Protective effects of intratracheally administered quercetin on lipopolysaccharide-induced acute lung injury

Background

Acute respiratory distress syndrome (ARDS) can result in a life-threatening form of respiratory failure, and established, effective pharmacotherapies are therefore urgently required. Quercetin is one of the most common flavonoids found in fruits and vegetables, and has potent anti-inflammatory and anti-oxidant activities. Quercetin has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO)-1. Here, we investigated whether the intratracheal administration of quercetin could suppress lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as well as the involvement of HO-1 in quercetin’s suppressive effects.

Methods

Mouse model of ALI were established by challenging intratracheally LPS. The wet lung-to-body weight ratio, matrix metalloproteinase (MMP)-9 activities, and pro-inflammatory cytokine productions, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in bronchoalveolar lavage fluid (BALF) were examined in ALI mice with or without quercetin pretreatment. We also examined the effects of quercetin on LPS stimulation in the mouse alveolar macrophage cell line, AMJ2-C11 cells.

Results

Intratracheal administration of quercetin decreased the wet lung-to-body weight ratio. Moreover, quercetin decreased MMP-9 activity and the production of pro-inflammatory cytokines in BALF cells activated by LPS in advance. We determined the expression of quercetin-induced HO-1 in mouse lung, e.g., alveolar macrophages (AMs), alveolar and bronchial epithelial cells. When AMJ2-C11 cells were cultured with quercetin, a marked suppression of LPS-induced pro-inflammatory cytokine production was observed. The cytoprotective effects were attenuated by the addition of the HO-1 inhibitor SnPP. These results indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects.

Conclusions

Our findings indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects. Intratracheal administration of quercetin will lead to new supportive strategies for cytoprotection in these serious lung conditions.     

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP''s behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell''s lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.     

Cloning and characterization of novel snake venom proteins that block smooth muscle contraction.

In this study, we isolated a 25-kDa novel snake venom protein, designated ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi). The amino-acid sequence of this protein was determined by peptide sequencing and cDNA cloning. The deduced sequence showed high similarity to helothermine from the Mexican beaded lizard (Heloderma horridum horridum), which blocks voltage-gated calcium and potassium channels, and ryanodine receptors. Ablomin blocked contraction of rat tail arterial smooth muscle elicited by high K+-induced depolarization in the 0.1-1 microm range, but did not block caffeine-stimulated contraction. Furthermore, we isolated three other proteins from snake venoms that are homologous to ablomin and cloned the corresponding cDNAs. Two of these homologous proteins, triflin and latisemin, also inhibited high K+-induced contraction of the artery. These results indicate that several snake venoms contain novel proteins with neurotoxin-like activity.     

Effects of Net and Local Charges on the Interaction between Chemically-Modified Horse Heart Cytochrome c and P700 in Photosystem 1-Enriched Subchloroplast Particles

Effects of salt and pH on the re-reduction of P700 by chemically-modifiedhorse heart cytochrome c after a flash illumination were examinedin Triton-treated P700- enriched subchloroplast particles (TSF-1particles). At low salt concentrations net charges on the membrane surfaceand native, guanidinated or succinylated cytochrome c were majorfactors that determined the reaction rates, as in the reactionbetween plastocyanin and P700 [Tamura et al. (1981) Plant &Cell Physiol. 22: 603]. The reaction rates also depended onreactant-specific factors, particularly the localized distributionof charges on macromolecules and their interaction over shortdistances, as well as on long-range Coulombic interaction. Theeffect of this type became clearer at high salt concentrations. (Received October 7, 1982; Accepted December 20, 1982)     

IGF-1 induction by acylated steryl β-glucosides found in a pre-germinated brown rice diet reduces oxidative stress in streptozotocin-induced diabetes

Background

The pathology of diabetic neuropathy involves oxidative stress on pancreatic β-cells, and is related to decreased levels of Insulin-like growth factor 1 (IGF-1). Acylated steryl β-glucoside (PR-ASG) found in pre-germiated brown rice is a bioactive substance exhibiting properties that enhance activity of homocysteine-thiolactonase (HTase), reducing oxidative stress in diabetic neuropathy. The biological importance of PR-ASG in pancreatic β-cells remains unknown.Here we examined the effects of PR-ASG on IGF-1 and glucose metabolism in β-cells exposed to oxidative stress.

