Quasi-isentropic compression of LiH above 400 GPa using magnetocumulative generator.

The knowledge of high-pressure behavior of LiH is significant for the validation of fundamental theoretical models and applications in thermonuclear materials and potential energy supplies. The compressibility of 7LiH under isentropic compression at high pressure was investigated experimentally and theoretically. The experimental technique for quasi-isentropic compression with low-density materials was developed using the magnetocumulative generator CJ-100 and x-ray flash radiography. The x-ray images and extracted interface of the sample target in dynamic flash radiography experiments were obtained. According to each interface size of the target both before and after compression, the compression ratio of 7LiH and reference material aluminum was obtained. The density of the reference and using its known isentropic curve provide the pressure in the reference. The pressure in 7LiH was deduced from the pressure in the reference and using the calculated gradient correction factor. The quasi-isentropic data point at 438 GPa was obtained experimentally. A semiempirical three-term complete equation of state was constructed and validated for 7LiH using the theory of Mie-Grüneisen-Debye with experimental data from the literature. The quasi-isentrope data point is reasonably consistent with the theoretical results. The quasi-isentropic experimental techniques and results broaden the existing research scope and are practical and helpful to further validate theoretical models in the future.

[Causes and management of frontal sinusitis after transfrontal craniotomy].

Objective:The aim of this study is to investigate the causes and the strategy of frontal sinusitis after transfrontal craniotomy by endoscopic frontal sinus surgery and traditional surgery with facial incision. Method:A total of thirty-four patients with frontal sinusitis after transfrontal craniotomy were admitted, with the symptom of purulence stuff, headache and upper eyelid discharging. The onset time was 2.6 years on average. The frontal sinus CT and MRI images showed frontal sinusitis. Twenty-seven patients were treated with endoscopic frontal sinus surgery, and seven patient was treated with combined endoscopic and traditional frontal sinus surgery. In the revision surgery, the bone wax and inflammatory granulation tissue were cleaned out in both operational methods. The cure standard was that the postoperative frontal sinus inflammation disappeared and the drainage of the volume recess was unobstructed. Result:Thirty-four patients had a history of transfrontal craniotomy, and there was a record of bone wax packing in every operation. Among twenty-seven patients with endoscopic frontal sinus surgery, Twenty-five cases cured and two cases were operated twice. Seven patients were cured with combined endoscopic and traditional frontal sinus surgery. Conclusion:The frontal sinusitis after transfrontal craniotomy may be related to the inadequate sinus management, especially bone wax to be addressed to the frontal sinus ramming leading to frontal sinus mucosa secretion obstruction and poor drainage. Endoscopic frontal sinus surgery is a way of minimally invasive surgery. The satisfying curative effect can be obtained by endoscopic removal of bone wax, inflammatory granulation tissue, and the enlargement of frontal sinus aperture after exposure to the frontal sinus, and some cases was treated with both operation method.

Intracellular pH-induced fluorescence used to track nanoparticles in cells.

A nanoparticle with pH-induced fluorescence was reported for intracellular tracking. The fluorescence was evoked by the isomerization of the ring-closed form spiropyran (SP) to the ring-open form merocyanine (MC) in the weak acidic environment of cells. The SP-MC switch accelerated the dissociation of nanoparticles to trigger the release of trapped paclitaxel.

Preparation of a HA/collagen film on a bioactive titanium surface by the electrochemical deposition method.

A hydroxyapatite (HA) film with or without collagen was electrochemically deposited on a bioactivated titanium metal prepared by acid-alkali treatment, so as to improve the biocompatibility of bioactive titanium metals. The cell response of the film was studied with MG63 osteoblasts culture. It was found that the hydroxyapatite formation in the film during the deposition process was inhibited when collagen was added in the electrolyte. More hydroxyapatite with and without collagen could be deposited on the bioactivated titanium than the control titanium metal without treatment, which indicated that the bioactivation process before the electrochemical deposition could accelerate the deposition. The abilities of cell attachment and proliferation were improved by the film especially in the group containing collagen, and the film on the bioactivated metal had higher cell response ability than that on the titanium without treatment. The results indicated that the hydroxyapatite/collagen film could improve the biocompatibility of the bioactive titanium metal surface, and the bioactivation surface modification could further regulate the film and its cell response. It is possible to get a titanium surface with higher bioactivity than the traditional bioactive titanium surface by combining the bioactivation surface modification and electrochemical deposition HA/collagen film.

