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PubMedDate not supplied

Determination of fat in raw and processed milks by the Gerber method: collaborative study.

The Gerber method is used worldwide as a simple and rapid method for determining fat in raw and processed milks. However, the volume of the test portion used in the method has not been internationally agreed upon. A collaborative study was conducted to evaluate performance of the Gerber method using either a weighed test portion (11.13 g) or by a 10.77 mL test portion delivered by pipet. For each method, laboratories received 10 test samples: 5 raw and 5 pasteurized homogenized milks, 2 of which were blind duplicate pairs. Eleven and 10 laboratories participated in the evaluation of aliquot addition by weight and pipet, respectively. Mojonnier ether extraction (Method 989.05) was used as the reference method. Interlaboratory study statistics were similar between methods of test portion addition and between raw and processed materials; therefore, summary interlaboratory study statistics were pooled. The fat content of milk samples ranged from 0.96 to 5.48%. Absolute reproducibility and repeatability were not affected by fat level, and pooled statistical performance (invalid and outlier data removed) was (g fat/100 g milk) s(r) = 0.026, s(R) = 0.047, r = 0.074, and R = 0.132. Relative standard deviations increased with decreasing fat content, and were summarized by fat level: 1-2% fat milk, mean = 1.437, RSD(r) = 1.809%, RSD(R) = 3.271%; 2-6% fat milk, mean = 4.156, RSD(r) = 0.626%, RSD(R) = 1.131%. Compared with ether extraction, test results by the Gerber method were slightly lower (0.02% fat) using a weighed test portion and significantly lower (0.06% fat) using a 10.77 mL volume addition by pipet. A trend toward underestimating fat content at lower fat concentrations (1-2% fat) was observed with the weighed test portion but not when a pipet was used. The Associate Referee recommends that the Gerber method using a weighed test portion be adopted as First Action with applicability limited to whole milk.

Animals↗

PubMed2001

Assessment of the virulence of Listeria monocytogenes: agreement between a plaque-forming assay with HT-29 cells and infection of immunocompetent mice.

Some Listeria monocytogenes strains not related to clinical cases have been found to exhibit a low virulence level in mice as well as in an in vitro test using Caco-2 cells. The purpose of this study was to validate a new in vitro test of virulence based on a plaque-forming assay (PFA) using a HT-29 cell monolayer with 118 Listeria strains. The use of HT-29 cells in 96-well tissue culture plates allowed the testing of 30 strains per day and providing results in 24 h. In addition. statistical analyses demonstrated the reproducibility and repeatability of the PFA. No quantitative relationship was observed between the virulence of the strains and the hemolytic titer or the cytotoxic effects on HT-29 cells. In contrast, good agreement was observed between virulence assessed after subcutaneous (SC) infection and virulence obtained by PFA. Three groups of L. monocytogenes strains (avirulent, hypovirulent and fully virulent) were established by comparison of the clinical origin of the strains, the number of immunocompetent contaminated mice and the numbers of Listeria strains recovered in the spleen after SC infection. With one exception, i.e. a clinical case of L. seeligeri (sensitivity 0.98), the PFA successfully detected the virulent strains only (specificity 1). Decision-tree algorithms performed by SAS and S-Plus demonstrated that this tissue culture assay discriminated between the avirulent and hypovirulent strains and the virulent strains. This test could therefore be an alternative to in vivo tests, allowing grading of virulence.

Animals↗

PubMed2003

Operational feasibility of routine bacterial monitoring of platelets.

Bacterial contamination of platelets poses the greatest risk of mortality and morbidity to platelet transfusion recipients. Some European countries have introduced routine bacterial monitoring of platelets to reduce the risk of transmission of bacteria. A pilot study was carried out at the Northern Ireland Blood Transfusion Service, using the BacT/ALERT automated culture system, to assess the operational feasibility of routine bacterial monitoring of platelets. About 4885 platelet concentrates (PCs) were tested in a 1-year period. Of the 28 (0.57%) initial reactive cultures, 13 (46%) were reproducible on repeat culturing. Of these, 10 were detected within 24 h of incubation either in aerobic or both aerobic and anaerobic culture bottles. A sample of time-expired units (423) that had initial negative culture results remained negative when retested on day 8. About 213 time-expired units were subjected to routine quality assessment and more than 85% were found to conform to quality standards specified in the UKBTS guidelines for platelet count (> or =240 x 10(9) per adult dose PC) and pH (6.4-7.4). There was a reduction in the platelet count because of the volume removed (15 mL) for sampling. Routine bacterial testing with day 2 sampling and a negative culture result after 24 h as a mandatory release criterion would improve product safety. Implementation of 100% testing would be operationally feasible but may require extension of the shelf life if unacceptable wastage is to be avoided.

