Determination of differences in ultrasound parameters for patellar tendons in males with unilateral patellar tendinopathy-An ancillary analysis of data from two randomized controlled trials.

PURPOSE: To investigate power Doppler (PD) activity and tendon structure (between the injured and contralateral limb) in patients with unilateral patellar tendinopathy (PT) using ultrasonography (US). Secondly, the aim was to determine the intra-rater reliability of the PD activity and tendon structure. METHODS: This study analyzed US baseline data from 57 male participants with symptomatic unilateral PT who had been enrolled in one of two randomized clinical trials. Data were analyzed to examine if systematic differences existed between injured and contralateral limbs using Fiji ImageJ. RESULTS: The PD activity of the symptomatic tendon was larger 25.6 (Q1 = 14.9; Q3 = 41.6) mm2 than the asymptomatic 0 (Q1 = 0.0; Q3 = 0.0) mm2 (p < 0.001). There was a significantly greater tendon thickness at the proximal (2.5 mm 95% CI [2.0; 3.0]), mid (0.8 mm 95% CI [0.5; 1.1]), and distal (0.2 mm 95% CI [0.1; 0.4]) part of the tendon for the symptomatic compared to the asymptomatic tendon. Intra-rater reliability for PD activity and tendon structure ranged from moderate-to-excellent (0.74; 0.99). CONCLUSION: These results provide mean estimates for tendon thickness of symptomatic and asymptomatic tendons, that can be used for clinicians to reliably estimate pathological tendon thickness.

Effects of genipin crosslinking on mechanical cell-matrix interaction in 3D engineered tendon constructs.

It is well known that cells can generate endogenous forces onto the extracellular matrix, but to what extent the mechanical properties of the matrix influences these endogenous cellular forces remains unclear. We therefore sought to quantify the influence of matrix rigidity on cell-matrix interactions by inducing cross-links using increasing concentrations of genipin (0.01-1 mM) or by blocking cross-link formation using beta-aminopropionitrile (BAPN) in engineered human tendon tissue constructs. The cell-matrix mechanics of the tendon constructs were evaluated as cell-generated tissue re-tensioning and stress-relaxation responses using a novel custom-made force monitor, which can apply and detect tensional forces in real-time in addition to mechanical failure testing. Genipin treatment had no influence on the biochemical profile (hydroxyproline, glycosaminoglycan and DNA content) of the constructs and cell viability was comparable between genipin-treated and control constructs, except at the highest genipin concentration. Endogenous re-tension after unloading was significantly decreased with increasing genipin concentrations compared to controls. Mechanical failure testing of tendon constructs showed increased (56%) peak stress at the highest genipin concentration but decreased (72%) with BAPN treatment when compared to controls. Tendon construct stiffness increased with high genipin concentrations (0.1 and 1 mM) and decreased by 70% in BAPN-treated constructs, relative to the controls. These data demonstrate that human tendon fibroblasts regulate their force exertion inversely proportional to increased cross-link capacity but did so independently of matrix stiffness. Overall, these findings support the notion of an interaction between cell force generation and cross-linking, and thus a role for this interplay in mechanical homeostasis of the tissue.

Application of the Multistate Tuberculosis Pharmacometric Model in Patients With Rifampicin-Treated Pulmonary Tuberculosis.

This is the first clinical implementation of the Multistate Tuberculosis Pharmacometric (MTP) model describing fast-, slow-, and nonmultiplying bacterial states of Mycobacterium tuberculosis. Colony forming unit data from 19 patients treated with rifampicin were analyzed. A previously developed rifampicin population pharmacokinetic (PK) model was linked to the MTP model previously developed using in vitro data. Drug effect was implemented as exposure-response relationships tested at several effect sites, both alone and in combination. All MTP model parameters were fixed to in vitro estimates except Bmax . Drug effect was described by an on/off effect inhibiting growth of fast-multiplying bacteria in addition to linear increase of the stimulation of the death rate of slow- and nonmultiplying bacteria with increasing drug exposure. Clinical trial simulations predicted well three retrospective clinical trials using the final model that confirmed the potential utility of the MTP model in antitubercular drug development.

