Changes In Total Protein Content Research Articles

Expression of an engineered acidic-subunit 11S globulin of amaranth carrying the antihypertensive peptides VY, in transgenic tomato fruits

The acidic-subunit of amarantin, main seed storage protein of Amaranthus hypochondriacus, carrying four antihypertensive biopeptides Val-Tyr was expressed in the fruit of transgenic tomato plants. Immunoblot analyses indicate that the expressed recombinant protein was stably accumulated at levels up to 12.71 % with respect to total protein content of transgenic fruits. There was a remarkable change in total protein content (5–22 % increase) of transgenic tomato fruits compared to non-transformed samples. Specific increases of the essential amino acids valine (31–40 %), tyrosine (29–34 %), isoleucine (21–31 %), leucine (28–31 %) and phenylalanine (28–29 %) were also detected in some transgenic lines versus wild type lines. Protein hydrolysates from transgenic tomato fruits showed in vitro inhibition of the angiotensin converting enzyme, with IC50 values that ranged from 0.376 to 3.241 μg ml−1; this represents an increase of up to 13-fold in the inhibitory activity compared with the protein hydrolysates of non-transformed fruits. These results suggest the possible application of transgenic tomato fruit for massive production of this engineered version of amarantin, which could be especially useful in the prevention and control of hypertension.

Protein Electrophoresis Values in Captive Adult Male Nile Crocodiles (Crocodilus niloticus) over Time

Abstract Protein electrophoresis is commonly used to assess the different protein fractions in vertebrates. This information can provide important clinical information regarding the health status of an individual patient or a population of animals. The objective of this study was to evaluate plasma protein fractions of clinically normal, captive, male adult Nile crocodiles (Crocodilus niloticus) over a 12 month period. Blood was collected from the ventral coccygeal vein into heparinized syringes and immediately placed into vacutainer tubes. Samples were centrifuged, plasma removed, and the total protein content of each sample determined using the biuret method. Plasma proteins were separated using gel electrophoresis and scanned using a laser densitometer. No changes in total protein concentration were detected over time. Albumin values were significantly higher in December through March and lower from April through September. Beta globulin values were significantly higher in August through September and ...

Embryonic development of Rhynchophorus palmarum (Coleoptera: Curculionidae): dynamics of energy source utilization.

Energy homeostasis is an essential process during oogenesis, nutrients are required for suitable embryonic development, and recently, studies have investigated metabolic activity during this process. This work aims the investigation of dynamics of energy source utilization of Rhynchophorus palmarum during embryogenesis. For this, we first evaluated the mobilization kinetics of the lipids and glycogen. Thereafter, the synthesis of RNA, protein, and the involvement of enzyme of the glycolytic and pentose-phosphate pathways. Results showed that lipid content decreased in contrast with the lipase activity. The total glycogen amounts it was partly consumed and the glucose content increased, but then values remained stable until hatching. Total RNA content increased, and no significant changes in total protein content were observed. A study of the glycolytic pathway data showed activity of hexokinase and pyruvate kinase at the beginning of embryogenesis. Furthermore, glucose-6-phosphate formed is driven into the pentose-phosphate pathway viewed the high activity of glucose-6-phosphate dehydrogenase. Finally, these results showed that mobilization of different energy sources together with different enzymatic activities has an important role in embryonic development of R. palmarum .

Chemical Composition, Quality Parameters, Exopeptidase and Oxidoreductase Activity Changes During Temporal Development of Wheat Grain Infestation by Sitophilus granarius

Changes in chemical composition, grain quality parameters, oxidoreductase and exopeptidase activities of wheat grains infested by Sitophilus granarius were monitored during 1 year of storage. Significant increase in flour titratable acidity of infested grains occurred by day 90. Subsequently, infestation was followed by decrease of grain hectoliter weight on day 180, decrease of carboxypeptidase activity on day 240, polyphenol oxidase and catalase activities increased and total starch decreased on day 270, while significant changes of total protein content, extractable protein concentration, phenylalanyl-aminopeptidase and Zeleny sedimentation occurred between 300 and 360 days of storage. The data indicate that S. granarius infestation influenced chemical composition, quality parameters, as well as oxidoreductase and exopeptidase activity in wheat grains, but the extent of these changes was mostly detectable at higher levels of grain infestation. Among the examined parameters, flour titratable acidity and carboxypeptidase activity showed potential to be used as early indicators of grain infestation. Practical Applications Granary weevil, S. granarius, is one of the major grain-damaging insects. This primary infester feeds on grains as adult, or on grain endosperm as larvae causing significant economic losses, due to unsuitability of damaged grains for human food production. Although various methods of S. granarius detection in stored grains have been applied worldwide, there is still a need for cheap indicative methods for detecting insect infestation. This study investigating changes in chemical composition, quality parameters, oxidoreductase and exopeptidase activities during temporal development of infestation by S. granarius shows that flour titratable acidity and carboxypeptidase activity could be used as early indicators of wheat grain infestation.