Aerobiosis↗

PubMed1993

Advantages and limitations of serial coronary arteriography for the assessment of progression and regression of coronary atherosclerosis. Implications for clinical trials.

BACKGROUND: Clinical trials with angiographic end points have been used to assess whether interventions influence the evolution of coronary atherosclerosis because sample size requirements are much smaller than for trials with hard clinical end points. Further studies of the variability of the computer-assisted quantitative measurement techniques used in such studies would be useful to establish better standardized criteria for defining significant change. METHODS AND RESULTS: In 21 patients who had two arteriograms 3-189 days apart, we assessed the reproducibility of repeat quantitative measurements of 54 target lesions under four conditions: 1) same film, same frame; 2) same film, different frame; 3) same view from films obtained within 1 month; and 4) same view from films 1-6 months apart. Quantitative measurements of 2,544 stenoses were also compared with an experienced radiologist's interpretation. The standard deviation of repeat measurements of minimum diameter from the same frame was very low (0.088 mm) but increased to 0.141 mm for measurements from different frames. It did not increase further for films within 1 month but increased to 0.197 mm for films 1-6 months apart. Diameter stenosis measurements were somewhat more variable. Measurement variability for minimum diameter was independent of vessel size and stenosis severity. Experienced radiologists did not systematically overestimate or underestimate lesion severity except for mild overestimation (mean 3.3%) for stenoses > or = 70%. However, the variability between visual and quantitative measurements was two to three times higher than the variability of paired quantitative measurements from the same frame. CONCLUSIONS: Changes of 0.4 mm or more for minimum diameter and 15% or more for stenosis diameter (e.g., 30-45%), measured quantitatively, are recommended as criteria to define progression and regression. Approaches to data analysis for coronary arteriographic trials are discussed.

Clinical Trials as Topic↗

PubMed1990

Two-dimensional electrophoresis of synovial tissue, synovial fluid and serum in patients with rheumatoid arthritis and related diseases.

Using the technique of two-dimensional (2D) electrophoresis with consecutive silver staining, we investigated samples of serum, synovial fluid and synovial tissue obtained from 19 patients suffering from rheumatoid arthritis (RA) or non-RA arthritis. From these experiments we have drawn the following conclusions. 2D electrophoresis of serum, synovial fluid and synovial tissue extracts taken from patients suffering from joint diseases is a reproducible method. Repeated runs of the same sample reveal an essentially constant protein spot pattern. The time period between surgery and tissue preparation did not influence the number of protein spots when less than 15 h was involved. The protein spot number is always lower in synovial fluid than in either synovial tissue or serum in RA and non-RA patients. The mean value for the number of spots is 68 for the inflamed tissue irrespective of the cause of arthritis (RA and non-RA group taken together) and 47 for the control group. This difference is significant. We were able to definitely identify 7 spots in the tissue extract. We did not find RA-specific protein spots in either serum, synovial fluid or tissue extracts from the synovial membrane. The only significant difference between RA patients and either non-RA or control group patients concerning the protein spot pattern is the increased size of the immunoglobulin spot (mainly IgG) in RA. In addition, we discuss possible reasons for failure of the 2D electrophoresis technique to detect disease-specific protein patterns.

Adult↗

PubMed2003

The reproducibility of independently measuring human regional hepatic arterial, portal and total hepatic blood flow using [15O]water and positron emission tomography.

The reproducibility of repeated human regional hepatic blood flow quantification using [15O]water and positron emission tomography (PET) was evaluated as a method of monitoring the effect of drug administration on hepatic blood flow. Nineteen patients underwent two measurements of hepatic blood flow by PET. Fifteen minutes after the first dynamic study using [15O]water, a second dynamic study was performed, and hepatic blood flow was calculated. The correlation between the first and second dynamic study of arterial blood flow was highly significant (P=6.31 x 10(-10), r=0.946). The regression line was y=1.08x. The mean error between studies was 0.158. The correlation between the first and second dynamic study of portal blood flow also was significant (P=1.29 x 10(-7), r=0.897). The regression line was y=1.03x. The mean error between the studies was 0.164. The correlation between total hepatic blood flow in the first and second dynamic study, too, was significant (P=2.68 x 10(-7), r=0.888). The regression line was defined by y=1.06x. The mean error between studies was 0.140. Hepatic blood flow has increased if the second measurement of hepatic arterial, portal, and total blood flow is more than 115%, 111% and 114% of baseline, and has decreased if the second measurement is less than 101%, 95% and 98% of the first measurement.

