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PubMed2004

Oligonucleotide incorporation of 8-thio-2'-deoxyguanosine.

[reaction: see text] 8-Thio-2'-deoxyguanosine (SdG) is a useful analogue of the abundant promutagen 8-oxo-2'-deoxyguanosine (OdG). Its synthesis and DNA incorporation using standard phosphoramidite chemistry is reported. To prevent oxidation during DNA synthesis, the sulfur was protected as a 2-(trimethylsilyl)ethyl sulfide. Subsequent treatment with TBAF yielded the desired 8-thiocarbonyl functionality. Melting studies with SdG revealed almost equal stabilities of SdG:dC and SdG:dA base pairs, lending insight into the base-pairing preferences of OdG.

Base Sequence↗
PubMed2006

Viewing single lambda site-specific recombination events from start to finish.

The site-specific recombination pathway by which the bacteriophage lambda chromosome is excised from its Escherichia coli host chromosome is a tightly regulated, highly directional, multistep reaction that is executed by a series of multiprotein complexes. Until now, it has been difficult to study the individual steps of such reactions in the context of the entire pathway. Using single-molecule light microscopy, we have examined this process from start to finish. Stable bent-DNA complexes containing integrase and the accessory proteins IHF (integration host factor) and Xis form rapidly on attL and attR recombination partners, and synapsis of partner complexes follows rapidly after their formation. Integrase-mediated DNA cleavage before or immediately after synapsis is required to stabilize the synaptic assemblies. Those complexes that synapsed (approximately 50% of the total) yield recombinant product with a remarkable approximately 100% efficiency. The rate-limiting step of excision occurs after synapsis, but closely precedes or is concomitant with the appearance of a stable Holliday junction. Our kinetic analysis shows that directionality of this recombination reaction is conferred by the irreversibility of multiple reaction steps.

Attachment Sites, Microbiological↗
PubMed1998

Determination of nafronyl in pharmaceutical preparations by means of stopped-flow micellar-stabilized room temperature phosphorescence.

The stopped-flow mixing technique was applied to micellar-stabilized room temperature phosphorimetry by measuring the fast appearance of the phosphorescent signal yielded by nafronyl in the presence of sodium dodecyl sulfate and thallium nitrate. This mixing system diminishes the time required for the deoxygenation of micellar medium by sodium sulfite, allowing a kinetic curve that levels off within only 5 s to be obtained. Phosphorescence enhancers thallium(I) nitrate, sodium dodecyl sulfate and sodium sulfite were optimized to obtain maximum sensitivity and selectivity. A pH value of 10.5 was selected as adequate for phosphorescence development. Two rapid, straightforward and automatic methods were proposed using the slope and amplitude of the kinetic curve, which are directly proportional to the nafronyl concentration, as analytical parameters. Calibration graphs were linear for the concentration range from 30 to 600 ng ml-1. Praxilene, the only commercial formulation containing nafronyl, was analysed by both proposed methodologies. Suitable recovery values were obtained.

Humans↗
PubMed2005

Synthesis, characterisation and theoretical calculations of 2,6-diaminopurine etheno derivatives.

Four derivatives of 2,6-diaminopurine (1) were synthesised and characterised. When 1 was reacted with chloroacetaldehyde, 5-aminoimidazo[2,1-i]purine (2), 9-aminoimidazo[2,1-b]purine (3), 9-aminoimidazo[1,2-a]purine (4) and diimidazo[2,1-b:2',1'-i]purine (5) were formed. The purified products (3-5) were fully characterised by MS, complete NMR assignments as well as fluorescence and UV spectroscopy. The purified, isolated yields of these products (3-5) varied from 2.5 to 30%. The relative stability of different tautomers was investigated by theoretical calculations. Fluorescence characteristics are also discussed and compared to the starting material 1 and a reference molecule 2-aminopurine.

2-Aminopurine↗
PubMed1994

Selection of catalytic antibodies for a biosynthetic reaction from a combinatorial cDNA library by complementation of an auxotrophic Escherichia coli: antibodies for orotate decarboxylation.

