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The effect of the oxidation of amino acid residues on albumin on its in vivo elimination was investigated using mutants and oxidized HSAs. The single-residue mutants (H146A, K199A, W214A, R218H, R410A, Y411A) and oxidized HSAs were produced by the recombinant DNA techniques and incubation with a metal ion-catalyzed oxidation (MCO) system for 12, 24, 48 or 72 h. Pharmacokinetics were evaluated in mice after labeling with 111In. Structural and functional properties were examined by several spectroscopic techniques. Time-dependent increase in carbonyl group content resulted in increase in the liver clearance of oxidized HSAs. Slight decreases in alpha-helical content as the result of oxidation was induced by the increases in accessible hydrophobic areas and the net negative charge on the HSA molecule. No significant change in the pharmacokinetics and structural properties was observed for the W214A, R218H and Y411A mutants, but the properties for the H146A, K199A and R410A mutants were affected (extent of effect: R410A > K199A > H146A). The liver clearance of these proteins is closely correlated to hydrophobicity (r = 0.929, P < 0.01) and the net charge of the proteins (r=0.930, P < 0.01). The rate of elimination of HSA is closely related to the hydrophobicity and net charge of the molecule. Further, the R410A mutants had a short half-life and structure similar to oxidized HSA after oxidation. Therefore, the modification of Arg-410 via oxidative stress may promote the elimination of HSA.

To determine the pharmacokinetic properties of advanced oxidation protein products (AOPP), we prepared oxidized human serum albumin (oxi-HSA) using chloramine-T (a hypochlorite analogue) in vitro. The AOPP and dityrosine content of oxi-HSA (AOPP content, 244.3+/-12.3 microM; dityrosine content, 0.7+/-0.11 nmol of dityrosine/mg protein) were similar to those of uremic patients. In structural analysis, the increases in AOPP and dityrosine content of HSA induced slight decreases in its alpha-helical content. In pharmacokinetic analysis, oxi-HSA left the circulation rapidly, and organ distribution of oxi-HSA 30 min after intravenous injection was 51% for the liver, 23% for the spleen, and 9% for the kidney, suggesting that the liver and spleen were the main routes of plasma clearance of oxi-HSA. The liver and spleen uptake clearance of oxi-HSA were significantly greater than those of normal HSA (CLliver, 5058+/-341.6 vs 24+/-4.2 microL/hr [p<0.01]; CLspleen, 2118+/-322.1 vs 32+/-2.7 microL/hr [p<0.01]). However, uptake by other organs was not significantly affected by oxidation. These results suggest that the liver and spleen play important roles in elimination of AOPP.

Transthyretin (TTR) amyloid fibril formation, which is triggered by the dissociation of tetrameric TTR, appears to be the causative factor in familial amyloidotic polyneuropathy and senile systemic amyloidosis. Binding of thyroxine (T(4)), a native ligand of TTR, stabilizes the tetramer, but the bioavailability of T(4) for TTR binding is limited due to the preferential binding of T(4) to globulin, the major T(4) carrier in plasma. Here, we show that Cr(3+) increased the T(4)-binding capacity of wild-type (WT) and amyloidogenic V30M-TTR. Moreover, we demonstrate that Cr(3+) and T(4) cooperatively suppressed in vitro fibril formation due to the stabilization of WT-TTR and V30M-TTR.

Hemodialysis (HD) patients are frequently in a state of increased oxidative stress, and hyperglycemia appears to be a major factor. We recently found that oxidized human serum albumin (HSA) is a reliable marker of oxidative stress in HD patients. However, the issue of whether oxidized HSA is associated with the progression of oxidative stress in HD patients with or without diabetes is not clear. In the present study, we examined the effect of a qualitative modification of HSA in HD patients with or without diabetes. Blood samples from 10 HD patients with diabetes, 7 HD patients without diabetes, and 10 healthy age-matched controls were examined. The increase in plasma protein carbonyl content and advanced glycation endproducts (AGEs) in HD patients was largely due to an increase in the levels of oxidized HSA. Furthermore, these increases were greatest in HD patients with diabetes. Purified HSA from HD patients (non-DM-HSA) was carbonylated and AGE-modified. The amount of modified HSA was the highest in HD patients with diabetes (DM-HSA). Carboxy methyl lysine (CML)-modified HSA triggered a neutrophil respiratory burst, and this activity was closely correlated with the increase in the CML/HSA ratio. These findings indicate that uremia plays an important role in the progression of oxidative stress in HD patients via an increase in CML-modified HSA. They also indicate that diabetic complications further exacerbate the progression of oxidative stress by further increasing the amount of these modified HSA molecules.

