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S Shen

Publications and source records attributed to S Shen.

At least 19 recordsLinked to original sources
PubMed1999

ESR study on the structure-antioxidant activity relationship of tea catechins and their epimers.

The purpose of this study is to examine the relationship between the free radical scavenging activities and the chemical structures of tea catechins ((-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC)) and their corresponding epimers ((-)-gallocatechin gallate (GCG), (-)-gallocatechin (GC) and (+)-catechin ((+)-C)). With electron spin resonance (ESR) we investigated their scavenging effects on superoxide anions (O-.2) generated in the irradiated riboflavin system, singlet oxygen(1O2) generated in the photoradiation-hemoporphyrin system, the free radicals generated from 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical. The results showed that the scavenging effects of galloylated catechins (EGCG and GCG) on the four free radicals were stronger than those of nongalloylated catechins (EGC, GC, EC, (+)-C), and the scavenging effects of EGC and GC were stronger than those of EC and (+)-C. Thus, it is suggested that the presence of the gallate group at the 3 position plays the most important role in their free radical-scavenging abilities and an additional insertion of the hydroxyl group at the 5' position in the B ring also contributes to their scavenging activities. Moreover, the corresponding phenoxyl radicals formed after the reaction with O-.2 were trapped by DMPO and the ESR spectra of DMPO/phenoxyl radical adducts were observed (aN=15.6 G and aHbeta=21.5 G). No significant differences were found between the scavenging effects of the catechins and their epimers when their concentrations were high. However, significant differences were observed at relatively low concentrations, and the lower their concentrations, the higher the differences. The scavenging abilities of GCG, GC and (+)-C were stronger than those of their corresponding epimers (EGCG, EGC and EC). The differences between their sterical structures played a more important role in their abilities to scavenge large free radicals, such as the free radicals generated from AAPH and the DPPH radical, than to scavenge small free radicals, such as O-.2 and 1O2, especially in the case with EGCG and GCG with more bulky steric hindrance.

Amidines↗
PubMed1999

Metabolic activation of diclofenac by human cytochrome P450 3A4: role of 5-hydroxydiclofenac.

Cytochrome P450 2C11 in rats was recently found to metabolize diclofenac into a highly reactive product that covalently bound to this enzyme before it could diffuse away and react with other proteins. To determine whether cytochromes P450 in human liver could catalyze a similar reaction, we have studied the covalent binding of diclofenac in vitro to liver microsomes of 16 individuals. Only three of 16 samples were found by immunoblot analysis to activate diclofenac appreciably to form protein adducts in a NADPH-dependent pathway. Cytochrome P450 2C9, which catalyzes the major route of oxidative metabolism of diclofenac to produce 4'-hydroxydiclofenac, did not appear to be responsible for the formation of the protein adducts, because sulfaphenazole, an inhibitor of this enzyme, did not affect protein adduct formation. In contrast, troleandomycin, an inhibitor of P450 3A4, inhibited both protein adduct formation and 5-hydroxylation of diclofenac. These findings were confirmed with the use of baculovirus-expressed human P450 2C9 and P450 3A4. One possible reactive intermediate that would be expected to bind covalently to liver proteins was the p-benzoquinone imine derivative of 5-hydroxydiclofenac. This product was formed by an apparent metal-catalyzed oxidation of 5-hydroxydiclofenac that was inhibited by EDTA, glutathione, and NADPH. The p-benzoquinone imine decomposition product bound covalently to human liver microsomes in vitro in a reaction that was inhibited by GSH. In contrast, GSH did not prevent the covalent binding of diclofenac to human liver microsomes. These results suggest that for appreciable P450-mediated bioactivation of diclofenac to occur in vivo, an individual may have to have both high activities of P450 3A4 and perhaps low activities of other enzymes that catalyze competing pathways of metabolism of diclofenac. Moreover, the p-benzoquinone imine derivative of 5-hydroxydiclofenac probably has a role in covalent binding in the liver only under the conditions where levels of NADPH, GSH, and other reducing agents would be expected to be low.