Controlled release of 9-nitro-20(S)-camptothecin from methoxy poly(ethylene glycol)-poly(D,L-lactide) micelles.

9-nitro-20(S)-camptothecin (9-NC) is a potent topoisomerase-I inhibitor, and it was applied for clinical trials in cancer treatment. However, the applications of 9-NC were limited by its poor solubility and instability. In order to overcome these disadvantages, 9-NC was encapsulated in amphiphilic copolymer micelles composed of methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-PDLLA, PELA). Three diblock copolymers with different PDLLA chain lengths were synthesized. The critical micelle concentration was varied from 10(-4) g L(-1) to 10(-2) g L(-1). The 9-NC loaded micelles were nanospheres with diameters ranging from 30 nm to 60 nm. The relationship between the composition of copolymers and the drug loading content was discussed. The encapsulation of micelles improved the solubility of 9-NC greatly. The solubility of 9-NC in micelle M1 was about 250 times higher than that of 9-NC in a phosphate buffer solution (PBS). The stability of 9-NC in micelles was also promoted. After being incubated in PBS for 160 min, 80% of 9-NC in micelles existed as an active lactone form, while 85% of 9-NC in PBS were transferred to an inactive carboxylate salt form. The release experiments were carried out in PBS and the results showed that the release processes were controllable.

Preparation of bioactive nanotitania ceramics with biomechanical compatibility.

In this article, bioactive nanotitania ceramics with biomechanical compatibility was prepared by using an additive of hydroxyapatite or MgO as particle growth inhibitor. After sintering at 1000 degrees C, the particle size of nanotitania ceramics prepared by using HA as additive (HT) was much smaller than that prepared by using MgO as additive (MT). In simulated body fluid (SBF), HT could induce apatite formation in 4 days, while no apatite could be found on MT even after it was soaked in SBF for 14 days. After Ros17/28 osteoblasts were cultured on the materials for 1, 4, and 6 days, MTT results showed that the osteoblasts on the HT differentiated faster than that on the MT. Mechanical tests results showed that the bending and compressive strength of HT were 160 and 200 MPa, while those of MT were 70 and 88 MPa, respectively. These results demonstrated that it is suitable to prepare bioactive nanotitania ceramics, with biomechanical compatibility, by using HA as particle growth inhibitor.

Nonselective cation current in rabbit ventricular myocytes.

Currents contributing repolarization in rabbit ventricular myocytes are very complex because the I(to.s) covers almost the whole repolarization phase of the action potential. The other components of repolarizing currents, such as I(Kr) and I(Ks), are small. In the present work, with whole-cell patch-clamp technique, a clear evidence was provided for the existence of a hitherto unreported, voltage-dependent, nonselective cation current (NSCC) in rabbit ventricular myocytes. Na(+), K(+), and Cs(+) can permeate through the nonselective cation channel and the NSCC can be blocked by Gd(3+). The channels are sensitive to Ca(2+), Mg(2+)-free, and insulin in bathing solution. Activation of NSCC may provide complex effects on action potential configuration depending on the basal conditions and the experimental situations. Considering the voltage dependence and rapid activation kinetics of this current, we speculate that this current can provide an important influence on all repolarization phases of the action potential as well as contribute to the resting membrane potential in rabbit ventricular myocytes. In addition, it is conceivable that, under certain pathophysiological conditions (e.g., ischemia or excessive mechanical stress), the sensitivity of the channels could be altered in such a way that the conductance opens even in the presence of physiological Ca(2+), Mg(2+), and insulin. It might be an important factor on the repolarization of the action potential. Changes in NSCC may lead to an induction or inhibition of arrhythmia.

Human fatty acid synthase: role of interdomain in the formation of catalytically active synthase dimer.

The human and animal fatty acid synthases are dimers of two identical multifunctional proteins (M(r) 272,000) arranged in an antiparallel configuration. This arrangement generates two active centers for fatty acid synthesis separated by interdomain (ID) regions and predicts that two appropriate halves of the monomer should be able to reconstitute an active fatty acid synthesizing center. This prediction was confirmed by the reconstitution of the synthase active center by using two heterologously expressed halves of the monomer protein. Each of these recombinant halves of synthase monomer contains half of the ID regions. We show here that the fatty acid synthase activity could not be reconstituted when the ID sequences present in the two recombinant halves are deleted, suggesting that these ID sequences are essential for fatty acid synthase dimer formation. Further, we confirm that the ID sequences are the only regions of fatty acid synthase monomers that showed significant dimer formation, by using the yeast two-hybrid system. These results are consistent with the proposal that the ID region, which has no known catalytic activity, associates readily and holds together the two dynamic active centers of the fatty acid synthase dimer, therefore playing an important role in the architecture of catalytically active fatty acid synthase.

