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PubMed2003

Monoclonal antibodies as a probe for the unfolding of porcine growth hormone.

Monoclonal antibodies (mAbs) were generated against pituitary porcine growth hormone (pGH). Ten mAbs were selected for their specificity and affinity for pGH. These mAbs were of the immunoglobulin G (IgG)(1) kappa subclass, with dissociation constants (K(d)) between 7.42 and 0.26 nM, and recognised seven non-overlapping epitopes. We measured whether the mAbs detected alterations of the pGH three-dimensional structure by comparing the antibody reactivity to native pGH and to pGH experimentally unfolded by heating at 50 degrees C, 75 degrees C and 100 degrees C or by reduction and S-carboxymethylation. The antibody-antigen interactions were studied with two enzyme-linked immunosorbent assays (ELISA), based either on a direct binding or inhibition format. The results show that: 1) one mAb, mAb D12, is a conformation-sensitive antibody that recognises an epitope present only in the native pGH. Because the intact three-dimensional structure is essential for the expression of biological activity, mAb D12 could be used to detect altered pGH molecules in biological samples (blood, pituitary extracts or material produced with recombinant technology), and for the one-step purification of biologically active pGH by immunoaffinity chromatography; 2) one mAb, mAb I4, binds to a linear epitope that is not significantly modified in the denatured hormone. This mAb was able to detect the hormone in assays where protein conformation is usually strongly altered, i.e. immunoblotting and immunohistochemistry; 3) the performances of the other eight mAbs differed significantly in the competitive and non-competitive ELISA.

Animals↗
PubMed2005

Altered calmodulin response to light in the suprachiasmatic nucleus of PAC1 receptor knockout mice revealed by proteomic analysis.

In mammals circadian rhythms are generated by a light-entrainable oscillator located in the hypothalamic suprachiasmatic nucleus (SCN). Light signals reach the SCN via a monosynaptic neuronal pathway, the retinohypothalamic tract, originating in a subset of retinal ganglion cells. The nerve terminals of these cells contain the classical neurotransmitter glutamate and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), and there is evidence that these two transmitters interact to mediate photoentrainment of the oscillator in the SCN. To elucidate light-provoked PACAP receptor signaling we used proteomic analysis. Wild-type mice and mice lacking the PAC1 receptor (PAC1-/-) were light stimulated at early night, and the SCN was examined for proteins that were differentially expressed using two-dimensional gel electrophoresis and identification by tandem mass spectrometry. The most striking finding, which was subsequently confirmed by Western blotting, was a significant reduction of calmodulin (CaM) in wild-type mice as compared with PAC1-/- mice. Analysis at the mRNA level by quantitative in situ hybridization histochemistry was inconclusive, indicating that a translational mechanism might be involved. The findings indicate that PAC1 receptor signaling in the SCN in response to light stimulation induces a down-regulation of CaM expression and that CaM is involved in the photic-entrainment mechanism.

Animals↗
PubMed2001

High-resolution two-dimensional electrophoresis separation of proteins from metal-stressed rice (Oryza sativa L.) leaves: drastic reductions/fragmentation of ribulose-1,5-bisphosphate carboxylase/oxygenase and induction of stress-related proteins.

Employing high-resolution two-dimensional electrophoresis (2-DE), we studied changes in the rice leaf protein patterns, in response to applied heavy and alkaline metals, important environmental pollutants in our surroundings. Drastic changes in 2-DE protein patterns after treatment with copper, cadmium, and mercury, over control were found, including changes in the morphology of the leaf segments. Changes in the major leaf photosynthetic protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, both suppression and fragmentation), and induction of proteins are reported. A total of 33 proteins, which were highly reproducible in repeated experiments, were visually identified as changed over the control, and taken for N-terminal or internal amino acid sequencing. Among these, nine proteins were N-terminally blocked, and six proteins could not be sequenced. Most of the proteins showed homology to RuBisCO protein, and some to defense/stress-related proteins, like the pathogenesis related class 5 protein (OsPR5), the probenazole-inducible protein (referred to as the OsPR10), superoxide dismutase, and the oxygen evolving protein. Results presented here strongly indicate a highly specific action of some of these metals in disturbing the photosynthetic machinery, as evidenced by prominent reductions/fragmentation of the major photosynthetic protein, RuBisCO, and resulting in stress.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2004

Identification of oxidized mitochondrial proteins in alcohol-exposed human hepatoma cells and mouse liver.