Animals↗
PubMed1999

Presence of multiple functional polyadenylation signals and a single nucleotide polymorphism in the 3' untranslated region of the human serotonin transporter gene.

The human serotonin transporter (hSERT) gene is a candidate for involvement in the aetiology of affective disorders. In humans, multiple transcripts of the gene have been detected by northern blot analysis of brain and other tissues. We performed 3' rapid amplification of cDNA ends to identify the common sites of polyadenylation in hSERT mRNA from human JAR cells and whole blood. Two major polyadenylation sites were identified: one 567 bp downstream of the stop codon, consistent with the usage of the polyadenylation signal AATGAA, and a second site 690 bp downstream of the stop codon. The putative polyadenylation signal upstream of this site contained a single nucleotide polymorphism (AG/TTAAC). However, allelic variation at this site did not influence polyadenylation site usage, and there were no significant differences in the abundance of the two alleles of this polymorphism between 329 control subjects, 158 individuals with major depression, and 130 individuals with bipolar affective disorder. This single nucleotide polymorphism in the 3' untranslated region of the hSERT gene should provide a useful genetic marker in the evaluation of hSERT as a candidate gene influencing susceptibility to mood disorders.

3' Untranslated Regions↗
PubMed1999

67Cu-2IT-BAT-Lym-1 pharmacokinetics, radiation dosimetry, toxicity and tumor regression in patients with lymphoma.

UNLABELLED: Lym-1, a monoclonal antibody that preferentially targets malignant lymphocytes, has induced therapeutic responses and prolonged survival in patients with non-Hodgkin's lymphoma when labeled with 1311. Radiometal-labeled antibodies provide higher tumor radiation doses than corresponding 1311 antibodies. 67Cu has an exceptional combination of properties desirable for radioimmunotherapy, including gamma and beta emissions for imaging and therapy, respectively, a biocompatible half-time and absence of pathways contributing to myelotoxicity. The radioimmunoconjugate, 67Cu-21T-BAT-Lym-1, has been shown to be efficacious in nude mice bearing human Burkitt's lymphoma (Raji) xenografts. Based on these results, a clinical study of the pharmacokinetics and dosimetry of 67Cu-21T-BAT-Lym-1 in patients with lymphoma was initiated. METHODS: Eleven patients with advanced stage 3 or 4 lymphoma were given a preload dose of unmodified Lym-1, then an imaging dose of 126-533 MBq (3.4-14.4 mCi) 67Cu-21T-BAT-Lym-1. Total Lym-1 ranged from 25 to 70 mg dependent on the specific activity of the radioimmunoconjugate and was infused at a rate of 0.5-1 mg/min. Imaging, physical examination, including caliper measurement of superficial tumors, and analysis of blood, urine and fecal samples were performed for a period of 6-13 d after infusion to assess pharmacokinetics, radiation dosimetry, toxicity and tumor regression. RESULTS: In 7 patients, in whom superficial tumors had been accurately measured, tumors regressed from 18% to 75% (mean 48%) within several days of 67Cu-21T-BAT-Lym-1 infusion. The uptake and biological half-time of 67Cu-21T-BAT-Lym-1 in tumors were greater than those of normal tissues, except the mean liver half-time exceeded the mean tumor half-time. The mean tumor-to-marrow radiation ratio was 32:1, tumor-to-total body was 24:1 and tumor-to-liver was 1.5:1. Images were of very good quality; tumors and normal organs were readily identified. Mild and transient Lym-1 toxicity occurred in 6 patients; 1 patient developed a human antimouse antibody. There were no significant changes in blood counts or serum chemistries indicative of radiation toxicity. CONCLUSION: Because of the long residence time of 67Cu-21T-BAT-Lym-1 in tumors, high therapeutic ratios were achieved and, remarkably, numerous tumor regressions were observed after imaging doses. The results indicate considerable therapeutic potential for 67Cu-21T-BAT-Lym-1.

Adult↗
PubMed1999

Immobilization of horseradish peroxidase in cross-linked phyllosilicates: conditions and characterizations.