In the present study, we investigated the esterase-like activity of human serum albumin (HSA) and the mechanism by which it hydrolyzes, and thereby activates, olmesartan medoxomil (CS-866), a novel angiotensin II receptor antagonist. CS-866 has previously been shown to be rapidly hydrolyzed in serum in which HSA appeared to play the most important role in catalyzing the hydrolysis. We found that the hydrolysis of CS-866 by HSA followed Michaelis-Menten kinetics. Compared with the release of p-nitrophenol from p-nitrophenyl acetate (PNPA), CS-866 showed lower affinity to HSA and a lower catalytic rate of hydrolysis. Thermodynamic data indicated that PNPA has a smaller value of activation entropy (deltaS) than CS-866; consequently, PNPA is more reactive than CS-866. Ibuprofen and warfarin acted as competitive inhibitors of hydrolysis of CS-866, whereas dansyl-L-asparagine, n-butyl p-aminobenzoate, and diazepam did not. These findings suggest that the hydrolytic activity is associated to parts of site I and site II for ligand binding. All chemically modified HSA derivatives (Tyr-, Lys-, His-, and Trp-modifications) had significantly lower reactivity than native HSA; Lys-HSA and Trp-HSA had especially low reactivity. All the mutant HSAs tested (K199A, W214A, and Y411A) exhibited a significant decrease in reactivity, suggesting that Lys-199, Trp-214, and Tyr-411 play important roles in the hydrolysis. Results obtained using a computer docking model are in agreement with the experimental results, and strongly support the hypotheses that we derived from the experiments.

The effect of telmisartan on ambulatory blood pressure, plasma neurohormonal parameters, and oxidation of serum albumin has not been investigated in hemodialysis (HD) patients. Thirteen hypertensive HD patients were treated with 40 mg telmisartan once daily, and 24-h ambulatory blood pressure monitoring was performed after 0, 4, and 8 weeks of treatment. Plasma renin activity, plasma aldosterone concentration (PAC), brain natriuretic peptide (BNP) level, and serum oxidized albumin level were determined at the same time points. Serum telmisartan concentration was also measured at 4 and 8 weeks. Telmisartan significantly reduced systolic blood pressure and diastolic blood pressure (both awake and sleeping) after 4 weeks, and these pressures showed a further significant decrease after 8 weeks. Plasma levels of aldosterone, BNP, and serum oxidized albumin were markedly decreased after 4 weeks and these lower levels were maintained at 8 weeks. The trough serum telmisartan concentration was not significantly different at 8 weeks compared with 4 weeks. Throughout the treatment period, there were no significant adverse effects. Telmisartan effectively lowers blood pressure and reduces PAC, BNP, and oxidative stress and is safe and well-tolerated by HD patients. A long-term study in a large population is required to determine the influence of telmisartan therapy on cardiovascular mortality and morbidity in HD patients.

Oxidized albumin is a reliable marker of oxidative stress in hemodialysis (HD) patients. However, oxidized albumin in vivo and its possible clinical significance has been rarely investigated. In the present study, the qualitative modification of albumin in HD patients (n = 20) was examined and their results were compared with healthy age-matched controls (n = 10). The increase in plasma protein carbonyl levels in HD patients was largely due to an increase in oxidized albumin. Human serum albumin (HSA) of HD patients, HSA of HD patients (HD-HSA) and normal subjects (Normal-HSA) were purified on a blue Sepharose CL-6B column. Spectroscopic analysis confirmed that the HD-HSA samples contained higher levels of carbonyls than Normal-HSA. An HPLC analysis also suggested that the state of the purified HSA used throughout the experiments accurately reflects the redox state of albumin in blood. HD-HSA was found to have a decreased the antioxidant activity, and was able to trigger the oxidative burst of human neutrophils, compared to Normal-HSA. HD-HSA was conformationally altered, with its hydrophobic regions more exposed and to have a negative charge. In binding experiments, HD-HSA showed impaired Site II-ligand binding capabilities. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in HD patients.

