[Surgical Treatment of Degenerative Lumbar Stenosis and Spondylolisthesis: Clinical Practice Guideline]. / Chirurgická lécba degenerativní lumbální stenózy a spondylolistézy: klinický doporucený postup.

PURPOSE OF THE STUDY This article presents the evidence and the rationale for the recommendations for surgical treatment of degenerative lumbar stenosis (DLS) and spondylolisthesis that were recently developed as a part of the Czech Clinical Practice Guideline (CPG) "The Surgical Treatment of the Degenerative Diseases of the Spine". MATERIAL AND METHODS The Guideline was drawn up in line with the Czech National Methodology of the CPG Development, which is based on the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. We used an innovative GRADE-adolopment method that combines adoption and adaptation of the existing guidelines with de novo development of recommendations. In this paper, we present three adapted recommendations on DLS and a recommendation on spondylolisthesis developed de novo by the Czech team. RESULTS Open surgical decompression in DLS patients has been evaluated in three randomized controlled trials (RCTs). A recommendation in favour of decompression was made based on a statistically significant and clinically evident improvement in the Oswestry Disability Index (ODI) and leg pain. Decompression may be recommended for patients with symptoms of DLS in the event of correlation of significant physical limitation and the finding obtained via imaging. The authors of a systematic review of observational studies and one RCT conclude that fusion has a negligible role in the case of a simple DLS. Thus, spondylodesis should only be chosen as an adjunct to decompression in selected DLS patients. Two RCTs compared supervised rehabilitation with home or no exercise, showing no statistically significant difference between the procedures. The guideline group considers the post-surgery physical activity beneficial and suggests supervised rehabilitation in patients who have undergone surgery for DLS for the beneficial effects of exercise in the absence of known adverse effects. Four RCTs were found comparing simple decompression and decompression with fusion in patients with degenerative lumbar spondylolisthesis. None of the outcomes showed clinically significant improvement or deterioration in favour of either intervention. The guideline group concluded that for stable spondylolisthesis the results of both methods are comparable and, when other parameters are considered (balance of benefits and risks, or costs), point in favour of simple decompression. Due to the lack of scientific evidence, no recommendation has been formulated regarding unstable spondylolisthesis. The certainty of the evidence was rated as low for all recommendations. DISCUSSION Despite the unclear definition of stable/unstable slip, the inclusion of apparently unstable cases of DS in stable studies limits the conclusions of the studies. Based on the available literature, however, it can be summarized that in simple degenerative lumbar stenosis and static spondylolisthesis, fusion of the given segment is not justified. However, its use in the case of unstable (dynamic) vertebral slip is undisputable for the time being. CONCLUSIONS The guideline development group suggests decompression in patients with DLS in whom previous conservative treatment did not lead to improvement, spondylodesis only in selected patients, and post-surgical supervised rehabilitation. In patients with degenerative lumbar stenosis and spondylolisthesis with no signs of instability, the guideline development group suggests simple decompression (without fusion). Key words: degenerative lumbar stenosis, degenerative spondylolisthesis, spinal fusion, Clinical Practice Guideline, GRADE, adolopment.

UHPLC-Orbitrap study of the first phase tacrine in vitro metabolites and related Alzheimer's drug candidates using human liver microsomes.