Heavy alcohol consumption can damage various cells and organs partly through production of reactive oxygen species (ROS) and mitochondrial dysfunction. Treatment with antioxidants can significantly reduce the degree of damage. Despite well established roles of ROS in alcohol-induced cell injury, the proteins that are selectively oxidized by ROS are poorly characterized. We hypothesized that certain cysteinyl residues of target proteins are oxidized by ROS upon alcohol exposure, and these modified proteins may play roles in mitochondrial dysfunction. A targeted proteomics approach utilizing biotin-N-maleimide (biotin-NM) as a specific probe to label oxidized cysteinyl residues was employed to investigate which mitochondrial proteins are modified during and after alcohol exposure. Human hepatoma HepG2 cells with transduced CYP2E1 (E47 cells) were used as a model to generate ROS through CYP2E1-mediated ethanol metabolism. Following exposure to 100 mM ethanol for 4 and 8 h, the biotin-NM-labeled oxidized proteins were purified with agarose coupled to either streptavidin or monoclonal antibody against biotin. The purified proteins were resolved by two-dimensional gel electrophoresis and protein spots that displayed differential abundances were excised from the gel, in-gel digested with trypsin and analyzed for identity utilizing either matrix-assisted laser desorption-time of flight mass spectrometry or microcapillary reversed-phase liquid chromatography-tandem mass spectrometry. The results demonstrate that heat shock protein 60, protein disulfide isomerase, mitochondrial aldehyde dehydrogenases, prohibitin, and other proteins were oxidized after alcohol exposure. The identity of some of the proteins purified with streptavidin-agarose was also confirmed by immunoblot analyses using the specific antibody to each target protein. This method was also used to identify oxidized mitochondrial proteins in the alcohol-fed mouse liver. These results suggest that exposure to ethanol causes oxidation of various mitochondrial proteins that may negatively affect their function and contribute to alcohol-induced mitochondrial dysfunction and cellular injury.

Alcohol Drinking↗
PubMed2006

Protein oxidation by chronic pulmonary diseases in children.

The oxidation of proteins may play an important role in the pathogenesis of chronic inflammatory lung diseases, and may contribute to lung damage. However, the extent of oxidation and the distribution among proteins are not known for most pediatric lung diseases. In this work, protein oxidation was assessed as protein carbonyls. Bronchoalveolar lavages (BAL) from children with chronic lung diseases were investigated by dot-blot assay for content and for pattern of distribution of oxidized proteins by two-dimensional (2D) electrophoresis and Western blotting. Significantly higher levels of protein oxidation than in healthy controls were determined in groups of patients with interstitial lung disease, gastro-esophageal reflux disease, and pulmonary alveolar proteinosis. The proteins most sensitive to oxidation were serum albumin, surfactant protein A, and alpha1-antitrypsin. Our data show increased oxidative stress in lungs of children with chronic pulmonary diseases, with significant interindividual variations. The extent of protein oxidation was proportional to the count of neutrophilic granulocytes in BAL fluid. These findings strongly support the concept that an abundance of reactive oxygen species produced during neutrophilic inflammation may be a deleterious factor, leading to pulmonary damage in these patients.

Adolescent↗
PubMed1995

Subunit sites of oxidative inactivation of Escherichia coli F1-ATPase by HOCl.

