Increase In Total Protein Content Research Articles

Ultrastructural and biochemical analysis of the effects of alendronate on salivary glands of young rats

IntroductionThe bisphosphonates are drugs known by their antiresorptive potency and are widely prescribed for treating and preventing osteoporosis. In the past years the employment of this class of drugs had spread to other pathologies, and it is being prescribed to patients in a wide range of ages. Some adverse effects of bisphosphonate treatment have been highlighted recently, however, little is known about its potential side effects in salivary glands. MethodsNewborn rats received daily doses of 2.5mg/kg/day of sodium alendronate during 30 days. On the thirtieth day the animals were stimulated with pilocarpine and their parotid and submandibular glands were collected, fixed and embedded for histological and ultrastructural analysis. Some glands were collected for analysis of protein content and amylase activity. ResultsAt light microscopy, the alendronate-treated animals presented an accumulation of secretion granules in their cytoplasm, which was confirmed by the ultrastructural examination. Biochemical analysis revealed an increase in total protein content and decreased amylase levels of both glands in the alendronate-treated animals in relation to the control. ConclusionBased on the current findings, alendronate is probably interfering in secretory pathways of parotid and submandibular glands.

Selected antibacterial factors in the saliva of diabetic patients

ObjectiveDiabetes mellitus leads to many systemic complications, including changes in the morphology, function of the salivary glands, and the composition of saliva. DesignThe study comprised a randomly selected 156 adults, of both genders, aged from 21 to 79, out of which patients with diabetes type 1 and 2, and healthy subjects forming two control age- and gender matched to the ill subjects. In unstimulated mixed saliva, total protein, peroxidase, myeloperoxidase and immunoglobulin A were measured as well as salivary flow rate. The periodontal condition was assessed with the use of GI, mSBI and PSR index. The obtained data were analysed with the use of U Mann–Whitney's test, Spearman's rang correlation and Chi-square test at a significant level of p<0.05 with use of Statistica 9.0 software. ResultsType 1 diabetics in comparison to healthy age and gender matched control group had a lower salivary flow rate (p<0.01), a higher content of total protein (p<0.01), myeloperoxidase (p<0.001) and immunoglobulin A (p<0.001). Similarly, type 2 diabetics in comparison to control subjects had a higher level of total protein concentration (p<0.01), myeloperoxidase (p<0.05) and immunoglobulin A (p<0.001). We also found worse periodontal condition. ConclusionWithin the limitation of the study it may be stated that diabetes type 1 and 2 can cause abnormalities in salivary glands function resulting in the diminishing of salivary flow rate and the increase in total protein content. Higher levels of myeloperoxidase and IgA in the saliva can be linked to worse periodontal condition in the diabetic patients.

Effects of copper exposure on the energy metabolism in juveniles of the marine clam Mesodesma mactroides

In freshwater osmoregulating mollusks, Cu can cause toxicity by inducing ionoregulatory disturbances. In mussels, it inhibits the activity of key enzymes involved in Na+ uptake and consequently induces ionic and osmotic disturbances. In snails, Cu induces disruption of the Ca2+ homeostasis leading to effects in shell deposition and snail growth. However, the mechanisms involved in Cu toxicity in osmoconforming sweater mollusks remain unclear. Recent findings from our laboratory have suggested that Cu toxicity in marine invertebrates can be associated with both ionic and respiratory disturbances. In the present study, metabolic changes induced by waterborne Cu exposure were evaluated in the osmoconforming clam Mesodesma mactroides, a bivalve species widely distributed along the South American sandy beaches. Juvenile clams were kept under control conditions (no Cu addition in the water) or acutely (96h) exposed to Cu (96-h LC10=150μgL−1) in artificial seawater (30ppt). ATP, protein, lipid, glycogen and glucose contents were analyzed in gills, digestive gland, pedal muscle and hemolymph. Dinucleotide (NAD+ and NADH) content was also analyzed in gills, digestive gland and pedal muscle while pyruvate and lactate content was determined in pedal muscle and hemolymph. In all tissues analyzed, Cu exposure did not affect ATP content and NAD+/NADH ratio, except in the hemolymph, where a decrease in ATP content was observed. These findings indicate that clam cells, except those from hemolymph, were able to maintain a constant level of free energy. A significant increase in total protein content was observed in the digestive gland, which could be a compensatory mechanism to counteract the higher level of protein oxidation previously observed in M. mactroides exposed to Cu under the same experimental conditions. Finally, reduced glucose content in the pedal muscle paralleled by increased lactate content in the pedal muscle and hemolymph was observed in Cu-exposed clams. Overall, these findings indicate that Cu exposure is leading to an increased reliance upon the anaerobic energy production to maintain the overall cellular ATP production in the clam M. mactroides.