Tacrine was the first drug used in the therapy of Alzheimer's disease (AD) and is one of the leading structures frequently pursued in the drug discovery of novel candidates for tackling AD. However, because tacrine has been withdrawn from the market due to its hepatotoxicity, ascribed to specific metabolites, concerns are high about the toxicity profile of newly developed compounds related to tacrine. From the point of view of drug safety, the formation of metabolites must be uncovered and analyzed. Bearing in mind that the main culprit of tacrine hepatotoxicity is its biotransformation to hydroxylated metabolites, human liver microsomes were used as a biotransformation model. Our study aims to clarify phase I metabolites of three potentially non-toxic tacrine derivatives (7-methoxytacrine, 6-chlorotacrine, 7-phenoxytacrine) and to semi-quantitatively determine the relative amount of individual metabolites as potential culprits of tacrine-based hepatotoxicity. For this purpose, a new selective UHPLC-Orbitrap method has been developed. Applying UHPLC-Orbitrap method, two as yet unpublished tacrine and 7-methoxytacrine monohydroxylated metabolites have been found and completely characterized, and the separation of ten dihydroxylated tacrine and 7-methoxytacrine metabolites was achieved for the first time. Moreover, the structures of several new metabolites of 7-phenoxytacrine and 6-chlorotacrine have been identified. In addition, the relative amount of these newly observed metabolites was determined. Based on the results and known facts about the toxicity of tacrine metabolites published so far, it appears that 7-phenoxytacrine and 6-chlorotacrine could be substantially less hepatotoxic compared to tacrine, and could potentially pave the way for metabolically safe molecules applicable in AD therapy.

A case series describing common radiographic and pathologic patterns of hard metal pneumoconiosis.

INTRODUCTION: Hard metal pneumoconiosis is a rare but serious disease of the lungs associated with inhalational exposure to tungsten or cobalt dust. Little is known about the radiologic and pathologic characteristics of this disease and the efficacy of treating with immunosuppression. OBJECTIVE: We describe the largest cohort of patients with hard metal pneumoconiosis in the literature, including radiographic and pathologic patterns as well as treatment options. METHODS: We retrospectively identified patients from the University of Pittsburgh pathology registry between the years of 1985 and 2016. Experts in chest radiology and pulmonary pathology reviewed the cases for radiologic and pathologic patterns. RESULTS: We identified 23 patients with a pathologic pattern of hard metal pneumoconiosis. The most common radiographic findings were ground glass opacities (93%) and small nodules (64%). Of 20 surgical biopsies, 17 (85%) showed features of giant cell interstitial pneumonia. Most patients received systemic corticosteroids and/or steroid-sparing immunosuppression. CONCLUSIONS: Hard metal pneumoconiosis is characterized predominately by radiographic ground glass opacities and giant cell interstitial pneumonia on histopathology. Systemic corticosteroids and steroid-sparing immunosuppression are common treatment options.

Prognostic role of serum C-reactive protein in patients with advanced-stage NSCLC treated with pemetrexed.

Pemetrexed is an intravenously administered antifolate cytostatic agent targeting several folate-dependent enzymatic pathways, widely used in the treatment of patients with advanced non-small cell lung cancer (NSCLC). It has been previously demonstrated that the superiority of pemetrexed is limited to patients with non-squamous histology. Aside from the non-squamous histology, there is still no available molecular biomarker predicting treatment efficacy of pemetrexed-based chemotherapy. The aim of our retrospective study was to evaluate the association of baseline serum levels of C-reactive protein (CRP) with outcomes in a large cohort of patients with non-squamous NSCLC treated with pemetrexed. Clinical data of 325 patients were analysed. Serum samples were collected within one week before the initiation of treatment. The median progression-free (PFS) and overall survival (OS) for patients with high CRP was 2.1 and 9.5 compared to 4.2 and 20.5 months for those with normal CRP (p=0.002 and p<0.001, respectively). The multivariable Cox proportional hazards model revealed that serum CRP (HR=1.46, p=0.002) was significantly associated with PFS and also with OS (HR=1.95, p<0.001). In conclusion, the study results suggest that pretreatment serum CRP is associated with poor outcome of non-squamous NSCLC patients treated with pemetrexed.

Serum albumin is a strong predictor of survival in patients with advanced-stage non-small cell lung cancer treated with erlotinib.

