Objectively measured physical activity and its influence on physical capacity and clinical parameters in patients with primary Sjögren's syndrome.

Objective The objectives of this paper are to objectively measure habitual physical activity levels in patients with primary Sjögren's syndrome (pSS) with mild disease activity and to determine to which extent it may be associated with physical capacity and function and clinical features. Methods In this cross-sectional study, 29 women with pSS were objectively assessed for habitual physical activity levels (using accelerometry) and compared with 20 healthy women (CTRL) frequency-matched for physical activity levels, age, body mass index, and body fat percentage with regard to physical capacity and function, fatigue, depression, pain, and health-related quality of life. Results pSS showed 8.5 min/day of moderate-to-vigorous physical activity (MVPA) when only MVPA accumulated in bouts ≥ 10 min was considered; when considering total MVPA (including bouts < 10 min), average levels were 26.3 min/day, with 62% of pSS patients achieving the recommendation (≥ 21.4 min/day). Moreover, pSS showed lower VO2peak, lower muscle strength and function, higher fatigue, and poorer health-related quality of life when compared with CTRL ( p < 0.05). These differences (except for aerobic capacity) were sustained even when only individuals achieving the minimum of 21.4 min/day of total MVPA in both groups were compared. Finally, MVPA time was significantly correlated with aerobic conditioning, whereas total counts and sedentary time were associated with lower-body muscle strength and the bodily-pain domain of SF-36 in patients with pSS. Conclusion When compared to physical activity-matched healthy controls, pSS patients showed reduced physical capacity and function, increased fatigue and pain, and reduced health-related quality of life. Except for aerobic conditioning, these differences were sustained when only more physically active participants were compared, indicating that minimum recommended levels of physical activity for the general population may not be sufficient to counteract pSS comorbidities.

Effects of acute aerobic exercise on leukocyte inflammatory gene expression in systemic lupus erythematosus.

UNLABELLED: Systemic lupus erythematosus (SLE) is an autoimmune disease with a persistent systemic inflammation. Exercise induced inflammatory response in SLE remains to be fully elucidated. The aim of this study was to assess the effects of acuteexercise on leukocyte gene expression in active (SLEACTIVE) and inactive SLE (SLEINACTIVE) patients and healthy controls(HC). METHODS: All subjects (n = 4 per group) performed a 30-min single bout of acute aerobic exercise (~70% of VO2peak) on a treadmill, and blood samples were collected for RNA extraction from circulating leukocyte at baseline, at the end of exercise, and after three hours of recovery. The expression of a panel of immune-related genes was evaluated by a quantitative PCR array assay. Moreover, network-based analyses were performed to interpret transcriptional changes occurring after the exercise challenge. RESULTS: In all groups, a single bout of acute exercise led to the down-regulation of the gene expression of innate and adaptive immunity at the end of exercise (e.g., TLR3, IFNG, GATA3, FOXP3, STAT4) with a subsequent up-regulation occurring upon recovery. Exercise regulated the expression of inflammatory genes in the blood leukocytes of the SLE patients and HC, although the SLE groups exhibited fewer modulated genes and less densely connected networks (number of nodes: 29, 40 and 58; number of edges: 29, 60 and 195; network density: 0.07, 0.08 and 0.12, for SLEACTIVE, SLEINACTIVE and HC, respectively). CONCLUSION: The leukocytes from the SLE patients, irrespective of disease activity, showed a down-regulated inflammatory geneexpression immediately after acute aerobic exercise, followed by an up-regulation at recovery. Furthermore, less organized gene networks were observed in the SLE patients, suggesting that they may be deficient in triggering a normal exercised-induced immune transcriptional response.

Inflammatory cytokine kinetics to single bouts of acute moderate and intense aerobic exercise in women with active and inactive systemic lupus erythematosus.