An innovative immobilization procedure was developed for intercalation of enzymes into dispersed phyllosilicates which were cross-linked with silicates resulting from the hydrolysis of tetramethyl orthosilicate. Donor:hydrogen-peroxide oxidoreductase intercalative immobilized in the cross-linked phyllosilicate exhibited a similar or higher activity than the free enzyme. The kinetic properties of peroxidase were unaffected by intercalative immobilization. Different factors, including drying methods, particle size, surface cations of the phyllosilicate and ratio of phyllosilicate to tetramethyl orthosilicate, were investigated to optimize immobilization conditions. The immobilized peroxidase exhibited similar kinetic properties to the free enzyme and good storage stability.

Bentonite↗
PubMed1999

67Cu-versus 131I-labeled Lym-1 antibody: comparative pharmacokinetics and dosimetry in patients with non-Hodgkin's lymphoma.

Antilymphoma mouse monoclonal antibody (MoAb) Lym-1, labeled with 67Cu or 131I, has demonstrated promising results in radioimmunotherapy (RIT) for lymphoma. Although 131I has played a central role in RIT thus far, some properties of 67Cu are preferable. A subset of our patients received both 67Cu- and 131I-labeled Lym-1, allowing a comparative evaluation of the two radiopharmaceuticals administered to a matched population of patients. Four patients with B-lymphocytic non-Hodgkin's lymphoma that had progressed despite standard therapy entered trials of 67Cu- and 131I-labeled Lym-1, which were injected 3-26 days apart. Lym-1 was conjugated to 6-[p-(bromoacetamido)benzyl]-1,4,7,11-tetraazacyclotetradecane-N,N ',N",N'"-tetraacetic acid (BAT) via 2-iminothiolane (2IT) and radiolabeled with 67Cu to prepare 67Cu-2IT-BAT-Lym-1; 131I-Lym-1 was preparred by the chloramine-T reaction. Planar imaging was used to quantitate 67Cu-2IT-BAT-Lym-1 or 131I-Lym-1 in organs and tumors daily for 3 days or longer. 67Cu-2IT-BAT-Lym-1 exhibited higher peak concentration in 92% (12 of 13) of tumors and a longer biological half-time in every tumor than 131I-Lym-1. The mean tumor concentration (%ID/g) of 67Cu-2IT-BAT-Lym-1 was 1.7, 2.2, and 2.8 times that of 131I-Lym-1 at 0, 24, and 48 h after injection, respectively. The mean biological half-times of 67Cu-2IT-BAT-Lym-1 and 131I-Lym-1 in tumor were 8.8 and 2.3 days, respectively. Consequently, the mean tumor radiation dose delivered by 67Cu-2IT-BAT-Lym-1 was twice that of 131I-Lym-1, 2.8 (range 0.8-6.7), and 1.4 (range 0.4-35) Gy/GBq, respectively. 67Cu-2IT-BAT-Lym-1 delivered a lower marrow radiation dose than 131I-Lym-1; hence, the tumor:marrow therapeutic indices were 29 and 9.7, respectively. Radiation doses from 67Cu-2IT-BAT-Lym-1 and 131I-Lym-1 to normal tissues were similar except for liver, which received a higher dose from 67Cu-2IT-BAT-Lym-1. Images obtained with 67Cu-2IT-BAT-Lym-1 were superior. Radiation dosimetry data for 67Cu-2IT-BAT-Lym-1 and 131I-Lym-1 agreed with corresponding data from the larger populations of patients from which the matched population for the current study was drawn. In conclusion, 67Cu-2IT-BAT-Lym-1 given to non-Hodgkin's lymphoma patients in close temporal proximity to 131I-Lym-1 exhibited greater uptake and longer retention in tumor, resulting in higher radiation dose and therapeutic index than 131I-Lym-1. These as well as other factors suggest that 67Cu-2IT-BAT-Lym-1 may be superior to 131I-Lym-1 for RIT.

Adult↗
PubMed1998

Cyclic AMP elevation is sufficient to promote the survival of spinal motor neurons in vitro.

