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At least 793 records · Page 44Linked to original sources
PubMed1998

PEG-Modified Horseradish Peroxidase and Its Properties in Nonaqueous Media.

Modification of enzymes can enhance their activities in organic solvents. Horseradish peroxidase (HRP) was modified with methoxy-PEG 5000 which was linked onto HRP peptidal free aminos or onto the aminos introduced through the oxidation of sugar side chains of HRP. It was found the enzyme with PEG linked to peptide chain expressed a nearly same activity as the native HRP in aqueous system. However, PEG modified HRP by oxidation of carbohydrate chains remained only about one third activity of the native HRP. The same results were observed in reversed micelles and in dioxane of less than 30%. Apparently, both PEG modified HRP had the higher activities in high concentration of dioxane. Especially, the activities of the modified HRP in toluene were both enhanced up to nearly 3 fold. When HRP was modified with PEG through the amino groups of peptide chain, it was more stable than the native enzyme in water, dioxane and toluene. However, the stability of PEG-modified HRP by carbohydrate chains was higher in water and lower in both dioxane and toluene in comparison with native HRP.

Journal Article↗
PubMed1999

[The effect of organic modifier on the capacity factor of the solute in the micellar electrokinetic capillary chromatography].

In this paper the mass action law has been used to study the relationship of solute, organic modifier and micelle in the micellar electrokinetic capillary chromatography. A series of equations were given showing the relationship of capacity factor(k) of the solute, equivalent constant, concentration of solute and migration speed under the condition of using only one organic modifier. The results showed that the organic modifier can, to some extent, be dissolved in micelles. It has almost no effect on micellar actual migration speed, but, the organic modifier influences the CMC of surfactants and the distribution of solutes between two phases and results in the change of the capacity factors of the solutes. No simple equations like those in HPLC can be used to show the effect of organic modifier and surfactants on k. At the first approximation, they have the following equation, lnk approximately a + bCB + clnCB where, CB is the total concentration of organic modifier, a, b and c are constants.

English Abstract↗
PubMed2002

[A randomized controlled study on the efficacy of modified sclerotherapy in treating angioma of ear, nose and throat].

OBJECTIVE: To evaluate the efficacy and security of modified and traditional sclerotherapy in treating angioma in ENT. METHOD: For the Angioma of derma and hypodermis (naso face, auricles, neck) 56 cases and 82 cases of mucosa and hypomucosa (the cavities of nose, throat and mouth) were randomized divided into 2 groups each.: The 5% Sodium Morrhate + Triamcinolone Acetonide (The Modified Sclerotherapy n = 28, n = 41) and only 5% Sodium Morrhate (Traditional Sclerotherapy n = 28, n = 41) were respectively injected into bodies of Angioma, as one time a week for 2-6 weeks, on average 19 days. RESULT: The rates of cure and effective for Modified and Traditional Sclerotherapies were 84.1%, 65.2% and 91.3%, 85.5% respectively. There is a significant in statistics for effective rates and there is a notable difference in cured rates between the two therapies. It shows that Modified Sclerotherapy has an obvious advantage (P < 0.05). Although the efficacy is similar between the two therapies for angioma of mucosa and hypomucosa, the difference has a significant in efficacy for angioma of derma and hypoderma between the two therapies (P < 0.05) and the side-effects were very slight for Modified Sclerotherapy. CONCLUSION: There is a more prominent efficacy and security for Modified Sclerotherapy than Traditional Sclerotherapy.

Adolescent↗
PubMed2003

[Study of the therapeutic effects of costimulatory molecules B7-1 and interleukin 12 modified tumor cells vaccines on ovarian epithelial carcinoma through induction of anti-cancer immunity of the body].