Molecular determination of genotoxic effects of cobalt and nickel on maize (Zea mays L.) by RAPD and protein analyses

Assessment of DNA damages stemming from toxic chemicals is an important issue in terms of genotoxicology. In this study, maize (Zea mays L.) seedlings were used for screening the genotoxic effects of cobalt (Co) and nickel (Ni) treatments at various concentrations (5 mM, 10 mM, 20 mM and 40 mM). For this purpose, randomly amplified polymorphic DNA (RAPD) technique was applied to genomic DNA extracted from metal-exposed and unexposed plant materials. Besides, changes in total protein contents were screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. For RAPD analysis, 16 RAPD primers were found to produce unique polymorphic band profiles on different concentrations of Co-/Ni-treated maize seedlings. Increased polymorphism resulting from the appearance of new bands or disappearance of normal bands was observed with increasing concentration of Co and Ni treatments. Genomic template stability, a qualitative measurement of changes in RAPD patterns of genomic DNA, decreased with increasing metal concentration. In SDS-PAGE analysis, it was observed that the total soluble protein content decreased by Co treatment, while it increased by Ni treatment. The results obtained from this study revealed that RAPD profiles and total soluble protein levels can be applied to detect genotoxicity, and these analyses can offer useful biomarker assays for the evaluation of genotoxic effects on Co- and Ni-polluted plants.

RESPONSE OF TRACHYDISCUS MINUTUS (XANTHOPHYCEAE) TO TEMPERATURE AND LIGHT(1).

The effects of different temperatures and light intensities on growth, pigments, sugars, lipids, and proteins, as well as on some antioxidant and proteolytic enzymes of Trachydiscus minutus (Bourr.) H. Ettl, were investigated. The optimum growth temperature and light intensity were 25°C and 2 × 132 μmol photons · m(-2 ) · s(-1) , respectively. Under these conditions, proteins were the main biomass components (33.45% dry weight [dwt]), with high levels of carbohydrates (29% dwt) and lipids (21.77% dwt). T. minutus tolerated temperatures between 20°C and 32°C, with only moderate changes in cell growth and biochemical composition. Extremely low (15°C) and high (40°C) temperatures decreased chl and RUBISCO contents and inhibited cell growth. The biochemical response of the alga to both unfavorable conditions was an increase in lipid content (up to 35.19% dwt) and a decrease in carbohydrates (down to 13.64% dwt) with much less of a change in total protein content (in the range of 30.51%-38.13% dwt). At the same time, the defense system of T. minutus was regulated differently in response to heat or cold treatments. Generally, at 40°C, the activities of superoxide dismutase (SOD), catalase (CAT), and proteases were drastically elevated, and three polypeptides were overexpressed, whereas the glutathione reductase (GR) and peroxidase (POD) activities were reduced. In contrast, at 15°C, all these enzymes except GR were suppressed. The effect of light was to enhance or decrease the temperature stress responses, depending on intensity. Our studies demonstrate the broad temperature adaptability of T. minutus as well as the potential for the production of valuable algal biomass.

Seasonal changes in antioxidant defence systems in seminal plasma and fluids of the boar reproductive tract

This study aimed to analyze seasonal variations in the antioxidant defence systems of the seminal plasma and fluids of the cauda epididymis and vesicular glands of the boar. The analyzed antioxidants included superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total L-glutathione (GSH+GSSG). Seasonal changes in total protein content and total antioxidant status (TAS) of the seminal plasma and reproductive fluids were also analyzed. Compared with the spring-summer period, total protein content in the seminal plasma was significantly higher during the autumn-winter period. Among the antioxidants analyzed, only SOD activity showed marked seasonal variations, being significantly higher during the spring-summer period. Likewise, the fluid of the cauda epididymis exhibited greater SOD and CAT activity during the spring-summer period, whereas TAS levels were markedly higher during the autumn-winter period. Neither GPx activity nor total GSH+GSSG content in the cauda epididymal fluid was significantly affected by the seasonal periods. The vesicular gland fluid exhibited an approximately 4-fold greater level of SOD activity during the autumn-winter period, as compared with the spring-summer period. By contrast, greater CAT and GPx activity, and a higher level of total GSH+GSSG were observed in the vesicular gland fluid during the spring-summer period. In conclusion, the findings of this study indicate that seasonal variations could have varying effects on the antioxidant defence systems in the seminal plasma and fluids of the boar reproductive tract.

