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1.
J Nanosci Nanotechnol ; 14(6): 4337-40, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24738393

ABSTRACT

Magnetic refrigeration is an alternative cooling technique with envisaged technological applications on micro- and opto-electronic devices. Here, we present a magnetic micro-refrigerator cooling device with embedded micro-channels and based on the magnetocaloric effect. We studied the influence of the coolant fluid in the refrigeration process by numerically simulating the heat transfer processes using the finite element method. This allowed us to calculate the cooling power of the device. Our results show that gallium is the most efficient coolant fluid and, when used with Gd5Si2Ge2, a maximum power of 11.2 W/mm3 at a working frequency of -5 kHz can be reached. However, for operation frequencies around 50 Hz, water is the most efficient fluid with a cooling power of 0.137 W/mm3.


Subject(s)
Computer-Aided Design , Magnetics , Micro-Electrical-Mechanical Systems/instrumentation , Models, Theoretical , Nanotechnology/instrumentation , Refrigeration/instrumentation , Solutions/chemistry , Computer Simulation , Energy Transfer , Equipment Design , Equipment Failure Analysis , Finite Element Analysis , Miniaturization , Thermal Conductivity , Thermodynamics
2.
Rhinology ; 49(3): 315-7, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21858262

ABSTRACT

INTRODUCTION: Silent sinus syndrome (SSS) is a rare idiopathic collapse of the maxillary sinus and orbital floor. We present the second largest series of sixteen patients with SSS and describe their management. METHODS: A cohort of 16 patients with spontaneous SSS between 1999 and 2009 were reviewed at the Royal National Throat Nose and Ear Hospital. All patients were initially referred from a postgraduate ophthalmic hospital, Moorfields Hospital. RESULTS: Fourteen patients required endoscopic sinus surgery to re-establish maxillary sinus drainage and the remaining two settled with intranasal medical therapy consisting of steroids and decongestions. Follow- up ranged from 6 months to 4 years with a mean of 2.6 years. An improvement in enophthalmos and was seen in all surgically treated patients with a mean improvement of 2.2mm and range 0.5-4mm. DISCUSSION: SSS arises from congestion of the ostiomeatal complex resulting in negative pressure within the maxillary sinus and a gradual implosion of the antral cavity. Endoscopic sinus surgery successfully re-establishes maxillary aeration in our series and leads to clinical and aesthetic improvement in the degree of enophthalmos and has avoided the need for orbital floor repair in all but two cases.


Subject(s)
Endoscopy , Maxillary Sinus , Orbital Diseases/surgery , Paranasal Sinus Diseases/surgery , Adolescent , Adult , Aged , Enophthalmos/etiology , Female , Humans , Male , Middle Aged , Orbital Diseases/pathology , Paranasal Sinus Diseases/diagnostic imaging , Syndrome , Tomography, X-Ray Computed , Young Adult
3.
J Frailty Aging ; 10(1): 38-43, 2021.
Article in English | MEDLINE | ID: mdl-33331620

ABSTRACT

BACKGROUND: Frailty is a biological syndrome that causes adverse events in the health of older adults. However, the Clinical Frailty Scale has not yet been culturally adapted and validated into Brazilian Portuguese language. OBJECTIVES: Our aim was to translate, reproduce and validate the Clinical Frailty Scale (CFS) for the Brazilian Portuguese language. DESIGN: An observational cross-sectional study with senior patients was conducted between Jan 2018 and Nov 2018. SETTING AND PARTICIPANTS: Volunteers aged >60 and living in Brazil. The translation and cultural adaptation of the CFS into the Portuguese language, the principles and good practices were followed. MEASUREMENTS: To conduct the validation and determine the reproducibility of an inter-observer evaluation, the patients answered the scale questions in Portuguese on two occasions, delivered by two separate examiners and separated by a 10-minute interval, on their first visit; the 36-item Short Form Survey quality-of-life questionnaire (SF-36) was also applied. Seven days later, a second visit was undertaken to perform an intra-observer reproducibility assessment. RESULTS: A total of 66 older individuals were enrolled (72 ± 8 years), the majority of which did not present frailty (63.6%) and reported a low physical limitation level in the SF-36. The CFS showed a significant correlation with the SF-36 quality-of-life questionnaire (r= -0.663; p<0.0001) and no statistical difference was observed between intra-rater (p=0.641) and inter-rater (p=0.350) applications, demonstrating the reproducibility and applicability of the instrument. The standard error estimate (SEE) was evaluated and there were no differences between the CFS and the SF-36 (SEE= 1.13 points). CONCLUSION: The Brazilian Portuguese language version of the CFS is a valid, reproducible and reliable instrument for evaluating the impact of frailty on the lives of senior patients.


