The Added Value of Different Data Types for Calibrating and Testing a Hydrologic Model in a Small Catchment.

This study investigated the added value of different data for calibrating a runoff model for small basins. The analysis was performed in the 66 ha Hydrological Open Air Laboratory, in Austria. An Hydrologiska Byråns Vattenbalansavdelning (HBV) type, spatially lumped hydrologic model was parameterized following two approaches. First, the model was calibrated using only runoff data. Second, a step-by-step approach was followed, where the modules of the model (snow, soil moisture, and runoff generation) were calibrated using measurements of runoff and model state variables and output fluxes. These measurements comprised laser-based measurements of precipitation, satellite and camera observations of snow, ultrasonic measurements of snow depth, eddy covariance measurements of evapotranspiration, time domain transmissometry-based soil moisture measurements, time-lapse photography of overland flow, and groundwater level measurements by piezometers. The two model parameterizations were evaluated on annual, seasonal, and daily time scales, in terms of how well they simulated snow, soil moisture, evapotranspiration, overland flow, storage change in the saturated zone, and runoff. Using the proposed step-by-step approach, the relative runoff volume errors in the calibration and validation periods were 0.00 and -0.01, the monthly Pearson correlation coefficients were 0.92 and 0.82, and the daily logarithmic Nash Sutcliffe efficiencies were 0.59 and 0.18, respectively. By using different sources of data besides runoff, the overall process consistency improved, compared to the case when only runoff was used for calibration. Soil moisture and evapotranspiration observations had the largest influence on simulated runoff, while the parameterization of the snow and runoff generation modules had a smaller influence.

Separation of Scales in Transpiration Effects on Low Flows: A Spatial Analysis in the Hydrological Open Air Laboratory.

The objective of this study was to understand whether spatial differences in runoff generation mechanisms affect the magnitudes of diurnal streamflow fluctuations during low flow periods and which part of the catchment induces the diurnal streamflow signal. The spatiotemporal variability of the streamflow fluctuations observed at 12 locations in the 66-ha Hydrological Open Air Laboratory experimental catchment in Austria was explained by differences in the vegetation cover and runoff generation mechanisms. Almost a quarter of the volume associated with diurnal streamflow fluctuations at the catchment outlet was explained by transpiration from vegetation along the tributaries; more than three quarters was due to transpiration by the riparian forest along the main stream. The lag times between radiative forcing and evapotranspiration estimated by a solar radiation-driven model increased from 3 to 11 hr from spring to autumn. The recession time scales increased from 21 days in spring to 54 days in autumn. Observations and model simulations suggest that a separation of scales in transpiration effects on low flows exists both in time and space; that is, the diurnal streamflow fluctuations are induced by transpiration from the riparian vegetation, while most of the catchment evapotranspiration, such as evapotranspiration from the crop fields further away from the stream, do not influence the diurnal signal in streamflow.

[Video-assisted transanal endoscopic microsurgery of rectal tumours - V-TEM]. / Videoassistierte transanale endoskopische Mikrochirurgie von Rektumtumoren - V-TEM.

BACKGROUND: Transanal endoscopic microsurgery (TEM) has become increasingly established as the method of choice for the local resection of endoscopically unresectable rectal adenomas and early-stage, low-risk rectal carcinomas. Multiple studies have shown that the single port-technique TEM results in significantly less trauma with comparable overall treatment outcome as compared to conventional radical surgical techniques. However, TEM is not widely used due to high initial set-up costs, the need for highly complex equipment and demanding surgical skill requirements. PATIENTS AND METHODS: To mitigate these challenges we have successfully developed a video-assisted TEM (V-TEM) method, which resulted in approximately 50 % lower initial set-up costs through the introduction of simplified original TEM surgical equipment. Between October 2003 and September 2011 we have completed 103 resections using the V-TEM method. RESULTS: The observed rates of complications and local recurrences are comparable to reported rates. CONCLUSIONS: We were able to mitigate the challenges of TEM through the establishment of the technically less demanding V-TEM method, which resulted in approximately 50 % lower initial set-up costs while maintaining overall treatment outcomes.