The aim of this study was to evaluate changes in the cytokines INF-γ, IL-10, IL-6, TNF-α and soluble TNF receptors (sTNFR1 and sTNFR2) in response to single bouts of acute moderate and intense exercise in systemic lupus erythematosus women with active (SLE(ACTIVE)) and inactive (SLE(INACTIVE)) disease. Twelve SLE(INACTIVE) women (age: 35.3 ± 5.7 yrs; BMI: 25.6±3.4 kg/m2), eleven SLE(ACTIVE) women (age: 30.4 ± 4.5 yrs; BMI: 26.1±4.8 kg/m2), and 10 age- and BMI-matched healthy control women (HC) performed 30 minutes of acute moderate (~50% of VO(2)peak) and intense (~70% of VO(2)peak) exercise bout. Cytokines and soluble TNF receptors were assessed at baseline, immediately after, every 30 minutes up to three hours, and 24 hours after both acute exercise bouts. In response to acute moderate exercise, cytokines and soluble TNF receptors levels remained unchanged in all groups (P>0.05), except for a reduction in IL-6 levels in the SLE(ACTIVE) group at the 60th and 180th minutes of recovery (P<0.05), and a reduction in sTNFR1 levels in the HC group at the 90th, 120th, 150th, 180th minutes of recovery (P<0.05). The SLE(INACTIVE) group showed higher levels of TNF-α, sTNFR1, and sTNFR2 at all time points when compared with the HC group (P<0.05). Also, the SLE(ACTIVE) group showed higher levels of IL-6 at the 60th minute of recovery (P<0.05) when compared with the HC group. After intense exercise, sTNFR1 levels were reduced at the 150th (P=0.041) and 180th (P=0.034) minutes of recovery in the SLE(INACTIVE) group, whereas the other cytokines and sTNFR2 levels remained unchanged (P>0.05). In the HC group, IL-10, TNF-α, sTNFR1, and sTNFR2 levels did not change, whilst INF-γ levels decreased (P=0.05) and IL-6 levels increased immediately after the exercise (P=0.028), returning to baseline levels 24 hours later (P > 0.05). When compared with the HC group, the SLE(INACTIVE) group showed higher levels of TNF-α and sTNFR2 in all time points, and higher levels of sTNFR1 at the end of exercise and at the 30th minute of recovery (P<0.05). The SLE(ACTIVE) group also showed higher levels of TNF-α at all time points when compared with the HC group (P<0.05), (except after 90 min, 120 min and 24 hours of recovery) (P>0.05). Importantly, the levels of all cytokine and soluble TNF receptors returned to baseline 24 hours after the end of acute exercise, irrespective of its intensity, in all three groups (P>0.05). This study demonstrated that both the single bouts of acute moderate and intense exercise induced mild and transient changes in cytokine levels in both SLE(INACTIVE) and SLE(ACTIVE) women, providing novel evidence that acute aerobic exercise does not trigger inflammation in patients with this disease.

Efficacy and safety of creatine supplementation in childhood-onset systemic lupus erythematosus: a randomized, double-blind, placebo-controlled, crossover trial.

INTRODUCTION: Creatine supplementation has emerged as a promising non-pharmacological therapeutic strategy to counteract muscle dysfunction and low lean mass in a variety of conditions, including in pediatric and rheumatic diseases. The objective of this study was to examine the efficacy and safety of creatine supplementation in childhood systemic lupus erythematosus (C-SLE). METHODS: C-SLE patients with mild disease activity (n = 15) received placebo or creatine supplementation in a randomized fashion using a crossover, double-blind, repeated-measures design. The participants were assessed at baseline and after 12 weeks in each arm, interspersed by an eight-week washout period. The primary outcomes were muscle function, as assessed by a battery of tests including one-maximum repetition (1-RM) tests, the timed-up-and-go test, the timed-stands test, and the handgrip test. Secondary outcomes included body composition, biochemical markers of bone remodeling, aerobic conditioning, quality of life, and physical capacity. Possible differences in dietary intake were assessed by three 24-hour dietary recalls. Muscle phosphorylcreatine content was measured through phosphorus magnetic resonance spectroscopy (31 P-MRS). The safety of the intervention was assessed by laboratory parameters, and kidney function was measured by (51)Cr-EDTA clearance. Additionally, self-reported adverse events were recorded throughout the trial. RESULTS: Intramuscular phosphorylcreatine content was not significantly different between creatine and placebo before or after the intervention (creatine-Pre: 20.5 ± 2.6, Post: 20.4 ± 4.1, placebo-Pre: 19.8 ± 2.0; Post: 20.2 ± 3.2 mmol/kg wet muscle; p = 0.70 for interaction between conditions). In addition, probably as a consequence of the lack of change in intramuscular phosphorylcreatine content, there were no significant changes between placebo and creatine for any muscle function and aerobic conditioning parameters, lean mass, fat mass, bone mass, and quality of life scores (p > 0.05). The (51)Cr-EDTA clearance was not altered by creatine supplementation and no side effects were noticed. CONCLUSION: A 12-week creatine supplementation protocol at 0.1 g/kg/d is well tolerated and free of adverse effects but did not affect intramuscular phosphorylcreatine, muscle function, free-fat mass or quality of life in non-active C-SLE patients. TRIAL REGISTRATION: Clinicaltrials.gov number: NCT01217320.