Adult↗

PubMed1995

Administration of nicotinamide during chart: pharmacokinetics, dose escalation, and clinical toxicity.

PURPOSE: To determine nicotinamide pharmacokinetics in patients undergoing accelerated radiotherapy with the CHART regimen (continuous, hyperfractionated, accelerated radiotherapy) and given nicotinamide on a daily basis. The aim was to establish the pharmacokinetic profiles and their reproducibility during repeated administration, the maximum tolerated dose with fractionated radiotherapy, whether such a dose achieves sufficiently high plasma levels for radiosensitization, the optimal time interval between nicotinamide and irradiation, and toxic side effects. METHODS AND MATERIALS: Nicotinamide plasma concentrations were determined using high performance liquid chromatography in 11 patients with advanced carcinomas of the head and neck and rectum being treated with CHART (36 fractions in 12 days). Kinetic profiles on the first day of radiotherapy and residual 24-h values were obtained in 10 patients; in four of these, full profiles were repeated two or three times during the course of treatment. In one other, a single sample per day was taken four times over the 12-day period. Doses of 80, 90, or 100 mg/kg/day were given 90 min prior to the second radiotherapy fraction on each day. RESULTS: A dose of 80 mg/kg/day was well tolerated by all the patients. However, an increase of 10-25% in dose led to significant drug accumulation and major clinical toxicity, and none of the patients in the dose-escalation arm completed the planned regimen. Large interpatient variations in absolute peak concentrations were seen from 0.4 to 1.4 mumol/ml (mean 0.9 +/- 0.3; standard deviation (SD)). Of the five samples with the lowest peak levels, four were obtained from one patient. The time taken to peak concentration was also very variable from 0.8 to 4 h (mean 2.1 +/- 1.3 h; SD). In 70% of the samples, absolute plasma levels > or = 0.7 mumol/ml were reached within 1-2 h after administration and maintained for up to 6 h (mean 2.8 +/- 1.8 h; SD). There was a small but nonsignificant increase in the half-life of nicotinamide when the dose was increased from 80 to 90 or 100 mg/kg (7.1 h and 8.6 h, respectively). CONCLUSIONS: In an accelerated regimen such as CHART, 80 mg/kg/day of oral nicotinamide is feasible and clinically tolerated, giving no or few side effects, and a 2-h interval between its oral administration and radiotherapy should achieve effective plasma levels in most patients.

Aged↗

PubMed2003

Effect of resiniferatoxin on the noxious heat threshold temperature in the rat: a novel heat allodynia model sensitive to analgesics.

1. An increasing-temperature hot plate (ITHP) was introduced to measure the noxious heat threshold (45.3+/-0.3 degrees C) of unrestrained rats, which was reproducible upon repeated determinations at intervals of 5 or 30 min or 1 day. 2. Morphine, diclofenac and paracetamol caused an elevation of the noxious heat threshold following i.p. pretreatment, the minimum effective doses being 3, 10 and 200 mg kg(-1), respectively. 3. Unilateral intraplantar injection of the VR1 receptor agonist resiniferatoxin (RTX, 0.048 nmol) induced a profound drop of heat threshold to the innocuous range with a maximal effect (8-10 degrees C drop) 5 min after RTX administration. This heat allodynia was inhibited by pretreatment with morphine, diclofenac and paracetamol, the minimum effective doses being 1, 1 and 100 mg kg(-1) i.p., respectively. 4. The long-term sensory desensitizing effect of RTX was examined by bilateral intraplantar injection (0.048 nmol per paw) which produced, after an initial threshold drop, an elevation (up to 2.9+/-0.5 degrees C) of heat threshold lasting for 5 days. 5. The VR1 receptor antagonist iodo-resiniferatoxin (I-RTX, 0.05 nmol intraplantarly) inhibited by 51% the heat threshold-lowering effect of intraplantar RTX but not alpha,beta-methylene-ATP (0.3 micromol per paw). I-RTX (0.1 or 1 nmol per paw) failed to alter the heat threshold either acutely (5-60 min) or on the long-term (5 days). The heat threshold of VR1 receptor knockout mice was not different from that of wild-type animals (45.6+/-0.5 vs 45.2+/-0.4 degrees C). 6. In conclusion, the RTX-induced drop of heat threshold measured by the ITHP is a novel heat allodynia model exhibiting a high sensitivity to analgesics.