[Indications for laparoscopic pancreas operations: results of a consensus conference and the previous laparoscopic pancreas register]. / Indikation zur laparoskopischen Pankreasoperation: Ergebnisse einer Konsensuskonferenz und des bisherigen laparoskopischen Pankreasregisters.

Laparoscopic pancreatic surgery is not common practice in Germany and is only carried out in approximately 20 clinics but with an increasing trend. The reasons for this are manifold, such as the current selection of patients and both skills in laparoscopic and pancreatic surgery are necessary to perform this operation safely. In 2008 a registry called "Laparoscopic pancreatic surgery" was implemented to collect enough data in Germany to find out whether the resection is safe, feasible and beneficial for the patient.For further development of new laparoscopic techniques new data is needed. A group of experts performing laparoscopic pancreatic surgery in Germany supplied their data for the German registry for laparoscopic pancreatic resection and a consensus conference about the indications became necessary. This consensus conference discussed in particular the indications for laparoscopic pancreatic resection. A consensus was found by all members of the conference utilizing currently available evidence-based data.It was suggested that all data of laparoscopic pancreatic surgery should be evaluated in the German Registry. A consensus was made which diseases were either suitable for laparoscopic resection or not suitable or suitable in selected cases.

[The missions of the Occupational Diseases Fund. Under-claim and recognition of occupational lung cancer, in particular those related to asbestos]. / Les missions du Fonds des Maladies Professionnelles. La sous-déclaration des cancers respiratoires professionnels, en particulier dus a l'amiante.

The missions of the Occupational Diseases Fund are defined in application of the law regarding the insurance against occupational diseases. The workers covered by this law are granted several rights, such as a financial compensation in case of temporary or permanent disability, a further compensation if they have to be taken away from the risk in the workplace, the reimbursement of health care costs related to the occupational disease, or the payment of an annuity to the widow(er) if death is its ultimate consequence. Among the compensable diseases, we shall focus on lung cancer, and especially the one related to asbestos exposure. This type of cancer is clearly under-registrated in Belgium as in most countries of the European Union, leading to an insufficient number of cases entitled to compensation by our institution. In this instance, the insurance against occupational diseases and all related social advantages are hugely under-exploited in our country. It is our duty to increase doctors' awareness of the problem and spread accurate information to reverse this trend and provide occupational cancer cases with a legitimate compensation, in particular those related to asbestos. A wider knowledge of the occupational history of cancer patients, thanks to occupational physicians, and a better use of mineralogical analyses on lung samples, would improve this situation inacceptable on any level : medical, social or even human.

[Actions of the Fund for Occupational Diseases, in terms of rehabilitation of work-related low-back pain]. / Le rôle du Fonds des Maladies Professionnelles en faveur de la réadaptation des patients lombalgiques.

The Fund for Occupational Diseases proposes a "rehabilitation programme for patients with work related low-back pain". The purpose of the programme is to accelerate by intensive physical training and a full recoverage of cognitive needs the return to work as well as preventing back pain becoming chronic. The programme is free for the patients and in addition supports measures being taken to reduce lower back related problems linked to work. Set up 4 years ago, the programme has become more and more in demand by workers.

Influence of moderate cycling on scrotal temperature.