OBJECTIVE: To study the biological characters of tumor cell vaccines modified by interleukin 12 (IL-12) and co-stimulatory molecule B(7 - 1) and their combination therapeutic effect on rat ovarian cancer animal model. METHODS: Retroviral vector pLmB(7 - 1)SN, which expressed murine B(7 - 1) was constructed. An epithelial ovarian cancer cell line NuTu-19 was infected with pLmIL-12SN and/or pLmB(7 - 1)SN by lipofectin-mediated gene transfer system. Cell lines that could highly express either or both of these cytokines were named NuTu-19/IL-12, NuTu-19/B(7 - 1) or NuTu-19/IL-12-B(7 - 1), according to the results of enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM). NuTu-19/Neo, the cell line that was transfected by vector pLXSN was used as a control. Various types of cytokine-modified tumor cells were injected subcutaneously into syngeneic rats Fischer 344 and their tumorigenecities were recorded. After being immunized twice by various types of mitomycin C treated gene modified tumor cells, the survival time of the intraperitoneal disseminating ovarian cancer animal model was observed, and the anti-tumor mechanisms of different gene modified tumor cell lines were discussed. RESULTS: The reconstruction of pLmB(7 - 1)SN was successful. Stable IL-12 and B(7 - 1) expression in cell lines NuTu-19/B(7 - 1), NuTu-19/IL-12 and NuTu-19/IL-12-B(7 - 1) were confirmed by ELISA and FCM. Tumorigenecities of various gene modified tumor cells decreased in syngeneic rats. The splenic lymphocytes proliferation indices (PI) in B(7 - 1) group (NuTu-19/B(7 - 1) immunized group) and IL-12 group (NuTu-19/IL-12 immunized group) were 2.4 and 4.6 (the letter P < 0.05 compared with that in control group), while PI in B(7 - 1) and IL-12 combination group (NuTu-19/IL-12-B(7 - 1) immunized group) increase to 10.2, being of significantly difference compared with those in control or B(7 - 1) or IL - 12 group (P < 0.01). More significant cytotoxic effects of cytotoxic lymphocytes (CTLs) on syngeneic tumor cells could be observed in B(7 - 1) group (15.0%) or IL-12 group (31.5%) (P < 0.05 or P < 0.01, compared with 2.6% in control group), while coexpression of IL-12 and B(7 - 1) was of great importance in enhancing CTL activity (52.4%), compared with that in control or B(7 - 1) or IL-12 group (P < 0.05, respectively). The life spans of ovarian cancer-bearing rats immunized by IL-12 (59.8 d) or B(7 - 1) (56.2 d) tumor cell vaccines were a little longer than that in control group (53.4 d), but no significant differences existed (P > 0.05), whereas in the animal models who had received IL-12 and B(7 - 1) co-immunization, prolonged survival time was showed which had statistical significance compared with that in control group (66.0 d, P < 0.05). CONCLUSION: B(7 - 1) and IL-12 modified tumor cell vaccines can induce anti-tumor immunity through stimulating lymphocytes proliferation and inducing recognition and cytotoxic effects of CTLs on tumor cells. An obvious synergistic effect exists when the two cytokines are combined. Combined immunogenic therapy with IL-12 and B(7 - 1) should prove to be a promising therapeutic strategy in ovarian cancer.

Abdominal Neoplasms↗
PubMed2000

Study on modified cold storage method of rat livers with self-made hyd solution.

OBJECTIVE: To investigate the cold preservation effect of rat livers by modified storage method with self-made HYD solution. METHODS: The modified method was that the vascular bed of rat livers was expanded with an additional 20 to 40ml self-made HYD solution/100g liver. After removing the liver, the extra HYD solution expressed as % liver weight was entrapped via portal infusion by tying off the supra-and infra hepatic inferior vena cava. According to the amount of extra HYD solution, 40 rats were randomly divided into four groups including: control group with conventional storage method, 20% group, 30% group and 40% group. The preservation effect of modified storage method with that of conventional storage method by using isolated perfused rat liver model was compared. RESULTS: Bile production and all the indices of hepatic microcirculation including portal perfusion pressure, endothelin-1 in the effluent, trypan blue distribution time and histology in modified method groups were significantly superior to those in control group (P < 0.05). The liver enzymes in 30% group were markedly lower than those in control group (P < 0.05). The preservation effect of rat liver in 30% group was the best among the modified method groups. CONCLUSION: The modified cold stage method is effective and may have potential for clinical application for liver preservation.