Clinical oral and salivary parameters of children with juvenile idiopathic arthritis

To evaluate clinical oral and salivary parameters of individuals with juvenile idiopathic arthritis (JIA). Clinical parameters and whole saliva were collected from children aged 6 to 12 years with JIA (n = 36) and from a healthy, matched control group (n = 36). The clinical and salivary parameters evaluated were the dental caries (decayed, missing, or filled teeth), gingival and simplified oral hygiene indices, salivary flow rate, pH, buffer capacity, total protein, and secretory immunoglobulin A concentrations. JIA individuals presented poorer oral hygiene (P ≤ .05) but no difference in the dental caries and gingival indices. JIA patients presented an increase in total protein concentration (86%) and buffer capacity in the range of pH 6.9 to 6.0 (10%) and a reduction in initial pH (6%), buffer capacity in the range of pH ≥ 7.0 (50%), and immunoglobulin A concentration (27%) (P ≤ .05). JIA is associated with poor oral hygiene and salivary changes, including reductions in immune factors and an altered profile of salivary buffer capacity.

Vaccine‐Mediated Protection against Helicobacter pylori is not Associated with Increased Salivary Cytokine or Mucin Expression

The development of an effective vaccine against Helicobacter pylori is impeded by the inability to reliably produce sterilizing immunity and our lack of knowledge regarding mechanisms of protective immunity against this pathogen. It has previously been described that salivary glands are essential for vaccine-mediated protection against H.pylori, but the mechanism responsible for this effect has not been identified. In this study we tested the hypothesis that vaccines reduce H.pylori colonization by inducing an immune-mediated change in salivary gland mucin secretion. Sublingual and submandibular salivary glands were removed from untreated mice, from mice infected with H.pylori and from mice vaccinated against H.pylori then challenged with live bacteria. Cytokine levels in these salivary glands were quantified by ELISA, and salivary mucins were quantified by real-time PCR. Salivary antibody responses were determined by Western blot. Vaccine-mediated protection against H.pylori did not produce any evidence of a positive increase in either salivary cytokine or mucin levels. In fact, many cytokines were significantly reduced in the vaccinated/challenged mice, including IL-17A, IL-10, IL-1ß, as well as the mucin Muc10. These decreases were associated with an increase in total protein content within the salivary glands of vaccinated mice which appeared to be the result of increased IgA production. While this study showed that vaccination increased salivary IgA levels, previous studies have demonstrated that antibodies do not play a critical role in protection against H.pylori that is induced by current vaccine formulations and regimes. The effector mechanism of protective immunity induced by vaccination of mice did not involve immune changes within the salivary glands, nor increased production of salivary mucins.

First report of the pituitary adenylate cyclase activating polypeptide (PACAP) in crustaceans: Conservation of its functions as growth promoting factor and immunomodulator in the white shrimp Litopenaeus vannamei

The high conservation of the pituitary adenylate cyclase activating polypeptide (PACAP) sequence indicates that this peptide fulfills important biological functions in a broad spectrum of organisms. However, in invertebrates, little is known about its presence and its functions remain unclear. Up to now, in non-mammalian vertebrates, the majority of studies on PACAP have focused mainly on the localization, cloning and structural evolution of this peptide. As yet, little is known about its biological functions as growth factor and immunomodulator in lower vertebrates. Recently, we have shown that PACAP, apart from its neuroendocrine role, influences immune functions in larval and juvenile fish. In this work, we isolated for the first time the cDNA encoding the mature PACAP from a crustacean species, the white shrimp Litopenaeus vannamei, corroborating its high degree of sequence conservation, when compared to sequences reported from tunicates to mammalian vertebrates. Based on this, we have evaluated the effects of purified recombinant Clarias gariepinus PACAP administrated by immersion baths on white shrimp growth and immunity. We demonstrated that PACAP improves hemocyte count, superoxide dismutase, lectins and nitric oxide synthase derived metabolites in treated shrimp related with an increase in total protein concentration and growth performance. From our results, PACAP acts as a regulator of shrimp growth and immunity, suggesting that in crustaceans, as in vertebrate organisms, PACAP is an important molecule shared by both the endocrine and the immune systems.