Testicular temperature highly correlates with scrotal temperature. It has been postulated that cycling is associated with increased scrotal temperatures with time and consecutively with impaired semen quality. The aim of this study was to evaluate the influence of moderate cycling on scrotal temperature during highly standardized conditions in an experimental lab. A total of 25 volunteers without a history of infertility and normal andrological examination were included for scrotal temperature evaluation. Scrotal temperatures were measured every minute with a portable data recorder connected with two thermistor temperature sensors, which were attached on either side of the scrotum. A further thermistor sensor was attached on the central surface of the bicycle saddle. Ambient temperature in the study room was adjusted to 22 degrees C throughout the whole experiment. All volunteers started the experiment at the same daytime. Clothing of the volunteers consisted of standardized cotton wool trousers and shirts fitting to body size. After acclimatization to the study room in a sitting posture, each volunteer cycled on an exercise cycle for 60 min with a power of 25 Watt representing a speed of 25.45 km/h respectively. The saddle surface temperature reached in the median 35.59 degrees C after 60 min cycling. Median values of scrotal temperatures increased from 35.75 degrees C at the beginning to 35.82 degrees C after 60 min for the left side and from 35.50 to 35.59 degrees C for the right side. No correlation between cycling duration and scrotal temperatures could be found using multivariate anova for repeated measurements. However, scrotal temperatures during cycling were significantly lower (p < 0.001) compared with the last 10 min in sitting posture before starting cycling with a difference of 1.31 degrees C for the left and 1.46 degrees C for the right side. The present study suggests that moderate cycling under standardized conditions with a power of 25 Watt is not a major genital heat stress factor.

Lessons learned from investigations on case study level for modelling of nutrient emissions in the Danube basin.

In the framework of the project daNUbs (Nutrient Management in the Danube Basin and its Impact on the Black Sea) the MONERIS emission model is used for the basin wide calculation of nutrient (nitrogen and phosphorus) emissions in the Danube Basin. The MONERIS model was developed and successfully applied for German river catchments. Based on investigations in selected test regions (case studies) the daNUbs approach is to check the applicability of the MONERIS emission model for the specific conditions of the Danube Basin in more detail than is possible with a basin wide application. Six case studies with areas of 400-3,500 km2 and several subcatchments have been selected in order to represent different conditions along the Danube Basin. In this study region intensive data collection and enhanced monitoring has been performed in order to raise the database significantly above the generally available data. Water balance as well as nutrient balance calculations have been performed with the MONERIS model as well as with other approaches. Results are compared to each other and to data from monitoring. Results up till now showed the applicability and sensitivity of the MONERIS approach in different conditions of the Danube Basin (e.g. emissions via groundwater). They indicated that the nitrogen retention in the catchments is well described with the MONERIS model.

Considerations on the influence of extreme events on the phosphorus transport from river catchments to the sea.

In this paper, results from rivers of different sizes in Romania, Hungary and Austria are presented. The paper shows the dynamics of extreme events and their contribution to the total P and suspended solids transported in these rivers. Special attention is paid to the influence of the size of the catchment and the event probability on the relative contribution of a single event to the total loads transported in the river. Further, the development of phosphorus loads along the Danube River at a flood event is shown. From the results it can be concluded that there is no immediate influence of high flow and flood events in upstream parts of the Basin on the transport of phosphorus from the catchment to the receiving Sea. Particle-bound phosphorus is mobilised from the catchment (through erosion) and the river bottom to a high extent at high flow events and transported at peak discharges to downstream, where retention by sedimentation of particles takes place. On the one hand this retention is a transport to flooded areas. In this case it can be considered as more or less long term retention. On the other hand sedimentation takes place in the riverbed, in case the tractive effort of the river is reduced. In this second case the P-pool in the sediments of the sedimentation area will be increased. If anaerobic conditions in the sediment appear, part of the phosphorus will be transformed to soluble ortho-phosphate and will continuously contribute to the phosphorus transport to the receiving sea. Part of the P-retained in the river sediment will be mobilised by resuspension at the next biggest high flow event. Altogether, these alternating processes of suspension, transport, export to flooded areas or sedimentation in the river bed with partly solution and partly resuspension at the next event decrease the share of the phosphorus transport during high flow events on the total loads transported in the more downstream parts of a catchments as compared to the more upstream parts. In the year of occurrence of an extreme flood event the P-transport of this year is dominated by the flood event. As an average over many years the contribution of high flow events to the total P-transport still may be between 7 and 20% in smaller catchments (around 1,000 km2). In a big catchment (e.g. river Danube) much smaller contributions of flood events on the total P-transport can be expected as an average over many years.