Alanine Transaminase↗
PubMed2003

[Nonlinear regression analysis of cardiac enzyme kinetic model with modified simplex method and DUD method using SAS software].

OBJECTIVE: To compare the efficacy of modified simplex method and DUD method using SAS software for nonlinear regression analysis of cardiac enzyme kinetic model. METHOD: By using cardiac enzyme kinetic one-compartment mathematical model, nonlinear regression results obtained from DUD methods using SAS software and modified simplex method were compared with the same starting values to fit the creatine kinase curves in 22 patients with acute myocardial infarction. RESULTS: The parameters calculated by DUD method and modified simplex method were the same in 4 cases, and smaller least square was resulted from DUD method in 4 cases. In the other 14 cases, modified simplex method yielded smaller least square. The mean value of the least square calculated by modified simplex method was less than that by DUD method (867 747 vs 6 712 989, P=0.013). CONCLUSION: Modified simplex method appears to be more adequate than DUD method in estimating circulatory parameters of the one-compartment model.

Humans↗
PubMed1992

Uptake, intracellular transport, and degradation of polyethylene glycol-modified asialofetuin in hepatocytes.

Polyethylene glycol (PEG) is attached to proteins in order to increase their half-life in the circulation and reduce their immunogenicity in vivo. For many applications involving "targeting" molecules, it is important to know how PEG modification of the molecule affects its interaction with a receptor and the subsequent internalization, intracellular transport, and lysosomal degradation. As a model system, we used asialofetuin, which binds to the galactose receptor of hepatocytes, because removal of sialic acid exposes galactose residues. We modified asialofetuin by attaching various amounts of PEG of molecular weight 1900 or 5000. The preparations were labeled with 125I so that endocytosis and degradation could be followed in suspended hepatocytes. Depending on the number of PEG molecules attached, receptor-mediated uptake was affected to varying degrees. If two-thirds of the exposed amino groups of the asialofetuin molecule were modified, the rate of uptake decreased to less than one-fourth of controls; degradation of endocytosed molecules was 12% of controls. The reduction in endocytic uptake was due to a reduced rate of formation of the receptor-ligand complex. Subcellular frationation in density gradients showed that PEG-modified asialofetuin is transported intracellularly and degraded in the same manner as the native protein, but the rate of proteolysis is reduced. This observation explains the paradoxical result of experiments with injection of modified asialofetuin into rats in vivo: even though the clearance of one preparation of PEG-asialofetuin was much slower than that of the native protein, accumulation of radioactivity in the liver from the modified protein was twice as high. The hepatocytes accounted for 85% of the hepatic accumulation of either PEG-modified or native asialofetuin in vivo.

Animals↗
PubMed2004

[Construction and immune potency of recombinant adenovirus containing codon-modified HIV-1 gp120].

BACKGROUND: To construct replication-deficient recombinant adenovirus expressing wild and codon-modified HIV-1 gp120. METHODS: The viral codons were changed to the codon usage of highly expressed mammal gene, the resulting modified gp120 gene was synthesized. The wild and modified gp120 genes were cloned into shuttle vector pShuttle-CMV respectively, and then the constructed plasmids containing gp120 gene was cotransformed with the backbone vector pADeasy-1 into E.coli BJ5183. Transfection of the recombinant AdEasy plasmid into 293 cells was performed to obtain recombinant adenoviruses. The mice were immunized with the recombinant adenoviruses. Their immunogenicity was evaluated by testing antibody and CTL levels of immunized mice. RESULTS: Two strains of recombinant adenovirus expressing wild and codon-modified HIV-1 gp120 were obtained. The protein expressing level of the recombinant adenoviruses containing modified genes was much higher than that containing wild genes. The mice immunized with recombinant adenoviruses elicited HIV-1 specific antibody and CTL response. The rAd-mod gp120 group was better than the rAd-wt gp120 group. CONCLUSION: Replication-deficient recombinant adenovirus expressing HIV-1 gp120 can elicit HIV-1 specific humoral and cellular response, the codon-modified recombinant virus was more efficient than the native.