We report the computed tomography (CT) and magnetic resonance imaging (MRI) findings of a rare case of synovial sarcoma of the sternum in an 86-year-old man. CT demonstrated an inhomogenously enhanced soft-tissue-density mass of the sternum that destroyed bone cortex and protruded anteriorly. On MRI, the tumor showed a multinodular mass with internal septation and heterogeneous enhancement. These CT and MRI findings were nonspecific, but were similar to those of soft tissue synovial sarcomas. The tumor was more clearly demarcated by MRI than CT. This is the first report concerning the CT and MRI findings of synovial sarcoma of the sternum. Synovial sarcoma should be added to the gamut of primary malignant neoplasms of the sternum.

Reversible thermal denaturation of 33 genetic variants of human serum albumin (HSA) appeared to be a two-state process when studied by circular dichroism (CD). Fourteen single-residue variants have Tm values (midpoint of denaturation) higher than, and nine have Tm values lower than, their endogenous, wild-type counterpart. Nine single-residue variants have DeltaHv values (van't Hoff enthalpy) higher than, and 14 have DeltaHv values lower than, normal albumin. All types of combinations of positive and negative DeltaTm values and Delta(DeltaHv) values were found. Good linear correlations between mutation-induced changes of alpha-helical content and Delta(DeltaHv) values, but not DeltaTm values, were found especially for the variants mutated in domains I and III. The effect of altered chain length and glycosylation on Tm and DeltaHv was also studied. For all variants, no clear relationship was found between the changes in the thermodynamic parameters and the type of substitution, changes in protein charge or hydrophobicity. However, the protein changes taking place in domain I have a rather uniform effect (almost all of the nine variants have positive DeltaTm values and negative Delta(DeltaHv) values, i.e., they denature more easily than normal albumin but they do so at a higher temperature). The present results can be of both protein chemical relevance and of clinical interest, because they could be useful when designing stable, recombinant HSAs for clinical applications.

The recombinant human serum albumin (rHSA) dimer, which was cross-linked by a thiol group of Cys-34 with 1,6-bis(maleimido)hexane, has been physicochemically characterized. Reduction of the inert mixed-disulfide of Cys-34 beforehand improved the efficiency of the cross-linking reaction. The purified dimer showed a double mass and absorption coefficient, but unaltered molar ellipticity, isoelectric point (pI: 4.8) and denaturing temperature (65 degrees C). The concentration dependence of the colloid osmotic pressure (COP) demonstrated that the 8.5 g dL(-1) dimer solution has the same COP with the physiological 5 g dL(-1) rHSA. The antigenic epitopes of the albumin units are preserved after bridging the Cys-34, and the circulation lifetime of the 125I-labeled variant in rat was 18 h. A total of 16 molecules of the tetrakis[(1-methylcyclohexanamido)phenyl]porphinatoiron(II) derivative (FecycP) is incorporated into the hydrophobic cavities of the HSA dimer, giving an albumin-heme hybrid in dimeric form. It can reversibly bind and release O2 under physiological conditions (37 degrees C, pH 7.3) like hemoglobin or myoglobin. Magnetic circular dichroism (CD) revealed the formation of an O2-adduct complex and laser flash photolysis experiments showed the three-component kinetics of the O2-recombination reaction. The O2-binding affinity and the O2-association and -dissociation rate constants of this synthetic hemoprotein have also been evaluated.

The capability of octanoate, N-acetyl-L-tryptophanate (N-AcTrp) and other ions of fatty acids and amino acids to stabilize human serum albumin (HSA) against thermal and oxidative stress was studied. Native-PAGE showed that octanoate, and more hydrophobic fatty acids anions, stabilizes the monomeric form of HSA during heating at 60 degrees C for 30 min. Heating in the presence of octanoate did not change the far-UV CD-spectrum. The stabilizing role of octanoate also showed as an increase in denaturation temperature and calorimetric enthalpy, determined by differential scanning calorimetry (DSC). N-AcTrp, which was found to compete with octanoate for a common high-affinity site, has only a minor stabilizing effect. By contrast, no effect was found for l-tryptophanate or N-acetyl-L-cysteinate. Any ligand effect on oxidation was examined by using 2,2' -azobis(2-amidino-propane)dihydrochloride (AAPH) as oxidizing agent. One hour of incubation resulted in the formation of the same number of carbonyl groups, whether octanoate or one of the above mentioned amino acids was present or not. However, the number of groups formed after 24 h of incubation was significantly decreased in the presence of L-tryptophanate and, especially, N-AcTrp. The effect of 1-h incubation with AAPH on the oxidative status of 34-Cys was studied by the HPLC technique. It was found that N-AcTrp, but not octanoate, has a large protecting effect on the sulfhydryl group. Thus, octanoate has the greatest stabilizing effect against heat, whereas the presence of N-AcTrp diminishes oxidation of HSA.