Oligonucleotides with bistranded abasic sites interfere with substrate binding and catalysis by human apurinic/apyrimidinic endonuclease.

Apurinic/apyrimidinic endonuclease (AP endo) is a key enzyme in oxidative damage DNA repair. The enzyme, which repairs abasic sites, makes a single nick 5' to the phosphodeoxyribose, leaving a free 3'-hydroxyl. We recently described single turnover kinetics for human recombinant AP endo acting on an oligonucleotide with a single abasic site. We hypothesized that the structural changes induced by the presence of a second abasic site might provide insight into how AP endo recognizes the first abasic site. Here we performed steady state and single turnover experiments using bistranded abasic site substrates, with the second site located on the complementary strand to the one being followed and either opposite to the first or displaced in the 5' direction. All sites on the complementary strand were within half a helical turn of the first. The catalytic efficiency was reduced 80 to 96% and the Kd for substrate binding and dissociation was elevated 40- to 125-fold. The smaller changes occurred when the second site was opposite the first site or displaced by four nucleotides. In addition, if the second abasic site was directly across the helix or displaced by 1 or 3 nucleotides from the first abasic site, cleavage of the first abasic site was subject to apparent substrate inhibition, which did not occur if the second abasic site was displaced by four nucleotides from the first. While a substrate containing a nick without a phosphodeoxyribose on the contralateral strand abasic site did not inhibit nicking of the first strand, a substrate with a nicked abasic site on the contralateral strand was an even stronger inhibitor of enzyme action than an oligonucleotide containing the corresponding abasic site on each strand. Consequently, the inhibitory effect of the second abasic site is probably the result of prior cleavage of the abasic site on the contralateral strand with resulting distortions to the DNA helix that interfere with enzyme binding and/or cleavage.

A spin-labeled abasic DNA substrate for AP endonuclease.

We report the first observation of a spin-labeled ds 23-mer oligonucleotide by high-field electron spin resonance (ESR) and demonstrate that it interacts with AP endonuclease, the key enzyme in DNA abasic site repair. The spin labeled 23-mer with a U at position 12 of the upper strand is processed by uracil DNA glycosylase to provide the abasic substrate. With a spin-label two nucleotides away from the abasic site, AP endo binds and cleaves when the label is 3' but not 5' to the abasic site. These results confirm that the disposition of the bases immediately upstream of the abasic site is particularly critical for cleavage by AP endo, and establish that DNA-protein interactions in this important enzyme can be examined using spin-labeled substrates.

Thioredoxin facilitates the induction of heme oxygenase-1 in response to inflammatory mediators.

Heme oxygenase (HO)-1 is a stress response protein that is regulated by oxidative stress. HO-1 catalyzes the generation of biliverdin, carbon monoxide, and iron from heme. Lipopolysaccharide (LPS) and interleukin (IL)-1beta induce HO-1 through the binding of nuclear proteins to AP-1 motifs in enhancer regions upstream from the transcription start site. The DNA binding activity of AP-1 proteins depends on the reduction of cysteines in their DNA-binding domains. We found that agents that disrupt free sulfhydryl groups abolish AP-1 binding activity in nuclear proteins obtained from rat aortic smooth muscle cells and macrophages stimulated with IL-1beta or LPS. Thioredoxin (TRX) may regulate the redox status of nuclear transcription factors in response to oxidative stimuli, thus we determined the role of TRX in the physiologic regulation of HO-1. TRX underwent nuclear translocation in cells stimulated with IL-1beta and LPS. We transfected macrophages with a heterologous promoter construct containing two AP-1 sites from an upstream enhancer region in the HO-1 promoter. Recombinant TRX induced promoter activity to a level analogous to that induced by LPS, and this TRX response was abolished by mutation of the AP-1 sites. An inhibitor of TRX reductase, used to prevent TRX translocation in the reduced state, decreased HO-1 induction by IL-1beta and LPS. These data provide the first evidence that TRX contributes to the induction of HO-1 by inflammatory mediators.