The short-term survival of highly purified embryonic spinal motor neurons (SMNs) in culture can be promoted by many peptide trophic factors, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), glial-derived neurotrophic factor (GDNF), and hepatocyte growth factor (HGF). We have asked whether these peptides are sufficient to promote the long-term survival of purified E15 SMNs. Contrary to previous reports, we find that when SMNs are cultured in serum-free medium containing a single peptide trophic factor only approximately one-third of the cells survive for 3 d in culture. When multiple factors are combined, additive effects on survival are observed transiently, but by 7 d of culture the majority of SMNs has died. Surprisingly, when cAMP levels are elevated, the majority of SMNs extend processes and survive for 1 week in culture in the absence of peptide trophic factors, even in low-density cultures. A combination of five peptide trophic factors, together with cAMP elevation, promotes the long-term survival of most of the SMNs in serum-free culture for 3 weeks. These findings provide useful culture conditions for studying the properties of SMNs and have implications for the treatment of motor neuron diseases.

1-Methyl-3-isobutylxanthine↗
PubMed1998

A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris.

In methylotrophic yeasts, glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the metabolism of methanol as a carbon source and certain alkylated amines such as methylamine as nitrogen sources. We describe the isolation and characterization of the FLD1 gene from the yeast Pichia pastoris. The gene contains a single short intron with typical yeast-splicing signals near its 5' end, the first intron to be demonstrated in this yeast. The predicted FLD1 product (Fld1p) is a protein of 379 amino acids (approx. 40 kDa) with 71% identity to the FLD protein sequence from the n-alkane-assimilating yeast Candida maltosa and 61-65% identity with dehydrogenase class III enzymes from humans and other higher eukaryotes. Using beta-lactamase as a reporter, we show that the FLD1 promoter (PFLD1) is strongly and independently induced by either methanol as sole carbon source (with ammonium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source). Furthermore, with either methanol or methylamine induction, levels of beta-lactamase produced under control of PFLD1 are comparable to those obtained with the commonly used alcohol oxidase I gene promoter (PAOX1). Thus, PFLD1 is an attractive alternative to PAOX1 for expression of foreign genes in P. pastoris, allowing the investigator a choice of carbon (methanol) or nitrogen source (methylamine) regulation with the same expression strain.

Alcohol Oxidoreductases↗
PubMed1998

Proteolytic mapping of the coronavirus infectious bronchitis virus 1b polyprotein: evidence for the presence of four cleavage sites of the 3C-like proteinase and identification of two novel cleavage products.

We have previously reported that the 3C-like proteinase of the coronavirus infectious bronchitis virus (IBV) is responsible for processing of the 1a and 1a/1b polyproteins to three mature products of 24, 10, and 100 kDa (Liu et al., 1994, 1997; Ng and Liu, 1998). The C-terminal cleavage site of the 100-kDa protein was defined to be the Q891(1b)-S892(1b) dipeptide bond encoded by nucleotides 15,129 to 15,134 (Liu and Brown, 1995). In this report, other cleavage sites of the 3C-like proteinase in the polyprotein encoded by the ORF 1b region were mapped by coexpression, deletion, and site-directed mutagenesis studies. Using two ORF 1b-specific antisera, V58 and V17, three more Q-S(G) dipeptide bonds, encoded by nucleotides 16,929 to 16,934, 18,492 to 18,497, and 19,506 to 19,511, respectively, were demonstrated to be the cleavage sites of the 3C-like proteinase. Cleavage at these four positions would result in the release of four mature products with molecular masses of approximately 68, 58, 39, and 35 kDa. Among them, the 39- and 35-kDa proteins were specifically identified in IBV-infected cells. Taken together with the 100-kDa protein previously identified, these results suggest that the ORF 1b region of IBV mRNA1 may be able to encode five mature products.

Animals↗
PubMed1998

Proteolytic processing of the polyprotein encoded by ORF1b of the coronavirus infectious bronchitis virus (IBV).