Methodology/Principal Findings

In the present study, a pre-germinated brown rice (PR)-diet was tested in streptozotocin (STZ)-induced diabetic rats. Compared with diabetic rats fed control diets, the PR-diet fed rats showed an improvement of serum metabolic and neurophysiological parameters. In addition, IGF-1 levels were found to be increased in the serum, liver, and pancreas of diabetic rats fed the PR-diet. The increased IGF-1 level in the pancreas led us to hypothesize that PR-ASG is protective for islet β-cells against the extensive injury of advanced or severe diabetes. Thus we examined PR-ASG to determine whether it showed anti-apoptotic, pro-proliferative effects on the insulin-secreting β-cells line, INS-1; and additionally, whether PR-ASG stimulated IGF-1 autocrine secretion/IGF-1-dependent glucose metabolism. We have demonstrated for the first time that PR-ASG increases IGF-1 production and secretion from pancreatic β-cells.

Conclusion/Significance

These findings suggest that PR-ASG may affect pancreatic β-cells through the activation of an IGF-1-dependent mechanism in the diabetic condition. Thus, intake of pre-germinated brown rice may have a beneficial effect in the treatment of diabetes, in particular diabetic neuropathy.     

Genetic isolation between the Western and Eastern Pacific populations of pronghorn spiny lobster Panulirus penicillatus

The pronghorn spiny lobster, Panulirus penicillatus, is a circumtropical species which has the widest global distribution among all the species of spiny lobster, ranging throughout the entire Indo-Pacific region. Partial nucleotide sequences of mitochondrial DNA COI (1,142-1,207 bp) and 16S rDNA (535-546 bp) regions were determined for adult and phyllosoma larval samples collected from the Eastern Pacific (EP)(Galápagos Islands and its adjacent water), Central Pacific (CP)(Hawaii and Tuamotu) and the Western Pacific (WP)(Japan, Indonesia, Fiji, New Caledonia and Australia). Phylogenetic analyses revealed two distinct large clades corresponding to the geographic origin of samples (EP and CP+WP). No haplotype was shared between the two regional samples, and average nucleotide sequence divergence (Kimura's two parameter distance) between EP and CP+WP samples was 3.8±0.5% for COI and 1.0±0.4% for 16S rDNA, both of which were much larger than those within samples. The present results indicate that the Pacific population of the pronghorn spiny lobster is subdivided into two distinct populations (Eastern Pacific and Central to Western Pacific), with no gene flow between them. Although the pronghorn spiny lobster have long-lived teleplanic larvae, the vast expanse of Pacific Ocean with no islands and no shallow substrate which is known as the East Pacific Barrier appears to have isolated these two populations for a long time (c.a. 1MY).     

Hybridization-dependent fluorescence of oligodeoxynucleotides incorporating new pyrene-modified adenosine residues

We report the synthesis and properties of oligonucleotides incorporating N(6)-[N-(pyren-1-ylmethyl)carbamoyl]-deoxyadenosine (dA(pymcm)). We designed the ODN which incorporated two consecutive dA(pymcm) residues. It was revealed that on hybridization with the target DNA and RNA oligomers, the fluorescence spectra of ODNs having two consecutive dA(pymcm) molecules near the 5'-terminal position can change from the pyrene monomer emission to the excimer, depending on the chain length of the target DNA and RNA. These results indicated that dA(pymcm)-modified ODNs can be used as interesting hybridization sensors that are sensitive to the size of the target strand.     

Effect of charge density of cationic polyelectrolytes on complex formation with DNA

The interaction between DNA and ionen polymers, -[N⁺(CH₃)₂(CH₂)_mN⁺(CH₃)₂(CH₂)_n], with m-n of 3–3, 6–6, and 6–10 were examined in order to know how the binding behavior of cationic polymers with DNA depends on the charge density of polycation. The ionen polymer has no bulky side chain and the binding forces with DNA would be attributed mainly to electrostatic interaction. When 3–3 ionen polymers were added to DNA solution, precipitable complexes with the ratio of cationic residue to DNA phosphate (+/?) of 1/1 and the free DNA molecules were segregated, while 6–6 and 6–10 ionen polymers formed soluble complexes with DNA molecules up to (+/?) = 0.5. This suggests that 3–3 ionen polymers bind cooperatively with DNA while 6–6 and 6–10 ionen polymers bind noncooperatively. The cooperative binding of 3–3 ionen polymer and the noncooperative binding of 6–6 ionen polymer were also supported by the thermal melting and recooling profiles from the midpoint between first and second meltings. It was concluded that the charge density of DNA phosphate is a critical value determining whether the ionen polymers bind to DNA by a cooperative or by a noncooperative binding, since the distance between successive cationic charges of 3–3 ionen polymer is shorter than that between successive phosphate charges on DNA double helix and those of 6–6 and 6–10 ionen polymers are longer.     

Cytotoxic enhancement of a bispecific diabody by format conversion to tandem single-chain variable fragment (taFv): the case of the hEx3 diabody

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.