Dynamics and ordering in a spin-labeled oligonucleotide observed by 220 GHz electron paramagnetic resonance.

The dynamics of a newly synthesized cytosine spin-label and the spin-labeled pentamer TTC*TT have been observed by high-frequency (220 GHz) electron paramagnetic resonance (EPR) in aqueous solution at ambient temperature using only nanomolar amounts of spin-label. Temperature studies were carried out for both labeled species in buffer containing glycerol. The motion of the spin-labeled monomer could be fitted using a model of fully anisotropic rotation (FAR) over the entire temperature range studied. In the single-stranded pentamer, the high-field spectra are best interpreted using a model of microscopic ordering with macroscopic disorder (MOMD) with the probe in a highly nonpolar environment. The observed local order parameters of 0.60-0.70 suggest a micelle-like structure in which the label is tightly packed with the hydrophobic bases. These preliminary studies illustrate how the excellent orientation selectivity of high-field EPR provides new dynamic information about local base motions in DNA, and also how high-field EPR of spin-labels allows one to discriminate accurately between the effects of local versus global motions in spin-labeled macromolecules.

Human apurinic/apyrimidinic endonuclease is processive.

Apurinic/apyrimidinic endonuclease (AP endo) is believed to play a critical role in repair of oxidative damage of DNA and is proposed to initiate repair of most abasic sites in the base excision repair pathway. AP endo makes a single nick 5' to an abasic site in double-stranded DNA. In this study, we investigated whether AP endo locates an abasic site through a processive or a distributive mechanism. We used a linear multi-abasic site substrate (concatemer), synthesized by ligating together identical 25-nucleotide monomeric units (25-mers). We first determined that the 25-mer monomer from which the concatemers were prepared was nicked by AP endo in a fashion similar to that of the previously published 49-mer substrate with a different sequence. Steady state parameters K(m) and k(cat) and single-turnover parameters for substrate binding were comparable to previously published values. Using the multi-abasic site concatemer, we demonstrated that AP endo was capable of cleaving approximately seven to eight abasic sites, traveling at least 200 nucleotides, before dissociating from its substrate. Thus, AP endo, like uracil DNA glycosylase, behaves in a quasi processive fashion. Processivity could be separated from catalysis, since processivity was maximal at 25 mM NaCl, while the rate of cleavage was maximal at 125 mM salt. In short, nicking activity was maximized close to physiological salt molarities while processivity was midrange at physiological salt concentrations. The latter is likely to be subject to tight regulation by small changes in ionic strength.

Changes in hair weight and hair count in men with androgenetic alopecia, after application of 5% and 2% topical minoxidil, placebo, or no treatment.

Quantitative estimation of hair growth using hair weight and number was recorded for 120 weeks in 4 groups of 9 men with androgenetic alopecia. Three double-blind groups applied either 2% or 5% minoxidil solution, or vehicle. The fourth group, unblinded, received no treatment. Measurements of hair weight and number were continued for 96 weeks, when treatment (if any) was stopped, though measurements were continued for another 24 weeks. Although not compared statistically, the placebo and untreated groups behaved in a similar fashion. In contrast, the 5% and 2% minoxidil treatment groups showed a statistically significant increase in mean percentage change in interval weight from baseline compared with placebo; results for number counts were usually less significant. Over 96 weeks, topical minoxidil induced and maintained an increase in interval weight over baseline of about 30%. After treatment was stopped, hair weight and number counts for the minoxidil groups returned to about the same levels as placebo in 24 weeks.

Single-turnover analysis of mutant human apurinic/apyrimidinic endonuclease.