Cellular inactivation of Escherichia coli by the neutrophil-generated toxin, hypochlorous acid, is accompanied by inactivation of its plasma membrane-localized F1-ATPase. The nature of oxidative damage leading to inactivation of this enzyme was probed by SDS-PAGE and 2D-gel electrophoresis and by hybrid reconstitution studies using purified subunits from untreated and extensively oxidized bacteria. The data indicate that inactivation is due to selective oxidation of a few highly vulnerable sites; although damage occurred to each of the alpha, beta, and gamma-subunits required for soluble ATP hydrolase activity, the extent of damage was insufficient to alter their electrophoretic properties.

Binding Sites↗
PubMed2004

Functional constraints of 6-phosphogluconate dehydrogenase (6-PGD) based on sequence and structural information.

The pentose phosphate cycle is considered as a major source of NADPH and pentose needed for nucleic acid biosynthesis. 6-Phosphogluconate dehydrogenase (6PGD), an enzyme participating in this cycle, catalyzes the oxidative decarboxylation of 6PGD to ribulose 5-phosphate with the subsequent release of CO2 and the reduction of NADP. We have determined the amino acid sequence of 6PGD of Bactrocera oleae and constructed a three-dimensional model based on the homologous known sheep structure. In a comparative study of 6PGD sequences from numerous species, all the conserved and variable regions of the enzyme were analyzed and the regions of functional importance were localized, in an attempt promoted also by the direct involvement of the enzyme in various human diseases. Thus, analysis of amino acid variability of 37 6PGD sequences revealed that all regions important for the catalytic activity, such as those forming the substrate and coenzyme binding sites, are highly conserved in all species examined. Moreover, several amino acid residues responsible for substrate and coenzyme specificity were also found to be identical in all species examined. The higher percentage of protein divergence is observed at two regions that accumulate mutations, located at the distant parts of the two domains of the enzyme with respect to their interface. These peripheral regions of non-functional importance are highly variable and are predicted as antigenic, thus reflecting possible regions for antibody recognition. Furthermore, locating the differences between diptera 6PGD sequences on the three-dimensional model suggests probable positions of different amino acid residues appearing at B. oleae fast, intermediate, and slow allozymic variants.

Amino Acid Sequence↗
PubMed2005

Methane-oxidizing bacteria in a Finnish raised mire complex: effects of site fertility and drainage.

Methane-oxidizing bacteria (MOB) are the only biological sinks for methane (CH4). Drainage of peatlands is known to decrease overall CH4 emission, but the effect on MOB is unknown. The objective of this work was to characterize the MOB community and activity in two ecohydrologically different pristine peatland ecosystems, a fen and a bog, and their counterparts that were drained in 1961. Oligotrophic fens are groundwater-fed peatlands, but ombrotrophic bogs receive additional water and nutrients only from rainwater. The sites were sampled in August 2003 down to 10 cm below the water table (WT), and cores were divided into 10-cm subsamples. CH4 oxidation was measured by gas chromatography (GC) to characterize MOB activity. The MOB community structure was characterized by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) and sequencing methods using partial pmoA and mmoX genes. The highest CH4 oxidation rates were measured from the subsamples 20-30 and 30-40 cm above WT at the pristine oligotrophic fen (12.7 and 10.5 micromol CH4 dm-3 h-1, respectively), but the rates decreased to almost zero in the vicinity of WT. In the pristine ombrotrophic bog, the highest oxidation rate at 0-10 cm was lower than in the fen (8.10 micromol CH4 dm-3 h-1), but in contrast to the fen, oxidation rates of 4.5 micromol CH4 dm-3 h-1 were observed at WT and 10 cm below WT. Drainage reduced the CH4 oxidation rates to maximum values of 1.67 and 5.77 micromol CH4 dm-3 h-1 at 30-40 and 20-30 cm of the fen and bog site, respectively. From the total of 13 pmoA-derived DGGE bands found in the study, 11, 3, 6, and 2 were observed in the pristine fen and bog and their drained counterparts, respectively. According to the nonmetric multidimensional scaling of the DGGE banding pattern, the MOB community of the pristine fen differed from the other sites. The majority of partial pmoA sequences belonged to type I MOB, whereas the partial mmoX bands that were observed only in the bog sites formed a distinct group relating more to type II MOB. This study indicates that fen and bog ecosystems differ in MOB activity and community structure, and both these factors are affected by drainage.

