Docosahexaenoic acid (DHA) ameliorates paraquat-induced pulmonary fibrosis in rats possibly through up-regulation of Smad 7 and SnoN.

Paraquat (PQ) poisoning has caused a large number of human fatalities due to the progressive and irreversible pulmonary fibrosis. Docosahexaenoic acid (DHA) is well-recognized as important modulators of multiple biological pathways that affect health and disease. A line of studies have shown that DHA supplementation is associated with the alleviation of some tissue fibrosis. In the current study, pulmonary fibrosis of rats was produced by a single oral dose of 50 mg/kg bw PQ treatment. Daily 500 mg/kg bw DHA supplementation was provided 7 days before PQ treatment and lasted for consecutive 35 days. DHA was found to ameliorate the pulmonary fibrotic alterations induced by PQ, which was evidenced by significant reduction of histological changes, hydroxyproline content and level of the transforming growth factor-ß1 (TGF-ß1) mRNA. Furthermore, the protein levels of Smad 7 and SnoN in the DHA supplemented rats were significantly increased compared with those in the rats of the PQ group. These results suggested that DHA ameliorated pulmonary fibrosis induced by PQ might be attributed to its enhancement of Smad 7 and SnoN expression.

Elevation of 4-hydroxynonenal and malondialdehyde modified protein levels in cerebral cortex with cognitive dysfunction in rats exposed to 1-bromopropane.

1-Bromopropane (1-BP), an alternative to ozone-depleting solvents (ODS), exhibits central nervous system (CNS) toxicity in animals and humans. This study was designed to relate CNS damage by Morris water maze (MWM) test and oxidative stress to 1-BP exposure in the rat. Male Wistar rats were randomly divided into 4 groups (n=10), and treated with 0, 200, 400 and 800 mg/kgbw 1-BP for consecutive 12 days, respectively. From day 8 to day 12 of the experiment, MWM test was employed to assess the cognitive function of rats. The cerebral cortex of rats was obtained immediately following the 24h after MWM test conclusion. Glutathione (GSH), oxidized glutathione (GSSG) and total thiol (total-SH) content, GSH reductase (GR) and GSH peroxidase (GSH-Px) activities, malondialdehyde (MDA) level, as well as 4-hydroxynonenal (4-HNE) and MDA modified proteins in homogenates of cerebral cortex were measured. The obtained results showed that 1-BP led to cognitive dysfunction of rats, which was evidenced by delayed escape latency time and swimming distances in MWM performance. GSH and total-SH content, GSH/GSSG ratio, GR activity significantly decreased in cerebral cortex of rats, coupling with the increase of MDA level. 4-HNE and MDA modified protein levels obviously elevated after 1-BP exposure. GSH-Px activities in cerebral cortex of rats also increased. These data suggested that 1-BP resulted in enhanced lipid peroxidation of brain, which might play an important role in CNS damage induced by 1-BP.

Docosahexaenoic acid (DHA) attenuated paraquat induced lung damage in mice.

CONTEXT: Accumulating evidences have proposed the critical roles of oxidative stress in the etiology of lung injury caused by paraquat (PQ). Docosahexaenoic acid (DHA) is an essential n-3 polyunsaturated fatty acid (PUFA), which has been proved to possess prominent antioxidative and anti-inflammatory effects. OBJECTIVE: The aim of this study was to evaluate effects of DHA against acute lung injury (ALI) induced by PQ in mice. MATERIALS AND METHODS: Male Kunming mice were randomly divided into three groups: control group, PQ group, and PQ+DHA group (n = 24). The mice of PQ+DHA group received 500 mg/kg bodyweight DHA by gavage daily for consecutive 14 days. On day 8, the mice in PQ and PQ+DHA groups received a single oral dose of 50 mg/kg bodyweight PQ. All the mice were sacrificed on day 15. The myeloperoxidase (MPO) activities, levels of the malondialdehyde (MDA) and glutathione (GSH), and the 4-hydroxynonenal (4-HNE) and MDA modified proteins of lung were investigated. RESULTS: DHA treatment significantly increased the survival rate of mice treated with PQ. Pulmonary MPO activities and MDA contents were elevated in the mice of the PQ group, while the GSH level was reduced. Furthermore, levels of 4-HNE and MDA modified protein in lungs of the PQ group mice were significantly increased. All the above changes were significantly inhibited by DHA pretreatment. Morphological examination revealed that DHA effectively attenuated the hyperemia, edema of ALI induced by PQ. CONCLUSION: These results demonstrated that DHA could effectively attenuate PQ-induced ALI in mice probably via its antioxidant activity.