Apurinic/apyrimidinic endonuclease (AP endo) is a key enzyme in the repair of oxidatively damaged DNA. Using single-turnover conditions, we recently described substrate binding parameters for wild type human AP endo. In this study, we utilized four enzyme mutants, D283A, D308A, D283A/D308A, and H309N, and assayed them under steady state and single-turnover conditions. The turnover number of the single aspartate mutants was decreased 10-30-fold in comparison to that of the wild type. The decrease in the turnover number was accompanied by a 17- and 50-fold decrease in the forward rate constant (kon) for substrate binding by D308A and D283A, respectively. The dissociation rate constant for substrate (koff) was unchanged for the D308A mutant but was 10 times faster for the D283A mutant than for the wild type. The apparent Km values for both of the single aspartate mutants were about equal to their respective KD values. To account for the kinetic behavior of the D308A mutant, it was necessary to insert a conformational change into the kinetic scheme. In contrast to the single aspartate mutants, the turnover number for the double mutant was 500-fold lower than that of the wild type, its apparent Km was 2.5-fold higher, and binding to substrate was weak. Mutation of His309 caused the greatest decrease in activity, resulting in a turnover number that was more than 30000-fold lower than that of the wild type and an apparent Km that was 13-fold higher, supporting the notion that His309 is intimately involved in catalysis. Molecular dynamics simulation techniques suggested that conversion of either aspartate to alanine resulted in major shifts in the spatial localization of key amino acids. Despite the fact that the two aspartates flank His309, the movement they engendered was distinct, consistent with the differences in catalytic behavior. We suggest that the conformation of the active site is largely maintained by the two aspartates, which enable efficient binding and cleavage of abasic site-containing DNA.

Establishment and characterization of osteoblast-like cell lines from retrovirus (RFB MuLV)-induced osteomas in mice.

Cell lines were established by a two-step method from osteomas which had been induced by infection of mice with RFB MuLV, a bone-pathogenic, replication-competent murine retrovirus. The benign tumors, consisting of mature lamellar bone and surrounded by a thin periosteum, were cultured on sponges of denatured collagen type I fibres for up to 4 weeks. At this time osteoma cells had grown into the collagenous matrix. After release and further cultivation in monolayers, the cell lines established from these cultures varied in morphology; they expressed T1, collagen type I and type III, alkaline phosphatase, osteonectin and osteopontin mRNAs at variable levels, but not osteocalcin/BGP. They also showed alkaline phosphatase activity, but lacked responsiveness to parathyroid hormone. All cell lines established from infected mice expressed retroviral and c-myc mRNA and viral protein. In contrast to cells from control mice they showed an extended life span in culture. After growth in a three-dimensional (3-D) collagen sponge culture the cells formed an extracellular matrix containing collagen type I, alkaline phosphatase and osteocalcin/BGP. These data indicate that the two-step method facilitates the establishment of osteoblast-like cell lines from osteomas and calvaria of old mice, and provides means for further analyses of retrovirus-induced skeletal pathogenesis and bone induction.

Domain mapping of human apurinic/apyrimidinic endonuclease. Structural and functional evidence for a disordered amino terminus and a tight globular carboxyl domain.