Direct hypertrophic effects of fibroblast growth factor 23 on cardiomyocytes

Fibroblast growth factor 23 (FGF23) is a secreted, bone derived hormone that regulates vitamin D and phosphate metabolism. Recent clinical data shows a correlation between elevated levels of serum FGF23 in patients with chronic kidney disease (CKD) and cardiac disease. Therefore, we investigated the direct effects of a pathophysiological concentration of FGF23 on cardiomyocyte hypertrophy. Using flow cytometry we observed an 18% increase in HL‐1 cardiomyocyte cell size after 48 hour exposure to 900 pg/mL FGF23 (FSC; p&lt;0.05; n=5). These hypertrophic effects on cells were verified in ventricular muscle tissue strips where treatment of FGF23 resulted in a 2 to5 fold increase in expression of common gene markers of cardiac hypertrophy (n=4–7; p&lt;0.05) and elevated ERK1/2 phosphorylation (n=4; p&lt;0.05). We also observed a 10% increase in total protein concentration which was blocked with an FGFR1 inhibitor (n=5–7; p&lt;0.05). These data show that FGF23 has direct effects on cardiomyocyte cell size and may be an important bone‐heart crosstalk regulator of cardiac hypertrophy. This may be of particular importance with certain bone diseases and CKD where FGF23 levels are elevated. This work was supported by AHA SDG 11SDG5330016 (MJW) and NIH NIAMS 1RC2AR058962 (MJW and LFB).

Mechano-Biology of Chondrocytes in Intact Joints Under Muscular Loading

It is well documented that onset and progression of OA is directly related to the mechanical loading of joints, and the associated mechano-biology of chondrocytes. However, work on the mechano-biology of chondrocytes has been performed with isolated cells or tissue explants subjected to confined and unconfined loading, conditions which neglect the role of the matrix and ignores the boundary conditions of cartilage-on-cartilage contact, and fails to reproduce physiological loading conditions.We developed a novel approach which allows quantification of chondrocyte mechanics, signaling and analysis of proteins, cytokines and fluid borne stem cells contained within the synovial fluid of intact knees in live mice while the joint is loaded using controlled muscular stimulation.Chondrocytes and their nuclei deform on average 18-25% for sub-maximal muscular loading in the intact mouse knee. Deformation occurs “instantaneously” upon loading, but requires minutes for full recovery (Figures 1a). High muscular loading of the mouse knee caused an increase in total protein content in the synovial fluid (Figure 1b) and an increase in PRG4 (also known as lubricin). When “high” intensity load conditions were repeated, the PRG4 and total protein release was stopped (Figure 1b).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Age-dependence of intracranial viscoelastic properties in living rats

To explore the effect of maturation on intracranial mechanical properties, viscoelastic parameters were determined in 44 live rats at ages 1–2, 10–12, 21, 56–70, and 180 days using instrumented indentation. With the dura mater intact, the apparent modulus of elasticity, the indentation modulus, and viscous behavior were measured in vivo, as well as 1 h after death. In a separate group of 25 rats, brain water, and protein content were determined. A significant increase of the elastic and indentation moduli beginning at 10–12 days after birth and continuing to 180 days was observed. The creep behavior decreased in the postnatal period and stabilized at 21 days. Changes in intracranial biomechanical properties corresponded to a gradual decrease of brain water, and an increase in total protein content, including glial fibrillary acidic protein, myelin basic protein, and neurofilament light chain. Elastic properties were not significantly different comparing the live and dead states. However, there were significant postmortem changes in viscous behavior. Viscoelastic properties of living rat intracranial contents are shown to be age dependent, reflecting the physical and biochemical changes during postnatal development. This may be important for understanding why young and mature brains respond differently in situations of brain trauma and hydrocephalus.