Subject(s)
Frailty , Language , Surveys and Questionnaires/standards , Aged , Brazil , Cross-Sectional Studies , Frailty/diagnosis , Humans , Reproducibility of Results , Translations
5.
Neuroscience ; 134(4): 1363-75, 2005.
Article in English | MEDLINE | ID: mdl-16054768

ABSTRACT

Substituted amphetamines such as p-chloroamphetamine and the abused drug methylenedioxymethamphetamine cause selective destruction of serotonin axons in rats, by unknown mechanisms. Since some serotonin neurones also express neuronal nitric oxide synthase, which has been implicated in neurotoxicity, the present study was undertaken to determine whether nitric oxide synthase expressing serotonin neurones are selectively vulnerable to methylenedioxymethamphetamine or p-chloroamphetamine. Using double-labeling immunocytochemistry and double in situ hybridization for nitric oxide synthase and the serotonin transporter, it was confirmed that about two thirds of serotonergic cell bodies in the dorsal raphé nucleus expressed nitric oxide synthase, however few if any serotonin transporter immunoreactive axons in striatum expressed nitric oxide synthase at detectable levels. Methylenedioxymethamphetamine (30 mg/kg) or p-chloroamphetamine (2 x 10 mg/kg) was administered to Sprague-Dawley rats, and 7 days after drug administration there were modest decreases in the levels of serotonin transporter protein in frontal cortex, and striatum using Western blotting, even though axonal loss could be clearly seen by immunostaining. p-Chloroamphetamine or methylenedioxymethamphetamine administration did not alter the level of nitric oxide synthase in striatum or frontal cortex, determined by Western blotting. Analysis of serotonin neuronal cell bodies 7 days after p-chloroamphetamine treatment, revealed a net down-regulation of serotonin transporter mRNA levels, and a profound change in expression of nitric oxide synthase, with 33% of serotonin transporter mRNA positive cells containing nitric oxide synthase mRNA, compared with 65% in control animals. Altogether these results support the hypothesis that serotonin neurones which express nitric oxide synthase are most vulnerable to substituted amphetamine toxicity, supporting the concept that the selective vulnerability of serotonin neurones has a molecular basis.


Subject(s)
3,4-Methylenedioxyamphetamine/analogs & derivatives , Brain/drug effects , Nerve Tissue Proteins/biosynthesis , Neurons/drug effects , Nitric Oxide Synthase/biosynthesis , Serotonin Agents/toxicity , p-Chloroamphetamine/toxicity , 3,4-Methylenedioxyamphetamine/toxicity , Animals , Blotting, Western , Brain/metabolism , Image Processing, Computer-Assisted , Immunohistochemistry , In Situ Hybridization , Male , Membrane Glycoproteins/drug effects , Membrane Glycoproteins/metabolism , Membrane Transport Proteins/drug effects , Membrane Transport Proteins/metabolism , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Nitric Oxide Synthase Type I , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Serotonin/metabolism , Serotonin Plasma Membrane Transport Proteins
6.
Br J Ophthalmol ; 89(10): 1348-50, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16170130