Systemic stiffening of mouse tail tendon is related to dietary advanced glycation end products but not high-fat diet or cholesterol.

Tendon pathology is related to metabolic disease and mechanical overloading, but the effect of metabolic disease on tendon mechanics is unknown. This study investigated the effect of diet and apolipoprotein E deficiency (ApoE(-/-)) on mechanical properties and advanced glycation end product (AGE) cross-linking of non-weight-bearing mouse tail tendons. Twenty ApoE(-/-) male mice were used as a model for hypercholesterolemia along with 26 wild-type (WT) mice. One-half of the mice from each group was fed a normal diet (ND) and the other half was fed a high-fat diet (HFD) to induce obesity. All were killed at 40 wk, and tail tendon fascicles were mechanically tested to failure and analyzed for AGEs. Diets were also analyzed for AGEs. ApoE(-/-) mice displayed a 14% increase in plateau modulus compared with WT mice (P < 0.05), whereas HFD mice displayed a 13% decrease in plateau modulus (P < 0.05) and a 12% decrease in total modulus (P < 0.05) compared with ND mice. Tail tendons of HFD mice had significantly lower concentrations of AGEs [carboxymethyllysine (CML): 26%, P < 0.0001; methylglyoxal-derived hydroimidazolone 1 (MG-H1): 15%, P < 0.005; pentosidine: 13%, P < 0.0005]. The HFD had ∼44-fold lower content of CML (P < 0.01), ∼29-fold lower content of carboxyethyllysine (P < 0.005), and ∼16-fold lower content of MG-H1 (P < 0.05) compared with ND. ApoE(-/-) increased, whereas HFD decreased mouse tail tendon stiffness. Dietary AGE content may be a crucial determinant for accumulation of AGE cross-links in tendons and for tissue compliance. The results demonstrate how systemic metabolic factors may influence tendon health.

Accuracy of MRI technique in measuring tendon cross-sectional area.

Magnetic resonance imaging (MRI) has commonly been applied to determine tendon cross-sectional area (CSA) and length either to measure structural changes or to normalize mechanical measurements to stress and strain. The ability to reproduce CSA measurements on MRI images has been reported, but the accuracy in relation to actual tendon dimensions has never been investigated. The purpose of this study was to compare tendon CSA measured by MRI with that measured in vitro with the mould casting technique. The knee of a horse was MRI-scanned with 1.5 and 3 tesla, and two examiners measured the patellar tendon CSA. Thereafter, the patellar tendon of the horse was completely dissected and embedded in an alginate cast. The CSA of the embedded tendon was measured directly by optical imaging of the cast impression. 1.5 tesla grey tendon CSA and 3 tesla grey tendon CSA were 16.5% and 13.2% lower than the mould tendon CSA, respectively. Also, 3 tesla tendon CSA, based on the red-green border on the National Institute of Health (NIH) colour scale, was lower than the mould tendon CSA by 2.8%. The typical error between examiners was below 2% for all the measured CSA. The typical error between examiners was below 2% for all the measured CSA. These data show that measuring tendon CSA on the grey-scale MRI images is associated with an underestimation, but by optimizing the measurement using a 3 tesla MRI and the appropriate NIH colour scale, this underestimation could be reduced to 2.8% compared with the direct measurements on the mould.

The value of selected in vitro and in silico methods to predict acute oral toxicity in a regulatory context: results from the European Project ACuteTox.

ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol-water partition coefficients and in silico prediction of intestinal absorption and blood-brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD50>2000 mg/kg b.w.).

Reported occupational injuries at Swedish recycling centres - based on official statistics.