Pollen Viability under a Stress Condition

RATIONALE: Experiments have suggested that pollen viability could be an indicator of air pollution. This testing could potentially replace the pesticides and heavy metals measurement currently performed to evaluate that parameter. The purpose of this exploratory study was to ascertain the efficacy of a stain to evaluate the viability of different pollen species under an artificial stress condition.METHODS: Timothy grass, short ragweed, and white oak pollen species were stored at 35oC for one month. Pollen viability was measured at time 0 and after 1, 5, 10, 20, and 30 days of storage using p-phenylenediamine to detect the presence of myeloperoxidase. A minimum of 150 pollen grains were examined by optical microscopy. Pollen grains were considered viable if they turned totally black. All samples were extracted at the end of the study. Bradford protein content was measured and qualitative protein profile was analyzed by SDS-PAGE.RESULTS: The discrimination between viable and non-viable pollen was efficient for oak and controversial for ragweed. The percents of viable Timothy grass, oak, and short ragweed pollen at time 0 were 93.1%, 58.8%, and 100%, respectively. The respective percents at the end of the study were 0.0%, 16.2%, and 18.3%. No substantial changes in total protein content and protein profile were observed at any testing time compared with the respective baseline levels.CONCLUSION: Myeloperoxidase staining could be a sensitive marker of pollen integrity for some species. This test could have potential applications in different areas. Additional experiments in the laboratory should be performed before initiating field experiments. RATIONALE: Experiments have suggested that pollen viability could be an indicator of air pollution. This testing could potentially replace the pesticides and heavy metals measurement currently performed to evaluate that parameter. The purpose of this exploratory study was to ascertain the efficacy of a stain to evaluate the viability of different pollen species under an artificial stress condition. METHODS: Timothy grass, short ragweed, and white oak pollen species were stored at 35oC for one month. Pollen viability was measured at time 0 and after 1, 5, 10, 20, and 30 days of storage using p-phenylenediamine to detect the presence of myeloperoxidase. A minimum of 150 pollen grains were examined by optical microscopy. Pollen grains were considered viable if they turned totally black. All samples were extracted at the end of the study. Bradford protein content was measured and qualitative protein profile was analyzed by SDS-PAGE. RESULTS: The discrimination between viable and non-viable pollen was efficient for oak and controversial for ragweed. The percents of viable Timothy grass, oak, and short ragweed pollen at time 0 were 93.1%, 58.8%, and 100%, respectively. The respective percents at the end of the study were 0.0%, 16.2%, and 18.3%. No substantial changes in total protein content and protein profile were observed at any testing time compared with the respective baseline levels. CONCLUSION: Myeloperoxidase staining could be a sensitive marker of pollen integrity for some species. This test could have potential applications in different areas. Additional experiments in the laboratory should be performed before initiating field experiments.