AIDS Vaccines↗
PubMed2005

[Sequence-dependent cleavage of HBV DNA by combining with triple helix-forming oligodeoxyribonucleotides modified with manganese porphyrin in vitro].

OBJECTIVE: To observe the ability of triple helix-forming oligonucleotides (TFO) modified with manganese porphyrin to combine with and cleave HBV DNA fractions. METHODS: The ends of TFO were modified with manganese porphyrin and acridine; At 37 degrees C and pH 7.4 condition in vitro, TFO modified with manganese porphyrin and acridine were bound with 32P labeled HBV DNA fragments, the affinity and specificity binding to target sequence were tested by electrophoretic mobility shift and DNase 1 footprinting assays, the ability to cleave HBV DNA fractions was observed with cleavage experiments. RESULTS: TFO modified with manganese porphyrin and acridine could bind to target sequence in a sequence-dependent manner with Kd values of 3.5 x 10(-7) mol/L and a relative affinity of 0.008. In the presence of KHSO5, TFO modified with manganese porphyrin and acridine could cleave target sequence in the region forming triple DNA. CONCLUSION: In the presence of KHSO5, TFO modified with manganese porphyrin and acridine could cleave target HBV-DNA in sequence-dependent manner.

Binding, Competitive↗
PubMed2005

Physician documentation in support of appropriate billing with modifier '-25'.

What is one of the surest ways for a practice to lose revenue and invite the unwanted attention of government auditors? By failing to select and document the appropriate use of modifiers to code for services provided. For example, the Office of Inspector General (OIG) has made it known that they will closely review professional billing with the use of modifier '-25.' The use of this particular modifier appeared on their radar screen, because in one recent year Medicare paid approximately 1.7 billion dollars for Evaluation and Management (E/M) services billed with modifier '-25'. The OIG feels an increase of this amount warrants further investigation to determine if these claims were billed and reimbursed appropriately. Additionally, Oklahoma Medicaid auditors are actively scrutinizing the use of modifier '-25' to a degree even more stringent than Medicare and the CPT code book's description allow. Therefore, it is important that physicians be knowledgeable in determining when it is appropriate to bill using this modifier.

Current Procedural Terminology↗
PubMed2005

[Degradation of nitrobenzene in aqueous solution by modified ceramic honeycomb-catalyzed ozonation].

Comparative experiments of modified ceramic honeycomb, ceramic honeycomb-catalyzed ozonation and ozonation alone were conducted with nitrobenzene as the model organic pollutant. It was found that the processes of modified ceramic honeycomb and ceramic honeycomb-catalyzed ozonation could increase the removal efficiency of nitrobenzene by 38.35% and 15.46%, respectively, compared with that achieved by ozonation alone. Under the conditions of this experiment, the degradation rate of modified ceramic honeycomb-catalyzed ozonation increased by 30.55% with the increase of amount of catalyst to 5 blocks. The degradation rate of three process all increased greatly with the increase of temperature and value of pH in the solution. But when raising the pH value of the solution to 10.00, the advantage of modified ceramic honeycomb-catalyzed ozonation processes lost. The experimental results indicate that in modified ceramic honeycomb-catalyzed ozonation, nitrobenzene is primarily oxidized by *OH free radical in aqueous solution. The adsorption of nitrobenzene is too limited to have any influence on the degradation efficiency of nitrobenzene. With the same total dosage of applied ozone, the multiple steps addition of ozone showed a much higher removal efficiency than that obtained by one step in three processes. Modified ceramic honeycomb had a relative longer lifetime.

Catalysis↗
PubMed2005

[Establishment of a modified larynx transplantation model with inbred F344 rat].