We present here evidence demonstrating that four previously predicted Q-S(G) cleavage sites, encoded by the IBV sequences from nucleotide 15,129 to 15,134, 16,929 to 16,934, 18,492 to 18,497, and 19,506 to 19,511, respectively, can be recognised and transcleaved by the 3C-like proteinase. Five mature products with sizes of approximately 100 kDa, 65 kDa, 63 kDa, 42 kDa and 35 kDa are released from the ORF1b polyprotein by the 3C-like proteinase-mediated cleavage at these positions. Meanwhile, expression of plasmids containing only the ORF1b region showed no autocleavage of the polyprotein encoded, suggesting that the 3C-like proteinase may be the sole proteinase involved in processing of the 1b polyprotein. These data may therefore represent a complete processing map of the polyprotein encoded by ORF1b of mRNA1.

Antibodies↗
PubMed1998

Quantification of iodine-131 in tumors using a threshold based on image contrast.

Accurate and reproducible quantification of tumor radioactivity by imaging requires definition of a region of interest (ROI) for the tumor. The use of a threshold for creating the tumor ROI based on tumor-to-background image contrast (image contrast) was examined. Quantification of iodine-131 in spheres in a phantom that simulated tumors in patients was investigated using planar imaging and geometric-mean and effective-point-source methods. Thresholds that provided the least quantitative error for spheres with different diameters (1-5 cm) and locations (0-11 cm deep in the body), 131I concentrations (0.037-3.2 MBq/ml), and sphere-to-background concentration ratios (1:0, 14:1 and 7:1) were investigated. The correlation between threshold and sphere image contrast was examined. The phantom study showed that an appropriate threshold value for quantification of tumor radioactivity could be determined using image contrast for a single view, provided that image contrast was >/=1.5. The error of quantification was less than 10% for spheres with high image contrast (>/=1.5) but was greater than 17% for spheres with low image contrast (<1.5). When image contrast-dependent thresholds were applied to patient studies, 131I concentrations determined by imaging were in good agreement with the concentrations determined by counting biopsy samples. Additionally, reproducibility was improved when compared with a visual boundary method. It is concluded that accurate and reproducible quantification of radioactivity in tumors is achievable using thresholds based on image contrast if image contrast is greater than or equal to 1.5. Optimal thresholds for quantification of tumor radioactivity were similar if image contrast was similar despite differing tumor diameters, locations and 131I concentrations. Under certain circumstances, the effective-point-source method was preferable to the geometric-mean method.

Humans↗
PubMed1998

Maximum-tolerated dose, toxicity, and efficacy of (131)I-Lym-1 antibody for fractionated radioimmunotherapy of non-Hodgkin's lymphoma.

PURPOSE: Lym-1, a monoclonal antibody that preferentially targets malignant lymphocytes, has induced remissions in patients with non-Hodgkin's lymphoma (NHL) when labeled with iodine 131 ((131)I). Based on the strategy of fractionating the total dose, this study was designed to define the maximum-tolerated dose (MTD) and efficacy of the first two, of a maximum of four, doses of (131)I-Lym-1 given 4 weeks apart. Additionally, toxicity and radiation dosimetry were assessed. MATERIALS AND METHODS: Twenty patients with advanced NHL entered the study a total of 21 times. Thirteen (62%) of the 21 entries had diffuse large-cell histologies. All patients had disease resistant to standard therapy and had received a mean of four chemotherapy regimens. (131)I-Lym-1 was given after Lym-1 and (131)I was escalated in cohorts of patients from 40 to 100 mCi (1.5 to 3.7 GBq)/m2 body surface area. RESULTS: Mean radiation dose to the bone marrow from body and blood (131)I was 0.34 (range, 0. 1 6 to 0.63) rad/mCi (0.09 mGy/MBq; range, 0.04 to 0.17 mGy/ MBq). Dose-limiting toxicity was grade 3 to 4 thrombocytopenia with an MTD of 100 mCi/m2 (3.7 GBq/m2) for each of the first two doses of (131)I-Lym-1 given 4 weeks apart. Nonhematologic toxicities did not exceed grade 2 except for one instance of grade 3 hypotension. Ten (71 %) of 14 entries who received at least two doses of (131)I-Lym-1 therapy and 11 (52%) of 21 total entries responded. Seven of the responses were complete, with a mean duration of 14 months. All three entries in the 100 mCi/m2 (3.7 MBq/m2) cohort had complete remissions (CRs). All responders had at least a partial remission (PR) after the first therapy dose of (131)I-Lym-1. CONCLUSION: (131)I-Lym-1 induced durable remissions in patients with NHL resistant to chemotherapy and was associated with acceptable toxicity. The nonmyeloablative MTD for each of the first two doses of (131)I-Lym-1 was 100 mCi/m2 (total, 200 mCi/m2) (3.7 GBq/m2; total, 7.4 GBq/m2).