ABSTRACT

AIM: Silicone oil is a useful tamponading material used in complex vitroretinal surgery. However, the use of silicone oil is associated with emulsification which can lead to vision threatening complications. The authors developed an experimental model to study the effect of encircling band on silicone oil emulsification. METHODS: Two identical artificial eye chambers were constructed with circumferential indentations placed at the sphere's equator (mimicking an encircling band indentation), and filled with varying amounts of Silicone Oil 1000 centistrokes (Adato, Bausch and Lomb, UK) and balanced salt solution. The chambers were then placed on a horizontal rotating shaker, mimicking physiological saccadic eye movements, which spun the chambers at 100 Hz for 5 days at 37 degrees C. Emulsification was then quantified by dark field microscopy, digital photography, and manual counting by a masked observer. RESULTS: The mean (standard deviation (SD)) values of silicone emulsification bubbles were as follows: in the 90% silicone oil filled chamber with no encircling band, 139.1 (SD 313.4); in the 90% silicone oil filled chamber with encircling band, 10.9 (SD 22.2) (p<0.0001); in the 75% silicone oil filled chamber with no encircling band, 103.6 (SD 272.6); in the 75% silicone oil filled chamber with encircling band, 18.5 (SD 32), (p = 0.001). CONCLUSIONS: The emulsification of silicone oil results from friction between the silicone oil and aqueous liquids. The results from this study suggest silicone oil emulsification is reduced by (1) more complete silicone oil fill and (2) indentation from an encircling band. The authors hypothesise that both these measures resulted in reduced emulsification by reducing silicone oil/aqueous movement and resulting shearing forces.


Subject(s)
Retinal Detachment/surgery , Scleral Buckling , Silicone Oils/chemistry , Emulsions , Humans , Models, Anatomic , Pressure , Rheology , Saccades , Silicone Oils/adverse effects
7.
J Cataract Refract Surg ; 31(7): 1351-6, 2005 Jul.
Article in English | MEDLINE | ID: mdl-16105606

ABSTRACT

PURPOSE: To compare the preoperative evaluation of secondary intraocular lens (IOL) implantation in aphakic adults following cataract extraction in childhood using slitlamp examination and high-frequency ultrasound (HFU). METHODS: In a prospective case series, patients who had had lensectomies for congenital cataracts without primary implantation IOL were evaluated for secondary IOL insertion. Slitlamp examination and HFU were performed to study the degree of ciliary sulcus support and iridocapsular adhesions. The choice of IOL (posterior sulcus supported or anterior chamber) was compared using the 2 techniques. RESULTS: Nine eyes of 5 patients (3 men and 2 women aged 15 to 40 years) were assessed for secondary IOL insertion. Clinical slitlamp examination suggested that 3 of 9 eyes had inadequate sulcociliary support, but HFU of these eyes revealed more than adequate capsular remnants. In all 9 eyes, sulcus-supported posterior chamber IOLs were implanted. No postoperative complications were observed, and no patients required surgery for dislocated IOL. CONCLUSIONS: High-frequency ultrasound is a useful adjunct for the preoperative assessment of secondary ciliary sulcus-supported IOL implantation in aphakic patients who had congenital cataract extraction without IOL implantation. In patients in whom inadequate dilation precludes the detection of capsular support, posterior ciliary sulcus-supported secondary IOL implantation should be considered preoperatively. Although the technique enhances surgical planning and informed patient consent, the final decision occurs at the time of surgery with direct visualization of the ciliary sulcus support.