The binding properties of the disulfide covalent bond between N-acetyl-L-cysteine (NAC) and human serum albumin (HSA) were investigated. HSA, purified from either healthy subjects or renal failure patients, was incubated with NAC in buffer and analyzed by 4VP-EG-Me column chromatography, which can distinguish between the redox states of the only free thiol of HSA. Although intact HSA was found to consist of mainly three sub-types, marcaptoalbumin (HMA), cysteine-bound nonmercaptoalbumin (HNA(Cys)) and a further oxidized form (HNA(oxy)), the formation of a new type of nonmercaptoalbumin (HNA(NAC)) was confirmed after incubation with NAC. Interestingly, NAC rapidly dissociated Cys from HNA(Cys) and NAC itself bound very slowly to HSA. These findings suggest that the interaction between NAC and HSA proceeds in a 2-step processes. The first-order binding and dissociation rate constants of NAC to healthy HSA (k(on,NAC)) and Cys from healthy HNA(Cys) (k(off,Cys)) were approximately 0.0032 and 1.3 (h(-1)), respectively. On the other hand, HSA from renal failure patients showed decreased HMA and increased HNA(Cys). The k(on,NAC) and k(off,Cys) were 0.0094 and 0.45 (h(-1)), respectively, suggesting that the pathological state may affect the binding properties of HSA and NAC.

Background: Intravenous iron administration (IVIR) is effective for correcting anemia in hemodialysis (HD) patients. However, it may also enhance the generation of hydroxyl radicals. Recently, plasma proteins have been demonstrated to be extremely susceptible to oxidative stress. Therefore, we investigated the effect of IVIR on the oxidative status of albumin, a major plasma protein, in HD patients., Methods: Eleven hemodialysis (HD) patients were treated with 40 mg of saccharated ferric oxide intravenously after every dialysis session for four weeks, and 11 age-/gender-matched HD patients were treated with vehicle. We performed high performance liquid chromatography (HPLC) analysis of serum albumin and determined the levels of reduced and oxidized albumin. Carbonyl formation of plasma proteins were also measured using an anti-2,4 dinitrophenylhydrazine antibody in patients with or without IVIR., Results: IVIR resulted in an increase in both disulfide form (f(HNA-1)) and oxidized form (f(HNA-2)) of albumin in HD patients (36.0 +/- 6.03 vs. 41.7 +/- 6.27; 5.46 +/- 1.50 vs. 8.7 +/- 2.22, respectively, P < 0.05). The findings here also show that IVIR substantially increased plasma protein carbonyl content by oxidizing albumin. In addition, we found a strong correlation between plasma carbonyl content and the levels of oxidized albumin (f(HNA-1) and f(HNA-2)) in HD patients (R= 0.674 and R= 0.724, respectively, P < 0.01)., Conclusion: The results of this study indicate that the HPLC analysis of serum albumin represents a potentially useful method for the quantitative and qualitative evaluation of oxidative stress in HD patients, and strongly suggest the possibility that oxidative stress, generated by IVIR, enhances the oxidation of albumin in those patients.

Objective: The standard operation for patients with stage IA lung adenocarcinoma is considered to be a lobectomy. Recently, some researchers have reported that patients with tumors showing greater proportions of ground-glass opacity (GGO) at computed tomography (CT) could be candidates for limited resection, because of its less aggressive nature. However, the lack of a precise definition or standard measuring method of GGO prevents its general use as an index for planning limited resection. Therefore, we attempted to define GGO based on CT number and measured it more objectively., Methods: Between 1998 and 2001, 90 patients with clinical stage IA adenocarcinoma, who underwent standard or intentional limited resection and whose images of chest high-resolution CT were preserved in Digital Imaging and Communications in Medicine (DICOM) format, constituted the study population. The tumor shadow seen on the solid window (WL, -160HU; WW, 2HU) was regarded as the central solid area of the tumor seen on the lung window, and GGO was defined as the whole tumor area with the exception of the central solid area. Each area was measured using Scion Image (Scion Corp., Frederick, MD). We analyzed the relationship between the proportion of GGO and both of pathologic findings and recurrence., Results: Among the 90 tumors, 31 (34.4%) were calculated to have a GGO area greater than or equal to 50%. Of these, 27 (87%) tumors were bronchioloalveolar carcinoma. Lymphatic and vascular invasions, or nodal involvement were found only in patients with a smaller proportion of GGO (<50%) (P < 0.05). During the follow-up period (median 36 months), recurrences occurred in eight patients who were diagnosed as having tumors showing smaller proportion of GGO (<50%)., Conclusions: Tumors with a greater proportion of GGO measured by our method are thought to have a less invasive nature. Our objective measuring method of GGO could be useful for future multicenter trials to elucidate the value of limited resection for clinical stage IA adenocarcinoma based on the proportion of GGO.