Sudden significant total protein concentration change: not an analytical problem

Sudden and significant total protein concentration changes in multiple myeloma (MM) can be associated with new treatment regimens. We present an example of a 66-year-old male with a total protein concentration of 74 g/L, albumin 27 g/L, and a monoclonal IgG concentration of 22.2 g/L before commencing lenalidomide treatment. The patient represented 8 days later and the total protein concentration was 66 g/L, albumin 32 g/L with a monoclonal IgG concentration of 10.3 g/L. The total protein and albumin measurements were performed on Beckman DxC800 analysers using bieuret and bromcresol purple methods, respectively (Beckman Coulter Diagnostics, Fullerton, CA). The IgG was performed on Beckman Image analyser (Beckman Coulter Diagnostics). It is regularly observed that patients with the new drug treatments have total proteins, more accurately the specific immunoglobulin decreasing significantly in a matter of days. Generally, the remaining biochemical profiles undergo minimal change. To laboratorians such a major significant change in total protein concentration will trigger alarms (including possibly delta check flags) of possible analytical errors: sampling accuracy or possible assay interference. This leads to additional investigations by laboratorians, which would include sample integrity checks (sufficient volume, microclots, bubbles), possible re-analysis, running additional quality control samples, changing total protein reagents, withholding of results until communication with other professionals including the clinical unit occur, and leading to delay in reporting of the results and added costs. MM is currently an incurable hematological malignancy. Monoclonal gammopathy, a common predisposing disease, occurs in 3% of persons 50 years of age or older (1). It is characterized by the clonal expansion of monoclonal immunoglobulin-secreting plasma cells within the bone marrow. The overall median survival for patients with MM without treatment is approximately 3 years. It is the second most common hematologic malignancy, and the most common cancer to involve bone. The cause of MM is unclear. Exposure to radiation, herbicides, insecticides, benzene, and other organic solvents may play a role. Until recently the standard treatment involved a combination of oral melphalan and prednisolone. Allogeneic transplantation was first attempted in the early 1980s, and it became routine therapeutic practice in the 1990s (2), but is not widely used due to high treatment-related mortality. Autologous stem cell transplant (ASCT) has become the treatment of choice. For patients unable to undergo ASCT, including elderly patients (465 years), or patients relapsing after ASCT there have been few treatment options. Singhal et al. in 1999 (3) indicated that thalidomide, an oral agent with anti-angiogenic and immunomodulatory properties, was able to induce objective responses in 30% of patients with refractory and advanced MM. In 2006, thalidomide was approved by the US FDA, and more recently lenalidomide (approved by FDA in 2006, European Union in June 2007), a thalidomide analog with up to 50,000-fold increased potency, (4) and bortezomib, a proteasome inhibitor (approved by US FDA in May 2003), have been added and used as salvage agents for the treatment of relapsed and refractory MM. Trials of these agents as frontline therapy are underway with very promising initial results. These new drugs are cytotoxic to MM cells in the bone marrow. Therefore, rapid and significant changes in total protein concentrations, globulin or specific immunoglobulin are expected.

The comparison of protease activity and total protein in three cultivars of kiwifruit of Northern Iran during fruit development

This study, for the first time, investigated the total protein changes and protease activity pattern of three cultivars of kiwifruit including Abbott, Allison and Hayward, cultivated in the north of Iran, during 6 months of fruit development from July to December 2008. During the last month of sampling, fruits were stored in ambient temperature. According to the result of fruit extracts, pattern of changes in total protein concentration showed three significant steps: low concentration of protein during the early stages of fruit development until middle of July for Allison and Hayward and early August for Abbott, increasing protein concentration until middle of October for Allison and Hayward and late October for Abbott, and steady state level of protein concentration until harvesting time. During storage time of fruits, all three cultivars showed increasing level of total protein concentration. Among the cultivars, Hayward and Allison showed the lowest and highest total protein concentration, respectively. Protease activity showed increasing pattern in all of the investigated cultivars significantly until harvesting time. During postharvest time, Hayward did not show any significant change in protease activity but, the other two cultivars had increasing pattern from 10 to 20th of storage and then decreased significantly. Among the cultivars, Abbott showed the lowest and Allison the highest level of total protease activity at harvesting and postharvesting time. SDS-PAGE, ion-exchange chromatography and zymogram native-PAGE of fruits extraction showed that dominant protein and protease activity in these cultivars during fruit development and ripening process is related to actinidin.

Protein Oligomerization: Thermodynamic and Structural Analysis of the Dimerization of Beta-lactoglobulin

Protein oligomers are widely found in living organisms and they are also crucial in some neurodegenerative diseases. The aim of this work is to analyze the thermodynamics and stability of protein oligomers employing β-lactoglobulin as the experimental model.β -lactoglobulin is a small, globular and compact protein which, in solution, is in equilibrium with its dimeric form. The association equilibrium can be affected through changes in total protein concentration, temperature and pH. The dimer is the prevalent species at neutral pH. We studied the dissociation process of β-lactoglobulin induced by temperature employing different techniques at pH = 6.7. We performed calorimetric experiments at different values of total protein concentration and we observed that the endotherms of β-lactoglobulin at pH = 6.7 are biphasic and they show a clear dependence with protein concentration. While the transition that appears at higher temperature is insensible to changes in protein concentration the first transition is stabilized when protein concentration is increased. This result allowed proposing a mechanism that involves dissociation of the dimer in the first instance and subsequent denaturing of the monomer. According to the thermodynamic model of a dimer dissociation presented in a previous work (1), it was determined through calorimetric profiles simulations, that only a dimer with negative dimerization enthalpy shows the dependence on protein concentration observed in these experiments. We also performed isothermal titration calorimetric studies and the association parameters of β-lactoglobulin were obtained at pH = 6.7. The dimerization process is exothermic in accordance with the model. Furthermore, temperature induced β-lactoglobulin dissociation at pH = 6.7 was monitored with infrared absorbance spectroscopy and we can state that there are structural changes related to the dissociation of the dimer.(1) J. Phys. Chem. B. 112 (45): 14325-14333, 2008.