[Development of peripheral neuropathy rat model induced by 1-bromopropane].

OBJECTIVE: To observe the peripheral neurotoxicity of 1-bromopropane (1-BP) by developing an animal model of peripheral neuropathy through oral administration of 1-BP. METHODS: Forty male Wistar rats were randomly and equally divided into low-dose group (200 mg/kg), medium-dose group (400 mg/kg), high-dose group (800 mg/kg), and control group. The rats in the low-dose, medium-dose, and high-dose groups were orally given 1-BP (dissolved in corn oil), while the rats in the control group were orally given an equal volume of corn oil. The oral administration (0.2 ml/100 g BW) was performed once per day, 5 days per week, for 16 consecutive weeks. Neurobehavioral indices including gait score, hindlimb grip strength, and hindlimb landing foot splay were recorded periodically. Hematological and biochemical parameters were also measured during and after 1-BP exposure. RESULTS: The gait scores were significantly higher in the high-dose group (after 8 ∼ 16 weeks of 1-BP exposure), medium-dose group (after 14 ∼ 16 weeks of 1-BP exposure), and low-dose group (after 15 ∼ 16 weeks of 1-BP exposure) than in the control group (P < 0.05, P < 0.01). Compared with the control group, the high-dose group showed significantly decreased hindlimb grip strength after 9, 12, and 14 weeks of 1-BP exposure (P < 0.05, P < 0.01), with the hindlimbs paralyzed after 16 weeks of 1-BP exposure. After 16 weeks of 1-BP exposure, the hindlimb grip strengths of rats in the medium-dose and low-dose groups were decreased to 72.6% and 91.2% of the control value (P < 0.01, P < 0.05). Compared with the control group, the high-dose group showed significantly increased hindlimb landing foot splay after 12, 14, and 16 weeks of 1-BP exposure, and the medium-dose group showed significantly increased hindlimb landing foot splay after 14 and 16 weeks of 1-BP exposure (P < 0.05, P < 0.01). The high-dose and medium-dose groups showed significantly higher serum alanine aminotransferase (ALT) activity than the control group after 8 weeks of 1-BP exposure, and so did the low-dose group after 16 weeks of 1-BP exposure (P < 0.01). CONCLUSION: The nervous system is sensitive to the toxic effect of 1-BP, and 1-BP exposure can induce peripheral neuropathy in rats.

Modeling nuclear blebs in a nucleoskeleton of independent filament networks.

Correlations between altered nuclear shape and disease are empirically observed, but the causes of nuclear dysmorphisms are poorly understood. The nucleoskeleton, which provides the majority of the mechanical stability of the nucleus, is composed primarily of intermediate filaments of lamin proteins. The nucleoskeleton forms a mostly-planar network between the inner nuclear membrane and chromatin. It is unclear if blebs and larger scale changes in nuclear morphology are consequences of reorganization of the nucleoskeleton alone or of other cellular processes. To test this, we computationally recapitulate the lamina network using a mechanical network model created as a network of Hookean springs. A- and B-type lamin filaments were distributed over a spherical surface into distinct networks linked to one another by lamin-associated proteins. Iterative force-based adjustment of the network structure, together with a stochastically modified Bell model of bond breakage and formation, simulates nucleoskeleton reorganization with blebs. The rate of bleb retraction into the nucleus depends on both initial size of the bleb and number of networks being deformed. Our results show that induced blebs are more stable when only one filament component is deformed or when the networks have no interconnections. Also, the kinetics of retraction is influenced by the composition of the bleb. These results match with our experiments and others.

[Protective effects of garlic oil on n-hexane-induced neurotoxicity in rats via inhibition of hepatic alcohol dehydrogenase activity].