The Expression and DNA-Binding Activity of NF-κB Nuclear Transcription Factor in the Tumors of Patients with Breast Cancer

The content and DNA-binding activity of NF-κB nuclear transcription factor subunit p65 (NF-κBp65) were evaluated by quantitative enzyme immunoassay in tumors and histologically intact tissues of 119 patients with breast cancer. DNA-binding activity of NF-κBp65 in the tumors was higher than in adjacent tissue in 97% cases. This elevation was paralleled by an increase in total protein content in the majority of cases. No significant relationship of the parameter with the disease stage, tumor size and histology, and degree of lymph node involvement was detected. However, the content of NF-κBp65 in tumors of malignancy degree III was significantly higher than in tumors of malignancy degree II. An increase in total expression of NF-κBp65 protein was found in HER-2+ tumors. This increase was not related to steroid hormone receptor status and was not paralleled by elevation of DNA-binding activity, which was maximum in tumors with the "triple negative" receptor status (RE-RP-HER-2-).

Next-generation protein-rich potato expressing the seed protein gene AmA1 is a result of proteome rebalancing in transgenic tuber

Protein deficiency is the most crucial factor that affects physical growth and development and that increases morbidity and mortality especially in developing countries. Efforts have been made to improve protein quality and quantity in crop plants but with limited success. Here, we report the development of transgenic potatoes with enhanced nutritive value by tuber-specific expression of a seed protein, AmA1 (Amaranth Albumin 1), in seven genotypic backgrounds suitable for cultivation in different agro-climatic regions. Analyses of the transgenic tubers revealed up to 60% increase in total protein content. In addition, the concentrations of several essential amino acids were increased significantly in transgenic tubers, which are otherwise limited in potato. Moreover, the transgenics also exhibited enhanced photosynthetic activity with a concomitant increase in total biomass. These results are striking because this genetic manipulation also resulted in a moderate increase in tuber yield. The comparative protein profiling suggests that the proteome rebalancing might cause increased protein content in transgenic tubers. Furthermore, the data on field performance and safety evaluation indicate that the transgenic potatoes are suitable for commercial cultivation. In vitro and in vivo studies on experimental animals demonstrate that the transgenic tubers are also safe for human consumption. Altogether, these results emphasize that the expression of AmA1 is a potential strategy for the nutritional improvement of food crops.

Morphine sulphate induced histopathological and histochemical changes in the rat liver

In this study, the histopathological and histochemical changes due to chronic usage of morphine sulphate in liver were assessed in rats with both light and electron microscopes. Twenty male albino rats ( Rattus norvegicus) (130–150 g) were included and divided into four groups. Normal saline (5 ml) was given orally as placebo in the control group ( N = 5). Morphine groups ( N = 5) received morphine orally at a single dose of 5 ml/kg/day for 10, 20 and 30 days (groups II, III and IV), respectively. Liver specimens from all groups were evaluated for histopathological and histochemical changes. Light microscopy revealed severe centrilobular congestion, portal fibrosis with bile ductal proliferation and an increased inflammatory infiltration and focal parenchymal necrosis. Histochemical study revealed a progressive depletion of general carbohydrates and an increase in total protein contents. These changes were confirmed at ultrastructural level, including the presence of accumulated lipid in the hepatocytes; deposits of a collagen-like fibrous material were seen in the space of Disse and a reduction in the number of endothelial cell fenestrations. Our findings pointed out the risk of increased lipid fibrosis and hepatic damage due to long-term use of morphine. Although opioids are reported to be effective in pain management, their toxic effects should be kept in mind during chronic usage.