Impaired aerobic exercise capacity and cardiac autonomic control in primary antiphospholipid syndrome.

Primary antiphospholipid syndrome (PAPS) is associated with increased risk of cardiovascular disease and mortality. Aerobic capacity and cardiac autonomic control are also associated with these risks. The aim of our study was to assess aerobic capacity and cardiac autonomic control in PAPS patients. Thirteen women with PAPS and 13 healthy controls matched for age, gender, and body mass index were enrolled for the study. Both groups were sedentary and were not under chronotropic, antidepressants and hypolipemiant drugs. All subjects performed a treadmill-graded maximal exercise. Aerobic capacity was assessed by peak oxygen uptake (VO2peak), time at anaerobic ventilatory threshold (VAT) and respiratory compensation point (RCP) and time-to-exhaustion, whereas cardiac autonomic control was assessed by chronotropic reserve (CR) and heart rate recovery at the first and second minutes after graded exercise (HRR1min and HRR2min, respectively). All aerobic capacity indexes were reduced more in PAPS patients than in healthy subjects: VO2peak (30.2 ± 4.7 vs 34.6 ± 4.3 ml.kg(-1).min(-1), p = 0.021), time at VAT (3.0 ± 1.5 vs 5.0 ± 2.0 min, p = 0.016), time at RCP (6.5 ± 2.0 vs 8.0 ± 2.0 min, p = 0.050), time-to-exhaustion (8.5 ± 2.0 vs 11.0 ± 2.5 min, p = 0.010). HRR1min (22 ± 9 vs 30 ± 7 bpm, p = 0.032) and HRR2min (33 ± 9 vs 46 ± 8 bpm, p = 0.002) were delayed in PAPS patients compared to healthy controls but CR was not significantly different (p = 0.272). In conclusion, an impaired aerobic capacity and cardiac autonomic control was identified in PAPS.

Gill histopathological and oxidative stress evaluation in native fish captured in Portuguese northwestern rivers.

The Northwestern Portuguese region is densely populated and highly industrialized, suffering from high anthropogenic pressure. To assess the biological effect of the several pollutants that are constantly released to the water, a biomarker-based biomonitoring is a promising approach that may provide early-warning signals of pollutants exposure. Fish gill is the first target of pollutants action, thus histopathological and biochemical changes may constitute potential biomarkers. To evaluate this hypothesis, three native fish species (barbel-Luciobarbus bocagei, chub-Squalius carolitertii and nase-Pseudochondrostoma sp.) were sampled in Northwestern Portuguese rivers, the gill histopathological changes were qualitative and quantitatively analyzed and the lipid peroxidation and glutathione-S-transferase activity were determined. A multivariate statistical analysis was performed to establish correlations between these biological responses, environmental variables and ecological status. The quantitative evaluation of the main histopathological changes and oxidative stress responses emphasize the differences, among species, in the responses to the presence of contaminants in water. Discriminant canonical analysis showed that filament epithelium proliferation, necrosis and GST activity were the main contributors to discriminate the ecological status classification. In addition, the results showed that a wide range of environmental factors are influencing fish physiology. In conclusion, the gill biological responses, although not reflecting specific contaminants, can be used as biomarkers of ecosystems perturbation.

Erratic control of breathing during exercise in patients with systemic lupus erythematosus: a pilot-study.