Antibodies capable of decarboxylating orotate were sought by immunization with a hapten designed to elicit antibodies with combining sites that resemble the orotate-binding and catalytic portion of the active site of the enzyme orotidine 5'-monophosphate (OMP) decarboxylase (orotidine-5'-monophosphate carboxy-lyase, EC 4.1.1.23). Active recombinant antibody fragments (Fabs) were selected from a combinatorial cDNA library by complementation of a pyrF strain of Escherichia coli and growth of the library-expressing cells on pyrimidine-free medium. In this biological screen, a sufficiently active antibody from the library would decarboxylate orotate to produce uracil, a pyrimidine source for the auxotroph, and would provide the cells with a growth advantage compared to cells without an active antibody. Six recombinant Fabs yielded identifiable colonies in a screen of 16,000 transformants. To enhance its stability and expression level, one of the six positive fragments was converted into single-chain form. In this form, the antibody fragment conferred a definite growth advantage to the auxotroph that was eliminated when the hapten was included in the medium. The purified single-chain antibody displayed orotate decarboxylase activity in vitro, as determined by a 14CO2 displacement assay. The specific activity of the antibody is approximately 10(-7) times that of naturally occurring OMP decarboxylase, but this antibody-catalyzed rate is estimated to be 10(8) times the background rate. The results offer the potential to use these methods to obtain catalytic antibodies for other biosynthetic reactions as well as to assess the effectiveness of the hapten transition state or active site analog in eliciting antibody catalysts.

Antibodies, Catalytic↗
PubMed1982

Immobilization of denatured DNA to macroporous supports: I. Efficiency of different coupling procedures.

Methods commonly used for covalent immobilization of single stranded DNA have been applied to several solid supports (Sephadex G-25 and Cellex 410) as well as to a number of macroporous materials (Sepharose C1-6B, C1-2B; Sephacryl S-500 and S-1000). Coupling efficiencies and stability of covalently bound DNA are compared for both classes of materials. The yields of the immobilization reaction for sonicated DNA are only 10-40% for G-25 and Cellex 410 in contrast to 60-80% for C1-6B and S-500. Under optimal conditions, up to 0.5 mg of DNA can be coupled initially per g of wet macroporous material. The immobilized DNAs are lost from the supports in a biphasic manner, with about 10-20% loss per day during the first 2-3 days at 45 degrees C, followed by only about 1% loss per day at the same temperature thereafter. The influence of the coupling procedure on the generation of mismatch effects has been studied in 2.4 M tetraethylammonium chloride solution for the hybrid formation between immobilized and mobile DNA. The degree of mismatch ranged from 0-3% and depended on the method of immobilization. The unspecific absorption of DNA on macroporous materials is sufficiently low to allow efficient hybrid selection. No size limitations have been observed when plastid mRNAs are selected by cloned fragments of plastid DNA immobilized to macroporous Sephacryl S-500.

Chromatography, Affinity↗
PubMed1996

Regioselective immobilization of short oligonucleotides to acrylic copolymer gels.

Four types of polyacrylamide or polydimethyl-acrylamide gels for regioselective (by immobilization at the 3' end) of short oligonucleotides have been designed for use in manufacturing oligonucleotide microchips. Two of these supports contain amino or aldehyde groups in the gel, allowing coupling with oligonucleotides bearing aldehyde or amino groups, respectively, in the presence of a reducing agent. The aldehyde gel support showed a higher immobilization efficiency relative to the amino gel. Of all reducing agents tested, the best results were obtained with a pyridine-borane complex. The other supports are based on an acrylamide gel activated with glutaraldehyde or a hydroxyalkyl-functionalized gel treated with mesyl chloride. The use of dimethylacrylamide instead of acrylamide allows subsequent gel modifications in organic solvents. All the immobilization methods are easy and simple to perform, give high and reproducible yields, allow long durations of storage of the activated support, and provide high stability of attachment and low non-specific binding. Although these gel supports have been developed for preparing oligonucleotide microchips, they may be used for other purposes as well.