Changes in the Salivary Biomarkers Induced by an Effort Test

Physical exercise induces biochemical changes in the body that modify analytes in blood and saliva among other body fluids. This study analyzed the effect of an incremental effort test on the salivary protein profile to determine whether any specific protein is altered in response to such stress. We also measured thresholds of salivary alpha amylase, total salivary protein and blood lactate and searched for correlations among them. Twelve male cyclists underwent a progressive test in which blood and saliva samples were collected simultaneously at each stage. The salivary total protein profile revealed that physical exercise primarily affects the polypeptide corresponding to salivary alpha-amylase, the concentration of which increased markedly during the test. We observed thresholds of salivary alpha-amylase (sAAT), total salivary protein (PAT) and blood lactate (BLT) in 58%, 83% and 100% of our sample, respectively. Pearson's correlation indicates a strong and significant association between sAAT and BLT (r= 0.84, P<0.05), sAAT and PAT (r= 0.83, P<0.05) and BLT and PAT (r= 0.90, P<0.05). The increased expression of the salivary alpha-amylase (sAA) polypeptide suggests that sAA is the main protein responsible for the increase in total protein concentration of whole saliva. Therefore, monitoring total protein concentration is an efficient tool and an alternative noninvasive biochemical method for determining exercise intensity.

Salivary Immunoglobulin A Response to a Match in Top-Level Brazilian Soccer Players

It has been suggested that several parameters of mucosal immunity, including salivary immunoglobulin A (s-IgA), are affected by heavy exercise either in field sports or in the laboratory environment. Few observations have been made during a true sporting environment, particularly in professional soccer. We tested the hypothesis that salivary IgA levels will be decreased after a 70-minute regulation in a top-level professional soccer friendly match. Saliva samples from 24 male professional soccer players collected before and after the match were analyzed. Salivary immunoglobulin A concentration was measured by enzyme-linked immunosorbent assay and expressed as the absolute concentration (s-IgAabs), s-IgA relative to total protein concentration (IgA-Pro), and the secretion rate of IgA (s-IgArate). Rate of perceived exertion (RPE) was used to monitor the exercise intensity. The paired t-test showed no significant changes in s-IgAabs and s-IgArate (p > 0.05) from PRE to POST match. However, a significant (p < 0.05) increase in total protein concentration (1.46 +/- 0.4 to 2.00 +/- 07) and a decrease in IgA-Pro were observed. The best and most significant correlation was obtained with the RPE and changes in IgA-Pro (rs = -0.43) and could indicate that this expression may be an interesting marker of intensity in a soccer match. However, further investigation regarding exercise intensity, protein concentration, and immune suppression, particularly in team sports, is warranted. From a practical application, the variability of the responses among the players leads us to suggest that there is a need to individually analyze the results with team sports. Some athletes showed a decrease in s-IgA expressions, suggesting the need for taking protective actions to minimize contact with cold viruses or even reducing the training load.

Changes in cerebrospinal fluid nerve growth factor levels during chick embryonic development

In the early stages of brain development, cells within the ependymal lining of the neural tube are thought to secrete cerebrospinal fluid (CSF), the so-called neural tube fluid (NTF), whereas before fusion of the neural folds, the neuroepithelium that lines the inside of the neural tube is in contact with amniotic fluid. As the neural tube closes, a membrane formed from these cells invaginates to form the specialized choroid plexus. The choroid plexus is a highly vascularized epithelial cell structure that secretes proteins, including growth factors, into the CSF. Embryonic CSF (e-CSF) contains high concentrations of proteins compared to adult CSF. CSF has been reported to contain nerve growth factor (NGF) and other neurotrophic factors. In this study, total protein concentration and NGF level in e-CSF samples from chick embryos were measured using a dye-based protein assay, enzyme-linked immunosorbent assay (ELISA) and Western blot. The total protein concentration and NGF levels in the CSF decreased from days E10 to E16. There was a rapid increase in total protein content on days E17 and E18, and thereafter the levels decreased from day E19 to day E21. Days E17 and E18 coincide with the onset of neuron migration, proliferation and organization of the cytoarchitecture of the developing cerebral cortex. After that time the total protein concentration and NGF levels decrease until hatching. Since CSF is in contact with the cerebral cortical germinal epithelium, changes in the protein concentration in the CSF could affect neuroepithelial cell proliferation, survival and migration. It is concluded that NGF is not only a constant component of CSF during chick embryogenesis but it might also be involved in cerebral cortical development.