PURPOSE: The aim of this study was to provide a comprehensive evaluation of the pattern and timing of breathing during incremental exercise in a sample of women living with systemic lupus erythematosus (SLE). METHODS: In this cross-sectional study, 20 women with SLE without pulmonary involvement were compared with 20 gender-, body mass index- (BMI), and age-matched healthy individuals. By using a cardiopulmonary incremental exercise test, the following parameters were assessed: tidal volume (VT); breathing frequency (BF); total respiratory time (TOT); inspiratory time (TI); expiratory time (TE); inspiratory time to total time (TI/TOT); mean inspiratory flow (VT/TI); ventilatory equivalent for carbon dioxide (VE/VCO2) and end-tidal carbon dioxide pressure (PETCO2). RESULTS: BF and BF/VT were significantly higher in patients with SLE versus controls, whereas VT, TE, TI and TOT were significantly lower in the former group ( p<0.05). Additionally, patients with SLE presented higher VE/VCO2 and lower PETCO2 than controls ( p<0.05), suggesting a ventilatory inefficiency. CONCLUSION: We reported compelling evidence of abnormal pattern and timing of breathing during incremental exercise in SLE. Considering that an erratic control of breathing may play an important role in exercise intolerance and fatigue, respiratory exercises emerge as a potential treatment for these symptoms in patients with SLE.

Aerobic exercise is safe and effective in systemic sclerosis.

Several studies have established that systemic sclerosis patients have a reduced exercise capacity when compared to healthy individuals. It is relevant to evaluate whether aerobic exercise in systemic sclerosis patients is a safe and effective intervention to improve aerobic capacity. Seven patients without pulmonary impairment and seven healthy controls were enrolled in an 8-week program consisting of moderate intensity aerobic exercise. Patients and controls had a significant improvement in peak oxygen consumption (19.72+/-3.51 vs. 22.27+/-2.53 and 22.94+/-4.70 vs. 24.55+/-3.00, respectively, p=0.006), but difference between groups was not statistically significant (p=0.149). This finding was reinforced by the fact that at the end of the study both groups were able to perform a significantly higher exercise intensity when compared to baseline, as measured by peak blood lactate (1.43+/-0.51 vs. 1.84+/-0.33 and 1.11+/-0.45 vs. 1.59+/-0.25, respectively, p=0.01). Patients improved the peak exercise oxygen saturation comparing to the baseline (84.14+/-9.86 vs. 90.29+/-5.09, p=0.048). Rodnan score was similar before and after the intervention (15.84+/-7.84 vs.12.71+/-4.31, p=0.0855). Digital ulcers and Raynaud's phenomenon remained stable. Our data support the notion that improving aerobic capacity is a feasible goal in systemic sclerosis management. The long term benefit of this intervention needs to be determined in large prospective studies.

U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.

Rating of the lesions in senile dementia of the Alzheimer type: concordance between laboratories. A European multicenter study under the auspices of EURAGE.

The study reported was intended to compare the impressions and analyses of investigators from 11 different laboratories on 2 slides, each from 6 cases with varying quantities of neuropathological change of the type found in Alzheimer's disease and normal ageing. The material came from 6 selected female patients over 75 years of age all of whom had been examined in detail and assessed by the Blessed Test Score. Two were severely demented, 2 mildly demented and 2 were considered to be normal. Unstained paraffin-embedded slides were sent to the investigators, the choice of the staining techniques being left to each laboratory. A quantitative evaluation of the changes was requested in 2 specified areas of the hippocampus and in the first temporal gyrus. Subjective scores of severity and a final guess about the pre mortem intellectual status (demented or not) were asked. The 11 replies were analyzed. A total of 14 different staining techniques were used. Absolute values of density differed much from one investigator to another, for senile plaques as well as for neurofibrillary tangles. Statistical analysis showed that concordance might be improved by the use of corrective factors which would standardize the scales of measurement. The ranking of the slides in increasing order of severity was in good agreement for 9 out of 11 observers concerning the neurofibrillary tangles and 3 out of 9 observers concerning the senile plaques. The correlation between the intellectual status and the density of lesions was higher for neurofibrillary tangles than for senile plaques. The subjective scores were in better agreement for the severely affected cases than for the mildly affected ones. The lowest correlation with intellectual deficit was obtained with the quantitative scores which took into account only the senile plaques or only the hippocampal lesions. The highest correlation coefficients were obtained with the subjective scores. The observers guessed correctly the intellectual status of the 2 most affected cases and often disagreed for the intermediate and normal cases. Neuropathology is mandatory for the diagnosis of definite Alzheimer's disease. Quantitative assessment is useful in cases with few lesions and light dementia but the neuropathological diagnostic procedure has to be more strictly standardized before quantitative histopathological criteria can be reliably transferred from one laboratory to another, especially when mildly affected cases are involved. Concordance seems presently easier to obtain by ranking the lesions and the cases in increasing order of severity than by using quantitative values of density.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipase production by Penicillium restrictum using solid waste of industrial babassu oil production as substrate.