Neonatal oxygen adversely affects lung function in adult mice without altering surfactant composition or activity

Despite its potentially adverse effects on lung development and function, supplemental oxygen is often used to treat premature infants in respiratory distress. To understand how neonatal hyperoxia can permanently disrupt lung development, we previously reported increased lung compliance, greater alveolar simplification, and disrupted epithelial development in adult mice exposed to 100% inspired oxygen fraction between postnatal days 1 and 4. Here, we investigate whether oxygen-induced changes in lung function are attributable to defects in surfactant composition and activity, structural changes in alveolar development, or both. Newborn mice were exposed to room air or 40%, 60%, 80%, or 100% oxygen between postnatal days 1 and 4 and allowed to recover in room air until 8 wk of age. Lung compliance and alveolar size increased, and airway resistance, airway elastance, tissue elastance, and tissue damping decreased, in mice exposed to 60-80% oxygen; changes were even greater in mice exposed to 100% oxygen. These alterations in lung function were not associated with changes in total protein content or surfactant phospholipid composition in bronchoalveolar lavage. Moreover, surface activity and total and hydrophobic protein content were unchanged in large surfactant aggregates centrifuged from bronchoalveolar lavage compared with control. Instead, the number of type II cells progressively declined in 60-100% oxygen, whereas levels of T1alpha, a protein expressed by type I cells, were comparably increased in mice exposed to 40-100% oxygen. Thickened bundles of elastin fibers were also detected in alveolar walls of mice exposed to > or = 60% oxygen. These findings support the hypothesis that changes in lung development, rather than surfactant activity, are the primary causes of oxygen-altered lung function in children who were exposed to oxygen as neonates. Furthermore, the disruptive effects of oxygen on epithelial development and lung mechanics are not equivalently dose dependent.

A Large Number of Protein Expression Changes Occur Early in Life and Precede Phenotype Onset in a Mouse Model for Huntington Disease

Huntington disease (HD) is fatal in humans within 15-20 years of symptomatic disease. Although late stage HD has been studied extensively, protein expression changes that occur at the early stages of disease and during disease progression have not been reported. In this study, we used a large two-dimensional gel/mass spectrometry-based proteomics approach to investigate HD-induced protein expression alterations and their kinetics at very early stages and during the course of disease. The murine HD model R6/2 was investigated at 2, 4, 6, 8, and 12 weeks of age, corresponding to absence of disease and early, intermediate, and late stage HD. Unexpectedly the most HD stage-specific protein changes (71-100%) as well as a drastic alteration (almost 6% of the proteome) in protein expression occurred already as early as 2 weeks of age. Early changes included mainly the up-regulation of proteins involved in glycolysis/gluconeogenesis and the down-regulation of the actin cytoskeleton. This suggests a period of highly variable protein expression that precedes the onset of HD phenotypes. Although an up-regulation of glycolysis/gluconeogenesis-related protein alterations remained dominant during HD progression, late stage alterations at 12 weeks showed an up-regulation of proteins involved in proteasomal function. The early changes in HD coincide with a peak in protein alteration during normal mouse development at 2 weeks of age that may be responsible for these massive changes. Protein and mRNA data sets showed a large overlap on the level of affected pathways but not single proteins/mRNAs. Our observations suggest that HD is characterized by a highly dynamic disease pathology not represented by linear protein concentration alterations over the course of disease.