[Study on preparation and pharmacodynamics of insulin-loaded polyester microparticles].

AIM: To investigate the possibility of using poly (epsilon-caprolactone-block-D, L-lactide) (PLCA) as a kind of materials to prepare the microparticles drug carrier. METHODS: PCLA-MP (microparticle, MP) was prepared by double-emulsification solvent evaporation method. Its morphology was examined by scanning electron microscope. Its size diameter was examined by particle analyser. Insulin (INS), as a model drug, was then encapsulated into PCLA-MP (INS-PCLA-MP). HPLC method was established for determining INS in INS-PCLA-MP. An "antibody-capture" procedure was devised for investigating encapsulation mechanism. The in vitro release behaviour of INS-PCLA-MP was determined in phosphatic buffer solution (pH 7.4). The diabetic rat model was established and blood glucose levels were measured using glucose oxidase (GOD-PAP) method to evaluate the hypoglycaemic effects after subcutaneous administration of INS-PCLA-MP. The pharmacological bioavailability (PBA) of INS-PCLA-MP was calculated from the area above the curve (AAC) in contrast with INS-solution. RESULTS: The mean diameter of INS-PCLA-MP was 1.9 microns, while the encapsulation ratio of INS reached to 76.46%. Only 18.25% encapsulated INS was on the surface of the microparticles, it could be measured by "antibody-capture" experiment. The in vitro release curve of INS-PCLA-MP consists of initial rapid release stage followed by slower exponential stage. In pharmacodynamic studies, after subcutaneous administration of INS-PCLA-MP 12 u.kg-1, the hypoglycaemic effect was significant. The PBA of INS-PCLA-MP was 123.08%. CONCLUSION: PCLA might become a new drug carrier material in the future.

Cooperativity between free and N-(2-hydroxypropyl) methacrylamide copolymer bound adriamycin and meso-chlorin e6 monoethylene diamine induced photodynamic therapy in human epithelial ovarian carcinoma in vitro.

The purpose of this study was to determine the interaction between free (unbound) and N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer bound adriamycin and meso-chlorin e6 monoethylene diamine (Mce6) induced photodynamic therapy in combination in their cytotoxic activities against human ovarian epithelial carcinoma (OVCAR-3) in vitro. The effects of each agent (free drugs and HPMA copolymer bound) alone and in combination were measured simultaneously utilizing two measures of cell viability: a) mitochondrial respiration via the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide reduction (MTT) assay; and b) thymidine incorporation via the tritiated thymidine incorporation (TI) assay. These were performed at 72 and 144 h after drug exposure. Forty-eight hours from time zero (24 h after drug addition), the cells treated with Mce6 (free and HPMA copolymer bound) and controls were exposed to 650 nm light (13 min at 15 mW/cm2, 11.7 J/cm2). The calculated ED50 values by the MTT 72 h assay for adriamycin (A) and Mce6/light (C) were 1.5 microg/ml and 209 ng/ml, respectively. Adriamycin demonstrated progressive cellular toxicity over time in both assays. Mce6/light demonstrated initial damage at 72 h by MTT and TI which recovered by 144 h. Adriamycin and Mce6/light acted cooperatively to increase the percentage of cells inhibited. In combination, 21.3+/-1.5% MTT reduction activity was observed by free adriamycin and Mce6/light compared to the expected 27+/-5% (p<0. 0001) based on additivity. Twice the ED50 of adriamycin (2A=3 microg/ml) or Mce6/light (2C=418 ng/ml) resulted in only 42+/-3.6% and 39.2+/-2.0% activity, respectively (both p<0.0001 vs. combination). When Mce6/light at 10x ED50 (10C) was combined with 1x ED50 of adriamycin (1A), or the reciprocal combination, additional cooperativity was demonstrated. Compared to free drugs, both HPMA copolymer bound adriamycin (P-A) and HPMA copolymer bound Mce6/light (P-C) required a 10-fold increase in drug concentration to show equivalency with free drugs (A or C). Dose response curves demonstrated a reduced slope compared to free drugs in the same dose ranges. When P-A was added (1-10x free adriamycin ED50) to an effective concentration of P-C (10P-C: equivalent to 10x free Mce6 ED50) an improved long-term inhibition of OVCAR-3 cell multiplication was noted in both the MTT and TI 144 h assays. P-C (1-10x free Mce6 ED50) added to an effective concentration of P-A (10P-A: equivalent to 10x free adriamycin ED50) did not appear to significantly improve the efficacy profile of P-A. A and C in vitro appear to act independently and are cooperative in their combined toxicity against the human ovarian epithelial carcinoma cell line OVCAR-3. HPMA copolymer-adriamycin and Mce6 conjugates (P-A and P-C, respectively) inhibited growth of OVCAR-3 in vitro. HPMA copolymer-adriamycin added to HPMA copolymer-Mce6 improved the efficacy of HPMA copolymer-Mce6.