Lipase, protease, and amylase production by Penicillium restrictum in solid-state fermentation was investigated. The basal medium was an industrial waste of babassu oil (Orbignya oleifera) production. It was enriched with peptone, olive oil, and Tween-80. The supplementation positively influenced both enzyme production and fungal growth. Media enriched with Tween-80 provided the highest protease activity (8.6 U/g), whereas those enriched with peptone and olive oil led to the highest lipase (27.8 U/g) and amylase (31.8 U/g) activities, respectively.

[Prevalence study of biotinidase deficiency in newborns]. / Estudo de prevalência em recém-nascidos por deficiência de biotinidase.

INTRODUCTION: Biotinidase deficiency is an inheritable disorder of biotin metabolism. This disorder fulfills major criteria for consideration for newborn screening: the affected children do no show clinical signs in the newborn period; the disease is highly disabling; treatment is effective in preventing neurological sequelae if undertaken promptly. MATERIAL AND METHODS: Screening of 125,000 infants born in Paraná State was carried out to establish the prevalence of biotinidase deficiency. A simple colorimetric procedure was used to detect two infants with biotinidase deficiency (1:62,500), one of them with profound deficiency (1:125,000) and the other with partial deficiency (1:125,000) of the enzyme. RESULTS: There were no known false-negative test results and 0.12% were false-positive, defined by further blood samples which were negative upon repeated testing. Sensitivity was 100% and specificity was 99.88%. Repeat blood samples could not be obtained in 63 (30%) suspected cases. CONCLUSIONS: Newborn screening for biotinidase is useful in identifying affected children, is inexpensive and allows early intervention, which may prevent irreversible neurological damage.

Reduced exercise capacity in systemic sclerosis patients without pulmonary involvement.

OBJECTIVE: To evaluate the exercise capacity of women with systemic sclerosis (SSc) without pulmonary involvement using a cardiopulmonary stress test. METHODS: Thirteen consecutive female SSc patients [mean age 40.8+/-14 years, mean body mass index (BMI) 25.5+/-3.7 kg/m2] without pulmonary and cardiac involvement and 13 healthy sedentary female controls (mean age 41.6+/-9.1 years, mean BMI 23.7+/-3.8 kg/m2) matched by age and BMI underwent a maximum cardiopulmonary stress test (Bruce protocol). The following parameters were analysed: peak oxygen uptake (VO2peak), anaerobic threshold (AT), respiratory compensation point (RCP) and metabolic equivalent (MET) of the VO2peak. Comparisons between groups were analysed using the Student t-test. RESULTS: Forced vital capacity (FVC; 92.2+/-14.2% predicted) and carbon monoxide diffusion lung capacity (DL CO; 85.8+/-5.8% predicted) were within the normal range in SSc patients. VO2peak of SSc patients was significantly reduced in comparison to the control group (19.8+/-4.6 vs. 23.7+/-4.5 mL/kg/min, p = 0.04). SSc patients also had a significant reduction in MET at peak exercise (5.6+/-1.3 vs. 6.7+/-1.3 MET, p = 0.04) and a significant shorter time interval between AT and RCP compared to the control group (112.6+/-95.6 vs. 164.0+/-65.3 s, p = 0.03). CONCLUSION: SSc patients without pulmonary impairment have reduced exercise capacity. Abnormal vascular response to exercise may account for this finding, as the vascular system is one of the major target organs in this pathological condition.