Swedish recycling centres are manned facilities for waste collection. There is no special category in the official injury statistics for employees at recycling centres, which precludes a straightforward analysis of reported occupational injuries. This study aimed at identifying the frequency of reported accidents and diseases and the type of events that contribute to such injuries at recycling centres, based on official injury statistics. The employees were identified as being affected by more than three to five times as many accidents compared with the total workforce in Sweden. The reported accidents had occurred during a wide range of situations, but most frequently during manual handling of waste. Reported work-related diseases were mostly associated with musculoskeletal disorders, mainly due to heavy lifting. A more detailed classification of sanitation professions and workplaces in the official injury statistics would facilitate future studies of injuries in a specific professional category, e.g. employees at recycling centres. Suggestions for prevention are given. STATEMENT OF RELEVANCE: The present article describes all reported work accidents and diseases among employees at recycling centres from 1992 to February 2005. It also highlights the problem of identifying new working groups in the official statistics and gives advice for a detailed classification to facilitate such future studies of injuries.

Joint investigation of working conditions, environmental and system performance at recycling centres--development of instruments and their usage.

Recycling is a new and developing industry, which has only been researched to a limited extent. This article describes the development and use of instruments for data collection within a multidisciplinary research programme "Recycling centres in Sweden - working conditions, environmental and system performance". The overall purpose of the programme was to form a basis for improving the function of recycling centres with respect to these three perspectives and the disciplines of: ergonomics, safety, external environment, and production systems. A total of 10 instruments were developed for collecting data from employees, managers and visitors at recycling centres, including one instrument for observing visitors. Validation tests were performed in several steps. This, along with the quality of the collected data, and experience from the data collection, showed that the instruments and methodology used were valid and suitable for their purpose.

Design and characterization of a tissue-equivalent CVD-diamond detector for clinical dosimetry in high-energy photon beams.

New solid-state detectors, based on chemical vapour deposited (CVD) polycrystalline diamonds produced by hot-filament (HF) or microwave plasma (MW) assisted deposition methods, were constructed for radiation therapy dosimetry. Properties of diamond crystals, such as high radiation sensitivity, resistance to radiation damage and tissue-equivalence giving a low-energy dependence are very advantageous for clinical dosimetry. Therefore the encapsulation was specially designed for these detectors to have as little influence as possible on the radiation response. The prototypes were irradiated with use of a wide range of photon beam qualities ((60)Co gamma-rays, 6 and 18 MV X-rays). The radiation sensitivity varied considerably between samples deposited with HF (9 nC Gy(-1)mm(-3)) and MW (66 and 144 nC Gy(-1)mm(-3)) methods. For all detectors the leakage current was of the order of 10% of the radiation-induced current (bias voltage 100 V, dose rate 0.3 Gy/min). When irradiated with (60)Co gamma-rays, the detectors showed a dose-rate linearity with an exponential Delta parameter close to unity. However, a difference of 8% was found between Delta values for the different beam qualities. A small energy dependence was observed, for which the most probable sources are interface effects due to the silver electrodes and partly the geometry of the encapsulation which needs to be further optimized. Despite some limitations in the performance of present prototype detectors, with an improved CVD technique producing crystals of better electrical and dosimetric properties, and with a well-designed tissue-equivalent encapsulation, CVD-diamonds could serve as very good dosimeters for radiotherapy.

Genetic diversity among Botulinum Neurotoxin-producing clostridial strains.

Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.

Development of dose delivery verification by PET imaging of photonuclear reactions following high energy photon therapy.

A method for dose delivery monitoring after high energy photon therapy has been investigated based on positron emission tomography (PET). The technique is based on the activation of body tissues by high energy bremsstrahlung beams, preferably with energies well above 20 MeV, resulting primarily in 11C and 15O but also 13N, all positron-emitting radionuclides produced by photoneutron reactions in the nuclei of 12C, 16O and 14N. A PMMA phantom and animal tissue, a frozen hind leg of a pig, were irradiated to 10 Gy and the induced positron activity distributions were measured off-line in a PET camera a couple of minutes after irradiation. The accelerator used was a Racetrack Microtron at the Karolinska University Hospital using 50 MV scanned photon beams. From photonuclear cross-section data integrated over the 50 MV photon fluence spectrum the predicted PET signal was calculated and compared with experimental measurements. Since measured PET images change with time post irradiation, as a result of the different decay times of the radionuclides, the signals from activated 12C, 16O and 14N within the irradiated volume could be separated from each other. Most information is obtained from the carbon and oxygen radionuclides which are the most abundant elements in soft tissue. The predicted and measured overall positron activities are almost equal (-3%) while the predicted activity originating from nitrogen is overestimated by almost a factor of two, possibly due to experimental noise. Based on the results obtained in this first feasibility study the great value of a combined radiotherapy-PET-CT unit is indicated in order to fully exploit the high activity signal from oxygen immediately after treatment and to avoid patient repositioning. With an RT-PET-CT unit a high signal could be collected even at a dose level of 2 Gy and the acquisition time for the PET could be reduced considerably. Real patient dose delivery verification by means of PET imaging seems to be applicable provided that biological transport processes such as capillary blood flow containing mobile 15O and 11C in the activated tissue volume can be accounted for.