Molecular targeted therapy based on tyrosine kinase inhibitors (TKI), directed at epidermal growth factor receptor (EGFR) is one of the novel effective agents in management of advanced-stage of Non Small Cell Lung cancer (NSCLC). However several candidate predictors have been extensively studied, apart from activating EGFR gene mutations, no reliable biochemical or molecular predictors of response to erlotinib have been validated. The aim of our retrospective study was to evaluate the association of baseline serum albumin with outcomes in a large cohort of patients with advanced-stage NSCLC treated with erlotinib. Clinical data of 457 patients with locally-advanced (III B) or metastatic stage (IV) NSCLC treated with erlotinib were analysed. Serum samples were collected and the measurement was performed one day before the initiation of erlotinib treatment. Before the treatment initiation, low albumin was (<35 g/l) measured in 37 (8.1%) patients and normal albumin (≥ 35 g/l) was measured in 420 (91.9%). The median PFS and OS for patients with low serum albumin was 0.9 and 1.9 months compared to 1.9 and 11.4 months for patients with normal serum albumin (p=0.001 and p<0.001). The multivariate Cox proportional hazards model revealed that EGFR mutation status (HR=2.50; CI: 1.59-3.92; p<0.001) and pretreatment serum albumin (HR=1.73; CI: 1.21-2.47; p=0.003) were significant independent predictive factors for PFS, whereas EGFR mutation status (HR=3.14; CI: 1.70-5.81; p<0.001), stage (HR=1.48; CI: 1.09-2.02; p=0.013), ECOG PS (HR=1.77; CI: 1.37-2.29; p<0.001) and pretreatment serum albumin (HR=4.60; CI: 2.98-7.10; p<0.001) were significant independent predictive factors for OS. In conclusion, the results of present retrospective study indicate that pretreatment hypoalbuminemia is associated with poor outcome of NSCLC patients treated with erlotinib. Based on these results, measuement of serum albumin is an objective laboratory method feasible for estimation of prognosis of patients with advanced-stage NSCLC.

[Determination of anti-Müllerian hormone in women]. / Stanovení anti-Müllerova hormonu u zen.

Anti-Müllerian hormone (AMH) is a glycoprotein and belongs to the TGF-ß growth factors family. Our review describes the method of AMH determination in serum and follicular fluid. The reference values and changes in AMH levels during a womans life are also discussed. In addition, it is also presented the relationship between AMH, obesity, smoking and use of hormonal contraceptives. The focus of the work is the importance of the determination of AMH in clinical practice. In assisted reproduction has become its determination one of the tools to detect ovarian reserve. It helps not only predict reduced response to stimulation with gonadotropins but also the risk of the ovarian hyperstimulation syndrome. Benefits of the ovarian reserve detection using AMH serum levels are discussed in comparison with the antral follicle count (AFC) determined by ultrasound. Several clinical indications of AMH determination are mentioned in the next section. These are primarily the polycystic ovary syndrome (PCOS), which is a great challenge not only for the AMH testing, but there is an open space for further interdisciplinary cooperation. Endometriosis has no direct effect on ovarian reserve and AMH levels in serum. AMH is very sensitive tumor marker in the diagnostics and monitoring of ovarian granulosa cells tumors. Treatment of cancer disease burdens entire body, including healthy cells. Ovarian follicles are very sensitive to chemotherapy and radiation. AMH is a good predictor of ovarian reserve damage during radio- and chemotherapy.

[HE4 a biomarker of ovarian cancer]. / HE4 - biomarker ovariálního karcinomu.

