the diagnosis of RAML should be made synthetically including angiography.

Purification and characterization of a novel calcium-binding protein, S100C, from porcine heart.

A novel Ca(2+)-binding protein, which we have named S100C (Ohta et al. (1991) FEBS Lett. 295, 93-96), was purified to homogeneity from porcine heart by Ca(2+)-dependent dye-affinity chromatography. S100C possesses some properties of S100 proteins, such as self-association and exposure of a hydrophobic site upon binding of Ca2+ but it differs from S100 proteins in forms of its isoelectric point (pI = 6.2), cross-reactivity with antibodies, staining by Stains-all, and its Ca(2+)-dependent interaction with the immobilized dye. S100C bound to cytoskeletal components at physiological concentrations of Ca2+. Moreover, it was found that 125I-labeled S100C interacted with annexin I in a Ca(2+)-dependent manner. S100C also inhibited the phosphorylation of annexin I by protein kinase C. These data suggest that S100C might act to regulate the cytoskeleton in a Ca(2+)-dependent manner via interactions with annexin I.