Assessing the difference between planned and delivered intensity-modulated radiotherapy dose distributions based on radiobiological measures.

AIMS: Because of the highly conformal distributions that can be obtained with intensity-modulated radiotherapy (IMRT), any discrepancy between the intended and delivered distributions would probably affect the clinical outcome. Consequently, there is a need for a measure that would quantify those differences in terms of a change in the expected clinical outcome. MATERIALS AND METHODS: To evaluate such a measure, cancer of the cervix was used, where the bladder and rectum are proximal and partially overlapping with the internal target volume. A solid phantom simulating the pelvic anatomy was fabricated and a treatment plan was developed to deliver the prescribed dose to the phantom. The phantom was then irradiated with films positioned in several transverse planes. The racetrack microtron at 50 MV was used in the treatment planning and delivery processes. The dose distribution delivered was analysed based on the film measurements and compared against the treatment plan. The differences in the measurements were evaluated using both physical and biological criteria. Whereas the physical comparison of dose distributions can assess the geometric accuracy of delivery, it does not reflect the clinical effect of any measured dose discrepancies. RESULTS: It is shown how small inaccuracies in delivered dose can affect the treatment outcome in terms of complication-free tumour cure. CONCLUSIONS: With highly conformal IMRT, the accuracy of the patient set-up and treatment delivery are critical for the success of the treatment. A method is proposed to evaluate the precision of the delivered plan based on changes in complication and control rates as they relate to uncertainties in dose delivery.

Influence of electrodes on the photon energy deposition in CVD-diamond dosimeters studied with the Monte Carlo code PENELOPE.

A new dosimeter, based on chemical vapour deposited (CVD) diamond as the active detector material, is being developed for dosimetry in radiotherapeutic beams. CVD-diamond is a very interesting material, since its atomic composition is close to that of human tissue and in principle it can be designed to introduce negligible perturbations to the radiation field and the dose distribution in the phantom due to its small size. However, non-tissue-equivalent structural components, such as electrodes, wires and encapsulation, need to be carefully selected as they may induce severe fluence perturbation and angular dependence, resulting in erroneous dose readings. By introducing metallic electrodes on the diamond crystals, interface phenomena between high- and low-atomic-number materials are created. Depending on the direction of the radiation field, an increased or decreased detector signal may be obtained. The small dimensions of the CVD-diamond layer and electrodes (around 100 microm and smaller) imply a higher sensitivity to the lack of charged-particle equilibrium and may cause severe interface phenomena. In the present study, we investigate the variation of energy deposition in the diamond detector for different photon-beam qualities, electrode materials and geometric configurations using the Monte Carlo code PENELOPE. The prototype detector was produced from a 50 microm thick CVD-diamond layer with 0.2 microm thick silver electrodes on both sides. The mean absorbed dose to the detector's active volume was modified in the presence of the electrodes by 1.7%, 2.1%, 1.5%, 0.6% and 0.9% for 1.25 MeV monoenergetic photons, a complete (i.e. shielded) (60)Co photon source spectrum and 6, 18 and 50 MV bremsstrahlung spectra, respectively. The shift in mean absorbed dose increases with increasing atomic number and thickness of the electrodes, and diminishes with increasing thickness of the diamond layer. From a dosimetric point of view, graphite would be an almost perfect electrode material. This study shows that, for the considered therapeutic beam qualities, the perturbation of the detector signal due to charge-collecting graphite electrodes of thicknesses between 0.1 and 700 microm is negligible within the calculation uncertainty of 0.2%.