OBJECTIVE: Verification of the importance of determination of HE4 and calculation of ROMA index for increasing the efficiency of diagnosis of ovarian cancer in a population of Czech women. DESIGN: Prospective study. SETTING: Department of Gynaecology and Obstetrics, Faculty Hospital in Pilsen. METHODS: In the period from 06/24/2010 to 12/01/2011 was at the Department of Gynaecology and Obstetrics, University Hospital Pilsen examined 552 patients with abnormalities in the pelvis. Patients were divided into two groups. There were 30 women with histologically confirmed malignant ovarian tumors. Another 522 women had benign findings. According to the levels of FSH were women in both groups divided into premenopausal and postmenopausal. At all women were measured CA 125, HE4 and FSH. HE4 and CA125 were determined using the chemiluminescent device Architect 1000 (Abbott, USA), FSH chemiluminescent method on the device DXI 800 (Beckman Coulter, USA). At all premenopausal women was calculated ROMA1 index and at all postmenopausal women ROMA2 index. SAS statistical software 9.2 were used for all statistical calculations. RESULTS: The highest diagnostic efficiency was achieved by a combination of HE4 and CA125 markers with the calculation ROMA2 index for postmenopausal women. In determining of menopausal status according to the values of FSH cut-off for menopause 40 IU/L and cut-off at 26.4% for ROMA2 reaches ROMA2 sensitivity of 92.3%, specificity of 88.5% and PV- of 99.3%. If we reduce the cut-off for laboratory diagnosis of menopause using FSH at 22 IU/L, and cut-off for ROMA2 was 26.3% reaches ROMA2 sensitivity of 95.2%, specificity of 87.8% and PV- of 99.5%. CONCLUSION: HE4 in combination with CA125 and current ROMA index calculation is a suitable methodology to improve the detection of ovarian cancer.

The retention behaviour of polar compounds on zirconia based stationary phases under hydrophilic interaction liquid chromatography conditions.

The most separations in HILIC mode are performed on silica-based supports. Nevertheless, recently published results have indicated that the metal oxides stationary phases also possess the ability to interact with hydrophilic compounds under HILIC conditions. This paper primarily describes the retention behaviour of model hydrophilic analytes (4-aminobenzene sulfonic acid, 4-aminobenzoic acid, 4-hydroxybenzoic acid, 3,4-diaminobenzoic acid, 3-aminophenol and 3-nitrophenol) on the polybutadine modified zirconia in HILIC. The results were simultaneously compared with a bare zirconia and a silica-based HILIC phase. The mobile phase strength, pH and the column temperature were systematically modified to assess their impact on the retention of model compounds. It was found that the retention of our model hydrophilic analytes on both zirconia phases was mainly governed by adsorption while on the silica-based HILIC phase partitioning was primarily involved. The ability of ligand-exchange interactions of zirconia surface with a carboxylic moiety influenced substantially the response of carboxylic acids on the elevated temperature as well as to the change of the mobile phase pH in contrast to the silica phase. However, no or negligible ligand-exchange interactions were observed for sulfanilic acid. The results of this study clearly demonstrated the ability of modified zirconia phase to retain polar acidic compounds under HILIC conditions, which might substantially enlarge the application area of the zirconia-based stationary phases.

[IGF1 and tumor markers in different breast cancer stages]. / IGF1 a nádorové markery ve stadiích nádoru prsu.

INTRODUCTION: In our work we asked ourselves whether it would be possible to use growth factors for a quick orientation in the clinical status of patients prior to biopsy and histological examination. MATERIAL AND METHODS: Our patient group included 82 patients with breast cancer. Serum samples were collected preoperatively. Histological examination findings were available for each patient. Our set was divided into three groups based on the disease stage. The values of analytes in different tumor stages were statistically evaluated and statistical comparisons of Stage I and II, and then of Stage II and III were performed. RESULTS: Tumor markers CEA, CA 15-3, TK, TPA-M and MonoTotal correlate with the disease severity. Serum levels of the growth factor IGFI negatively correlated with the severity of cancer. There was aa statistically significant increase in the EGF growth factor serum levels between Stage I and II. No statistically significant differences between Stage I vs. II and Stage II vs. III were detected when HGF and VEGF growth factor serum levels were assessed. CONCLUSION: The growth factor EGF is one of the candidates to become a tumor growth marker in early disease stages. The IGFI, HGF and VEGF growth factors can not be used for quick and correct orientation in the clinical condition of patients in the early stages of tumor growth.