Sequence-dependent termination of in vitro DNA synthesis by cis- and trans-diamminedichloroplatinum (II).

Inhibition of DNA replication by the antitumor drug cis-diamminedichloroplatinum (II) (cis-DDP) has been proposed to be responsible for its cytotoxicity. Treatment of primed phage M13 mp8 viral DNA templates with the drug followed by second-strand synthesis using large fragment DNA polymerase I reveals that cis-DDP forms an adduct with DNA that inhibits DNA synthesis in vitro. This inhibition occurs at all (dG)n (n greater than or equal to 2) sequences in the template strand, confirming that these regions are the major cis-DDP binding sites on DNA. trans-Diamminedichloroplatinum (II), which is inactive as a drug, also forms adducts that inhibit DNA synthesis. Although considerably lower specificity is observed with the trans isomer, there appears to be a preference for d(GpNpG) sequences, where N is any intervening nucleotide. The monofunctional adduct formed between chlorodiethylenetriamineplatinum(II) chloride and DNA does not inhibit DNA synthesis in this system.

Management of nasal trauma--widespread misuse of radiographs.

OBJECTIVE: To assess the reasons behind the widespread use of X-ray in the management of nasal trauma despite the fact that it has no useful purpose, comparing the responses of doctors in Accident and Emergency (A&E) departments between the District General Hospitals (DGH) and the Teaching Hospitals. METHOD: A multiple-choice questionnaire was sent to all doctors in Accidents and Emergency departments in the North-West Region of England. RESULT: 212 questionnaires were sent out and 159 were returned. Amongst the 92 (57.9 per cent) doctors who use nasal radiographs, the overall most common reason is medico-legal in 48 (52.1 per cent). A high proportion of DGH doctors use radiographs for diagnostic purposes and 35 (28.9 per cent) will refer patients based on X-ray demonstration of nasal bone fracture. Other stated reasons included detection of unsuspected facial fracture, diagnosis of compound nasal fracture and foreign body detection. CONCLUSION: Doctors need to be better informed that nasal radiography has no useful value. A clear clinical guideline should be set up nationwide to protect patients from unnecessary exposure to radiation. This will also save the time of the doctors, radiographers and patients. It will prevent inappropriate referrals. Money and other resources will therefore be better utilized.

The mammalian analogue of the yeast PRP8 splicing protein is present in the U4/5/6 small nuclear ribonucleoprotein particle and the spliceosome.

HeLa cell nuclear extracts contain a protein reactive with antibodies against PRP8, a polypeptide essential for pre-mRNA splicing in yeast and a specific component of the yeast U5 small nuclear ribonucleoprotein (snRNP) [Lossky, M., Anderson, G. J., Jackson, S. P. & Beggs, J. (1987) Cell 51, 1019-1026]. The mammalian protein appears as a doublet at approximately 200 kDa, smaller than the 260-kDa yeast protein, and possesses an Sm epitope as determined by immunoblotting. Its association with a snRNP of the Sm class other than U1 or U2 is indicated by its immunoprecipitation by anti-Sm and anti-trimethylguanosine antibodies but not by anti-(U1) or anti-(U2) RNP sera. Gradient fractionation of splicing extracts demonstrates that the 200-kDa protein is a component of the U4/5/6 snRNP complex and of U5 snRNPs. It is also present in affinity-purified spliceosomes.

Construction of a catalytically active iron superoxide dismutase by rational protein design.

The rational protein design algorithm DEZYMER was used to introduce the active site of nonheme iron superoxide dismutase (SOD) into the hydrophobic interior of the host protein, Escherichia coli thioredoxin (Trx), a protein that does not naturally contain a transition metal-binding site. Reconstitution of the designed protein, Trx-SOD, showed the incorporation of one high-affinity metal-binding site. The electronic spectra of the holoprotein and its N3- and F- adducts are analogous to those previously reported for native {Fe3+}SOD. Activity assays showed that {Fe3+}Trx-SOD is capable of catalyzing the dismutation of the superoxide anion; comparative studies with the unrelated wild-type E. coli iron SOD indicated that {Fe3+}Trx-SOD catalyzes the dismutation reaction at a rate on the order of 10(5) M-1s -1. The ability to design catalytically competent metalloenzymes allows for the systematic investigation of fundamental mechanistic questions concerning catalysis at transition metal centers.