BACKGROUND & OBJECTIVE: As a non-vital organ, transplantation of larynx lags behind that of the other organs, but it is supposed that if induction of immunologic tolerance of the host to the allograft comes true, larynx transplantation would be an optimal approach for management of advanced laryngeal cancer as well as functional reconstruction of the larynx. This study was designed to establish a modified rat model of larynx transplantation to improve survival rate of the recipients and vitality of the allografts, and provide an optimal rat model for further research on immunology and induction of immunologic tolerance in larynx transplantation. METHODS: A Strome's model of larynx transplantation was repeated with inbred F344 rats (40 rats in Stroma model A and 40 rats in Stroma model B). A modified model of larynx transplantation was developed (40 rats), in which both superior thyroid arteries and ascending pharyngeal arteries were preserved, combining with the hyoid, base of the tongue, hypopharynx, larynx, thyroid, part of the cervical trachea and cervical esophagus as an allograft complexity. The blood supply of the allograft was reconstructed through end-to-end anastomosis of the donors' carotid arteries with the recipients' carotid artery and anterior jugular vein. The recipients were injected with 15 ml of 5% glucose normal saline (GNS) through contralateral external jugular vein intraoperatively, and injected subcutaneously with 5 ml of 5% GNS daily for 3-5 days postoperatively. Morphology of the allografts and patency of the arteries and veins were observed under microscope 7 days after operation, and vitality of the allogafts was assessed pathologically. Survival of the recipients and vitality of the allografts in Strome model and modified model were compared. RESULTS: Survival rates of the recipient rats were 70% in Strome model A, 85% in Strome model B, and 95% in modified model; the survival rates of the allografts were 30% in Strome model A, 40% in Strome model B, and 80% in modified model, respectively. CONCLUSIONS: Compare with Strome models, the modified model of larynx transplantation reduces mortality of the recipients through enhancing perioperative care, improves vitality of the allografts through including both ascending pharyngeal arteries and superior thyroid arteries in the allograft complexity, which increases blood supply of the allografts and reduces micro-circulation resistance, therefore, reduces the micro-circulation malfunction of the allografts.

Anastomosis, Surgical↗
PubMed2006

Modified techniques for adult-to-adult living donor liver transplantation.

BACKGROUND: Because of critical organ shortage, transplant professionals have utilized living donor liver transplantation (LDLT) in recent years. We summarized our experience in adult-to- adult LDLT with grafts of right liver lobe by a modified technique. METHODS: From January 2002 to August 2005, 24 adult patients underwent living donor liver transplantation with grafts of the right liver lobe at West China Hospital, Sichuan University, China. Twenty-two patients underwent modified procedures designed to improve the reconstruction of the right hepatic vein and the tributaries of the middle hepatic vein by interposing a great saphenous vein (GSV) graft and the anastomosis of the hepatic arteries and bile ducts. RESULTS: No severe complications and death occurred in all donors. In the first 2 patients, (patients 1 and 2), operative procedure was not modified. One patient suffered from "small-for-size syndrome" and the other died of sepsis with progressive deterioration of graft function. In the rest 22 patients (patients 3 to 24), however, the procedure of venous reconstruction was modified, and better results were obtained. Complications occurred in 7 recipients including acute rejection (2 patients), hepatic artery thrombosis (1), bile leakage (1), intestinal bleeding (1), left subphrenic abscess (1), and pulmonary infection (1). One patient with pulmonary infection died of multiple organ failure (MOF). The 22 patients underwent direct anastomosis of the right hepatic vein to the inferior vena cava (IVC), 9 direct anastomosis plus the reconstruction of the right inferior hepatic vein, and 10 direct anastomosis plus the reconstruction of the tributaries of the middle hepatic vein by interposing a GSV graft to provide sufficient venous outflow. Trifurcation of the portal vein was met in 3 patients. Venoplasty or separate anastomosis was performed. The ratio of graft to recipient body weight ranged from 0.72% to 1.17%. Among these patients, 19 had the ratio <1.0% and 4 <0.8%, and the ratio of graft weight to recipient standard liver volume was between 31.86% and 62.48%. Among these patients, 10 had the ratio <50% and 2 <40%. No "small-for-size syndrome" occurred in the 22 recipients who were subjected to modified procedures. CONCLUSIONS: With the modified surgical techniques for the reconstruction of the hepatic vein to obtain an adequate outflow and provide a sufficient functioning liver mass, living donor liver graft in adults using the right lobe can be safe to prevent the "small-for-size syndrome".