An innovative approach to the analysis of 3-[4-(2-methylpropyl)phenyl]propanoic acid as an impurity of ibuprofen on a carbon-coated zirconia stationary phase.

3-[4-(2-Methylpropyl)phenyl]propanoic acid has been introduced as impurity F to the European Pharmacopoeia in its Supplement 4.2. In contrast to other impurities, which are evaluated by HPLC, the content of impurity F is determined by gas chromatography after previous derivatization. Thus a novel reversed-phase HPLC method was developed to simplify the evaluation of pharmacopoeial impurity F of ibuprofen. Favourable properties of zirconia stationary phases were employed for this purpose. The HPLC separation was achieved on a Zr-CARB column (150 mm x 4.6mm i.d., 5 microm) using the mobile phase acetonitrile-phosphate buffer (pH 3.5, 25 mM) (38:62, v/v), temperature 80 degrees C and the flow rate 1.2 ml min(-1). The fluorescence detection was employed to enhance the sensitivity of the method. Optimal detection parameters were chosen on the basis of fluorescence spectra of the analytes. The excitation and emission wavelengths were 220 nm and 285 nm, respectively. The analysis was completed within 25 min. The subsequent validation of the method confirmed the applicability of method for the analytical assay of impurity F.

Using of HPLC coupled with coulometric detector for the determination of biotin in pharmaceuticals.

The method for the determination of biotin by high performance liquid chromatography (HPLC) coupled with coulometric detector is presented here. Chromatographic and detection conditions were tested. A LiChrospher 60RP-select B column (250 mm x 4 mm; 5 microm) and the mobile phase containing 0.24 mol/L aqueous solution of acetic acid and acetonitrile in the ratio 85:15 (v/v) were found as the most suitable. The flow rate was 1 mL/min and the injected volume of the sample was 20 microL. The hydrodynamic voltammogram of biotin was measured and according to obtained data the detection parameters were set--channel I 600 mV, channel II 900 mV, sensitivity 1 microA. The developed method has been validated. The calibration curve is linear in the range 15-3600 ng/mL, correlation coefficient is 0.9998, limits of detection and quantification are 5 and 15 ng/mL, respectively. Recovery of the spiked samples was 98.67% with R.S.D. 0.255% on average. The developed method has been successfully applied for determination of biotin in pharmaceutical preparations.

Determination of a series of quinolones in pig plasma using solid-phase extraction and liquid chromatography coupled with mass spectrometric detection Application to pharmacokinetic studies.

A simple and sensitive liquid chromatography (LC) method was developed for the simultaneous determination of eight quinolones in pig plasma samples. The following two methods of detection were used: ultraviolet (UV) and mass spectrometry with electrospray ionization (ESI/MS). Sample preparation consisted of solid-phase extraction (SPE) on Strata X cartridges prior to the analysis by LC/UV or LC/ESI/MS. The recovery, linearity, limit of detection (LOD) and limit of quantification (LOQ), precision and accuracy of the method were evaluated using spiked pig plasma samples. The suitability of the method for pharmacokinetic studies was evaluated by determining the concentrations of enrofloxacin (ENR) and ciprofloxacin (CIP) also in pig plasma, after administration of 200mg of enrofloxacin per kilogram of fodder during 5 consecutive days.

Use of the zirconia-based stationary phase for separation of ibuprofen and its impurities.

A new reversed-phase liquid chromatographic method using zirconia-based stationary phase was developed for determination of ibuprofen, its related compounds and its main degradation products. The chromatographic separation was successfully achieved on the Discovery Zr-PS column (150 mm x 4.6 mm i.d., 5 microm), using a mobile phase methanol-phosphate buffer (pH 4.5; 0.05 M)-tetrahydrofurane (21:74:5, v/v/v) and the flow rate 0.5 ml min(-1). The UV detection was performed in dual wavelength mode (219 and 258 nm) to detect all compounds of interest. The column temperature was set on 60 degrees C to shorten the analysis time and improve the peak symmetry. The method is simple, rapid and cuts down the amount of hazardous waste produced in the analysis. The assay is completed within 22 minutes.