Selective modification of apoB-100 in the oxidation of low density lipoproteins by myeloperoxidase in vitro.

Oxidative modification of LDL may be important in the initiation and/or progression of atherosclerosis, but the precise mechanisms through which low density lipoprotein (LDL) is oxidized are unknown. Recently, evidence for the existence of HOCl-oxidized LDL in human atherosclerotic lesions has been reported, and myeloperoxidase (MPO), which is thought to act through production of HOCl, has been identified in human atherosclerotic lesions. In the present report we describe the formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive modifications in the apolipoprotein (apo) by exposure of LDL to myeloperoxidase in vitro. In contrast with the complex mixture of peptides from oxidation of LDL with reagent HOCl, oxidation with MPO in vitro produced a major tryptic peptide showing absorbance at 365 nm. This peptide was isolated and characterized as VELEVPQL(*C)SFILK..., corresponding to amino acid residues 53-66...on apoB-100. Mass spectrometric analyses of two tryptic peptides from oxidation of LDL by HOCl indicated formation of the corresponding methionine sulfoxide (M=O), cysteinyl azo (*C), RS -N= N-DNP, derivatives of EEL(*C)T(M=O)FIR and LNDLNS VLV(M=O)PTFHVPFTDLQVPS(*C)K, which suggest oxidation to the corresponding sulfinic acids (RSO2H) by HOCl. The present results demonstrate that DNPH-reactive modifications other than aldehydes and ketones can be formed in the oxidation of proteins and illustrate how characterization of specific products of protein oxidation can be useful in assessing the relative contributions of different and unexpected mechanisms to the oxidation of LDL and other target substrates. The data also suggest a direct interaction of the LDL particle with the active site on myeloperoxidase and indicate that effects of the protein microenvironment can greatly influence product formation and stability.

Identification of modified tryptophan residues in apolipoprotein B-100 derived from copper ion-oxidized low-density lipoprotein.

Oxidative modifications of low-density lipoproteins (LDL) may contribute to the pathogenesis of atherosclerosis. Although the oxidation products of the lipid components of LDL have been studied extensively, less is known about the oxidation products of the apoprotein, apolipoprotein B-100. To identify the specific oxidative modifications, we oxidized LDL in the presence of Cu(2+), treated with DNPH, precipitated and delipidated the protein, digested the protein with trypsin, and analyzed the peptides by high-performance liquid chromatography. We isolated nine peptides that exhibited measurable absorbance at 365 nm, which is characteristic of hydrazones derived from DNPH and is not observed in peptides derived from unoxidized LDL. Unexpectedly, we obtained the same peptides with absorbance at 365 nm in Cu(2+)-oxidized LDL not treated with DNPH. N-terminal sequence analyses and mass spectrometry indicated that the peptides isolated from the Cu(2+)-oxidized LDL all contained kynurenine residues in place of Trp residues found in the native apoprotein. The product profile we observed in Cu(2+)-oxidized LDL was remarkably different from the profiles observed in LDL oxidized by HOCl or myeloperoxidase in vitro, and the preferential oxidation of Trp to kynurenine in Cu(2+)-catalyzed oxidation of LDL contrasts with the products observed following oxidation of LDL with HOCl or myeloperoxidase. Our studies to date support the working hypothesis that the specific products of protein oxidation are sufficiently distinct to be developed as biomarkers of proposed mechanisms of oxidation of LDL and biological molecules in other toxicities and diseases.