Adult↗
PubMed1998

Yttrium-90-DOTA-peptide-chimeric L6 radioimmunoconjugate: efficacy and toxicity in mice bearing p53 mutant human breast cancer xenografts.

UNLABELLED: The novel radioimmunoconjugate, 90Y-DOTA-peptide-chimeric L6 (ChL6), was designed to reduce radiation to critical normal tissues with an exceptionally stable 90Y chelate moiety and a biodegradable linker. Human breast cancer tumors (HBT 3477) in mice were treated with 90Y-DOTA-peptide-ChL6 to examine the effects of increasing dose on the therapeutic efficacy and toxicity of this new agent. METHODS: Groups of athymic mice bearing HBT 3477 xenografts received 4.1- to 14.1-MBq doses of 90Y-DOTA-peptide-ChL6 intravenously. The lethal dose (LD)(50/30), general well-being (weight loss), hematotoxicity and therapeutic efficacy were studied. RESULTS: The LD(50/30) was 12.8 MBq, which corresponded to doses of 17.9 and 50.9 Gy to the total body and tumor (200 mm3), respectively. Deaths were associated with hematotoxicity; no deaths occurred at doses of 9.6 MBq or less. At sublethal doses, the rate of tumor response (cures +/- complete responses + partial responses) increased with increasing dose: 4.1 MBq, 27%; 5.9 MBq, 41%; 8.5 MBq, 69%; and 9.6 MBq, 79% (maximum tolerated dose, MTD). In mice receiving doses of 4.1-9.6 MBq, 6 of 74 (8%) of tumors were cured. Increasing the 90Y dose led to smaller tumor size at nadir and longer tumor regrowth delay but no increase in cure. Although the HBT 3477 p53 gene was found to be mutant resulting in p53 protein not binding DNA breaks, tumors at MTD demonstrated evidence of apoptosis. CONCLUSION: In the human breast cancer athymic mouse model, 90Y-DOTA-peptide-ChL6 had a high therapeutic index and LD(50/30) leading to a 79% response rate at the MTD. The evidence of apoptosis as a mechanism for this tumor response in p53 mutant breast cancer warrants further studies because these observations are relevant to the treatment of lethal breast cancer.

Adenocarcinoma↗
PubMed1998

A preliminary cell kinetics model of thrombocytopenia after radioimmunotherapy.

UNLABELLED: Thrombocytopenia is often the dose-limiting toxicity for radionuclide therapy. Prediction of platelet counts after therapy is important for treatment planning. Simple prediction methods based on linear correlation between radiation dose and blood count nadir have been insufficient because they have not considered time, because of the complicated hierarchical structure of the hematopoietic system in which platelets are not directly injured by low dose rate radiation and because of changing radiation dose rates to marrow with time. This study addresses these problems using a cell kinetics model. METHODS: The model consists of compartments for progenitor cells, megakaryocytes, platelets and stromal cells. A linear quadratic formula was used for progenitor cell survival. Stromal cells were described by a model based on a maximum likelihood estimate for cellular damage, repair and proliferation. Reported values for murine cellular turnover rates and radiosensitivity of progenitor cells were used in the model calculations. Experimental mice received 4 Gy of external beam radiation for tumor implantation and 12.4-23.3 MBq 67Cu-2-iminothiolane-BAT-Lym-1 (BAT = 6-[p-(bromoacetamido) benzyl]-1,4,8,11-tetra-azacyclotetradecane-N,N',N'',N'''-tetraacet ic acid) 19-30 days later. Blood counts were measured three times each week. RESULTS: The model predicted the severity of thrombocytopenia, and the time of the nadir corresponded to measured values in mice. For a dose of 14.2 MBq 67Cu-2-iminothiolane-BAT-Lym-1 that induced a platelet nadir of 20% of baseline (Grade II), the model predicted that at least 20 days were needed before a second 14.2-MBq injection if a subsequent nadir of <10% of baseline (Grade IV) was to be avoided. CONCLUSION: The nadir and duration of thrombocytopenia predicted by the model were similar to those observed in the mice. Predicted information could be useful for planning the dose and timing of fractionated radionuclide therapy. This model provides a stepping stone for future development of a predictive model for patients.