Acetaminophen↗

PubMed2005

Use of real-time three-dimensional echocardiography to measure left atrial volume: comparison with other echocardiographic techniques.

OBJECTIVES: Left atrial (LA) volume (LAV) is a prognostically important biomarker for diastolic dysfunction, but its reproducibility on repeated testing is not well defined. LA assessment with 3-dimensional (3D) echocardiography (3DE) has been validated against magnetic resonance imaging, and we sought to assess whether this was superior to existing measurements for sequential echocardiographic follow-up. METHODS: Patients (n = 100; 81 men; age 56 +/- 14 years) presenting for LA evaluation were studied with M-mode (MM) echocardiography, 2-dimensional (2D) echocardiography, and 3DE. Test-retest variation was performed by a complete restudy by a separate sonographer within 1 hour without alteration of hemodynamics or therapy. In all, 20 patients were studied for interobserver and intraobserver variation. LAVs were calculated by using M-mode diameter and planimetered atrial area in the apical 4-chamber view to calculate an assumed sphere, as were prolate ellipsoid, Simpson's biplane, and biplane area-length methods. All were compared with 3DE. RESULTS: The average LAV was 72 +/- 27 mL by 3DE. There was significant underestimation of LAV by M-mode (35 +/- 20 mL, r = 0.66, P < .01). The 3DE and various 2D echocardiographic techniques were well correlated: LA planimetry (85 +/- 38 mL, r = 0.77, P < .01), prolate ellipsoid (73 +/- 36 mL, r = 0.73, P = .04), area-length (64 +/- 30 mL, r = 0.74, P < .01), and Simpson's biplane (69 +/- 31 mL, r = 0.78, P = .06). Test-retest variation for 3DE was most favorable (r = 0.98, P < .01), with the prolate ellipsoid method showing most variation. Interobserver agreement between measurements was best for 3DE (r = 0.99, P < .01), with M-mode the worst (r = 0.89, P < .01). Intraobserver results were similar to interobserver, the best correlation for 3DE (r = 0.99, P < .01), with LA planimetry the worst (r = 0.91, P < .01). CONCLUSIONS: The 2D measurements correlate closely with 3DE. Follow-up assessment in daily practice appears feasible and reliable with both 2D and 3D approaches.

Adult↗

PubMed1981

Human squamous cell carcinoma. Establishment and characterization of new permanent cell lines.

Squamous cell carcinoma is the most common of human cancers, and yet because it is poorly represented by cultured cell lines, little is known about the characteristic cell biology and the cell-surface antigenic phenotypes of such tumors. To develop a continuously available source of squamous cell carcinoma for repeated and reproducible serologic analysis and for better understanding of its biologic characteristics, tissue culture methods and nude mice were used to establish new cell lines of squamous carcinoma. Special media, serum supplements from several sources, and methods of handling fresh tissue specimens were all examined as a means of improving the survival of tumor cell lines. Several new cell lines were established. Features characteristic of a squamous cell origin, eg, microvilli, desmosomes, tonofilaments, and the squamous cell differentiation antigen (pemphigus antigen), were found. The clinical course of disease in individual donor patients has been examined.

Aged↗

PubMed1996

Factors affecting urethrocystographic parameters in urinary continent women.

To evaluate the urethrocystographic changes in different conditions, 154 women were evaluated by using introital sonography. Patients were divided into three groups: group 1 (n = 103) normal, including 10 postmenopausal women; group 2 (n = 46) pregnant, including 16 women in the first trimester, 15 in the second trimester, and 15 in the third trimester; group 3 (n = 15) severe genitourinary prolapse. None of the 154 women had a history of urinary incontinence. The following parameters were measured at rest: urethral thickness, uretheral length, urethral inclination, and posterior urethrovesical angle. On maximum straining, urethral inclination, posterior urethrovesical angle, and rotational angle were measured. In general, age, parity, and menopause did not affect the urethrocystographic parameters in Group 1 patients. Postmenopausal women had a significant decrease in the urethral thickness compared with the premenopausal women (p = 0.026). Patients in Groups 2 and 3 had a significantly lower urethral position than those in group 1. However, hypermobility of the urethra was found only in Group 3. Different menstrual ages did not affect the urethral position but could affect the posterior urethrovesical angle at rest in the first trimester. Introital sonography, without the risk of radiation exposure, enables the observation of static and dynamic changes in the lower urinary tract, both repeatedly and reproducibly.