Oxidative modifications of apoB-100 by exposure of low density lipoproteins to HOCL in vitro.

Although the products of oxidation of the lipid components of LDL have been studied extensively, much less is known about the specific products of oxidative modification of the apoprotein. We reacted native LDL and LDL that had been treated with HOCl with 2,4-dinitrophenylhydrazine (DNPH), delipidated and trypsinized the protein, and analyzed the products by HPLC. Although tryptic digests of native LDL and LDL oxidized by limited quantities of HOCl showed similar patterns by HPLC with detection at 220 nm, oxidized LDL showed several discrete peaks at 365 nm, which is characteristic of hydrazones formed with aldehydes and ketones, commonly termed protein carbonyls. Native LDl showed no peaks in the chromatograms at 365 nm. Peptides absorbing at 365 nm were isolated by HPLC and characterized. In most cases, the probable sites of modification on the peptides could be implied by failure of an anticipated amino acid to appear in the expected sequence. Of the 14 peptides isolated and characterized to date, eight peptides contained Cys residues. In other peptides, Lys, Trp, and Met were identified as amino acid residues apparently modified by HOCl treatment of LDL. Thirteen of the peptides identified are from trypsin-releasable peptides located on the surface of unoxidized native LDL. Our studies suggest a selective process of modification of apoB-100 by HOCl and the approaches used in the present studies should be useful for the characterization of the mechanisms of oxidation of this and other proteins.

Oxidation of bovine beta-casein by hypochlorite.

We recently observed two 2,4-dinitrophenylhydrazine (DNPH)-reactive proteins of 40 and 120 kDa in the bronchoalveolar lavage fluids of rats exposed to >95% O(2) for 48 h. The N-terminal sequences of these proteins were both identical over 16 amino acids with rat beta-casein, which, in addition to its more common association with milk, is produced by cytotoxic T-lymphocytes, and has been found to have proinflammatory properties. Because of the inflammatory response that accompanies hyperoxic lung injury, we investigated the oxidation of bovine beta-casein by HOCl. Following exposure to HOCl at 4 degrees C for 15 min, derivatization with DNPH, washing, and digestion with trypsin, the resultant peptides were separated by reverse-phase HPLC. One peptide isolated from a peak absorbing at 365 nm was identified as AVP(Y*)PQR, corresponding to amino acids 177-183 of bovine beta-casein. Analysis of the peptide by both electrospray and matrix assisted laser desorption ionization (MALDI) mass spectrometry identified a molecular ion MH+ of 1008.5 Da, which represents an increase of 178 Da from the calculated monoisotopic MH+ of the unmodified peptide of 830.45 Da. Daughter ion spectra of the doubly charged parent ion of the peptide further support the oxidation of the tyrosine to the quinone methide, with subsequent conversion to the corresponding hydrazone with DNPH. A second pair of products were identified as arising from oxidation of Y(193) within the tryptic peptide constituted by amino acids 184-202, and the corresponding chymotryptic cleavage side product, 191-202. Exposure of beta-casein to increasing amounts of HOCl revealed that M and Y residues were the most susceptible, although bovine beta-casein contains no C, and a single W, which would not be detected by our methods. The approach described in the present report can be used to evaluate the contributions of distinct mechanisms of oxidation in other experimental or pathological models.

Effects of gemfibrozil on very-low-density lipoprotein composition and low-density lipoprotein size in patients with hypertriglyceridemia or combined hyperlipidemia.

To examine the effects of gemfibrozil on very-low-density lipoprotein (VLDL) composition and low-density lipoprotein (LDL) size, five men with hypertriglyceridemia (HTG) alone and five men with HTG and hypercholesterolemia (combined hyperlipidemia, CHLP) were randomized for 8 weeks to Lopid SR (slow-release gemfibrozil; two 600-mg tablets once per day) or placebo in a crossover study. Drug therapy versus placebo significantly decreased plasma triglyceride (68%), and VLDL (77%), and significantly increased high-density lipoprotein cholesterol (25%); total cholesterol, apolipoprotein B and lipoprotein[a] concentrations did not change significantly. With drug, mean total apoE in plasma was 53% lower in patients with HTG and 39% lower in patients with CHLP. Gemfibrozil significantly affected VLDL composition: protein increased 26%, molar ratio of apoE to apoB reduced 48%, apoC-II increased 19%, and apoC-III decreased 9%. LDL cholesteryl ester significantly increased with drug treatment. VLDL subfractions were separated and classified as heparin binding (VLDLR, apoE rich) or nonbinding (VLDLNR-1 and VLDLNR-2, both apoE poor). All VLDL subfractions were significantly lower with drug therapy, and the differences for total VLDL and for VLDL subfractions were greater in patients with HTG. With placebo, VLDLR accounted for 41.8% of VLDL in HTG and 49.0% of VLDL in CHLP, reduced to 27.6% and 38.6%, respectively, with gemfibrozil. Taken together, these results suggest that treatment with gemfibrozil reduces plasma concentrations of VLDL and alters the apoprotein composition of VLDL in a manner that may favor LDL- and VLDL-receptor-mediated clearance of the apoE-rich VLDL subfraction, thereby reducing TG-rich particle concentrations, and possibly reducing risk for coronary heart disease.