Acrylamides↗
PubMed1994

Metallic spinal artifacts in magnetic resonance imaging.

STUDY DESIGN: The magnetic resonance artifact susceptibility of traces of surgical aluminum, titanium, and stainless steel in a human spine model was investigated. Metallic filings were deposited in noncontiguous disc spaces in five human thoracic spines before magnetic resonance imaging with spin echo and gradient echo sequences. OBJECTIVES: Spin echo and gradient echo sequences were used for quantitate and compare void artifact produced by commonly used surgical metals. This was compared to a liquid paraffin control. SUMMARY OF BACKGROUND DATA: No significant susceptibility artifact was seen with any metal in all spin echo sequences, including T1 (TR 600, TE 12), T2 (TR 2000, TE 30), proton density (TR 2000, TE 80), and fast T2 scanning (TR 3800, TE 96, Ef). METHODS: Sagittal magnetic resonance imaging permitted void artifact quantification and comparison between different metallic alloys. Two neuroradiologists, working on a blinded basis, evaluated all data and rated the void susceptibility artifact on a scale of 1 (least) to 4 (greatest). RESULTS: In general, the magnitude of an imaging artifact during magnetic resonance imaging correlated with the magnetism of the metal. Nickel, found in a larger concentration in 316L than in 304 stainless steel, decreases the magnetic resonance artifact of specific metals because of its ability to stabilize iron in a non-magnetic state. Therefore, the 316L stainless steel yielded less artifact production than 304 stainless steel on gradient echo imaging. CONCLUSION: If upon gradient echo imaging in the postoperative period significant artifact production is noted, stainless steel deposition should be suspected as the causative agent. In this situation, spin echo techniques should be the first approach for attempting optimal visualization of the spinal cord and soft tissue structures.

Aged↗
PubMed1987

Transformation in vitro and covalent modification with biotin of steroid-affinity-purified rat-liver glucocorticoid-hormone-receptor complex.

The molybdate-stabilized rat liver glucocorticoid receptor complex was purified 9000-fold with a 46% yield by steroid-affinity chromatography and DEAE-Sephacel ion-exchange chromatography. The purified glucocorticoid receptor was identified as a 90-92-kDa protein by SDS/polyacrylamide gel electrophoresis. Raising the temperature to 25 degrees C in the absence of molybdate resulted in increased binding of the receptor complex to DNA-cellulose or nuclei, similar to the effect on the cytosolic complex. The purified complex has a sedimentation coefficient of 9-10 S before and after heat treatment in the absence of molybdate. The appearance of smaller 3-4-S species was unrelated to the extent of DNA-cellulose binding of the complex. The process termed 'transformation', i.e. increasing the affinity for DNA, is not concomitant with subunit dissociation or loss of RNA. Highly purified glucocorticoid receptor could be covalently modified with biotin to retain its steroid-binding activity but with a 50% decrease in nuclear binding capacity. The biotin-modified complex reacts with streptavidin in solution without losing its steroid.

Animals↗
PubMedDate not supplied

NADP-specific isocitrate dehydrogenase from the simian malaria parasite Plasmodium knowlesi: partial purification and characterization.

Cell-free schizonts of Plasmodium knowlesi, a simian malaria parasite, possess significant isocitrate dehydrogenase (IDH) activity, about 90% of which is contributed by the NADP-specific enzyme that is localized in the cytosolic fraction. The enzyme has been partially purified by affinity chromatography using Blue sepharose CL-6B. Although unstable in nature, it is stabilized by citrate and glycerol. Kinetic studies with DL-isocitrate and NADP yielded hyperbolic curves with Michaelis constants of 0.210 and 0.038 mM, respectively. Manganous or magnesium ions are essential for activity. The enzyme is thermosensitive, shows maximum activity at pH 8.0, and has a molecular mass of about 48.5 kDa. It is strongly inhibited by thiol-blocking agents but protected against them by thiol-providing agents. Cupric and argentic ions also have a marked inhibitory effect on its activity. The enzyme is significantly inhibited by chloroquine and oxytetracycline in vitro, but to a lesser degree by tetracycline.