Use of chiral liquid chromatography for the evaluation of stereospecificity in the carbonyl reduction of potential benzo[c]fluorene antineoplastics benfluron and dimefluron in various species.

Benfluron (B) [5-(2-dimethylaminoethoxy)-7H-benzo[c]fluorene-7-one hydrochloride] is a potential antineoplastic agent. In the organism, B undergoes a rapid phase I biotransformation through oxidative and reductive metabolic pathways. The carbonyl reduction of B leads to reduced benfluron, red-B, this is one of the principal pathways for the deactivation of this compound. The structure of B was modified to suppress its rapid deactivation via the carbonyl reduction on C7. Dimefluron, D (3,9-dimethoxy-benfluron) is one of the derivatives of B, in which an alternative metabolic pathway (O-desmethylation) prevails over the carbonyl reduction. The goal of this study was to develop HPLC methods enabling chiral separations of the red-B and -D enantiomers. The separation of red-B enantiomers was successful done on a Chiralcel OD-R column (250 mm x 4.6 mm ID, 5 microm) using a mobile phase acetonitrile-1 M NaClO4 (40:60, v/v). Another mobile phase, methanol-1 M NaClO4 (75:25, v/v), had to be employed for the sufficient resolution of red-D enantiomers. Flow rate was 0.5 ml min(-1) in both cases. Red-B was detected at 340 nm, red-D at 370 nm. The above chiral HPLC methods were used for the study of the biotransformation of B and D in the microsomal fractions of liver homogenates prepared from various species (rat, rabbit, pig, guinea pig, goat and human). The enantiospecificity of the respective carbonyl reductases was evaluated and discussed for both prochiral compounds, B and D.

The energetics of the interaction of BamHI endonuclease with its recognition site GGATCC.

The interaction of BamHI endonuclease with DNA has been studied crystallographically, but has not been characterized rigorously in solution. The enzyme binds in solution as a homodimer to its recognition site GGATCC. Only six base-pairs are directly recognized, but binding affinity (in the absence of the catalytic cofactor Mg(2+)) increases 5400-fold as oligonucleotide length increases from 10 to 14 bp. Binding is modulated by sequence context outside the recognition site, varying about 30-fold from the bes t (GTG or TAT) to the worst (CGG) flanking triplets. BamHI, EcoRI and EcoRV endonucleases all have different context preferences, suggesting that context affects binding by influencing the free energy levels of the complexes rather than that of the free DNA. Ethylation interference footprinting in the absence of divalent metal shows a localized and symmetrical pattern of phosphate contacts, with strong contacts at NpNpNpGGApTCC. In the presence of Mg(2+), first-order cleavage rate constants are identical in the two GGA half-sites, are the same for the two nicked intermediates and are unaffected by substrate length in the range 10-24 bp. DNA binding is strongly enhanced by mutations D94N, E111A or E113K, by binding of Ca(2+) at the active site, or by deletion of the scissile phosphate GpGATCC, indicating that a cluster of negative charges at the catalytic site contributes at least 3-4 kcal/mol of unfavorable binding free energy. This electrostatic repulsion destabilizes the enzyme-DNA complex and favors metal ion binding and progression to the transition state for cleavage.

Structure of free BglII reveals an unprecedented scissor-like motion for opening an endonuclease.

Restriction endonuclease BglII completely encircles its target DNA, making contacts to both the major and minor grooves. To allow the DNA to enter and leave the binding cleft, the enzyme dimer has to rearrange. To understand how this occurs, we have solved the structure of the free enzyme at 2.3 A resolution, as a complement to our earlier work on the BglII-DNA complex. Unexpectedly, the enzyme opens by a dramatic 'scissor-like' motion, accompanied by a complete rearrangement of the alpha-helices at the dimer interface. Moreover, within each monomer, a set of residues--a 'lever'--lowers or raises to alternately sequester or expose the active site residues. Such an extreme difference in free versus complexed structures has not been reported for other restriction endonucleases. This elegant mechanism for capturing DNA may extend to other enzymes that encircle DNA.