Adult↗
PubMed2006

[Effect of modified MAID regimen for patients with advanced soft tissue sarcoma].

BACKGROUND & OBJECTIVES: Clinical study suggests that 72-hour continuous infusion (CIV) of MAID regimen is more effective and achieves longer time of no progression than ADR-based two-drug regimen in advanced soft tissue sarcoma (ASTS) treatment, but has no improvement on the long-term survival. Because of the severe grade 3/4 toxicities as well as treatment-related deaths, the regimen has not been widely applied in ASTS. This study was to investigate the efficacy and toxicity of the modified MAID regimen in ASTS treatment. METHODS: In the modified regimen, adriamycin (ADR) was substituted with tetrahydropyranyl adriamycin (THP-ADR) and the application of ifosfamide (IFO) was modified. All enrolled patients received chemotherapy (IFO 2,000 mg . m(-2), 4h, day 1-3; mesna 1,200 mg . m(-2) at 0, 4 and 8 hours of IFO infusion, day 1-3; THP-ADR 20 mg . m-2 and dacarbazine (DTIC) 333.3 mg . m(-2) were mixed in the same bag or pump, CIV for 3 days). The therapy was repeated every 3 weeks for at least 2 cycles before evaluating the effects and toxicities. The patients received follow-up every 2 months after completing 2 cycles until the study was finished. Life table was used to calculate long-term survival rates and time to progression. RESULTS: Fifty-four cases of evaluable patients had completed at least 2 cycles of modified MAID chemotherapy. The overall response rate was 42.59%. The toxicities were mild. Grade 3/4 neutropenia and thrombocytopenia were 25.93% and 16.17%, respectively. Neutropenia fever was 11.11%. There were no other toxicities, such as hepatic and renal toxicities; no central nervous system toxicity and treatment-related deaths. During 2 year follow-up, time to progression was 7 months, 1- and 2- year survival rates were 61.11% and 36.36%, respectively. CONCLUSIONS: Modified MAID regimen simplifies the application of treatment procedure compared with original regimen, which three drugs have to be CIV simultaneously. Moreover the modified MAID regimen has better survival rates in ASTS, with milder toxicity and better tolerance.

Adolescent↗
PubMed1992

rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids.

p21ras and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxyl-terminal sequence motif, Caa(M/S/L), where a is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modified preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rab1B (GGCC), rab2 (GGCC), and rab5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Caa(M/S/L) motif. We now show that these proteins function as specific acceptors for geranylgeranyl in vitro and are modified by 20-carbon isoprenyl groups in COS cells metabolically labeled with [3H]mevalonate. Proteins encoded by rab4 and rab6, with yet another distinct carboxyl-terminal motif (xCxC), are similarly modified by 20-carbon isoprenoids in vitro and in vivo. The geranylgeranyl modification of rab5 protein (CCSN) is catalyzed by an enzyme in brain cytosol but not by a purified geranylgeranyltransferase that modifies GTP-binding proteins with the CaaL motif. Unlike the prenylation of proteins with Caa(M/S/L) termini, the prenylation of rab5 protein is not inhibited by a synthetic peptide based on its carboxyl-terminal sequence (TRNQCCSN). When cellular isoprenoid synthesis is blocked by treatment of cells with lovastatin, rab proteins that are normally localized in membranes of the endoplasmic reticulum, Golgi apparatus, and endosomes accumulate in the cytosol. This change in rab protein localization is reversed by providing cells with mevalonate. These findings suggest that geranylgeranyl modification underlies the ability of rab GTP-binding proteins to associate with intracellular membranes, where they are postulated to function as mediators of vesicular traffic.

Animals↗
PubMed1991

Acetaldehyde-modified proteins and associated antibodies in ethanol-fed rats.