OBJECTIVE: To study the protective effects of garlic oil (GO) on the peripheral nerve injuries induced by n-hexane. METHODS: Male Wistar rats were randomly divided into four groups (10 rats in each group): the control, the n-hexane treatment (2000 mg/kg), the low dose GO, and the high dose GO groups. The rats in the low and high doses of GO groups were pretreated with GO (40 and 80 mg/kg) before exposure to n-hexane (2000 mg/ kg), while the animals of the n-hexane treatment group were given normal saline and then 2000 mg/ kg n-hexane. The rats were exposed to GO and n-hexane 6 times a week for 10 weeks. The gait scores and staying time on the rotating rod for all rats were detected every two weeks. The rats were sacrificed at the end of ten weeks, then the levels of alcohol dehydrogenase (ADH), maleic dialdehyde (MDA), reduced glutathione (GSH), glutathione peroxidase(GSH-Px), total antioxidation capacity(T-AOC) and the ability of inhibition of *OH in livers were examined. RESULTS: The gait scores increased significantly and the time staying on the rotating rod obviously decreased in rats of n-hexane treatment group, as compared with control group (P < 0.05 or P < 0.01). In the hepatic tissues of n-hexane group, the levels of MDA and ADH significantly increased, the activities of GSH-Px, T-AOC and the ability of inhibition of *OH obviously decreased, as compared to control group (P < 0.05 or P < 0.01). In 2 GO groups, the gait scores and the staying time on the rotating rod were significantly improved, the levels of MDA and ADH significantly decreased, the activities of GSH-Px, T-AOC and the ability of inhibition of *OH obviously increased, as compared with n-hexane group (P < 0.05 or P < 0.01 ). CONCLUSION: ADH could play an important role in the protective effects induced by garlic oil on the peripheral nerve injuries produced by n-hexane.

[Effects of 1-bromopropane exposure on cognitive function in rats].

OBJECTIVE: To study the effects of 1-bromopropane (1-BP) on the functions of learning-memory and the central cholinergic system in rats. METHODS: Forty male Wistar rats were randomly divided into four groups: low 1-BP group (200 mg/kg), middle 1-BP group (400 mg/kg), high 1-BP group (800 mg/kg) and control group, and the exposure time was 7 days. The Morris water maze (MWM) test was applied to evaluate the learning-memory function in rats. After the MWM test, the rats were sacrificed, the cerebral cortex and hippocampus were quickly dissected and homogenized in ice bath. The activity of acetylcholine esterase (AChE) and choline acetyltransferase (ChAT) in supernatant of homogenate were detected. RESULTS: The latency and swim path-length of rats in middle and high 1-BP groups prolonged significantly in place navigation test and the efficiency of searching strategy obviously decreased, as compared with control group (P < 0.05 or P < 0.01). In spatial probe test, the number of crossing platform in three 1-BP groups decreased significantly, as compared with control group (P < 0.05 or P < 0.01). The cortical AChE activity of rats in middle and high 1-BP groups was significantly higher than that of control and low 1-BP group (P < 0.05 or P < 0.01). The AChE activity in rat hippocampus of high 1-BP group obviously increased, as compared with control group as compared with control group (P < 0.05). There was no significant difference of cortical ChAT activity between three 1-BP groups and control group (P > 0.05). In the hippocampus, there was no difference of ChAT activity among the groups (P > 0.05). CONCLUSION: 1-BP exposure could significantly influence the learning-memory function in rats due to the increase of AChE activity.

Beyond lamins other structural components of the nucleoskeleton.

The nucleus is bordered by a double bilayer nuclear envelope, communicates with the cytoplasm via embedded nuclear pore complexes, and is structurally supported by an underlying nucleoskeleton. The nucleoskeleton includes nuclear intermediate filaments formed by lamin proteins, which provide major structural and mechanical support to the nucleus. However, other structural proteins also contribute to the function of the nucleoskeleton and help connect it to the cytoskeleton. This chapter reviews nucleoskeletal components beyond lamins and summarizes specific methods and strategies useful for analyzing nuclear structural proteins including actin, spectrin, titin, linker of nucleoskeleton and cytoskeleton (LINC) complex proteins, and nuclear spindle matrix proteins. These components can localize to highly specific functional subdomains at the nuclear envelope or nuclear interior and can interact either stably or dynamically with a variety of partners. These components confer upon the nucleoskeleton a functional diversity and mechanical resilience that appears to rival the cytoskeleton. To facilitate the exploration of this understudied area of biology, we summarize methods useful for localizing, solubilizing, and immunoprecipitating nuclear structural proteins, and a state-of-the-art method to measure a newly-recognized mechanical property of nucleus.