Animals↗
PubMed1998

Characterization of soybean lipoxygenase immobilized in cross-linked phyllosilicates.

Lipoxygenase (LOX) is an enzyme that regioselectively introduces the hydroperoxide functionality into polyunsaturated fatty acids, such as linoleic acid (LA). Hydroperoxide derivatives of polyunsaturated fatty acids are of interest because they can serve as important intermediates in the synthesis of chemical and pharmaceutical compounds. In this study, LOX was immobilized in dispersed phyllosilicate layers that were cross-linked with silicate polymers formed by the hydrolysis of tetramethyl orthosilicates. The effects of substrate concentration, reaction temperature and solvent participation were studied on the oxidation of LA by LOX. The temperature optimum for the oxidation of LA by immobilized LOX was 25 degrees C and values of Km and Vmax for this reaction were 1.7 mM and 0.023 micromol/min respectively. Enzymic activity was stimulated by the addition of 10% (v/v) iso-octane to the reaction mixture. The immobilized LOX preparation showed a degree of substrate preference that demonstrated that 1,3-dilinolein was a better substrate than LA in the oxidation reaction, followed in order by 1-monolinolein, methyl oleate and trilinolein. In general, LOX immobilized in cross-linked phyllosilicates retained the physical and chemical characteristics of free LOX.

Diglycerides↗
PubMed1998

Comparison of 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-peptide-ChL6, a novel immunoconjugate with catabolizable linker, to 2-iminothiolane-2-[p-(bromoacetamido)benzyl]-DOTA-ChL6 in breast cancer xenografts.

Radioimmunotherapy using 131I-ChL6 antibody has shown promise in patients with breast cancer. To enhance this potential, a novel ChL6 immunoconjugate that is catabolizable and tightly binds 90Y and (111)In was developed. The immunoconjugate, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-peptide-ChL6, consists of the macrocyclic chelator DOTA linked to ChL6 by a peptide that is preferentially catabolized in the liver. The pharmacokinetic and dosimetric properties of the radioimmunoconjugates (RICs) (111)In- and 90Y-DOTA-peptide-ChL6 and (111)In- and 90Y-2-iminothiolane (2-IT)-2-[p-(bromoacetamido)benzyl]-DOTA-ChL6 were compared in athymic mice bearing HBT3477 human breast cancer xenografts. Each of the RICs was stable in vivo and concentrated well in the xenografts. Liver concentration, cumulative radioactivity (activity over time), and radiation dose of the DOTA-peptide-ChL6 RICs were one-third to one-half of those of the corresponding 2-IT-2-[p(bromoacetamido)benzyl]-DOTA-ChL6 RICs. Indium-111 RICs were imperfect tracers for corresponding 90Y RICs, although their pharmacokinetics and radiation dosimetries were similar. The results of this study were consistent with previously published in vitro data, which indicated that the peptide linker of DOTA-peptide-ChL6 was catabolized by cathepsin B. The cumulative activities and radiation doses to the liver of DOTA-peptide-ChL6 RICs were one-half of those of corresponding RICs with the 2-IT linker. Preliminary data from pilot studies in patients with breast cancer are in accord with these observations. These novel DOTA-peptide RICs seem to have excellent clinical potential for radioimmunotherapy associated with marrow transplantation, for which liver radiation is likely to be dose limiting for 90Y.

Animals↗