Subject(s)
Anterior Eye Segment/diagnostic imaging , Aphakia, Postcataract/surgery , Ciliary Body/diagnostic imaging , Iris/diagnostic imaging , Lens Implantation, Intraocular , Preoperative Care , Adolescent , Adult , Cataract/congenital , Cataract Extraction , Diagnostic Techniques, Ophthalmological , Female , Humans , Lenses, Intraocular , Male , Prospective Studies , Ultrasonography
8.
Curr Opin Drug Discov Devel ; 2(4): 365-76, 1999 Jul.
Article in English | MEDLINE | ID: mdl-19649965

ABSTRACT

Carbohydrates and glycoconjugates have attracted a great deal of attention in recent years because of the key roles they play in biological processes. The development of carbohydrate-based libraries is a crucial step towards investigating the details of carbohydrate-mediated interactions, exploring the molecular diversity intrinsic to carbohydrates and identifying novel therapeutically interesting carbohydrate-containing drugs. This review describes recent advancements in solid- and solution-phase methods that relate to the generation of carbohydrate and glycoconjugate libraries. Special attention will be dedicated to the increasing use of carbohydrates as molecular scaffolds for library synthesis.

9.
Braz J Biol ; 74(3): 523-8, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25296198

ABSTRACT

Forest fragmentation affects animal population dynamics mainly by loss of habitat and disruption of animal movement. Lizard assemblages are affected by environmental changes, but, depending on their ecological needs, some species might be more vulnerable than others. The southern Amazon suffers accelerated anthropic actions replacing natural environments by farmland (crops and pasture). This region is considerably drier than most of the northern Amazon, with stational semi-deciduous forests fragmented and isolated by pasture, and crops to a lesser extent. Here we report data on lizard assemblages using semi-deciduous forests, forest edge and the surrounding pasture in the southern Amazon in Mato Grosso, Brazil. Lizards were collected in 21 forest fragments (41 to 7,035 ha) surrounded by pasture; using pitfall traps placed on a degradation gradient - from pasture inwards forest fragment (up to 200 m). We collected 242 individuals (14 species, seven families) in 6,300 trap-days. The pattern of species occurrence was largely nested and this nesting was associated with three habitat guilds (generalist, edge-tolerant, and forest species). Although there was no obvious fragmentation effect on lizards diversity community-wise, Hoplocercus spinosus, Bachia dorbignyi, Micrablepharus maximiliani and Kentropyx calcarta were more vulnerable to such effects than all other ten species collected. We verified that assemblages inhabiting pasture and forest edge are a nested subset of assemblages from the forest core. The remnant native vegetation is not distributed homogeneously and lizards species can persist in different parts of the landscape, therefore we recommend the protection of forest remnants as an important conservation action for lizards of the southern Amazon.


Subject(s)
Environmental Monitoring , Forests , Lizards/classification , Animals , Brazil , Conservation of Natural Resources , Population Density , Population Dynamics
14.
Br J Ophthalmol ; 92(7): 954-8, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18556423

ABSTRACT

AIM: To evaluate outcome following retinectomy surgery and to identify factors that predict visual outcome. METHODS: This was a retrospective uncontrolled interventional case-series of patients who underwent retinectomy surgery at Moorfields Eye Hospital (London, UK) during a 2-year period. We recorded peri-operative factors with the potential to influence functional outcome including aetiology of retinal detachment; type of ocular trauma; preoperative visual acuity and intraocular pressure; grade of PVR; extent and position of retinectomy; peri-operative complications; tamponade agent and prophylactic 360 degrees laser retinopexy. RESULTS: The authors identified 145 patients who underwent retinectomy surgery. After a mean follow-up period of 23.2 months (6-58 months) 16% of eyes had visual acuity of 20/60 or better, 33% had visual acuity of between 20/60 and 20/400, and 51% had visual acuity less than 20/400. Visual acuity was improved or stable in 76% of eyes and the rate of complete retinal reattachment was 68%. For each stepwise increase in the grade of PVR there was an approximately 15% increased risk of final visual acuity of less than 20/40. The use of additional 360 degree prophylactic laser retinopexy prior to removal of silicone oil was associated with a higher rate of final retinal reattachment. CONCLUSIONS: This study provides important information to help surgeons inform and counsel their patients about visual prognosis following retinectomy surgery. Furthermore, the study demonstrates that the aetiology of retinal detachment, the stage of PVR and the use of 360 degrees laser retinopexy have significant predictive value for visual outcome. SUMMARY: Retinectomy is a valuable technique in the management of complex retinal detachment but is associated with a significant risk of retinal redetachment and poor visual outcome. In this large study, we identified that the aetiology of retinal detachment, the stage of proliferative vitreoretinopathy and the use of 360 degrees prophylactic laser retinopexy are predictive of functional outcome.