Stabilization of the spectrin-like domains of nesprin-1α by the evolutionarily conserved "adaptive" domain.

Nesprins are located at the outer and inner membranes of the nuclear envelope and help link the cytoskeleton to the nucleoskeleton. Nesprin-1α, located at the inner nuclear membrane, binds to A-type lamins and emerin and has homology to spectrin-repeat proteins. However, the mechanical and thermodynamic properties of the spectrin-like repeats (SLRs) of nesprin-1α and the potential structural contributions of the unique central domain were untested. In other spectrin superfamily proteins, tandem spectrin-repeat domains undergo cooperatively coupled folding and unfolding. We hypothesized that the large central domain, which interrupts SLRs and is conserved in other nesprin isoforms, might confer unique structural properties. To test this model we measured the thermal unfolding of nesprin-1α fragments using circular dichroism and dynamic light scattering. The SLRs in nesprin-1α were found to have structural and thermodynamic properties typical of spectrins. The central domain had relatively little secondary structure as an isolated fragment, but significantly stabilized larger SLR-containing molecules by increasing their overall helicity, thermal stability and cooperativity of folding. We suggest this domain, now termed the 'adaptive' domain (AD), also strengthens dimerization and inhibits unfolding. Further engineering of the isolated AD, and AD-containing nesprin molecules, may yield new information about the higher-order association of cooperative protein motifs.

Enhanced expression and primary purification of soluble HBD3 fusion protein in Escherichia coli.

Human beta-defensin-3(HBD3) is a low molecular weight cationic peptide with a broad antimicrobial spectrum. A recombinant Escherichia coli (pET32-smHBD3) was constructed to produce HBD3 fusion protein (TrxA-HBD3) before, but the productivity is relatively low. In the present work, the effects of different expression conditions were systematically investigated to improve the expression level of the fusion protein. With regard to the volumetric productivity, the optimal conditions were determined as follows: cultivation at 34 degrees C in MBL medium, induction at middle stage of the exponential growth phase with 0.4 mM isopropylthio-D-galactoside, and postinduction expression for 8 h. Under these conditions, the volumetric productivity of the fusion protein reached 2.55 g/L, i.e., 0.55 g mature HBD3/L, which was about 2.6 times of that obtained under the unoptimized conditions. And the target protein still maintained high solubility (> or =97.9%) and accounted for 66% of the total soluble protein. A cationic exchange purification step was employed to obtain high-purity target protein (90%) with a recovery ratio of 78%. This soluble expression level of HBD3 fusion protein was the highest among all the reported literature and facilitated the development of high efficient purification of HBD3.

[Efficient expression and purification of human beta-defensin-2 in E.coli].

OBJECTIVE: To demonstrate the possibility of high-level expression of bioactive human beta-defensin-2 (hBD2) in E.coli, and to purify the recombinant hBD2. METHODS: DNA fragment containing mature hBD2 coding region (smhBD2-cDNA) was amplified by PCR, multiple copies of smhBD2-cDNA were linked using Bgl II and BamH I enzymes, pET32-nsmHBD2-cDNA with 1, 2, 4, or 8 copies of smhBD2-cDNA was constructed. The soluble and insoluble hBD2 proteins were separated and analyzed by SDS-PAGE analysis. The soluble protein underwent a separation process containing affinity chromatography, enterokinase digestion and ion exchange chromatography to get the recombinant hBD2 peptide. The bioactivity of recombinant hBD2 was examined by bacteria-inhibition tests in liquid culture. RESULT: The plasmids pET32-nsmHBD2-cDNA with 1, 2, 4 copies of smhBD2-cDNA were constructed and the expressed soluble protein accounted for 52 %, 48 %, and 31 % respectively. The plasmids with 8 copies expressed mainly insoluble protein with few in soluble form. The growth of E.coli K12D31 was dramatically suppressed with a inhibition rate of 90 %, when the final concentration of recombinant hBD2 reached between 0.4 to 0.5 mug/ml. CONCLUSION: Fusion expression of human beta-defensin-2 with multiple joined genes in E.coli could increase the expression of hBD2.

