Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Yield stability”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,099 records · Page 61Linked to original sources
PubMed2003

Stability of protein production from recombinant mammalian cells.

One of the most important criteria for successful generation of a therapeutic protein from a recombinant cell is to obtain a cell line that maintains stability of production. If this is not achieved it can generate problems for process yields, effective use of time and money, and for regulatory approval of products. However, selection of a cell line that sustains stability of production over the required time period may be difficult to achieve during development of a therapeutic protein. There are several studies in the literature that have reported on the instability of protein production from recombinant cell lines. The causes of instability of production are varied and, in many cases, the exact molecular mechanisms are unknown. The production of proteins by cells is modulated by molecular events at levels ranging from transcription, posttranscriptional processing, translation, posttranslational processing, to secretion. There is potential for regulation of stability of protein production at many or all of these stages. In this study we review published information on stability of protein production for three industrially important cell lines: hybridoma, Chinese hamster ovary (CHO), and nonsecreting (NS0) myeloma cell lines. We highlight the most likely molecular loci at which instability may be engendered and indicate other areas of protein production that may affect stability from mammalian cells. We also outline approaches that could help to overcome the problems associated with unpredictable expression levels and maximized production, and indicate the consequences these might have for stability of production.

Animals↗
PubMed1996

Plasma bovine placental lactogen concentration throughout pregnancy in the cow; relationship to stage of pregnancy, fetal mass, number and postpartum milk yield.

This study characterized the peripheral plasma placental lactogen (bPL) profile throughout gestation and examined the relationship between the stage of gestation, fetal mass, number, and postpartum lactation with circulating levels of bPL in Holstein cows after nonsurgical embryo transfer. Cows (n = 12) were divided into two groups: Group 1 = single embryo recipient cows (n = 5); Group 2 = twin embryo recipient cows (n = 7). Blood was collected about every third day from Day 0 (Day 0 was defined as the first day of standing estrus), then daily for the last 10 d of gestation, and sampling was stopped 1 d postpartum. The cows were milked twice daily at 0800 and 1800 hr. Two twin-embryo recipient cows had abnormal pregnancies; therefore, their data were excluded from that of the group and reported separately. The time trend concentrations of plasma bPL were significantly affected by the stage of gestation (P < 0.01) but not fetal number (P < 0.21). In both groups bPL levels remained low during the first two trimesters, then increased rapidly (P < 0.01) to peak concentrations between Days 200 and 220, and stabilized at this elevated level until parturition. Postpartum milk yields were indistinguishable between the singleton and twin bearing cows. Calf birth weight and postpartum lactation were both correlated (P < 0.01) to peripheral bPL concentration in singleton cows, however, this relationship decreased with a subsequent increase in fetal number. Cows giving birth prematurely to stillborn calves or to a schistosomus reflexus calf exhibited a deviating pBL profile. These results indicate that peripheral bPL levels are positively associated with the stage of gestation but not with fetal number. Otherwise, the peripheral pattern of bPL is a valuable index for predicting feto-placental viability.

Animals↗
PubMed2005

Multiflash experiments reveal a new kinetic phase of photosystem II manganese cluster assembly in Synechocystis sp. PCC6803 in vivo.