Subject(s)
Retina/surgery , Retinal Detachment/surgery , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Follow-Up Studies , Humans , Male , Middle Aged , Postoperative Complications , Prognosis , Retinal Detachment/etiology , Retinal Detachment/physiopathology , Retrospective Studies , Treatment Outcome , Visual Acuity
15.
Braz J Biol ; 68(4): 703-10, 2008 Nov.
Article in English | MEDLINE | ID: mdl-19197487

ABSTRACT

A community of small mammals was studied in seasonal semideciduous submontane forest in the state of Mato Grosso, Brazil. This study evaluated the use of edge and matrix pasture, by different small mammal species. Overall, 31 areas were studied, with a total sampling effort of 33,800 trap x nights. Only seven of the 25 species captured in the study sites were able to use the pasture matrix; we classified these species as generalists. Fourteen species were found to be intermediate in habits, being able to use forest edges. We found only four species habitat specialists, occurring only on transect lines in the interior of the fragment, at least 150 m from the edge. Transects located in the pasture matrix and 50 m from the edge had significantly lower species richness and abundance than transects located in the fragment edge or in the interior of the fragment. All transects located within the fragment had similar species richness and abundance, but transects located 50 m from the edge had slightly lower, but non-significant, species richness than transects located 100 m apart from edges. Rarefaction curves demonstrated that only medium-sized fragments (100-300 ha) reached an asymptote of species accumulation. The other areas require further sampling, or more sampling transect, before species accumulation curves stabilize, due to a continued increase in species number.


Subject(s)
Biodiversity , Ecosystem , Mammals/physiology , Trees , Animals , Brazil , Mammals/classification , Population Density
16.
Eye (Lond) ; 21(2): 246-8, 2007 Feb.
Article in English | MEDLINE | ID: mdl-16410813

ABSTRACT

Two patients with chronic Wegener's granulomatosis presented with worsening proptosis and visual acuity. Both patients had been maintained on long-term corticosteroids, which led to masking of the signs of orbital sepsis with potentially life-threatening implications.


Subject(s)
Abscess/complications , Granulomatosis with Polyangiitis/complications , Orbital Diseases/complications , Administration, Oral , Aged , Chronic Disease , Exophthalmos/complications , Exophthalmos/pathology , Female , Glucocorticoids/administration & dosage , Granulomatosis with Polyangiitis/drug therapy , Granulomatosis with Polyangiitis/pathology , Humans , Middle Aged , Orbital Diseases/pathology , Orbital Diseases/surgery , Prednisolone/administration & dosage , Treatment Outcome , Visual Acuity/physiology
17.
Rev. bras. plantas med ; Rev. bras. plantas med;13(1): 24-29, 2011. tab
Article in Portuguese | LILACS | ID: lil-582758

ABSTRACT

Este trabalho objetivou avaliar a divergência genética entre nove acessos de mentrasto (Ageratum conyzoides) por métodos multivariados utilizando caracteres morfológicos, em quatro épocas de colheita. Os nove acessos de mentrasto foram obtidos de plantas que ocorrem naturalmente nas cinco regiões climáticas do Estado do Tocantins. Em cada época de colheita foram avaliadas as características como biomassa fresca da parte aérea, biomassa seca, área foliar, número de folhas, floração e altura. Pela análise de agrupamento (método de Tocher) foram formados dois grupos na primeira e quarta épocas de colheita, três grupos na segunda época e quatro grupos na terceira época de colheita. Observou-se variação na constituição dos grupos pelos acessos nas épocas de colheita. A divergência genética entre acessos de mentrasto, avaliada por caracteres morfológicos, foi influenciada pelo estádio de desenvolvimento da espécie. A existência de variabilidade entre os acessos coletados em diferentes localidades fornece subsídio para a coleta sistematizada de germoplasma em A. conyzoides no Estado do Tocantins.