Animals↗
PubMed1972

Production and degradation of serogroup B Neisseria meningitidis polysaccharide.

Polysaccharide produced from cultures of serogroups A, B, and C Neisseria meningitidis was assayed by the serogroup-specific hemagglutination inhibition (HAI) test. The polysaccharide produced by all serogroups was found to increase during the exponential phase of growth. For serogroups A and C, the HAI activity was stable during the stationary phase; for serogroup B, however, the HAI decayed rapidly. The degradation of the serogroup B polysaccharide was not caused by enzymatic degradation, but was due to acid accumulation in the culture medium. The large-molecular-size serogroup B polysaccharide was stabilized by increasing the buffering capacity of the medium, which also increased the yield of this antigen.

Antigens, Bacterial↗
PubMed1993

Characterization of cytR mutations that influence oligomerization of mutant repressor subunits.

In Escherichia coli, the transport and catabolism of nucleosides require expression of the genes composing the CytR regulon. The role of the CytR repressor in transcriptional regulation has been examined through a study of mutant CytR proteins. Two important and interrelated CytR mutants are encoded by cytR delta M149, a dominant negative allele, and cytRC289R. Studies with CytR delta M149 indicated that the native, repression-competent CytR protein is multimeric while the CytR amino acid substitution C-289-->R has been proposed to affect subunit oligomerization on the basis of its ability to suppress the transdominance of CytR delta M149. The present study identifies other CytR amino acid residues proximal to Cys-289 that may also participate in normal subunit oligomerization. Mutations in these CytR residues, cytRA307P, cytRM308R, and cytRL309P, encoded inactive repressors in a CytR- background and, when combined with cytR delta M149, yielded hybrid repressors that were recessive in a CytR+ genetic background. Because the stability and solubility observed for the new, mutant CytR proteins and the wild-type CytR protein were indistinguishable, these residue replacements, like the C-289-->R substitution, are envisaged as being located at the subunit interface and thus suppress the CytR delta M149 transdominance by blocking efficient and stable assembly of wild-type and hybrid CytR subunits. The assignment of CytR amino acids to a protein region involved in subunit association is also consistent with the observations that these CytR amino acids are roughly colinear with regions of the LacI repressor that influence monomer-dimer association and would be surface located by alignment to the E. coli galactose-binding protein crystal structure.

Amino Acid Sequence↗
PubMed2001

Potentiation of kinin analogues by ramiprilat is exclusively related to their degradation.

The potentiation of kinin actions represents a cardioprotective property of ACE inhibitors. Although a clear contribution to this effect is related to the inhibition of bradykinin (BK) breakdown, the high efficacy of potentiation and the ability of ACE inhibitors to provoke a B(2)-receptor-mediated response even after receptor desensitization has also triggered hypotheses concerning additional mechanisms of kinin potentiation. The application of kinin analogues with enhanced metabolic stability for the demonstration of degradation-independent mechanisms of potentiation, however, has yielded inconsistent results. Therefore, the relation between the susceptibility of B(2)-agonists to ACE and the potentiation of their actions by ACE inhibitors was investigated with the use of minimally modified kinin derivatives that varied in their degree of ACE resistance. The B(2)-agonists BK, D-Arg-[Hyp(3)]-BK, [Hyp,(3) Tyr(Me)(8)]-BK, [DeltaPhe(5)]-BK, [D-NMF(7)]-BK, and [Phe(8)psi(CH(2)-NH)Arg(9)]-BK were tested for degradation by purified rabbit ACE and for their potency in contracting the endothelium-denuded rabbit jugular vein in the absence and presence of ramiprilat. Purified ACE degraded D-Arg-[Hyp(3)]-BK and [Hyp,(3) Tyr(Me)(8)]-BK at 81% and 71% of BK degradation activity, respectively, whereas other peptides were highly ([DeltaPhe(5)]-BK) or completely ([D-NMF(7)]-BK, [Phe(8)psi(CH(2)-NH)Arg(9)]-BK) resistant. The EC(50) of BK-induced venoconstriction (1.15+/-0.2 nmol/L) was reduced by a factor of 5.7 in the presence of ramiprilat. Likewise, D-Arg-[Hyp(3)]-BK and [Hyp,(3) Tyr(Me)(8)]-BK were both significantly potentiated by a factor of 4.4, whereas the activities of the other agonists were not affected. Ramiprilat exerted no influence on the maximum contraction induced by any of the agonists. It is concluded that the potentiation of kinin analogues during ACE inhibition correlates quantitatively with the susceptibility of each substance to degradation by ACE. As such, no evidence of degradation-independent potentiating actions of ACE inhibitors could be obtained.