Isobolographic assessment of the interaction between adriamycin and photodynamic therapy with meso-chlorin e6 monoethylene diamine in human epithelial ovarian carcinoma (OVCAR-3) in vitro.

OBJECTIVE: Considering the differing mechanisms of cytotoxicity produced by adriamycin and the photosensitizer meso-chlorin e6 monoethylene diamine (Mce6) with light, the interaction of these agents in combination on human ovarian epithelial carcinoma (OVCAR-3 in vitro) was evaluated by dose and effect addition isobole analysis. METHODS: Mitochondrial respiration via the 3-(4,5-dimethyl thiazol-2 yl)-2,5-diphenyl tetrazolium bromide cleavage assay (MTT) and reproductive capacity via the tritiated thymidine incorporation assay (TI) were assessed 72 and 144 hours after exposure to adriamycin, Mce6, and light (650 nm), and to their combinations, in OVCAR-3 cells grown in vitro (20,000 cells per well). RESULTS: In the majority of assays, reproductive capacity was more sensitive to the drug(s) than was mitochondrial respiration (2-10x). Dose-addition isobole analysis showed synergy for the combination of 50% median effective dose (ED50) adriamycin with 50% ED50 Mce6/light in all assays (all P < or = .027). Antagonism was noted with the combination 25% ED50 adriamycin with 75% ED50 Mce6/light. Additivity and synergy were the predominant interactions for 75% ED50 adriamycin with 25% ED50 Mce6/light by dose-addition isobole analyses. Effect-addition isoboles showed a predominance of synergy, particularly for the combination 50% ED50 adriamycin with 50% ED50 Mce6/light. CONCLUSION: Synergy and additivity are the primary in vitro interactions for the combination of adriamycin and Mce6/light in the dosage range tested. Reproductive capacity is more sensitive to these agents than is mitochondrial respiration.

The influence of the extent of target organs on sensitivities of methods for screening rodent carcinogens.

Two hundred and thirty-three rodent carcinogens from the Carcinogenic Potency Database (CPDB) were analyzed with CASE (Computer Automated Structure Evaluation), and a comparison of the extents of target organs with the sensitivities for long-term carcinogenic bioassays in rats and mice, Salmonella assay (Sty), electrophilic substructure alert analysis (ESAA) and CASE was made. The carcinogenicity of 233 chemicals was evaluated in both rat and mouse bioassays. The present study showed that the sensitivities of the five methods for screening carcinogens were related to the extents of target organs of carcinogens. Among the carcinogens that did not induce tumors (extent = 0) in rats, the sensitivities of Sty and ESAA were 46 and 53, respectively. Among the carcinogens which induced tumors at a single organ (extent = 1) in rats, the sensitivities were 57 and 64 respectively; and 71 and 80 at multiple organs (extent > 1) respectively. The sensitivities of CASE were 76, 82, and 89 respectively at these three different extents. Similar results were obtained with these carcinogens in mice. The results indicate that mutagenic or electrophilic carcinogens are more likely to induce tumors at multiple target organs; in contrast, most carcinogens which induced tumors at only a single target organ in one species are rarely mutagenic or electrophilic. The sensitivities of Sty and ESAA were lower than that of the CASE method in these carcinogens. CASE analyzed chemical structures of many carcinogens and non-carcinogens and then established a database of key fragments, and its parameters are not only based on mutagenicity or electrophilicity of chemicals, and this resulted in a more exact detection of the carcinogenicity of chemicals with the CASE method.

Micronucleus formation induced by three polycyclic aromatic hydrocarbons in rat bone marrow and spleen erythrocytes following intratracheal instillation.