The assembly of Mn(2+) ions into the H(2)O oxidation complex (WOC) of the photosystem II (PSII) reaction center is a light-driven process, termed photoactivation. According to the "two-quantum" model, photoactivation involves two light-driven charge separations coupled to the photooxidation of Mn(2+) in order to form the first stable intermediate in a process that culminates in the oxidative assembly of four Mn(2+) ions and one Ca(2+) ion to form the active, higher valence (Mn(4)-Ca) center of the WOC. To better define the kinetics of the dark rearrangement and to gain some understanding of the basis for the very low quantum yield of the overall process, photoactivation experiments, involving different flash patterns, were conducted with Synechocystis sp. PCC6803. It was found that even the so-called first stable intermediate is readily lost during protracted (1-10 s) dark periods during photoactivation of Synechocystis cells. Low concentrations of the electron acceptor, DCBQ, improved the stability of the dark intermediates. The unstable photoactivation intermediates formed early in the photoactivation process were not, however, stabilized by the addition of Ca(2+), although the overall yield of photoactivation is enhanced by the additional Ca(2+). Measurements of the kinetics of fluorescence yield verify that Q(A)(-) to Q(B) electron transfer rates change during the course of photoactivation as the high potential form of Q(A)(-) is converted to the low potential form and show that DCBQ acts as an efficient electron acceptor from Q(A)(-) even while in its high potential form. In addition the approximately 150 ms phase corresponding to the originally described dark rearrangement of photoactivation, repetitive, double flash experiments, with a 10 s intervening dark period, reveals a faster, 15 ms phase that is accentuated by DCBQ.

Benzoquinones↗
PubMed2001

Active site structure and mechanism of human glyoxalase I-an ab initio theoretical study.

The structure of the active site of human glyoxalase I and the reaction mechanism of the enzyme-catalyzed conversion of the thiohemiacetal, formed from methylglyoxal and glutathione, to S-D-lactoylglutathione has been investigated by ab initio quantum chemical calculations. To realistically represent the environment of the reaction center, the effective fragment potential methodology has been employed, which allows systems of several hundred atoms to be described quantum mechanically. The methodology and the active site model have been validated by optimizing the structure of a known enzyme-inhibitor complex, which yielded structures in good agreement with the experiment. The same crystal structure has been used to obtain the quantum motif for the investigation of the glyoxalase I reaction. The results of our study confirm that the metal center of the active site zinc complex plays a direct catalytic role by binding the substrate and stabilizing the proposed enediolate reaction intermediate. In addition, our calculations yielded detailed information about the interactions of the substrate, the reaction intermediates, and the product with the active site of the enzyme and about the mechanism of the glyoxalase I reaction. The proton transfers of the reaction proceed via the two highly flexible residues Glu172 and Glu99. Information about the structural and energetic effect of the protein on the first-shell complex has been attained by comparison of the structures optimized in the local protein environment and in a vacuum. The environment of the zinc complex disturbs the Cs symmetry found for the complex in a vacuum, which suggests an explanation for the stereochemical behavior of glyoxalase I.

Catalytic Domain↗
PubMed1989

Stabilization, purification, and characterization of glutamate synthase from Clostridium pasteurianum.

Clostridium pasteurianum possesses a high level of glutamate synthase (EC 1.4.1.14) activity and cell yield when grown on 4 mM ammonium chloride and molasses as the sole nitrogen and carbon sources, respectively. The enzyme activity is stabilized by addition of alpha-ketoglutarate, EDTA, and 2-mercaptoethanol. Ammonium sulfate precipitation and single-step combined gel and ion-exchange chromatography followed by fractional dialysis yield a homogeneous protein with 40% recovery of the glutamate synthase activity. The native enzyme (Mr congruent to 590,000) gives five different subunits (as dimers) upon SDS gel electrophoresis. The enzyme has been characterized for pH and temperature optimum, substrate specificity, Kmapp values, energy of activation, half-life, and thermal stabilization. Metal ions and citric acid cycle metabolites do not affect the enzyme activity. Glutamate synthase shows fluorescence maximum at 370 nm when excited at 280 nm. The fluorescence is quenched upon the addition of NADH. Spectroscopic examination of the enzyme gave absorption maximum at 280 and none at 380 and 440 nm, indicating the absence of iron and flavin. The absence of iron and flavin was also confirmed by atomic absorption, chemical analysis, and fluoroscopy, respectively. The C. pasteurianum enzyme differs from that of other aerobic bacterial sources.

Chemical Precipitation↗
PubMed2002

Synthesis and characterization of a series of novel monoacylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids, as skin antioxidants.