Chemical and biological studies of the major DNA adduct of cis-diamminedichloroplatinum(II), cis-[Pt(NH3)2(d(GpG]], built into a specific site in a viral genome.

A duplex Escherichia coli bacteriophage M13 genome was constructed containing a single cis-[Pt(NH3)2(d(GpG]] intrastrand cross-link, the major DNA adduct of the anticancer drug cis-diamminedichloroplatinum(II). The duplex dodecamer d(AGAAGGCCTAGA).d(TCTAGGCCTTCT) was ligated into the HincII site of M13mp18 to produce an insertion mutant containing a unique StuI restriction enzyme cleavage site. A genome with a 12-base gap in the minus strand was created by hybridizing HincII-linearized M13mp18 duplex DNA with the single-stranded circular DNA of the 12-base insertion mutant. The dodecamer d(TCTAGGCCTTCT) was synthesized by the solid-phase phosphotriester method and platinated by reaction with cis-[Pt(NH3)2(H2O)2]2+ (yield 39%). Characterization by pH-dependent 1H NMR spectroscopy established that platinum binds to the N7 positions of the adjacent guanosines. The platinated oligonucleotide was phosphorylated in the presence of [gamma-32P]ATP with bacteriophage T4 polynucleotide kinase and incorporated into the 12-base gap of the heteroduplex, thus situating the adduct specifically within the StuI site in the minus strand of the genome. Approximately 80% of the gapped duplexes incorporated a dodecanucleotide in the ligation reaction. Of these, approximately half did so with the dodecanucleotide covalently joined to the genome at both 5' and 3' termini. The site of incorporation of the dodecamer was mapped to the expected 36-base region delimited by the recognition sites of XbaI and HindIII. The cis-[Pt(NH3)2(d(GpG]] cross-link completely inhibited StuI cleavage, which was fully restored following incubation of the platinated genome with cyanide to remove platinum as [Pt(CN)4]2-. Gradient denaturing gel electrophoresis of a 289-base-pair fragment encompassing the site of adduction revealed that the presence of the cis-[Pt(NH3)2(d(GpG]] cross-link induces localized weakening of the DNA double helix. In addition, double- and single-stranded genomes, in which the cis-[Pt(NH3)2(d(GpG]] cross-link resides specifically in the plus strand, were constructed. Comparative studies revealed no difference in survival between platinated and unmodified double-stranded genomes. In contrast, survival of the single-stranded platinated genome was only 10-12% that of the corresponding unmodified single-stranded genome, indicating that the solitary cis-[Pt(NH3)2(d(GpG]] cross-link is lethal to the single-stranded bacteriophage.

The major adduct of the antitumor drug cis-diamminedichloroplatinum(II) with DNA bends the duplex by approximately equal to 40 degrees toward the major groove.

We used gel electrophoresis methods to show that reaction of DNA with cis-diamminedichloroplatinum(II) results in a substantial (approximately equal to 40 degrees) bend in the double helix at the intrastrand crosslink between the N7 atoms of adjacent guanosine nucleosides, which bond to the platinum(II) complex with loss of two chloride ions. Multimers of a 22-base-pair (bp) oligonucleotide platinated at a single site show strong anomalies in their electrophoretic mobilities as a consequence of coherent addition of in-phase platinum-induced bends. Increase of the sequence repeat of the platinated site to 27 bp yields multimers of nearly normal mobility because the bends are out of phase. The direction of Pt-induced bends relative to adenosine-tract bends was determined from the electrophoretic mobility of multimers in which repeated platination sites are interdigitated at various phasings relative to repeated adenosine tracts. Optimum bending occurs when 1.5 helical turns separate the cis-[Pt(NH3)2[d(pGpG)]](N7,N7) adducts from the center of the adenosine tracts. This phasing produces coherent addition of adenosine tract bends toward the minor groove at their centers and Pt-induced bends toward the major groove, where the platinum is located.