Bacteria↗
PubMed2005

[Multidisciplinary treatment rheumatoid arthritis. Statistical and clinical change in pain experience].

INTRODUCTION: To date only few studies have been reported on the effect of multidisciplinary inpatient treatment on pain experience in patients with rheumatoid arthritis (RA). Aims of the present research were: to asses the development of different pain qualities at the beginning, at the end of treatment and at a follow-up three months later in a RA-patient sample as a whole, to determine statistically and clinically significant changes on individual base, and to study pain coping behaviors as predictive and pain-change associated variables. METHODS: Subjects were 66 patients with a diagnosis of RA. They were treated with non-steroidal antiphlogistic and disease modifying drugs as well as with physical therapy. Measurement instruments were the pain experience scale with 5 subtests, the four-dimensional questionnaire of pain behavior, the functional disability scale, and a joint-index. RESULTS: At follow-up the reduction was strongest in the quality of persistent pain experience (effect size d: 0.54). Among sensory components a marked reduction was found for the experience of thermal pain. A statistically and clinically reliable change was assessed in 18% of he RA-patients, further 26% indicated a statistical only change. 33% remained stable at a functional level of pain experience. 12% did not change a high level of pain, and 11% deteriorated. Change in the coping behaviors of avoidance and support were associated with success in pain reduction. CONCLUSION: In the assessment of patient improvement different qualities of pain experience should be taken into account. It was suggested that inpatient multidisciplinary treatment was beneficial with regard to pain reduction in nearly on half of the RA-patients. The coping behaviors of avoidance and social support deserve attention as pain change associated variables.

Adult↗
PubMed1990

Endogenous electrical currents and the resultant voltage gradients in the chick embryo.

We have studied some of the electrophysiological properties of 2 1/2- to 4-day-old (stage 14-22) chick embryos. Using a recently developed two-dimensional vibrating probe, large currents were found to exit the posterior intestinal portal (p.i.p.) during the period of tail gut reduction. During this period, epithelial cells lining cloacal regions of the hindgut are dying, thus creating a low-resistance pathway for current flow out of the embryo. Currents entered the intact epithelium over other regions of the embryo. The outward currents at the p.i.p. were first detected at stage 15 and reached their average maximum current density of 112 +/- 10 microA/cm2 at stage 17. After stage 17, the magnitude of the currents decreased, dropping to 16 +/- 0.3 microA/cm2 by stage 22. The currents were reversibly reduced by about 50% when Na+ was replaced by choline in the bathing solution. The magnitude of the currents leaving the p.i.p. suggested the existence of a measurable intraembryonic voltage gradient. The transepithelial potential (TEP) of stage 14-21 embryos was measured lateral to the neural tube through the dorsal ectoderm. For all stages, the combined average TEP was 16 +/- 0.5 mV. Differences in the TEP between various regions of the embryo were used to calculate an intraembryonic voltage gradient. At stage 14, before outward current was found at the p.i.p., no significant intraembryonic voltage gradient was detected. At stage 17, when the outward current at the p.i.p. was maximum, a voltage gradient of 21 +/- 5 mV/mm (mean +/- SEM; N = 6) was measured in the caudal end of the embryo. This gradient in some cases was as steep as 33 mV/mm. This is well above the minimum level needed to affect the direction of embryonic cell migration in vitro. We hypothesize that this endogenous electrical field acts as a directional cue for neural crest cell movements in the developing chick embryo.

Amiloride↗
PubMed1995

Chronic stress tolerance levels for human articular cartilage: two nonuniform contact models applied to long-term follow-up of CDH.