We recently described the pre-steady state enzymatic binding kinetics of apurinic/apyrimidinic endonuclease (AP endo). In this report we describe the domain structure of the enzyme in solution determined by mild protease digestion in the presence and absence of substrate, product, and an efficient competitive inhibitor (HDP). AP endo is a 35.5-kDa protein with a high degree of homology to its prokaryotic counterpart, exonuclease III (Exo III), except for the amino terminus, which is lacking in the prokaryotic enzyme. The entire conserved region plus an additional 20 residues unique to the eukaryotic enzyme was inaccessible to trypsin and V8 protease, indicating that it forms a tight globular structure. In contrast, the amino-terminal 35 residues were readily accessible to all the proteases investigated, leading us to conclude that they associate poorly with the rest of the structure and constitute a highly fluid region. When AP endo was boiled with SDS and cooled prior to the addition of V8 protease, several acidic residues within the globular domain became protease-accessible, indicating rapid renaturation except along the nuclease fold with restoration of globular conformation for the carboxyl two-thirds of the molecule. Of all the proteases tested, only chymotrypsin was able to cleave internal to the globular portion without prior denaturation. Although AP endo cleaved with chymotrypsin retained full enzymatic activity, the activity was lost when the digested peptides were recovered after denaturation by heat and/or boiling in SDS, precipitation, and renaturation or when fragments were recovered from an SDS gel and renatured. Thus, the protein is probably held together strongly by noncovalent interactions that maintain enzymatic function after protease nicking. The three major chymotrypsin cleavage sites, Tyr-144, Leu-179, and Leu-205, became strikingly less accessible to protease digestion in the presence of abasic site-containing DNA. Since the three residues form a spherical triangle on the surface of the molecule on one side of the nuclease fold, there must be multiple means by which DNA containing an abasic site associates with the enzyme. The most likely explanation is that substrate and product, both of which were present during proteolysis, bind differently to the enzyme. Finally, the two cysteine residues thought to be involved in the redox reaction of AP endo with Jun protein were entirely inaccessible to proteolysis even after prolonged exposure of AP endo to reducing agents. Consequently, if AP endo plays a role in the physiological function of Jun, it must undergo major conformational changes in the process. Alternatively, the two cysteines could maintain an appropriate conformation such that other residues participate directly in the redox activity.

Human DNA polymerase beta deoxyribose phosphate lyase. Substrate specificity and catalytic mechanism.

DNA polymerase beta (beta-pol) cleaves the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity). The same bond is cleaved even if the AP site has been previously 5'-incised by AP endonuclease, resulting in a 5' 2-deoxyribose 5-phosphate (i.e. dRP lyase activity). We characterized these lyase reactions by steady-state kinetics with the amino-terminal 8-kDa domain of beta-pol and with the entire 39-kDa polymerase. Steady-state kinetic analyses show that the Michaelis constants for both the dRP and AP lyase activities of beta-pol are similar. However, kcat is approximately 200-fold lower for the AP lyase activity on an intact AP site than for an AP endonuclease-preincised site. The 8-kDa domain was also less efficient with an intact AP site than on a preincised site. The full-length enzyme and the 8-kDa domain efficiently remove the 5' dRP from a preincised AP site in the absence of Mg2+, and the pH profiles of beta-pol and 8-kDa domain dRP lyase catalytic efficiency exhibit a broad alkaline pH optimum. An inhibitory effect of pyridoxal 5'-phosphate on the dRP lyase activity is consistent with involvement of a primary amine (Lys72) as the Schiff base nucleophile during lyase chemistry.

Effects of extremely low frequency electromagnetic field (EMF) on collagen type I mRNA expression and extracellular matrix synthesis of human osteoblastic cells.

Human osteoblastic cells were grown in a three-dimensional (3-D) cell culture model and used to test the effects of a 20 Hz sinusoidal electromagnetic field (EMF; 6 mT and 113 mV/cm max) on collagen type I mRNA expression and extracellular matrix formation in comparison with the effects of growth factors. The cells were isolated from trabecular bone of a healthy individual (HO-197) and from a patient presenting with myositis ossificans (MO-192) and grown in a collagenous sponge-like substrate. Maximal enhancement of collagen type I expression after EMF treatment was 3.7-fold in HO-197 cells and 5.4-fold in MO-192 cells. Similar enhancement was found after transforming growth factor-beta (TGF-beta) and insulin-like growth factor-I (IGF-I) treatment. Combined treatment of the cells with EMF and the two growth factors TGF-beta and IGF-I did not act synergistically. MO-192 cells produced an osteoblast-characteristic extracellular matrix containing collagen type I, alkaline phosphatase, and osteocalcin, together with collagen type III, TP-1, and TP-3, two epitopes of an osteoblastic differentiation marker. The data suggest that the effects of EMFs on osteoblastic differentiation are comparable to those of TGF-beta and IGF-I. We conclude that EMF effects in the treatment of skeletal disorders and in orthopedic adjuvant therapy are mediated via enhancement of collagen type I mRNA expression, which may lead to extensive extracellular matrix synthesis.