Characterisation of polymeric surfactants that are glutathione transferase mimics.

Catalysts that can detoxify reactive organic chemicals (electrophiles) could be of potential beneficial use. Electrophilic compounds are common toxic agents that are conjugated to endogenous nucleophiles (i.e. glutathione) in an enzyme catalysed reaction (by glutathione transferases). Here, the properties of newly synthesised polymeric surfactant catalysts, which are glutathione transferase mimics, are described (which are not limited to the glutathione thiol donor). Reactions studied were nucleophilic aromatic substitution with 1-chloro-2,4-dinitrobenzene (CDNB) and thiolysis of p-nitrophenyl acetate. Polymeric quaternary ammonium salts synthesised starting from 2-(dimethyl-amino)ethylmethacrylate or 1,3-bis(dimethylamino)isopropylmethacrylate were used as surfactants. Five polysoaps were studied possessing different charge density and different density of hydrophobic chains. In comparison with cetyltrimethylammonium bromide, the polymeric surfactants were clearly more efficient catalysts (i.e. 4.9 vs. 150 (10(3) per M(2)/s) with benzyl hydrosulfide and CDNB). Polymers with high charge and hydrophobic density were most efficient. With a given catalyst, increasing hydrophobicity of the thiol substrate parallels increasing reaction rates (e.g. 0.7- > or = 37 (10(3) per M(2)/s) with CDNB). Concentration of the substrate in the micellar pseudophase together with solvent shielding is suggested as the underlying rate enhancement mechanism. Dead-end Meisenheimer complex stabilisation, where an extremely electrophilic compound (1,3,5-trinitrobenzene) reversibly interacts with glutathione is seen both with glutathione transferases and the polymeric surfactant catalysts. The degree of stabilisation follows catalytic efficiency and thus supports the above structure activity relationships. In conclusion, polymeric materials that can perform biological functions in detoxication are described, as well as their optimal properties.

Kinetic analysis of the slow ionization of glutathione by microsomal glutathione transferase MGST1.

An important aspect of the catalytic mechanism of microsomal glutathione transferase (MGST1) is the activation of the thiol of bound glutathione (GSH). GSH binding to MGST1 as measured by thiolate anion formation, proton release, and Meisenheimer complex formation is a slow process that can be described by a rapid binding step (K(GSH)d = 47 +/- 7 mM) of the peptide followed by slow deprotonation (k2 = 0.42 +/- 0.03 s(-1). Release of the GSH thiolate anion is very slow (apparent first-order rate k(-2) = 0.0006 +/- 0.00002 s(-)(1)) and thus explains the overall tight binding of GSH. It has been known for some time that the turnover (kcat) of MGST1 does not correlate well with the chemical reactivity of the electrophilic substrate. The steady-state kinetic parameters determined for GSH and 1-chloro-2,4-dinitrobenzene (CDNB) are consistent with thiolate anion formation (k2) being largely rate-determining in enzyme turnover (kcat = 0.26 +/- 0.07 s(-1). Thus, the chemical step of thiolate addition is not rate-limiting and can be studied as a burst of product formation on reaction of halo-nitroarene electrophiles with the E.GS- complex. The saturation behavior of the concentration dependence of the product burst with CDNB indicates that the reaction occurs in a two-step process that is characterized by rapid equilibrium binding ( = 0.53 +/- 0.08 mM) to the E.GS- complex and a relatively fast chemical reaction with the thiolate (k3 = 500 +/- 40 s(-1). In a series of substrate analogues, it is observed that log k3 is linearly related (rho value 3.5 +/- 0.3) to second substrate reactivity as described by Hammett sigma- values demonstrating a strong dependence on chemical reactivity that is similar to the nonenzymatic reaction (rho = 3.4). Microsomal glutathione transferase 1 displays the unusual property of being activated by sulfhydryl reagents. When the enzyme is activated by N-ethylmaleimide, the rate of thiolate anion formation is greatly enhanced, demonstrating for the first time the specific step that is activated. This result explains earlier observations that the enzyme is activated only with more reactive substrates. Taken together, the observations show that the kinetic mechanism of MGST1 can be described by slow GSH binding/thiolate formation followed by a chemical step that depends on the reactivity of the electrophilic substrate. As the chemical reactivity of the electrophile becomes lower the rate-determining step shifts from thiolate formation to the chemical reaction.