Two computer models of nonuniform contact stress on the articular surface of the human hip were used to study the relationship between chronically excessive articular cartilage contact stress and long-term clinical outcome in a series of patients with congenital dislocation of the hip (CDH). The analyzed database consisted of 409 stylus digitized radiographs from 83 patients with unilateral CDH, who had been treated by closed reduction, and whose average follow-up time was 29.2 y. The first model (nonuniform Legal) involved a three-dimensional contact stress distribution function whose pole was coincident with the resultant force acting through the hip, and which acted over a contact area whose borders were determined soley by bony landmarks. In the second model (Brinckmann), the direction of the pole of the contact stress distribution function was initially unknown; one border of the contact region was determined by radiographic landmarks, while the other border depended upon the pole of the iteratively determined contact stress distribution function. In both models, the contact stress distributions were converted to area engagement histograms, corresponding to the fractional areas of cartilage experiencing specific ranges of stress (0.5 MPa increments). These histograms were integrated over time to calculate a cumulative contact stress overdose, which was then compared to clinical outcome. Reasonable correlations (Spearman rho = 0.63-0.66) with patient outcomes were obtained for optimally chosen damage thresholds, although these thresholds were appreciably different (2.0 versus 4.5 MPa) due to the respective modelling assumptions.

Biomechanical Phenomena↗
PubMed1991

Characterization and purification of the hyaluronan-receptor on liver endothelial cells.

In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.

Animals↗
PubMed2005

Protein oxidation in plant mitochondria detected as oxidized tryptophan.

The formation of N-formylkynurenine by dioxygenation of tryptophan was detected in peptides from rice leaf and potato tuber mitochondria. Proteins in matrix and membrane fractions were separated by two-dimensional gel electrophoresis and identified using a Q-TOF mass spectrometer. N-Formylkynurenine was detected in 29 peptides representing 17 different proteins. With one exception, the oxidation-sensitive aconitase, all of these proteins were either redox active themselves or subunits in redox-active enzyme complexes. The same site was modified in (i) several adjacent spots containing the P protein of the glycine decarboxylase complex, (ii) two different isoforms of the mitochondrial processing peptidase in complex III, and (iii) the same tryptophan residues in Mn-superoxide dismutase in both rice and potato mitochondria. This indicates that Trp oxidation is a selective process.

Aconitate Hydratase↗
PubMed2006

Redox proteomics in some age-related neurodegenerative disorders or models thereof.

Neurodegenerative diseases cause memory loss and cognitive impairment. Results from basic and clinical scientific research suggest a complex network of mechanisms involved in the process of neurodegeneration. Progress in treatment of such disorders requires researchers to better understand the functions of proteins involved in neurodegenerative diseases, to characterize their role in pathogenic disease mechanisms, and to explore their roles in the diagnosis, treatment, and prevention of neurodegenerative diseases. A variety of conditions of neurodegenerative diseases often lead to post-translational modifications of proteins, including oxidation and nitration, which might be involved in the pathogenesis of neurodegenerative diseases. Redox proteomics, a subset of proteomics, has made possible the identification of specifically oxidized proteins in neurodegenerative disorders, providing insight into a multitude of pathways that govern behavior and cognition and the response of the nervous system to injury and disease. Proteomic analyses are particularly suitable to elucidate post-translational modifications, expression levels, and protein-protein interactions of thousands of proteins at a time. Complementing the valuable information generated through the integrative knowledge of protein expression and function should enable the development of more efficient diagnostic tools and therapeutic modalities. Here we review redox proteomic studies of some neurodegenerative diseases.

Aged↗
PubMed2002

Assessment of cardiac performance using Tei indices in patients undergoing pulmonary thromboendarterectomy.