Objective: The long-term results of the surgical treatment for patients with pulmonary metastases from uterine malignancies were clarified., Methods: A total of 133 patients who underwent pulmonary metastasectomy for uterine malignancies were enrolled in the Metastatic Lung Tumor Study Group of Japan between March 1984 and February 2002. These patients constituted the study population, and their clinical, pathologic, and prognostic data were retrospectively analyzed., Results: The morbidity and mortality rates related to the operation were minimal (1% and 1%, respectively). The 5- and 10-year survivals after the surgical resection in all cases were 54.6% and 44.9%, respectively. The 5-year survivals for each histologic type were estimated to be 46.8% for squamous cell carcinoma (n = 58), 40.3% for cervical adenocarcinoma (n = 13), 75.7% for endometrial adenocarcinoma (n = 23), 86.5% for choriocarcinoma (n = 16), and 37.9% for leiomyosarcoma (n = 11). In the univariate analysis, the following were shown to be associated with poor survival: primary tumor in the cervix, short disease-free interval (<12 months), large number of resected metastases (> or =4), and large tumor size (> or =3 cm). After mutual adjustment, short disease-free interval (<12 months) alone was related to risk of death (hazard ratio = 2.26, 95% confidence interval = 1.06-4.78) for 105 patients, excluding patients with choriocarcinoma and miscellaneous histologic types., Conclusion: Pulmonary metastasectomy for uterine malignancies is a safe and acceptable treatment to improve survival. Patients with a disease-free interval of 12 months or more are good candidates for this treatment if there is adequate control of the primary tumor without extrapulmonary metastasis.

Schwannoma is a benign neurogenic tumor, and no cases of metastasis or implantation from this tumor have been reported. We first describe a case of port site recurrence after video-assisted thoracoscopic resection of chest wall schwannoma. She has been well with no evidence of disease for 2 years after resection of the recurrent tumor. We stress the importance of using a specimen bag and careful manipulation during video-assisted thoracic surgery, even for benign tumors.

Purpose: The validity of using chloramine-T as a model compound for mimicing oxidative stress was examined using human serum albumin (HSA) as a model. Important sites of oxidation were studied by mild treatment with chloramine-T and by mutating 34Cys for a serine (C34S)., Methods: High-performance liquid chromatography (HPLC) combined with fluorescence detection to confirm the validity of chloramine-T as an oxidizing agent was used. Oxidized amino acid residues were detected by reaction with 5,5'-dithiobis(2-nitro benzoic acid), digestion with cyanogen bromide, followed by capillary electrophoresis. Protein conformation was examined by spectroscopic techniques., Results: From the HPLC analysis of human serum, the validity of using chloramine-T as an oxidizing agent was confirmed. At low chloramine-T concentrations (CT0.1-HSA, CT1-HSA), 34Cys and Met residues were oxidized, at medium concentrations (CT10-HSA), the tryptophan residue also appeared to be oxidized, and at the highest concentration (CT50-HSA), the net charge of Site II of HSA was found to be more negative. The two highest levels of oxidation of HSA (CT10-HSA, CT50-HSA) resulted in conformational changes with an increased exposure of hydrophobic regions, decreased high-affinity bindings of warfarin and ketoprofen and a reduced esterase-like activity. The latter protein also has a shorter plasma half-life and an increased liver clearance., Conclusions: We succeeded in imitating oxidative damage to HSA using chloramine-T and the findings show that Site II is more affected than Site I and 34Cys, when HSA is exposed to oxidative stress.