Transcriptional responses to environmental metal exposure in wild yellow perch (Perca flavescens) collected in lakes with differing environmental metal concentrations (Cd, Cu, Ni)

To investigate the mechanisms involved in metal stress in wild fish, yellow perch (Perca flavescens) were collected in eight lakes of the Rouyn-Noranda and Sudbury regions (Canada). Due to mining and smelting activities, these two regions indeed present a broad contamination gradient in metal concentrations (Cd, Cu, Zn and Ni; water, sediment and prey) and offer a unique research opportunity to investigate relationships between metal bioaccumulation and resulting deleterious effects in indigenous biota chronically exposed to metal mixtures. The expression level of genes encoding for proteins involved in metal detoxification (metallothioneins, mts), protein protection (heat shock protein-70, hsp-70), growth (insulin-like growth factor-1, igf-1), aerobic energy metabolism (cytochrome c oxydase, cco-1) and protection against oxidative stress (Cu/Zn superoxide dismutase, sod-1) were assessed in fish liver and muscle in association with protein and enzymatic assays for cytochrome c oxidase (CCO). Bioaccumulation of both Cd and Cu increased in response to higher ambient metal concentrations, but the two metals clearly have different modes of action. For Cd, changes in gene expression levels were more marked in the liver than in the dorsal muscle, whereas for Cu the opposite trend was observed. Hepatic Cd accumulation was linked to decreased cco-1 and sod-1 gene expression, whereas Cu accumulation was associated with a decrease in CCO enzymatic activity and an increase in total protein concentration and in cco-1, mts and hsp-70 gene expression levels. For Ni, no significant correlations were observed at the transcriptional level, but increasing hepatic Ni concentrations were significantly and positively correlated with protein concentrations and CCO activity. By coupling gene expression to biochemical and physiological endpoints, this work provides new insights into the mechanisms involved in metal stress and the adaptive response of fish chronically exposed to metal mixtures.

Metabolic and structural remodeling of heart‐derived H9c2 cells by AMPK activation

AMP‐activated protein kinase (AMPK), a kinase involved in control of cellular metabolism, inhibits pathologic cardiac hypertrophy development but also plays a role in skeletal muscle adaptation to exercise. We tested the hypothesis that AMPK activation per se results in structural and metabolic remodeling of heart muscle cells characteristic of that seen in physiologic cardiac hypertrophy. Adenoviral‐mediated over expression of a constitutively active form of AMPK (CA‐AMPK) was used to increase AMPK activity in H9c2 cells, a cell line from embryonic rat heart ventricle. Then, indices of cellular hypertrophy and substrate use were measured 48 hrs later. AMPK activity was ~40‐50% higher in CA‐AMPK treated cells than in untreated cells. This was accompanied by a ~20‐25% increase in total protein content and phenylalanine incorporation without a measurable increase in atrial natriuretc peptide expression. CA‐AMPK stimulated fatty acid oxidation and glycolysis, but did not significantly alter glucose oxidation. Thus, AMPK activation causes structural and metabolic remodeling in heart muscle cells different from that seen in pathologic settings, indicating AMPK activation alone leads to a physiologic form of hypertrophy in heart muscle cells and may alter the reciprocal relationship between fatty acid and glucose utilization.This research project is funded by Canadian Institute of Health Research.

Generation and analysis of an androgen-responsive myoblast cell line indicates that androgens regulate myotube protein accretion

Androgens have anabolic actions in skeletal muscle and could potentially act to: (a) increase proliferation of myoblasts; (b) delay differentiation to myotubes; and (c) induce protein accretion in post-proliferative myofibers. To identify the site of androgens action, we investigated the proliferative response of the C2C12 mouse myoblast cell line to testosterone and dihydrotestosterone (DHT) treatment. Neither androgens affected cell proliferation after up to 7 days treatment, nor was there a synergistic effect of androgens on the proliferative response of C2C12 cells to IGF-I treatment. However, proliferating C2C12 cells expressed 0.1% of the level of androgen receptor (AR) mRNA found in adult mouse gastrocnemius muscle (p<0.01). Therefore, we generated mouse C2C12 myoblast cell lines stably transfected with the mouse AR cDNA driven by the SV40 promoter (C2C12-AR). C2C12-AR cell proliferation, differentiation, and protein content were analyzed in response to androgen treatment. Our data demonstrated that androgen treatment does not alter either proliferation rate or differentiation rate of C2C12-AR cells. However, treatment of differentiated C2C12-AR myotubes with 100 nM DHT for 3 days caused a 20% increase in total protein content vs vehicle treatment (p<0.05). This effect was not observed in control C2C12 cells transfected with empty vector. These data suggest that androgens act via the AR to upregulate myotube protein content. This model cell line will be useful to further investigate the molecular mechanisms via which androgens regulate protein accretion.