BACKGROUND: This study was designed to evaluate left and right ventricular performance using Tei indices in patients with severe chronic thromboembolic pulmonary hypertension undergoing pulmonary thromboendarterectomy (PTE). The Doppler-derived indices are easily measurable indicators of ventricular function based on nongeometric assessment, which helps overcome some of the difficulties entailed in the geometric assessment of left ventricular (LV) and right ventricular (RV) function in pulmonary hypertension. METHODS: The indices were derived for 24 patients (aged 54+/-14 years) before and after PTE. Calculation of these indices was based on the duration of two time intervals using the formula (A - B)/B, where A is the interval between cessation and onset of mitral inflow (or tricuspid inflow) and B is LV or RV ejection time. In addition, LV and RV end-diastolic and end-systolic chamber areas were determined using two-dimensional echocardiography, and systolic function was calculated. Mean pulmonary artery pressure was determined invasively. RESULTS: PTE led to a significant reduction of mean pulmonary artery pressure (46+/-10 versus 25+/-6 mm Hg; p < 0.05). LV and RV indices were abnormally high before surgery, declined significantly afterwards, and then almost matched normal values (0.61+/-0.26 versus 0.37+/-0.18; p < 0.05 and 0.55+/-0.22 versus 0.37+/-0.13; p < 0.05). Geometric assessment of the left and right ventricle also showed impaired systolic function before PTE, with significant improvement after surgery. CONCLUSIONS: LV and RV Tei indices allow a quantitative assessment of ventricular function in patients undergoing PTE. Lower indices after surgery reflect an improvement of the previously impaired cardiac function. Our results emphasize the value of PTE in the treatment of chronic thromboembolic pulmonary hypertension.

Adult↗
PubMed2002

Dynamics of nucleotides in VDAC channels: structure-specific noise generation.

Nucleotide penetration into the voltage-dependent mitochondrial ion channel (VDAC) reduces single-channel conductance and generates excess current noise through a fully open channel. VDAC channels were reconstituted into planar phospholipid membranes bathed in 1.0 M NaCl. At a given nucleotide concentration, the average decrease in small-ion channel conductance induced by mononucleotides ATP, ADP, AMP, and UTP and dinucleotides beta- and alpha-NADH, NAD, and NADPH are very close. However, the excess current noise is about seven times higher in the presence of NADPH than in the presence of ATP and is about 40 times higher than in the presence of UTP. The nucleotide-generated low-frequency noise obeys the following sequence: beta-NADPH > beta-NADH = alpha-NADH > ATP > ADP > beta-NAD > or = AMP > UTP. Measurements of bulk-phase diffusion coefficients and of the effective charge of the nucleotides in 1.0 M NaCl suggest that differences in size and charge cannot be the major factors responsible for the ability to generate current noise. Thus, although the ability of nucleotides to partition into the channel's pore, as assessed by the reduction in conductance, is very similar, the ability to generate current noise involves a detailed recognition of the three-dimensional structure of the nucleotide by the VDAC channel. A possible mechanism for this selectivity is two noise-generating processes operating in parallel.

Adenine Nucleotides↗
PubMed2001

A comparison of the mechanical properties of a gallium-based alloy with a spherical high-copper amalgam.

OBJECTIVES: The aim of the present study was to investigate how the mechanical properties of a palladium free gallium-based alloy (Galloy) compare with a leading spherical high-copper amalgam (Tytin). METHODS: Cylindrical specimens were mechanically condensed, according to the ISO 1559:1986 standard, to measure compressive strength, Vickers hardness, static creep and dimensional change on setting. Disc and beam shaped specimens were manually prepared to assess the diametral tensile and flexural strengths of the investigated alloys. RESULTS: The mean hardness, 1h compressive fracture strength, 24 h diametral tensile and 24h flexural strengths of Galloy were significantly lower (P<0.001) than Tytin. No significant differences in modulus of elasticity, creep, dimensional change on setting, 24 and 168 h compressive fracture strength for the two alloys were identified. SIGNIFICANCE: The significant reduction in the 1 h mean compressive fracture strength and hardness identified for Galloy compared with Tytin possibly indicate a slower setting reaction in the gallium-based alloy. Manual condensation of the gallium-based alloy produced specimens with inferior mechanical properties possibly due to the increased likelihood of introducing voids within the test specimens. Previous reports indicating poor corrosion resistance and moisture sensitivity of gallium-based alloys highlight the need for further research to investigate the effect of the oral environment on the gallium-based alloy.

Compressive Strength↗