A series of novel monoacylated vitamin C derivatives were chemically synthesized with a stable ascorbate derivative, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), and acid anhydrides in pyridine. Their solubility in organic phase, thermal stability, radical scavenging activity, and in vitro skin permeability was evaluated. These monoacylated derivatives were identified as 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids (6-Acyl-AA-2G) by UV spectra, elemental analyses, and nuclear magnetic resonance spectroscopy. The reactions afforded 6-Acyl-AA-2G in high yields (30-60%). 6-Acyl-AA-2G exhibited satisfactory stability in neutral solution comparable to that of a typical stable derivative, AA-2G, and also showed the radical scavenging activity. The lipid solubility of 6-Acyl-AA-2G was increased with increasing length of their acyl group. Increased skin permeability was superior to those of AA-2G and ascorbic acid (AsA). 6-Acyl-AA-2G that is susceptible to enzymatic hydrolysis by tissue esterase and/or alpha-glucosidase produces AA-2G and AsA, which is in the skin tissues. Thus, these findings indicate that the novel vitamin C derivatives presented here, 6-Acyl-AA-2G, may be effective antioxidants in skin care and medicinal use.

1-Octanol↗
PubMedDate not supplied

Radiohalogenation of proteins: an overview of radionuclides, labeling methods, and reagents for conjugate labeling.

Direct labeling of proteins with radionuclides of iodine will continue to be the method of choice to answer questions addressed in many future studies. However, it seems likely that a increasing number of applications of radiohalogenated proteins will require, or benefit from, conjugate labeling. While many radiohalogen conjugates have been studied, a large proportion of them have only undergone preliminary studies to date, leaving a question of their overall utility. Phenolic conjugates give good radioiodination labeling yields, but mixtures of radiohalogenated products and problems with in vivo stability can be expected. This fact, along with the fact that phenolic compounds do not have a general application to radiohalogens, makes them less attractive than other alternatives. Radiohalogen labeling through the use of organometallic intermediates has proven to be facile, resulting in high yields of high specific activity labeled small-molecule conjugates. Although the choice of which organometallic intermediate to use may depend somewhat on the radionuclide employed, arylstannanes appear to have the most general applicability. Fluorine-18 labeling of small-molecule conjugates has been best accomplished by ipso aromatic nucleophilic substitution (exchange) reactions. Radiohalogenated small molecules have been prepared which can be conjugated with specific functional groups (e.g. amines, sulfhydryl groups, and carbohydrates) or conjugated nonspecifically with groups in the proximity of the conjugate when it is photolyzed. On the basis of previous studies, good conjugation yields (i.e. 60-90%) can be expected for reactions with specific groups, whereas low yields (i.e. 1-5%) can be expected for conjugations with reactive nitrenes and carbenes. However, recent developments in the chemistry of conjugates that produce nitrenes and carbenes will likely improve the radiolabeling yields. There have been too few comparative studies to readily assess which is the best approach to take when beginning a study involving radiohalogenation of a protein or peptide. However, it is clear that radiohalogenated conjugates of proteins can offer an advantage over direct labeling in that conjugates may be designed which provide some control over in vivo stability and secondary distribution of metabolites. Conjugates can be prepared which are designed to utilize in vivo biochemical processes to release a radiohalogenated small molecule from a tissue (i.e. kidney or liver) or retain the radioactivity at the target tissue (e.g. tumor). Aside from the designing of conjugates with linking molecules for desired biological effects, the ultimate future goal for the radiolabeling chemical should be to prepare protein conjugates which can be radiohalogenated in a single one-step procedure.

Animals↗
PubMed2004

Increased thermal stability of a poled electro-optic polymer using high-molar-mass fractions.