PGF 2α-associated vascular smooth muscle hypertrophy is ROS dependent and involves the activation of mTOR, p70 S6k, and PTEN

Prostaglandin F 2α (PGF 2α) increases reactive oxygen species (ROS) and induces vascular smooth muscle cell (VSMC) hypertrophy by largely unknown mechanism(s). To investigate the signaling events governing PGF 2α-induced VSMC hypertrophy we examined the ability of the PGF 2α analog, fluprostenol to elicit phosphorylation of Akt, the mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70 S6k), glycogen synthase kinase-3β (GSK-3β), phosphatase and tensin homolog (PTEN), extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) in growth arrested A7r5 VSMC. Fluprostenol-induced hypertrophy was associated with increased ROS, mTOR translocation from the nucleus to the cytoplasm, along with Akt, mTOR, GSK-3β, PTEN and ERK1/2 but not JNK phosphorylation. Whereas inhibition of phosphatidylinositol 3-kinase (PI3K) by LY-294002 blocked fluprostenol-induced changes in total protein content, pre-treatment with rapamycin or with the MEK1/2 inhibitor U0126 did not. Taken together, these findings suggest that fluprostenol-induced changes in A7r5 hypertrophy involve mTOR translocation and occur through PI3K-dependent mechanisms.

Hematologic and biochemical changes during canine agility competitions

Normal response to different types of exercise needs to be established before performance, training level, or exercise intolerance can be assessed. The aim of this research was to describe the hematologic and biochemical changes that dogs undergo during an agility competition. Fifteen dogs were subjected to an agility test that covered a distance of 360-400 m with 40 obstacles. Basal venous blood samples were drawn 1 week before the competition, immediately after exercise, and at 5, 15, and 30 minutes of recuperation. A CBC, plasma biochemical profile, and lactate concentration were determined by standard methods using automated analyzers. Serum cortisol concentration was measured by competitive immune-enzyme analysis; insulin was measured by sandwich ELISA. Hematologic response to the agility test consisted of significant increases in RBC count, hemoglobin concentration, and HCT, with no change in total protein concentration. Serum triglyceride concentrations were increased postexercise, with highest values observed at 30 minutes of recuperation. Postexercise lactate concentrations exceeded the anaerobic threshold of 4 mmol/L. Serum lactate dehydrogenase activity was significantly higher after 30 minutes of recuperation. Significant differences were not observed in other analytes. Our results indicate that agility competitions induce mild to moderate changes in hematologic and biochemical results consistent with splenic contraction, increased lipolysis, and utilization of anaerobic pathways involved in energy resynthesis in muscle.

Age-dependent changes in the total protein concentrations in the brain regions and hypophyses of the pig

88 crossbred Large White male day-old piglets were used to investigate age-related changes in total protein concentrations in ten regions of the pig brain and hypophyses from birth to 36 months of age. Age-related changes in protein concentrations in all the brain regions except the pons and cerebral cortex were not significant (P>0.05). However, the linear and quadratic curves and coefficients revealed substantial increase in total protein content in all the brain regions from day-old to between 12 and 20 months, thereafter declining rapidly and, in certain regions, gradually to much lower levels. In the pons, a rise in total protein of 1,091% was observed from day-old to 12 months, declining thereafter by 88% to 36 months. In the cerebellum, values rose by 642% from day-old to 10 months progressively falling by 75% in subsequent months to 36 months. Similar trends were observed in the amygdala and hippocampus. In the hypothalamus, midbrain and medulla oblongata, total protein levels rose progressively by 212%, 229% and 316% respectively from day-old to 36 months with a few non-specific fluctuations in- between. Significant falls in total protein levels with advancing age were observed in the adenohypophysis (P

The genotoxic effects of logran onHordeum vulgareL. andTriticum aestivumL.

In the present study, the cytogenetic effects of the herbicide Logran on root tip cells of Triticurn aestivum L. and Hordeum vulgare L. and changes of total protein content in root tip meristems were studied. The seeds of plants were treated with various concentrations of Logran (125, 250, 500 microg/ml) for 3 and 6 h. The percentages of abnormal cells were seen to increase with increasing treatment period and concentrations. The most dominant types of observed abnormalities were C-mitosis, distributed metaphase and anaphase, stickiness. All the used concentrations of Logran significantly induced a number of chromosomal aberrations in root tip cells of Hordemrn vulgare L. and Triticum aestivum L. Logran also decreased mitotic index. The decrease of protein content in root tips of Triticum aestivum L. is significant at all the treated concentrations and treatment periods when compared with control.