High-level expression of a soluble functional antimicrobial peptide, human beta-defensin 2, in Escherichia coli.

In this work, taking human beta-defensin-2 (HBD2) as a demonstrative molecule, the strategies for high efficient production of functional human beta-defensins in E. coli were studied. Fusion mature HBD2 (TrxA-mHBD2) showed high solubility and productivity without the need for lowering the cultivation temperature. The solubility of target fusion protein could attain 81.3% even at 37 degrees C with a volumetric productivity as high as 235 mg/L in a rich medium MBL at the same temperature and reached 346 mg/L at 28 degrees C. The His-Tag in the fusion protein enabled the application of affinity chromatography separation to obtain high purity of the overexpressed recombinant fusion protein. After digestion by enterokinase, purification via cationic exchange chromatography, and desalting by ultrafiltration, mature HBD2 product was obtained with a purity of 95% in an overall recovery of 29.2%. The antimicrobial activity of the recombinant mature HBD2 and the influence factors were tested using E. coli K12D31 as a sensitive strain.

Production of bioactive human beta-defensin-3 in Escherichia coli by soluble fusion expression.

A codon optimized mature human beta-defensin-3 gene (smHBD3) was synthesized and fused with TrxA to construct pET32-smHBD3 vector, which was transformed into E. coli BL21(DE3) and cultured in MBL medium. The volumetric productivity of fusion protein reached 0.99 g fusion protein l(-1), i.e. 0.21 g mature HBD3 l(-1). Ninety-six percentage of the fusion protein was in a soluble form and constituted about 45% of the total soluble protein. After cell disruption, the soluble fusion protein was separated by affinity chromatography and cleaved by enterokinase, and then the mature HBD3 was purified by cationic ion exchange chromatography. The overall recovery ratio of HBD3 was 43%. The purified mature HBD3 demonstrated antimicrobial activity against E. coli.

High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression.

Human beta-defensin-4 (hBD4) is a cationic 50-amino acid antimicrobial peptide with three conserved cysteine disulfide bonds. It exhibits a broad antimicrobial spectrum. This study describes the synthesis of hBD4 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released hBD4. A PCR-based gene SOEing (splicing by overlap extension) synthesis method was used in the synthesis of the hBD4 gene with optimized codons. By constructing the expression plasmid (pET32-smhBD4), high concentration of soluble hBD4 fusion protein (1.9 g/l) can be obtained in E. coli. Further optimization studies showed that the expression system was very efficient to produce soluble target protein, and the solubility of the target protein could attain more than 99% even when the culture temperature was as high as 37 degrees C. The highest productivity (2.68 g/l) of the hBD4 fusion protein was achieved by cultivating the E. coli (pET32-smhBD4) in MBL medium at 34 degrees C, inducing the culture at the mid-exponential phase with 0.4-mM isopropyl beta-D-galactopyranoside (IPTG), and collecting the broth after 6-h expression. The soluble target protein accounted for 64.6% of the total soluble proteins, and the mature hBD4 expression level was stoichiometrically estimated to be 0.689 g/l. This fusion protein was then purified and cleaved to get the mature hBD4 peptide that showed antimicrobial activity against E. coli and Pseudomonas aeruginosa.

Tandem repeat mhBD2 gene enhance the soluble fusion expression of hBD2 in Escherichia coli.

Human beta-defensin-2 (hBD2) is a cysteine-rich cationic antimicrobial peptide with low molecular weight that exhibits a broad range of antimicrobial activity. To improve the expression level of hBD2 in Escherichia coli, tandem repeats of mature hBD2 gene were constructed and expressed as fusion proteins (TrxA-nmhBD2, n = 1, 2, 4, 8) by constructing the vectors of pET32-nsmhBD2 (n = 1, 2, 4, 8). The results showed that the tandem repeats of mhBD2 gene were highly expressed in our constructed system. Comparing the expression levels of soluble mhBD2, BL21(DE3)/pET32-2smhBD2 was selected as an ideal recombinant strain for mature hBD2 production. Under the optimized conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the maximum expression level of soluble mature hBD2 (0.76 g/l) with the highest percentage of fusion protein in soluble proteins (62.2%) was obtained in the present work, which was the highest yield of hBD2 reported so far.