Analysis of regulatory phosphorylation sites in ZAP-70 by capillary high-performance liquid chromatography coupled to electrospray ionization or matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A methodology for the rapid and quantitative analysis of phosphorylation sites in proteins is presented. The coupling of capillary high-performance liquid chromatography (HPLC) to electrospray ionization mass spectrometry (ESI-MS) allowed one to distinguish phosphorylation sites based on retention time and mass difference from complex peptide mixtures. The methodology was first evaluated and validated for a mixture of non-, mono-, and dityrosine-phosphorylated synthetic peptides, corresponding to the tryptic fragment 485-496 (ALGADDSYYTAR) of the human protein tyrosine kinase ZAP-70. The limits of detection for the non-, mono- and diphosphorylated peptides were about 15, 40 and 100 fmol, respectively, when using a 300 microm I.D. column. Application of the method was extended to identify phosphopeptides generated from a trypsin digest of recombinant autophosphorylated ZAP-70, in particular with respect to quantifying the status at the regulatory phosphorylation sites Tyr-492 and Tyr-493. Combination of chromatographic and on-line tandem mass spectrometry data allowed one to ascertain the identity of the detected peptides, a prerequisite to analyses in more complex biological samples. As an extension to the methodology described above, we evaluated the feasibility of interfacing capillary HPLC to matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), using a micromachined piezoelectric flow-through dispenser as the interface. This enabled direct arraying of chromatographically separated components onto a target plate that was precoated with matrix for subsequent analysis by MALDI-TOF-MS without further sample handling.

Photon scatter kernels for intensity modulating radiation therapy filters.

The most important beam property while optimizing photon therapy is the ability to modulate the intensity of the beam. The use of photon absorbers for intensity modulation of beam profiles requires special attention to be paid to the alteration of beam properties due to scatter and spectral changes, in addition to the desired intensity modulation. In this study the influence of photon scatter in high-density filters irradiated with very narrow photon pencil beams was investigated. A simple analytical relation is developed to quantify the contribution by scattered photons. A scatter kernel was derived by convolving the first Compton scatter distribution with an approximate expression for the second-order scattered photons. The calculations were validated experimentally with film dosimetry and also by using Monte Carlo simulations. Results show that the difference in photon scatter estimation by different methods is relatively small when higher order scattering is accounted for. At 6 MV x-rays the agreement is slightly better than that for 18 MV x-rays results. The simple relation presented in this paper can be used to account for the scattered photon contribution in filter optimization codes to deliver biologically or physically optimized intensity modulated treatments.

Reactivity of cysteine-49 and its influence on the activation of microsomal glutathione transferase 1: evidence for subunit interaction.