Angiotensin-Converting Enzyme Inhibitors↗
PubMedDate not supplied

Anterior cruciate ligament reconstruction. A prospective randomized study of three surgical methods.

A prospective randomized study was performed to determine the differences in results between three methods of anterior cruciate ligament reconstruction: autogenous bone-patellar tendon-bone graft (group 1), semitendinosus and gracilis tendon graft reconstruction combined with an extraarticular procedure (group 2), and semitendinosus and gracilis tendon graft reconstruction alone (group 3). Preoperatively, there were no significant differences between groups. At a mean of 35.4 +/- 11.6 months postoperatively, 102 patients returned for evaluation. International Knee Documentation Committee knee evaluation revealed no significant differences in symptoms, function, return to pre-injury activity, harvest site abnormalities, or limitation of motion between groups 1 and 3. Patients in group 2 had a higher incidence of patellofemoral crepitation and loss of motion than did patients in group 3. The mean manual maximum KT-1000 arthrometer side-to-side difference was 2.1 +/- 2.0 mm in group 1, which was statistically significantly better than the difference in group 3 (3.1 +/- 2.3 mm). Final knee rating showed that 34 of 35 patients in group 1, 23 of 34 patients in group 2, and 26 of 33 patients in group 3 had a normal or nearly normal overall knee rating. Anterior cruciate ligament reconstruction with a semitendinosus and gracilis or a patellar tendon autograft may yield similar subjective results; however, the patellar tendon autograft may provide better objective stability in the long term. In addition, there appears to be no benefit to combining an intraarticular anterior cruciate ligament reconstruction with an extraarticular procedure.

Adolescent↗
PubMed2004

Timing effects and the interpretation of period fertility.

Low fertility levels and later childbearing in many developed countries have reinvigorated the debate between period and cohort perspectives on fertility and on the meaningfulness of the period total fertility rate (TFR). Here, fertility-timing effects are defined as level changes in period fertility that do not reflect level changes in the completed fertility of cohorts. That definition leads to the average cohort fertility (ACF) as a measure of period fertility adjusted for timing effects. In an influential paper, Bongaarts and Feeney (1998) presented an alternative approach and a different measure, TFR*, to adjust for timing effects. Here, the two measures are compared. In the context of model populations, the ACF performs well, reflecting an average of the fertility of the active cohorts. The Bongaarts-Feeney TFR*, however, is frequently unreliable and can be erratic when there are cycles in period timing. When applied to twentieth-century U.S. experience, the TFR* behaves like a period measure and yields adjustments that are often wide of the mark. The ACF shows the stability associated with cohort measures and quantifies the substantial impact that timing effects had during the "birth dearth" of the 1970s. The period TFR reached a low of 1.74 in 1976, but the ACF never went below 2.06 during the 1970s.

Adult↗
PubMed1996

Decrease in high density lipoprotein cholesterol during prolonged storage. CELL Study Group.