Polymer fractionation yields materials with higher average molar masses and thus substantially increased glass transition temperatures. We apply this technique to a photoaddressable polymer that exhibits high electro-optic coefficients, in order to enhance the thermal stability of the poled material. The degree of orientation of the dipolar chromophores is investigated with both pyroelectric and electro-optic measurements. The pyroelectric measurements at elevated temperatures show no significant contribution of beta relaxation as compared to alpha relaxation.

Journal Article↗
PubMed2002

Effects of stabilized rice bran, its soluble and fiber fractions on blood glucose levels and serum lipid parameters in humans with diabetes mellitus Types I and II.

Stabilized rice bran (SRB), a source of complex carbohydrates, tocols, gamma-oryzanols, and polyphenols, was treated with carbohydrases and heat to yield two fractions, rice bran water solubles (RBWS), and rice bran fiber concentrates (RBFC). Stabilized rice bran and its fractions were fed for 60 days to insulin-dependent and noninsulin-dependent diabetes mellitus (IDDM = Type I and NIDDM = Type II) subjects to determine possible effects on serum hemoglobin, carbohydrate and lipid parameters. The Type I subjects (n = 22, 26, and 20) fed Stabilized rice bran, rice bran water solubles, and rice bran fiber concentrates plus AHA Step-1 diet reduced glycosylated hemoglobin 1%, 11%, and 10%, respectively. The fasting serum glucose levels were also reduced significantly (P < 0.01) with stabilized rice bran (9%), rice bran water solubles (29%), and rice bran fiber concentrates (19%).The Type II subjects (n = 31, and 26) fed rice bran water solubles and rice bran fiber concentrates plus AHA Step-1 diet had decreased levels of glycosylated hemoglobin (15% and 11%) and fasting glucose (33% and 22%; P < 0.001), respectively. Serum insulin levels were increased (4%) with rice bran water solubles in both types of diabetes. The reduction of glycosylated hemoglobin and a slight increase in insulin levels indicate that consumption of rice bran water solubles can control blood glucose levels in human diabetes. Serum total cholesterol, LDL-cholesterol, apolipoprotein B, and triglycerides levels were reduced with rice bran fiber concentrates in the Type I (10, 16, 10, 7%) and Type II groups (12, 15, 10, 8%), respectively. These results indicate that rice bran water solubles significantly reduces hyperglycemia (P < 0.01), whereas rice bran fiber concentrates reduces hyperlipidemia (P < 0.05) in both types of diabetes. Therefore, these natural products can be used as nutritional supplements for the control of both types of diabetes mellitus in humans.

Journal Article↗
PubMed2004

Radiolabelling morpholinos with 188Re tricarbonyl provides improved in vitro and in vivo stability to re-oxidation.

BACKGROUND: For pretargeting and other nuclear medicine applications, it may eventually be useful to radiolabel phosphodiamidate morpholinos (MORFs) with therapeutic radionuclides such as 188Re. However, by preparing 188Re-MORFs labelled conventionally with MAG3 as chelator, we have observed unacceptable levels of oxidation to perrhenate in vitro and in vivo in mice. OBJECTIVE: To improve upon stability, tricarbonyl labelling was considered since tricarbonyl complexes are thought to stabilize metals in low oxidation states. METHODS: An amine derivatized 25 mer MORF was conjugated with either NHS-MAG3 or NHS-Hynic. The MAG3 conjugated MORF was radiolabelled conventionally with 188Re while the Hynic conjugated MORF was radiolabelled through its tricarbonyl intermediate. Using a commercial kit modified with additional reducing agent over that required for the preparation of the 99mTc tricarbonyl complex [99mTc(CO)3(H2O)3]+, we demonstrated that the equivalent 188Re tricarbonyl, [188Re(CO)3(H2O)3]+, could be prepared. Simple incubation at elevated temperatures with the Hynic conjugated MORF then provided 188Re-(CO)3-Hynic-MORF. Confirmation was achieved by a shift assay using a complementary MORF conjugated polymer and size exclusion HPLC. To evaluate the relative stability of the tricarbonyl labelled MORF compared to the MAG3 labelled MORF in vitro, the radiolabelled MORFs were incubated in phosphate buffer and the presence of perrhenate measured periodically by strip chromatography. Stability in vivo was evaluated by biodistribution studies in normal mice. RESULTS: The overall yields for tricarbonyl intermediates averaged greater than 90% for 99mTc and 60-80% for 188Re. Yields following subsequent labelling to Hynic-MORF were about 60-80% for 99mTc and 15-20% for 188Re. The in vitro stability results in phosphate buffer showed that 188Re-MAG3-MORF was fully oxidized by 48 h while 188Re-(CO)3-Hynic-MORF was less than 20% oxidized at that time. Similarly, the 188Re-(CO)3-Hynic-MORF biodistribution in normal mice showed lower radioactivity level in stomach, intestines and thyroid compared with 188Re-MAG3-MORF. CONCLUSION: 188Re-tricarbonyl labelling of Hynic conjugated MORFs may be considerably more stable to oxidation than the MAG3 labelled MORFs and therefore more suitable for radiotherapy trials.