Adult↗

PubMed1998

Determination of four anabolic steroid metabolites by gas chromatography/mass spectrometry with negative ion chemical ionization and tandem mass spectrometry.

A gas chromatography/mass spectrometry method is described which uses negative ion chemical ionization and tandem mass spectrometry for the determination of anabolic steroid metabolites. Four anabolic steroid metabolites to be derivatized by Pentafluoropropionic anhydride (PFPA) were determined using gas chromatography/mass spectrometry (GC/MS) with negative chemical ionization (NCI) and NCI/MS/MS. The repeatability and reproducibility of this procedure were in the range of 5.3-9.7% and 6.1-10.2%, respectively. This method of derivatization with PFPA for NCI and NCI/MS/MS was useful to determine four metabolites of nandrolone, dromostanolone, methenolone and boldenone. The derivatized metabolites of boldenone could be detected to 2 ppb and the other three steroids could be detected to 25 ppb in urine at a signal-to-noise ratio of S/N = 3.

Anabolic Agents↗

PubMedDate not supplied

Application of covalent affinity chromatography with thiol-disulphide interchange for determination of environmental exposure to heavy metals based on the quantitative isolation of Cd-thionein from human breast milk.

The aim of this study was to test a new chromatographic method for the quantitative determination of Cd-thionein (Cd-Th) in human breast milk, in order to determine the level of exposure to heavy metals. Cd-thionein was isolated by covalent affinity chromatography with thiol-disulphide interchange, which is a modern separation technique of high affinity, good repeatability and reproducibility, allowing specific isolation of the thiolproteins. The fundamentals of indirect determination of the contents of metallothionein protein from human milk were worked out through estimation of the quantities of cadmium bound with Cd-thionein and adsorbed on covalent affinity chromatography gel during a separation process.

Chromatography, Affinity↗

PubMedDate not supplied

Application of covalent affinity chromatography with thiol-disulphide interchange for determination of environmental exposition to heavy metals based on the quantitative determination of Zn-thionein from physiological human fluids by indirect method based on analysis of metal contents.

Intoxication with heavy metals results in numerous poisonings and diseases. They disturb metabolism of the system, are the source of cancer, degeneration changes and others. As a result of kidney damage the urine of people exposed to heavy metals contains different low molecular weight proteins, oligopeptides and amino acids, indicating pathological changes. One of the proteins is a very specific metallopolythiopolypeptide--metallothionein (MT). Based on earlier investigations, a very good correlations has been found between the contents of metallothionein in urine and plasma and the concentration of heavy metals in the blood, urine, kidneys, liver and brain and general in level of exposition to heavy metals. The aim of our investigations was to carry out quantitative isolation of Zn-thionein (Zn-Th), in order to determine the level of exposition to heavy metals. For Zn-Th protein isolation by covalent affinity chromatography with thiol-disulphide interchange (CAC-TDI) was applied, which is a modern technique of separation of a high affinity, good repeatability and reproducibility, allowing specific isolation of the thiol-proteins CAC-TDI gel was used as a solid-phase extraction (SPE) support for preconcentration of Zn-Th protein and Zn bonded with Zn-Th from water, rine, plasma and breast milk samples. The investigations showed unfavourable effect of the support on separation of thiol proteins and good correlation between the concentration of MTs protein added to water, plasma and urine and the concentration of protein indirectly determined via atomic absorption spectrometric (AAS) method, by preconcentration on SPE support metals formerly bound with MT protein and absorbed on CAC-TDI gel and calculated from metals concentration. The present paper is a continuation of earlier experiments on quantitation of Hg-thionein and Cd-thionein in physiological fluids and homogenates.

Chromatography, Affinity↗

PubMed2000

Direct determination of chlorpyrifos and its main metabolite 3,5, 6-trichloro-2-pyridinol in human serum and urine by coupled-column liquid chromatography/electrospray-tandem mass spectrometry.