Different studies on the stability of high density lipoprotein cholesterol (HDL) in frozen serum or plasma have yielded conflicting results, namely increase, decrease, or no change at all during prolonged storage under freezing conditions. As part of a major trial on lipid-lowering strategies we statistically demonstrated a time-related decrease in HDL cholesterol during storage up to 46 months at -20 degrees C. We therefore re-analysed 85 frozen samples that had been analysed fresh and then stored from 26 to 46 months, using the dextran sulphate 500/Mg2+ method. A linear regression analysis of change in HDL cholesterol on time was performed. The slope was significantly negative (p < 0.0005). The regression equation was (decrease in HDL) = 0.05 - 0.008 x (time in months), i.e. after 6 months' storage at -20 degrees C there was almost a 1% decrease in the HDL cholesterol concentration per month of storage.

Cholesterol, HDL↗
PubMedDate not supplied

Neutrase entrapment in stable multilamellar and large unilamellar vesicles for the acceleration of cheese ripening.

We report the encapsulation of neutrase in liposomes for the acceleration of cheese ripening. The liposome preparation procedure consisted of repeated freeze-thaw cycles of multilamellar vesicles followed by extrusion through polycarbonate filters. The neutrase encapsulation efficiency in the liposomes was influenced by the number of freeze-thaw cycles, achieving the highest value after seven cycles. Filtration through 200-nm polycarbonate membranes yielded homogenous size liposome populations with trapping efficiencies of about 65%. The vesicle stability and low neutrase release during the first stages of the cheese-making procedure, coupled with an almost quantitative retention of neutrase-loaded liposomes in cheese curd, ensured a proteolysis rate that was twice that observed in the control cheese.

Capsules↗
PubMedDate not supplied

Organ blood flow redistribution in response to hypoxemia in neonatal piglets.

This study was designed to determine the effects of severe hypoxemia on newborn piglet visceral blood flow. While the hemodynamic effects of a severe hypoxemic insult are well characterized in newborn animals, its impact on organ perfusion in premature infants is not well characterized. Cannulas were placed in the femoral vessels and left atrium of term (1-14 days old) and prematurely delivered (cesarean section at 90% of term gestation) piglets. After stabilization, some animals were subjected to 1 h of ventilator-controlled hypoxia (yielding PaO2 approximately = 30-40 torr) followed by 30 min of reoxygenation; the remaining animals served as unchallenged controls. Radiolabeled microspheres were injected in all animals at times 0 min (baseline), 5 and 60 min (hypoxia), and 90 min (reoxygenation). Blood flows (mL/min/g tissue) to organs were determined using reference organ techniques. Control animals displayed no alterations in any of the variables monitored. Throughout the experimental period, organ blood flows were almost uniformly lower (p<.05, ANOVA) in premature versus term animals. The trend toward increased cerebral and cardiac blood flows during hypoxia observed in the premature piglets was similar to that of term animals, but of lower magnitude. In term piglets, hypoxia produced an immediate and significant (*p<.05) decline in small-intestinal blood flow followed by autoregulatory escape (2.02+/-0.17 mL/min/g at time 0, 1.56+/-0.15 mL/min/g at 5 min hypoxia, 1.88+/-0.18 mL/min/g at 60 min hypoxia, 2.26+/-0.19 mL/min/g at 30 min reoxygenation), an effect not readily observed in the premature piglets (0.48+/-0.10 mL/min/g at time 0, 0.44+/-0.07 mL/min/g at 5 min hypoxia, 0.46+/-0.10 mL/min/g at 60 min hypoxia, 0.42+/-0.08 mL/min/g at 30 min reoxygenation). However, mucosal blood flows measured in these younger animals declined throughout the experimental period to almost 50% of baseline, compared to a complete restoration to baseline blood flow observed following reoxygenation of term piglets. Intestinal blood flow in premature infants is small when compared to term animals, and alterations in small intestinal blood mucosal flow induced by hypoxia appear less well tolerated by the premature animals. Taken together, this may in part account for the increased risk of developing intestinal ischemic diseases in premature infants who are even temporarily exposed to a severe hypoxic challenge.

Analysis of Variance↗