Benz[a]anthracene (BA), dibenz[a,h]anthracene (DBA) and dibenzo[a,i]pyrene (DBP) are polycyclic aromatic hydrocarbons (PAHs) found in incomplete combustion products of fossil fuels, coal tar, and other organic materials. Workers in related industries may be exposed to these chemicals by inhalation. The information related to the potential health hazards of these chemicals to the exposed workers, however, is very limited. In the present study, micronucleus (MN) formation in rat bone marrow and spleen polychromatic erythrocytes (PCEs) was determined following three intratracheal instillations within a 24-h period with either BA, DBA or DBP. Three doses with five rats per dose were used for each chemical. Bone marrow and spleen cells were harvested 24 h after the first dosing. Results showed that the order of toxicity for the three PAHs was DBP > DBA > BA. BA induced MN in a dose-related manner in both bone marrow and spleen PCEs at doses above 25 mg/kg. DBA caused significant increases in the frequencies of MN in both spleen and bone marrow PCEs at the dose of 8.5 mg/kg or higher. At 10 mg/kg, DBP significantly increased MN frequency in spleen PCEs, but the increase in bone marrow PCEs was not significantly different from the control. These results indicate that: (1) all three PAHs studied are absorbed through the respiratory tract and their genotoxic metabolites reach the bone marrow and/or spleen; (2) except for DBP which does not induce MN in the bone marrow, all three PAHs induced MN in both bone marrow and spleen PCEs; and (3) the sensitivity of the spleen to the three PAHs is comparable to or higher than that of the bone marrow.

Structure of human apolipoprotein D: locations of the intermolecular and intramolecular disulfide links.

We have determined the primary structure of human apolipoprotein D (apoD) by aligning peptides derived from digestions by cyanogen bromide, trypsin, and chymotrypsin. Our results confirm the primary structure derived from cDNA [Drayna et al. (1986) J. Biol. Chem. 261, 16535-16539]. ApoD consists of 169 amino acid residues, including 5 cysteines. Tryptic peptide analysis indicated that Cys41 and Cys16 are joined by a disulfide bridge. Using a combination of manual Edman degradations and mass spectrometric analysis on a purified cluster of chymotryptic fragments, we identified an intramolecular disulfide bridge between Cys8 and Cys114 and an intermolecular bridge between Cys116 of apoD and Cys6 of apoA-II. In addition, sites of N-glycosylation were found at Asn45 and Asn78. Because apoD contains two intramolecular disulfide linkages and has a high content of proline to disrupt alpha-helical structures, formation of the amphipathic helical regions that characterize the other soluble apolipoproteins is unlikely. We conclude that apoD binds to lipoprotein surfaces through structures other than alpha-helices, such as disulfide links.

Immunoreactivity of apolipoprotein B-100 in oxidatively modified low density lipoprotein.

Thirteen monoclonal antibodies (MAbs) against apolipoprotein B-100 (apo B) were used to analyze changes in immunoreactivity of human LDL resulting from oxidation mediated by cupric ions and oxygen. Decrease in immunoreactivity of oxidized LDL was demonstrated by competitive ELISA with MAbs 5F8, BL3, Mb43, 2G8, B3, B5, and BL7 for which the epitopes are located within residues 1-1297, 4235-4355, 4027-4081, 3728-4306, 2239-2331, 1854-1878, and in the vicinity of residue 2331, respectively. Immunoreactivity of the epitope B6 (2239-2331) increased during first 4 hours of oxidation and then diminished gradually. Epitope B1 (405-539) had slightly reduced immunoreactivity during first 8 h of LDL oxidation and then its minor increase was observed. MAb 12G10, specific to the epitope within apo B thrombin-digest fragment T4 (1-1297), displayed either weak or strong binding to LDL. LDL with weak binding pattern demonstrated significant increase in immunoreactivity upon oxidation. In contrast, LDL with strong binding pattern showed little to no change. Epitopes Mb47 (3441-3569) and 8G4 (1-1297) remained unchanged in oxidized LDL. Immunoreactivity of apo B-100 epitope recognized by MAb 4C11 (residues 2377-2658) was shown to be a function of oxidation time: it increased progressively up to 16 h and was stabilized for another 24 h of LDL oxidation. This epitope may be unmasked by LDL oxidation and may provide a useful immunochemical marker to monitor the extent of LDL oxidation.