Animals↗
PubMed1993

Diagnostic methods and treatment modalities of dry eye conditions.

One may view dry eye conditions as a group of diseases in which the ocular surface is adversely affected. Tear film instability invariably leads to some degree of cellular surface damage over the cornea and conjunctiva. In turn, ocular epitheliopathy may adversely affect tear film stability. The clinical presentation of the disease may not yield a clue as to its etiology. In recent years considerable progress was made both in the diagnosis and the treatment of the disease and promising studies are planned or are underway. The diagnostic techniques can be divided into four groups. The first is concerned with the clinical presentation. The second is concerned with the bulk properties of the aqueous tears including dynamic characteristics, composition, and colligative properties. The third is tear-film related and includes the film break-up time, evaporation rate, and lipid abnormality. The fourth is concerned with the ocular surface and includes vital staining, impression cytology, and surface microscopy. The most promising attempts are being made in the second group by attempting to elucidate the role of enzyme and enzyme activator activity and inhibitor contents as well as the tear protein profiles and correlating them with the specific disease states. The treatment modalities belong to three major groups aside from surgical intervention; the supplementation, preservation, and the stimulation of tears. The modern version of tear supplementation is expected to include the topical use of efficacious aqueous formulations that typically contain film stabilizing polymers, nutrients, and/or--in the future--biochemically active ingredients such as enzyme activators and inhibitors.

Dry Eye Syndromes↗
PubMed2006

Thermofluor-based high-throughput stability optimization of proteins for structural studies.

Production of proteins well suited for structural studies is inherently difficult and time-consuming. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins' probability of yielding crystals, and optimization of these properties will improve success rates of crystallization. In the current study, we applied the thermofluor method as a high-throughput approach for identifying optimal protein formulation for crystallization. The method also allowed optimal stabilizing buffer compositions to be rapidly identified for each protein. Furthermore, the method allowed the identification of potential ligands, physiological or non-physiological, that can be used in subsequent crystallization trials. For this study, the thermally induced melting points were determined in different buffers as well as with additives for a total of 25 Escherichia coli proteins. Crystallization trials were set up together with stabilizing and destabilizing additives identified using thermofluor screening. A twofold increase in the number of crystallization leads was observed when the proteins were cocrystallized with stabilizing additives as compared with experiments without these additives. This suggests that thermofluor constitutes an efficient generic high-throughput method for identification of protein properties predictive of crystallizability.

Buffers↗
PubMed2004

(123)I-Labeled chitinase as specific radioligand for in vivo detection of fungal infections in mice.