A rapid and sensitive automated coupled-column liquid chromatography/electrospray tandem mass spectrometry (LC/LC/ES-MS/MS) method has been developed for the quantitation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol (TCP) in both human serum and urine. Human serum was first protein precipitated with acetonitrile, while urine was directly injected into the coupled-column system. A 10 microL aliquot was then analyzed using as first separation column a Discovery C18 5 microm 50 x 2.1 mm; the fraction containing the analyte was transferred on-line to the second column consisting of a ABZ+ 5 microm 100 x 2.1 mm, which was connected to the electrospray source (Z-spray) of a Quattro LC triple-quadrupole instrument. Chlorpyrifos was detected in positive ion mode using four multi reaction monitoring (MRM) transitions while TCP was measured in negative ion mode using three pseudo-MRM transitions. The clean-up performed by the coupled-column approach avoids the use of an internal standard for the correct quantitation of both analytes, and the highly automated procedure renders a sample throughput of more than 100 samples per day. Both compounds can be determined using the same set-up, the only difference in the procedure being the composition of the first mobile phase. The method has proved to be fast, reliable and sensitive, yielding calibration curves for both analytes with correlation coefficients greater than 0.9995. The repeatability and reproducibility at 5 and 50 ng/mL was lower than 8%. The accuracy and precision were evaluated by means of recovery experiments from fortified serum (5-50 ng/mL) and urine (1-10 ng/mL) samples, obtaining satisfactory recoveries for both compounds (87-113% in serum, and 98-109% in urine), with coefficients of variation (CVs) less than 10%. The detection limits were similar for chlorpyrifos and metabolite: 1.5 ng/mL in serum, and 0.5 ng/mL in urine, where no sample handling took place. The validated procedures provide excellent tools for the specific assessment of occupational exposure to the organophosphorus pesticide chlorpyrifos, throughout the analysis of both human serum and urine, and it is more selective and sensitive than the current assay based on the measurement of the decrease in the cholinesterase activity.

Chlorpyrifos↗

PubMed2001

Rapid separation of tetracycline derivatives and their main degradation products by capillary zone electrophoresis.

A mixture of five tetracycline (TC) derivatives: minocycline (MC), demeclocycline (DMCTC), doxycycline (DC), and sancycline (SC), as well as each TC derivative from its main degradation product were separated by capillary zone electrophoresis (CZE). The influence of the pH and the concentration and nature of the background electrolyte (BGE) on the separations was investigated. Ethylenediaminetetraacetic acid (EDTA; 1 mM) was used as additive in a 25 mM phosphate buffer (pH 2.3) because this BGE enabled the rapid separation of the TC derivatives and of each TC derivative from its respective degradation product in less than 6 min. After optimization of the separation conditions, the analytical characteristics of the method were investigated. The parameters involved were linearity, precision (repeatability and reproducibility), and limits of detection (LODs). LODs obtained for the five TC derivatives studied were about 3 microg/mL. Finally, the CZE method developed was applied to study the stability of TC derivatives and to analyze the TC derivative content in three different pharmaceutical preparations.

Anti-Bacterial Agents↗

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↗

PubMed2002

Proteome analysis of differentially displayed proteins as a tool for investigating ozone stress in rice (Oryza sativa L.) seedlings.

Employing classical two-dimensional electrophoresis (2-DE), amino acid sequencing and immunoblot analysis, we examine for the first time the effect of ozone, a highly notorious environmental pollutant, on rice seedling proteins. Drastic visible necrotic damage to leaf by ozone and consequent increase in ascorbate peroxidase protein(s) was accompanied by rapid changes in the 2-DE protein profiles, over controls. Out of a total of 56 proteins investigated, which were reproducible in repeated experiments, 52 protein spots were visually identified as differentially expressed over controls. Six proteins were N-terminally blocked, and the sequence of 14 proteins could not be determined, whereas 36 proteins were N-terminally and one was internally sequenced. Ozone caused drastic reductions in the major leaf photosynthetic proteins, including the abundantly present ribulose-1, 5-bisphosphate carboxylase/oxygenase, and induction of various defense/stress related proteins. Most prominent change in leaves, within 24 h post-treatment with ozone, was the induced accumulation of a pathogenesis related (PR) class 5 protein, three PR 10 class proteins, ascorbate peroxidase(s), superoxide dismutase, calcium-binding protein, calreticulin, a novel ATP-dependent CLP protease, and an unknown protein. Present results demonstrate the highly damaging effect of ozone on rice seedlings at the level of the proteome.

Air Pollutants↗