Objective: In patients with clinical T1 N0 M0 lung adenocarcinoma, we investigated whether the proportion of ground-glass opacity area measured on high-resolution computed tomography was valuable for predicting the existence of lymph node metastasis, lymphatic invasion, or vascular invasion., Methods: Between 1994 and 1999, 111 patients with clinical stage IA adenocarcinoma underwent surgical resection of the lung at our hospital. Of these, 96 patients received high-resolution computed tomography of the chest, and they constituted the study population. The tumors were semiquantitatively classified into 5 groups on the basis of the proportion of ground-glass opacity area to whole tumor shadow on high-resolution computed tomography: group I, 0%; group II, 1% to 25%; group III, 26% to 50%; group IV, 51% to 75%; and group V, 76% to 100%. Correlations of computed tomographic findings, pathologic results of lymph node metastasis and lymphatic and vascular invasion, and the histologic subtype according to the new World Health Organization classification were examined. We also investigated the characteristics of the patients with ground-glass opacity areas on high-resolution computed tomography and their value for predicting lymph node metastasis., Results: Among the 96 patients, 15 (15.6%) had mediastinal lymph node metastases, and 3 (3.1%) had hilar node metastases. Regarding the proportion of the ground-glass opacity area of the tumors, 15 (15.6%) tumors were classified as group V, 11 (11.5%) as group IV, 9 (9.3%) as group III, 22 (22.9%) as group II, and 39 (40.6%) as group I, respectively. Of the 18 patients with lymph node metastases, no patients were found in groups IV and V, 2 (22.2%) were found in group III, 4 (18.2%) were found in group II, and 12 (30.8%) were found in group I (trend P =. 003), respectively. Twenty-six patients classified into groups IV and V also showed neither lymphatic invasion nor recurrence. All the smaller tumors (< or =2.0 cm) in group IV or V were histologically proved to be bronchioloalveolar carcinoma. Adjusted for smoking status and other characteristics, patients without ground-glass opacity on high-resolution computed tomography had a significantly increased risk of concurrent lymph node metastasis compared with those with ground-glass opacity., Conclusion: In patients with clinical T1 N0 M0 adenocarcinoma, the proportion of ground-glass opacity area on thin-section computed tomography scans was a strong predictor for tumor aggressiveness and thus could be a useful index for planning limited surgical resection for these patients.

We report a case of synchronous tumors consisted of bronchial carcinoid and adenocarcinoma of the lung. A 58-year-old female was referred to our hospital after screening, because an abnormal shadow was noted in the right lung on her computed tomography (CT) of the chest. CT scans showed a peripheral pulmonary mass in the right middle lobe and a nodule around the right lower lobe bronchus. The nodular lesion like swollen lymph node was diagnosed as bronchial carcinoid originated in B6 by bronchoscopy. The pulmonary mass was diagnosed as adenocarcinoma by using core needle biopsy during operation. Right middle and lower bilobectomy and mediastinal lymph node dissection were performed. Coincidence of a bronchial carcinoid and an adenocarcinoma of the same side of the lung is a rare occurrence.

Purpose: Human serum albumin (HSA) was mildly oxidized by a metal-catalyzed oxidation system (MCO-HSA), chloramine-T (CT-HSA) or H2O2 (H2O2-HSA), and the effects of these treatments on the structural, drug-binding and esterase-like properties were studied., Methods: Protein conformation was examined by calorimetric, chromatographic, electrophoretic and spectroscopic techniques. Drug binding was studied by ultrafiltration method, and esterase-like activity was determined using p-nitrophenyl acetate as a substrate., Results: Far-UV and near-UV CD spectra indicated that significant structural changes had occured as the result of treatment with MCO-HSA and CT-HSA but not with H2O2-HSA. However, SDS-PAGE analysis does not provide precise information on gross conformational changes such as fragmentation, cross-linking and SDS-resistant polymerisation. The results of differential scanning calorimetry, the fluorescence of the hydrophobic probe 1,1-bis-4-anilino-naphthalene-5,5-sulfonic acid and the elution time from a hydrophobic HPLC column indicated that MCO-HSA and CT-HSA in particular, have a more open structure and a higher degree of exposure of hydrophobic areas than unoxidized HSA. In all cases, high-affinity binding of warfarin remained unchanged for all the oxidized HSAs. However, high-affinity binding of ketoprofen to CT-HSA and, especially, MCO-HSA was diminished. In addition, the esterase-like activity of these proteins were all decreased to the same low level., Conclusions: Mild oxidation of HSA has no detectable effect on the binding of drugs to site I in subdomain IIA. In contrast, both the ligand binding property of site II and the esterase-like activity of oxidized HSAs are decreased, most probably due to conformational changes in subdomain IIIA.