UNLABELLED: Given the scarcity of diagnostic tools for invasive fungal infections, the aim of this project was to develop new, specific radiopharmaceuticals for diagnosis of fungal infections. Chitin, which is expressed in the fungal cell wall but is absent in mammalian and bacterial cells, represents a potentially selective target for development of tracers for fungal infections. ChiB_E144Q (ChiB = chitinase B) from Serratia marcescens was labeled with (123)I, and in vitro and in vivo studies were assessed. METHODS: (123)I labeling of ChiB_E144Q from S. marcescens by direct iodination was characterized by high-pressure liquid chromatography (HPLC), and stability was evaluated. The in vitro binding properties of the compound to living bacteria, Candida albicans, and Aspergillus fumigatus were examined. Scintigraphy was performed and in vivo characteristics were studied in mice with infected thigh muscles. RESULTS: An average radiochemical yield of 35% was obtained. Radiochemical purity was >97% with a stability of >24 h as determined by HPLC and instant thin-layer chromatography. The average specific activity of the noncarrier-free (123)I-chitinase was 9.25 MBq/ micro g of enzyme. Binding assays showed virtually no binding to Eschericha coli and Staphylococcus aureus, and 2.4 x 10(3) Bq per 1 x 10(7) cells for A. fumigatus and 3.0 x 10(3) Bq per 1 x 10(7) cells for C. albicans (P < 0.05). Binding of the tracer dropped to almost zero for organisms previously incubated with a 50-fold excess of unlabeled enzyme. At 24 h after injection, target-to-nontarget (T/NT) ratios in mice were 20.6 +/- 3.6 for C. albicans and 15.2 +/- 3.7 for A. fumigatus infections, respectively, whereas T/NT ratios for S. aureus -and E. coli-infected thigh muscles or thigh muscles with a sterile inflammation did not exceed 4.9 +/- 2.6, 3.0 +/- 2.3, and 5.3 +/- 2.8, respectively (P < 0.05). Target-to-blood ratios for fungus-infected thighs were always >1. CONCLUSION: Our results show that (123)I-ChiB_E144Q has affinity in vitro for fungi. In vivo, the tracer accumulates in tissue infected with C. albicans and A. fumigatus but not in tissue infected with gram-positive or gram-negative bacteria, or in sterile inflammations, proving it to be a valuable SPECT diagnostic.

Animals↗
PubMed2004

Formation and regeneration of protoplasts in Sclerotium rolfsii ATCC 201126.

AIMS: Different cultural conditions for forming and reverting protoplasts were systematically studied to establish a rapid and efficient protocol for Sclerotium rolfsii ATCC 201126. METHODS AND RESULTS: Osmotic stabilizer, lytic enzymes and mycelial age were the main factors influencing protoplast yields. An optimized protocol involving 1-h hydrolysis of 45-h-old mycelium with Trichoderma harzianum enzymes in a 1 : 1 (w/w) biomass : enzyme ratio and 0.6 mol l-1 MgSO4 as osmotic stabilizer was designed to produce approx. 2 x 109 protoplasts per gram biomass dry weight, with 99% viability. Differences on the lytic activity between batches of commercial enzymes were clearly evidenced. Protoplast release was highly efficient showing no remaining cell wall material as witnessed by fluorescent brightener 28. Up to 26% of purified protoplasts developed into the typical filamentous form after 50 h of incubation on 0.6 mol l-1 sucrose agar media. CONCLUSIONS: The methodology herein proposed allowed a rapid, inexpensive and efficient protoplast production. Optimum yields were higher or in the order of that elsewhere reported for other S. rolfsii strains and the required lytic time was significantly shorter. Purified protoplasts successfully reverted to the filamentous morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: The present research reports the former protocol for the isolation and reversion of protoplasts in S. rolfsii ATCC 201126 providing key factors to ensure optimum results. In addition, the described procedure constitutes a starting point for downstream genetic manipulation.

Basidiomycota↗
PubMed1996

Iron Carbonyl-Promoted Isomerization of Olefin Esters to Their alpha,beta-Unsaturated Esters: Methyl Oleate and Other Examples.