Microsomal glutathione transferase 1 is a homotrimeric detoxication enzyme protecting against electrophiles. The enzyme can also react with electrophiles, and when modification occurs at a unique Cys49 the reaction often results in activation. Here we describe the characterization of the chemical properties of this sulfhydryl (kinetic pK(a) was 8.8 +/- 0.3 and 9.0 +/- 0.1 with two different reagents) and we conclude that the protein environment does not lower the pK(a). Upon a direct comparison of the reactivity of Cys49 and low molecular weight thiols [L-Cys and glutathione (GSH)], the protein sulfhydryl displayed a 10-fold lower reactivity. The reactivity was correlated to reagent concentration in a linear fashion with a polar reagent, whereas the reactivity toward a hydrophobic reagent displayed saturation behavior (at low concentrations). This finding indicates that Cys49 is situated in a hydrophobic binding pocket. In a series of related quinones, activation occurs with the more reactive and less sterically hindered compounds. Thus, activation can be used to detect reactive intermediates during the metabolism of foreign compounds but certain intermediates can (and will) escape undetected. The reactivities of the three cysteines in the homotrimer were shown not to differ dramatically as the reaction of the protein with 4, 4'-dithiodipyridine could be fitted to a single exponential. On the basis of this result, a probabilistic expression could be used to relate the overall degree of modification to fractional activation. When N-ethylmaleimide activation (determined by the 1-chloro-2, 4-dinitrobenzene assay) was plotted against modification (determined with 4,4'-dithiodipyridine), a nonlinear relation was obtained, clearly showing that subunits do not function independently. The contribution to activation by single-, double-, and triple-modified trimers, were 0 +/- 0.06, 0.74 +/- 0.09, and 0.97 +/- 0.06, respectively. The double-modified enzyme appears partly activated, but this conclusion is more uncertain due to the possibility of independent modification of the purified enzyme upon storage. It is, however, clear that the single-modified enzyme is not activated whereas the triple-modified enzyme is fully activated. These observations together with the fact that MGST1 homotrimers bind only one substrate molecule (GSH) strongly support the view that subunits must interact in a functional manner.

Cerebrospinal fluid and plasma viral load in HIV-1-infected patients with various anti-retroviral treatment regimens.

Highly active anti-retroviral therapy (HAART) effectively decreases HIV-1 RNA in cerebrospinal fluid (CSF) and plasma in controlled clinical trials. To study the virological effect in CSF and plasma achieved in routine practice, HIV-1 RNA levels were analysed retrospectively in 27 patients on mono-nucleoside reversed transcriptase inhibitor (NRTI) treatment, 27 on dual-NRTI-treatment and 45 on HAART using a Roche Amplicor HIV-1 monitor quantitative PCR. A significant difference was found in the proportion of patients with a CSF viral load below 20 copies/ml between patients treated with 1 (0%) and 2 NRTIs (41%) as well as between those treated with 2 NRTIs and HAART (69%). The proportion of patients with plasma viral load below 20 copies/ml differed significantly between patients on HAART (47%) and those on 2 NRTIs (0%), but not between those with 1 (0%) or 2 NRTIs. In multivariate regression analysis, treatment regimen and prior anti-retroviral experience (but not treatment time) were independently associated with the CSF viral load. Plasma viral load was independently associated with treatment regimen and treatment time, but not with anti-retroviral experience. Dual-NRTI-treatment affects the CSF viral load substantially, while HAART is required to achieve an essential decline in plasma viral load.

Factors underlying the cell growth-related bystander responses to alpha particles.

Increases in cell proliferation are widely viewed as being of importance in carcinogenesis. We report that exposure of normal human lung fibroblasts to a low dose of alpha particles like those emitted by radon/radon progeny stimulates their proliferation in vitro, and this response also occurs when unirradiated cells are treated with supernatants from alpha-irradiated cells. We attribute the promitogenic response to superoxide dismutase- and catalase-inhibitable a particle-induced increases in the concentrations of transforming growth factor beta1 (TGF-beta1) in cell supernatants. TGF-beta1 at concentrations commensurate with those in the supernatants capably induces increases in intracellular reactive oxygen species (ROS) in unirradiated cells. Furthermore, the addition of supernatants from alpha-irradiated cells to unirradiated cells decreases cellular levels of TP53 and CDKN1A and increases CDC2 and proliferating cell nuclear antigen in the latter. Like the increased intracellular ROS bystander effect, this "decreased TP53/CDKN1A response" can be mimicked in otherwise untreated cells by the addition of low concentrations of TGF-beta1. Our results indicate that alpha particle-associated increases in cell growth correlate with intracellular increases in ROS along with decreases in TP53 and CDKN1A, and that these cellular responses are mechanistically coupled. As well, the proliferating cell nuclear antigen and CDC2 increases that occur along with the decreased TP53/CDKN1A bystander effect also would expectedly favor enhanced cell growth. Such processes may account for cell hyperplastic responses in the conducting airways of the lower respiratory track that occur after inhalation exposure to radon/ radon progeny, as well as, perhaps, other ROS-associated environmental stresses.