Nitric oxide (NO) acts as a neurotransmitter and neuromodulator in the nervous system of many vertebrates and invertebrates. The effects of extracellularly applied sodium nitroprusside (SNP) and diethylamine NO (C(2)H(5))(2)N[N(O)NO]-Na(+) (DEA/NO), NO donors, on a glutamate (Glu)-induced K(+) current in identified Onchidium neurons were investigated using voltage clamp and pressure ejection techniques. Bath-applied SNP (10 microM) and DEA/NO (5-10 microM) reduced the Glu-induced K(+) current without affecting the resting membrane conductance and holding current. The Glu-induced K(+) current also was inhibited by the focal application of SNP to the neuron somata. The suppressing effects of NO donors were concentration-dependent and completely reversible. Pretreatment with hemoglobin (50 microM), a nitric oxide scavenger, and 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), a specific inhibitor of NO-stimulated guanylate cyclase, decreased the SNP-induced inhibition of the Glu-induced current. Bath-applied 50 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, or intracellular injection of 1 mM guanosine 3',5'-cyclic monophosphate (cGMP) inhibited the Glu-induced current, mimicking the effect of NO donors. These results demonstrate that SNP and DEA/NO inhibit the Glu-induced K(+) current and that the mechanism of NO inhibition of the Glu-induced current involves cGMP-dependent protein kinase., (Copyright 2000 Wiley-Liss, Inc.)

The present study was undertaken to improve the oral absorption of KCA-098, an antiosteoporosis drug. In this study, the form 2 of KCA-098 was used as a desirable crystal form for pharmaceutical formation among three kinds of crystal forms, 1, 2, and 3. Solid dispersions of KCA-098 with hydroxypropylcellulose (HPC) or poly(vinylpyrrolidone) (PVP) were prepared by the solvent method. The physicopharmaceutical properties of the solid dispersions were characterized by powder x-ray diffraction, FTIR spectroscopy, and differential scanning calorimetry (DSC). The powder x-ray diffractograms suggest that KCA-098 in the HPC-SL solid dispersion existed in a partial crystalline state as a new crystal form that could be produced by recrystallization from the solvent. Dissolution from the solid dispersions was markedly enhanced in comparison with that of the drug alone. The dissolution enhancement was observed to be greater for the solid dispersion with HPC-SL than for that with PVP. The KCA-098/HPC-SL (1:2) solid dispersion capsule showed a 3.5-fold increase in the initial concentration and 2.5-fold increase in initial concentration of dissolved drug after 60 min, compared with the values for a physical mixture of KCA-098 (form 2)/lactose (1:2). The in vivo absorption of the drug was investigated after oral administration of KCA-098 or its solid dispersion. The area under the plasma concentration curve of KCA-098 after oral administration of the KCA-098/HPC-SL (1:2) solid dispersion capsule was three-fold greater than that for the drug itself.

The inhibition of rabbit heart carbonyl reductase (RHCR) by fatty acids was examined using 4-benzoylpyridine (4BP) as a substrate. The inhibitory potency of saturated fatty acids increased with elongation in the carbon chain from caprylic acid to myristic acid, but decreased with further elongation. Myristic acid with 14 carbon atoms most strongly inhibited RHCR. All of the unsaturated fatty acids tested strongly inhibited RHCR; the cis-isomers were more potent inhibitors than the corresponding trans-isomers. The methyl esters and alcohols, which lack a carboxyl group, derived from fatty acids did not exert a significant inhibitory effect on RHCR. These results indicate that the existence of a proper length of carbon chain, double bond(s), and a carboxyl group in a fatty acid molecule is important for RHCR inhibition. We also propose the possibility that myristic acid at low concentrations inhibits the reduction of 4BP by interacting with a binding site other than the coenzyme- and substrate-binding sites of RHCR.