Ultraviolet photolysis of stoichiometric amounts of methyl oleate and Fe(CO)(5) in hexanes solvent at 0 degrees C gives Fe(CO)(3)(eta(4)-alpha,beta-ester) in which the alpha,beta-unsaturated ester isomer of methyl oleate is stabilized by eta(4)-oxadiene pi coordination of the olefin and ester carbonyl groups to the Fe(CO)(3) unit. Treatment of the Fe(CO)(3)(eta(4)-alpha,beta-ester) with pyridine or CO liberates the free alpha,beta-ester, methyl octadec-trans-2-enoate, in 70% yield. The Fe(CO)(3) unit both catalyzes the olefin isomerization and stabilizes the alpha,beta-unsaturated ester, which results in the formation of the alpha,beta-ester in a yield far above that (3.5%) observed for simple catalyzed methyl oleate isomerization. The much smaller olefin esters, methyl 3-butenoate and ethyl 4-methyl-4-pentenoate, are isomerized under the same conditions to their alpha,beta-unsaturated esters in 94 and 90% yields, respectively. The effects of reaction conditions on the yield, the use of Fe(CO)(3)(cis-cyclooctene)(2) as a nonphotolytic catalyst, and the mechanism of this useful synthetic process are discussed.

Journal Article↗
PubMed1989

UvrABC incision of N-methylmitomycin A-DNA monoadducts and cross-links.

The Escherichia coli UvrABC endonuclease is a multisubunit enzyme that initiates the repair of a wide variety of DNA lesions in vivo by making dual incisions on a damaged strand at the eighth or ninth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the modified base. It has been hypothesized that UvrABC is able to recognize a broad spectrum of lesions because it does not recognize the lesion per se but rather gross helical distortions that the lesion induces in the DNA. Several lesions have recently been studied which are thermal stabilizing and are not believed to distort the DNA grossly, including the CC-1065-N-3-adenine and anthramycin-N-2-guanine adducts. We have studied the activity of UvrABC in vitro on another thermal stabilizing and nondistortive adduct, N-methylmitomycin A (NMA), a bifunctional DNA-alkylating agent that reacts with guanine on the side facing the minor groove, yielding either monoadducts or interstrand cross-links. NMA adducts increase the thermal stability of DNA, and theoretical calculations indicate that NMA adducts do not grossly distort the DNA helix. Our results show that UvrABC makes incisions at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to an NMA monoadduct, consistent with the incision pattern observed for the majority of other lesions that are also recognized by UvrABC. DNA containing a site-specific NMA cross-link was also recognized and incised by UvrABC. The rate of incision of NMA cross-linked DNA was about 200-fold higher in supercoiled molecules than in relaxed molecules, whereas the rate of incision of DNA containing NMA monoadducts was stimulated approximately 2-fold by supercoiling. The signal for UvrABC recognition and incision of damaged DNA is discussed in relation to the ability of UvrABC to incise NMA adducts as well as other nondistortive lesions.

Base Sequence↗
PubMed2003

Radiosynthesis and in vitro stability evaluation of various radioiodine-labelled beta-iodoalkylether prosthetic groups linked to model compounds.

A systematic comparative investigation into the in vitro radiochemical stabilities of model compounds containing radioiodinated beta-iodoethoxyl units and derivatives thereof, as well as those of similar compounds lacking a beta-oxygen to serve as control references, was undertaken. The radioiodinations were carried out in fair to modest yields by means of substitution of a tosyl group by iodide. Stability evaluations were carried out by incubating the labeled compounds in human blood serum at 37 degrees C and measuring free radioiodide by means of radio-HPLC and radio-TLC. The compounds containing beta-iodoethoxyl units displayed much superior stabilities than those without, while the presence of small alkyl or aryl groups in such a unit rendered an additional degree of stability to the carbon-iodine bond, especially over a long period.

Chromatography, High Pressure Liquid↗