This study aimed to analyze the genetic divergence among nine mentrasto (Ageratum conyzoides) accessions by multivariate methods using morphologic traits for four harvesting periods. The nine accessions of mentrasto were obtained from plants that occur naturally in five climatic regions of the state of Tocantins. At each harvesting period, the following traits were evaluated: fresh biomass of the aerial part, dry biomass, leaf area, number of leaves, flowering and height. The grouping analysis (Tocher's method) identified two groups, in the first and fourth harvest periods, three groups in the second harvest period and four groups in the third harvest period. Variation was observed for the group constitutions among the different harvesting periods. The genetic divergence among mentrasto accessions, evaluated by morphologic traits, was influenced by developmental stage of specie. The existence of genetic variability among accessions from different regions supports the systematic collection of A. conyzoides germplasm in the state of Tocantins.


Subject(s)
Ageratum/anatomy & histology , Ageratum/growth & development , Ageratum/physiology , Ageratum/genetics , Brazil , Multivariate Analysis , Natural Resources Management
18.
Biochemistry ; 32(2): 463-71, 1993 Jan 19.
Article in English | MEDLINE | ID: mdl-8422355

ABSTRACT

Chromomycin A3 (CRA3) is a glycosylated antitumor antibiotic that binds as a dimer to the minor groove of DNA, with a Mg2+ cation (or another divalent cation with a radius less than 0.85 A) forming the center of the dimer. It has been shown that the chromose sugars are necessary for DNA binding [Kaziro & Kamiyama (1967) J. Biochem. (Tokyo) 62, 424-429; Kamiyama (1968) J. Biochem. (Tokyo) 63, 566-572], although the reason for this has not been addressed. We have investigated the role that the chromose sugars play in metal complexation in solution (methanol) by comparing the optical behavior of CRA3 and its aglycon, CRN, in the presence of various divalent metals (Mg2+, Ni2+, and Ca2+). The results show that CRA3 forms a dimeric complex [i.e., (CRA3)2M, where M is a metal ion] in the presence of 1 mol equiv of either Ni2+ or Mg2+ but a 1:1 complex in the presence of the much larger Ca2+. In contrast, CRN forms a 1:1 complex (CRN.M)+ with all three metals under identical conditions (1:1 mole ratio of drug to metal). Thus, for the smaller metal ions the sugars stabilize the 2:1 CRA3-metal complex in solution. NMR data on the 2:1 CRA3-Mg2+ complex show that the trisaccharide of one CRA3 molecule lies in close proximity to the chromophore of the other CRA3 molecule. This interaction, which is also present in the Mg(2+)-CRA3-DNA complex [Gao & Patel (1989) Biochemistry 28, 751-762], appears to be related to the stability of the dimer in solution.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Carbohydrates/chemistry , Chromomycin A3/chemistry , Magnesium/chemistry , Anthracenes/chemistry , Calcium/chemistry , Carbohydrate Sequence , Circular Dichroism , DNA/chemistry , Magnetic Resonance Spectroscopy , Methanol/chemistry , Molecular Sequence Data , Nickel/chemistry , Spectrometry, Mass, Fast Atom Bombardment , Spectrophotometry, Ultraviolet , Water/chemistry
19.
Bioorg Med Chem ; 2(11): 1251-9, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7757421