The present study was undertaken to assess the effects of sodium nitroprusside (SNP) and diethylamine NO (C2H5)2N[N(O)NO]-Na+ (DEA/NO), NO donors, on an acetylcholine (ACh)-induced Cl- current in identified Onchidium neurons using voltage-clamp and pressure ejection techniques. Bath-applied SNP (10 microM) and DEA/NO (5-10 microM) reduced the ACh-induced Cl- current in the neurons without affecting the resting membrane conductance and holding current. The suppressing effect of NO donors were concentration-dependent and completely reversible. Pretreatment with 1H-[1,2,4]oxadiazolo-[4,3-a] quinoxalin-1-one (1 micro M), a specific inhibitor of NO-stimulated guanylate cyclase, and hemoglobin (50 micro M), a nitric oxide scavenger, decreased the SNP-induced inhibition of the ACh-induced current. Intracellular injection of guanosine 3',5'-cyclic monophosphate (cGMP) or bath-application of 3-isobutyl-1-methylxanthine (50 micro M), a non-specific phosphodiesterase inhibitor, inhibited the ACh-induced current, mimicking the effect of NO donors. These results suggest that SNP and DEA/NO inhibit the ACh-induced Cl- current and that this effect is mediated by an increase in intracellular cGMP.

The authors extracted a dialyzable substance from sea urchin (Diadematidae) collected in Fiji, and investigated its effect on the frequency of miniature endplate potentials (MEPPs) in the sartorius muscle of the frog. This substance was shown to be different from previously known biologically active substances in many respects. The increase of MEPP frequency by the application of toxin was significant and dose-dependent. The frequency began increasing immediately after application and returned to normal after washing. This increasing action of toxin on MEPP frequency was strengthened by increased calcium concentration, but even in Ca-depleted solution toxin action was significant. Toxin action in Ca-free Mg2+ solution was found to be just as remarkable as in Ca2+ solution, and was dependent on magnesium concentration. Toxin action was also found to depend significantly on sodium concentration. The toxin did not change membrane potential of the muscle appreciably. From these results, the depolarization of the nerve terminal was not considered to be the main effect of toxin on MEPP frequency. The increasing action of toxin on MEPP frequency was interpreted as being the result of increased permeability of the nerve terminal to both divalent cations and Na+.

The ability of various organic cations to depolarize the ACh-activated endplate membrane in the absence of Na ions was examined on frog sartorius muscle by measuring the endplate potential on the muscle surface with the moving electrode technique. The ACh-activated endplate membrane was very permeable to ammonium and its methyl and hydroxy derivatives, and moderately permeable to guanidine derivatives and Tris (hydroxymethyl) aminomethane. The permeability of alkylol derivatives of ammonium diminished progressively with increase in molecular size. The present results suggested that the endplate ionic channels can be represented by a pore of about 6.4 A in diameter.

In the 4-aminopyridine (4AP) Ringer in which the concentration of 4AP is above 0.005 mM, the frog muscle contracted repetitively by rapid cooling. This contraction was named AP-RCC. It took more than 20 min for 4AP to show the fall effect on the AP-RCC. During the AP-RCC action potentials of the muscle membrane which synchronized with the repetitive contractions were observed. Tetrodotoxin abolished the AP-RCC. In muscle pretreated with 400 mM glycerol, no AP-RCC was obtained. During the AP-RCC no action potential of the nerve was seen. d-Tubocurarine abolished both the AP-RCC and the accompanying action potential of the muscle. Mn2+ and, to a lesser extent, Mg2+, inhibited both the AP-RCC and the twitch by indirect stimulation. The repetitive nerve stimulation which lasted long enough to exhaust acetylcholine at the nerve terminal abolished the AP-RCC. 4AP restored the contraction suppressed by dantrolene sodium. The AP-RCC was masked by caffeine rapid cooling contracture when the concentration of caffeine was high. From these results, the AP-RCC is considered to be generated by acetyl-choline release from the nerve terminal by the combined action of 4AP and depolarization during cooling.

In the isolated frog muscle, the proteinaceous venom extracted from jellyfish (genus Aurelia) produced 1) a complete and irreversible block of indirectly and directly elicited muscle twitch and 2) an irreversible depolarization of the muscle membrane. This venom-induced depolarization was effectively reversed or prevented by the substitution of choline for sodium in Ringer solution, but not by the introduction of tetrodotoxin (TTX), a sodium channel blocker. The mechanism of muscle membrane depolarization appears to involve probably an increase in membrane permeability to sodium ion as shown by the decrease in membrane resistance. These results suggest that the venom forms a pore which has sodium selectivity or activates a TTX-insensitive sodium channel which is different from the known sodium channel.