Influence of pH, buffer species, and storage temperature on physicochemical stability of a humanized monoclonal antibody LA298

The purpose of this work is to study the effect of pH, buffer species, and temperature on the physicochemical stability of a humanized monoclonal antibody LA298. The study was carried out in solution state of the antibody in the presence of different buffer species at different pH values and storage temperature. No significant changes in total protein content were observed for any of the solutions with different buffers at different pH values when stored for 8 weeks at both 5 °C and 25 °C or at 37 °C for 1 week. Known asparagines (Asn55) deamidation of LA298 was found to be dependent on pH, buffer type, and temperature. The estimated rate constant of the double heavy chain Asn55 deamidation in phosphate buffer at pH 6.5 and 7.0 was much higher than that in citrate buffer under the same storage conditions. However, comparable results were obtained for single heavy chain Asn55 deamidation in citrate and phosphate buffer. Aggregation of LA298 was not significant for samples at different pH values, buffers, and temperatures as the monomer of LA298 decreased dramatically over time. Less decrease in monomeric LA298 was observed in citrate buffer, pH 5.0–5.5. In conclusion, to minimize deamidation and loss of LA298 monomer, it is important to optimize its solution pH, buffer species, and storage temperature.

Flow cytometric analysis of protein content in Taxus protoplasts and single cells as compared to aggregated suspension cultures.

Plant suspension cultures are highly aggregated, preventing the direct application of flow cytometry for the study of population dynamics. The utility of single cells to accurately represent aggregated suspension cultures was tested through the analysis of total protein content. Specifically, protein content of two Taxus cuspidata suspension culture lines was studied using the Bradford assay for aggregated suspension cultures, and flow cytometry with fluorescein isothiocyanate staining for protoplasts and single cells. Taxus protein levels were measured at 75-160 mg per gram dry weight via the Bradford assay. Aggregated suspension cultures, protoplasts, and single cells predicted the same trend of protein content over the culture period (21 days). Normalized protein content of isolated single cells was statistically equivalent to aggregated suspensions for both cell lines. However, normalized protein content of isolated protoplasts showed significant differences from aggregated suspensions for one of the two cell lines. Elicitation with methyl jasmonate (MJ) is commonly utilized to increase paclitaxel accumulation in suspension cultures, and therefore the effect of MJ elicitation on protein content in aggregated suspensions, isolated single cells and protoplasts was assessed. Aggregated suspension cultures, protoplasts, and single cells did not show any change in total protein content following elicitation with MJ at 200 microM on day 7. This study illustrates the usefulness of flow cytometry for obtaining culture population information and the value of using intact single cells for the study of plant metabolism.

Direct actions of urotensin II on the heart: implications for cardiac fibrosis and hypertrophy.

Urotensin II (UII) is a somatostatin-like peptide recently identified as a potent vasoconstrictor. In this study, we examined whether UII promotes cardiac remodeling through nonhemodynamic effects on the myocardium. In a rat model of heart failure after myocardial infarction (MI), increased UII peptide and UII receptor protein expression was observed in both infarct and noninfarct regions of the left ventricle compared with sham. Moreover, post-MI remodeling was associated with a significant 75% increase in UII receptor gene expression in the heart (P<0.05 versus sham controls), with this increase noted in both regions of the left ventricle. In vitro, UII (10-7 mol/L) stimulation of neonatal cardiac fibroblasts increased the level of mRNA transcripts for procollagens alpha1(I), alpha1(III), and fibronectin by 139+/-15% (P<0.01), 59+/-5% (P<0.05), and 141+/-14% (P<0.01), respectively, with a concomitant 23+/-2% increase in collagen peptide synthesis as determined by 3H-proline incorporation (P<0.01). UII had no effect on cellular hypertrophy, as determined by changes in total protein content in isolated neonatal cardiomyocytes. However, expression of recombinant rat UII receptor in neonatal cardiomyocytes resulted in significant UII-dependent activation of hypertrophic signaling as demonstrated by increased total protein content (unstimulated, 122.4+/-4.0 microg/well; rat UII, 147.6+/-7.0 microg/well; P<0.01) and activation of the hypertrophic phenotype through Galpha(q)- and Ras-dependent pathways. These results indicate that, in addition to potent hemodynamic effects, UII may be implicated in myocardial fibrogenesis through increased collagen synthesis by cardiac fibroblasts and may also be an important determinant of pathological cardiac hypertrophy in conditions characterized by UII receptor upregulation.