Rats fed ethanol for periods of 3-27 months were found to produce an immune response to acetaldehyde-modified proteins, whereas treatment for a shorter period (3 weeks) did not lead to such a response. The antibodies from the ethanol-fed animals were shown to be reactive with a series of proteins modified by acetaldehyde in vitro. This suggests that reactivity towards the modified proteins is independent of the carrier protein, with antibodies being raised which interact solely with the acetaldehyde-containing adduct and not the protein itself. We have also been able to demonstrate the presence of modified proteins in the liver cytosol of these ethanol-fed rats. However, unlike previous reports which suggested that only one protein was modified, we found that many cytosolic proteins were modified. This suggests that the modification of proteins by acetaldehyde is less specific than was previously thought.

Acetaldehyde↗
PubMed1991

Reversible inhibition of neutrophil elastase by thiol-modified alpha-1 protease inhibitor.

We have modified the single cysteine residue of alpha 1-protease inhibitor (alpha 1-PI) with HgCl2, methylmethane thiosulfonate, oxidized glutathione (GSSG), and N-(1-anilinonaphthyl-4)maleimide (ANM). Whereas native alpha 1-PI combines rapidly and quasi-irreversibly with neutrophil elastase, the thiol-modified alpha 1-PI derivatives are dissociable reversible competitive inhibitors of the enzyme, with values of Ki in the range of 6-7 nM. Removal of the thiol modifications restores the rapid irreversible mode of inhibition. Once native alpha 1-PI has combined with neutrophil elastase, the enzyme-inhibitor complex retains a reactive thiol group, but the two proteins can no longer be dissociated by subsequent reaction with ANM, even after exposure to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From kinetic measurements of fluorescence, ANM-modified alpha 1-PI combines with neutrophil elastase via an apparent biomolecular process with a second order rate constant on the order of 10(5) M-1 S-1. We estimate a dissociation rate constant on the order of 10(-3) S-1. The emission of ANM-modified alpha 1-PI is increased in intensity and blue shifted from the maximum in ANM-modified cysteine, consistent with a predominantly nonpolar environment. Association with neutrophil elastase results in an additional blue shift with further increase in intensity, consistent with a further decrease in polarity of the environment of the cysteine. Modification with methylmethane thiosulfonate or GSSG results in a small decrease in quantum yield and a red shift in the tryptophan emission spectrum of the modified inhibitor, suggestive of increased polarity of the environment of at least 1 of the 2 tryptophan residues in alpha 1-PI. These changes are reversed by dithiothreitol and are consistent with a conformational change which transforms the inhibitory activity from a rapid, irreversible mode in native alpha 1-PI to a dissociable competitive mode in the mixed disulfide derivatives.

Binding, Competitive↗
PubMed1989

Direct evidence for a protein recognized by a monoclonal antibody against oxidatively modified LDL in atherosclerotic lesions from a Watanabe heritable hyperlipidemic rabbit.

Low density lipoproteins (LDL) that have been oxidatively modified have been implicated in the pathogenesis of atherosclerosis. Monoclonal antibodies were generated against oxidatively modified human low density lipoproteins (OxLDL); these antibodies reacted with OxLDL, but did not react with native LDL, either in an enzyme-linked immunosorbent assay (ELISA) or a Western blot analysis. The anti-OxLDL antibodies did react with other modified forms of LDL (eg, acetylated LDL, malondialdehyde-modified LDL, and cell-modified LDL) that were recognized by the scavenger receptor on macrophages. Single- and double-label immunofluorescence of atheromatous lesions from a Watanabe heritable hyperlipidemic (WHHL) rabbit demonstrated some colocalization of proteins detected by anti-LDL and anti-OxLDL antibodies. However, clearly there were also areas stained by the anti-OxLDL antibodies that did not stain with anti-LDL. Staining of the lesion by the anti-OxLDL antibody was abolished by adsorption of the antibody with OxLDL, but not by adsorption with LDL or bovine serum albumin. Arterial tissue from a control New Zealand White rabbit did not show staining with anti-LDL or anti-OxLDL antibodies. These observations suggest that OxLDL (or possibly other proteins recognized by the anti-OxLDL antibody) is present in atheromatous lesions of WHHL rabbits, and are consistent with oxidatively modified lipoproteins having a role in atherogenesis.

Animals↗