Crystallization and preliminary X-ray diffraction analysis of MspI restriction endonuclease in complex with its cognate DNA.

The MspI restriction endonuclease is a type II restriction enzyme. Unlike all other restriction enzymes with known structures, MspI recognizes the palindromic tetranucleotide sequence 5'-C/CGG and cleaves it as indicated by the '/' to produce DNA products with 5' two-base overhangs. Owing to the nature of its cleavage pattern, it is likely that MspI would represent a new structural class of restriction endonucleases. Crystals of the dimeric MspI restriction enzyme bound to a duplex DNA molecule containing the specific recognition sequence have been obtained by vapor-diffusion techniques in the presence of polyethylene glycol as precipitant. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 50.2, b = 131.6, c = 59.3 A, beta = 109.7 degrees. The crystals contain one dimeric complex in the asymmetric unit. A complete native data set has been collected to a resolution of 2.05 A by cryo-crystallographic methods, with an R(merge) of 4.0%.

Crystallization of restriction endonuclease BamHI with nonspecific DNA.

Restriction endonucleases show extraordinary specificity in distinguishing specific from nonspecific DNA sequences. A single basepair change within the recognition sequence results in over a million-fold loss in activity. To understand the basis of this sequence discrimination, it is just as important to study the nonspecific complex as the specific complex. We describe here the crystallization of restriction endonuclease BamHI with several nonspecific oligonucleotides. The 11-mer, 5'-ATGAATCCATA-3', yielded cocrystals with BamHI, in the presence of low salt, that diffracted to 1.9 A with synchrotron radiation. The cocrystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 114.8 A, b = 91.1 A, c = 66.4 A, alpha = 90 degrees, beta = 90 degrees, gamma = 90 degrees. This success in the cocrystallization of BamHI with a nonspecific DNA provides insights for future attempts at crystallization of other nonspecific DNA-protein complexes.

Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 A resolution.

Restriction endonucleases are remarkably resilient to alterations in their DNA binding specificity. To understand the basis of this immutability, we have determined the crystal structure of endonuclease BglII bound to its recognition sequence (AGATCT), at 1. 5 A resolution. We compare the structure of BglII to endonuclease BamHI, which recognizes a closely related DNA site (GGATCC). We show that both enzymes share a similar alpha/beta core, but in BglII, the core is augmented by a beta-sandwich domain that encircles the DNA to provide extra specificity. Remarkably, the DNA is contorted differently in the two structures, leading to different protein-DNA contacts for even the common base pairs. Furthermore, the BglII active site contains a glutamine in place of the glutamate at the general base position in BamHI, and only a single metal is found coordinated to the putative nucleophilic water and the phosphate oxygens. This surprising diversity in structures shows that different strategies can be successful in achieving site-specific recognition and catalysis in restriction endonucleases.

Crystallization and preliminary diffraction analysis of a hyperthermostable DNA polymerase from a Thermococcus archaeon.

The hyperthermostable DNA polymerase from a marine Thermococcus archaeon has been crystallized in space group P212121, with unit-cell dimensions a = 94.8, b = 98.2, c = 112.2 A with one molecule per asymmetric unit. Conditions for data collection at 98 K have been identified, and a complete data set was collected to 2.2 A resolution. Strategies employed here may facilitate crystallization of other hyperthermostable proteins. The structure of this enzyme will provide the first structural data on the archaeal and hyperthermostable classes of DNA polymerases. Sequence homology to human polymerase alpha (polymerase B family) may make it a model for studying eukaryotic and viral polymerases and for the development of anti-cancer and anti-viral therapeutics.