Toxicity of imidacloprid to the terrestrial isopod Porcellio scaber (Isopoda, Crustacea)

Imidacloprid is a neonicotinoid insecticide with neurotoxic action that, as a possible alternative for commonly used organophosphorus pesticides, has gained registration in about 120 countries for use in over 140 agricultural crops. Only few data are available on its toxicity for soil invertebrates. We therefore assessed the effects of imidacloprid on survival, weight gain, feeding rate, total protein content, glutathione S-transferase activity (GST), and digestive gland epithelial thickness in juveniles and adults of the terrestrial isopod Porcellio scaber. After two weeks of feeding on imidacloprid-dosed food, weight gain (NOEC 5μg/g dry food) and feeding rate (NOEC 10μg/g) in juveniles, and feeding rate (NOEC<10μg/g) and digestive gland epithelial thickness (NOEC<10μg/g) in adults were most affected. In juveniles induction of GST activity and increase of total protein content per wet animal weight was detected at 5μg/g dry food, whereas in adults a reduction of GST was observed at 25μg/g (NOEC 10μg/g). An estimate of actual intake rates suggests that imidacloprid affects isopods at similar exposure concentrations as insects. The toxicity of imidacloprid was similar to that of the organophosphorus pesticide diazinon, tested earlier using the same methods [Stanek, K., Drobne, D., Trebse, P., 2006. Linkage of biomarkers along levels of biological complexity in juvenile and adult diazinon fed terrestrial isopod (Porcellio scaber, Isopoda, Crustacea). Chemosphere 64, 1745–1752]. At actual environmental concentrations, diazinon poses a higher risk to P. scaber. Due to its increasing use in crop protection and higher persistence in soil, imidacloprid might however, be potentially more dangerous after long-term application. We conclude that toxicity testing with P. scaber provides relevant, repeatable, reproducible and comparable toxicity data that is useful for the risk assessment of pesticides in the terrestrial environment.

Src Tyrosine Kinases Mediate Activations of NF-κB and Integrin Signal during Lipopolysaccharide-Induced Acute Lung Injury

Src tyrosine kinases (TKs) are signaling proteins involved in cell signaling pathways toward cytoskeletal, membrane and nuclear targets. In the present study, using a selective Src TK inhibitor, PP1, we investigated the roles of Src TKs in the key pulmonary responses, NF-kappaB activation, and integrin signaling during acute lung injury in BALB/C mice intratracheally treated with LPS. LPS resulted in c-Src phosphorylation in lung tissue and the phospho-c-Src was predominantly localized in recruited neutrophils and alveolar macrophages. PP1 inhibited LPS-induced increases in total protein content in bronchoalveolar lavage fluid, neutrophil recruitment, and increases in the production or activity of TNF-alpha and matrix metalloproteinase-9. PP1 also blocked LPS-induced NF-kappaB activation, and phosphorylation and degradation of IkappaB-alpha. The inhibition of NF-kappaB activation by PP1 correlated with a depression of LPS-induced integrin signaling, which included increases in the phosphorylations of integrin beta(3), and of the focal adhesion kinase (FAK) family members, FAK and Pyk2, in lung tissue, and reductions in the fibrinogen-binding activity of alveolar macrophages. Moreover, treatment with anti-alpha(v), anti-beta(3), or Arg-Gly-Asp-Ser (RGDS), inhibited LPS-induced NF-kappaB activation. Taken together, our findings suggest that Src TKs play a critical role in LPS-induced activations of NF-kappaB and integrin (alpha(v)beta(3)) signaling during acute lung injury. Therefore, Src TK inhibition may provide a potential means of ameliorating inflammatory cascade-associated lung injury.