ABSTRACT

Chromomycin A3 (CRA3) is an antitumor antibiotic that binds to DNA. It contains an acac-like metal binding site and forms a 2:1 complex with Mg2+. Interestingly, acac ligands similar to CRA3 form 1:1 complexes with Mg2+. We have previously shown that the unusual stability of the 2:1 CRA3-Mg2+ complex is related to a favorable intermolecular interaction between the CDE trisaccharide of one CRA3 molecule and the chromophore of the other. We have used this knowledge to design and synthesize a very simple molecule in which a triethylene glycol chain mimics the CDE trisaccharide of CRA3. This minimalist ligand behaves like CRA3 with respect to dimer formation. This result sheds light on how the CRA3 sugars function to stabilize the dimer. At the same time, the work provides a starting point for investigating the relationship between dimer formation and DNA binding. Starting from these relatively simple metal complexes, it should be possible to develop a better understanding of the structural requirements for DNA binding by CRA3 and related molecules.


Subject(s)
Chromomycin A3/chemistry , Oligosaccharides/chemistry , Polyethylene Glycols/chemistry , Anti-Bacterial Agents , Antineoplastic Agents , Carbohydrate Sequence , Chromomycin A3/metabolism , DNA/metabolism , Ligands , Magnesium/metabolism , Magnetic Resonance Spectroscopy , Molecular Mimicry , Molecular Sequence Data , Spectrophotometry
20.
Biochemistry ; 37(16): 5528-35, 1998 Apr 21.
Article in English | MEDLINE | ID: mdl-9548936

ABSTRACT

Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii utilizes adenosylcobalamin and catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. One equivalent of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate, F2dCTP, rapidly inactivates RTPR. Analysis of the reaction products reveals that inactivation is accompanied by release of two fluoride ions and 0.84 equiv of 5'-deoxyadenosine and attachment of 1 equiv of corrin covalently to an active-site cysteine residue of RTPR. No cytosine release was detected. Proteolysis of corrin-labeled RTPR with endoproteinase Glu-C and peptide mapping at pH 5.8 revealed that C419 was predominantly modified. The kinetics of the inactivation have been examined by stopped-flow (SF) UV-vis spectroscopy and rapid freeze quench (RFQ) electron paramagnetic resonance (EPR) spectroscopy. Monitoring DeltaA525 nm shows that cob(II)alamin is formed with an apparent kobs of 50 s-1, only 2. 5-fold slower than a similar experiment carried out with cytidine 5'-triphosphate (CTP). The same reaction mixture was thus quenched at times from 22 ms to 30 s and examined by EPR spectroscopy. At early time points the EPR spectrum resembled a thiyl radical exchange coupled to cob(II)alamin. From 22 to 255 ms the total spin concentration remained unchanged at 1.4 spins/RTPR, twice that predicted by the amount of cob(II)alamin determined by SF. However, with time the signal attributed to the thiyl radical-cob(II)alamin disappears and new signal(s) with broad feature(s) at g = 2.33 and a sharp feature at g = 2.00 appeared, suggesting formation of cob(II)alamin and a nucleotide-based radical with only dipolar interactions. These studies have been interpreted to support the proposal that an RTPR-based thiyl radical can give rise to a nucleotide-based radical.


Subject(s)
Deoxycytidine/analogs & derivatives , Enzyme Inhibitors/pharmacology , Lactobacillus/enzymology , Ribonucleotide Reductases/antagonists & inhibitors , Sulfhydryl Compounds/pharmacology , Cytosine/metabolism , Deoxyadenosines/metabolism , Deoxycytidine/chemistry , Deoxycytidine/metabolism , Deoxycytidine/pharmacology , Electron Spin Resonance Spectroscopy , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Stability/drug effects , Fluorides/metabolism , Free Radicals/metabolism , Peptides/metabolism , Protein Denaturation , Ribonucleotide Reductases/metabolism , Spectrophotometry, Ultraviolet , Vitamin B 12/metabolism , Gemcitabine
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