The presence of serotonin in cigarette smoke – a possible mechanistic link to 5-HT-induced airway inflammation

We previously reported the involvement of serotonin (5-HT) metabolism in cigarette smoke-induced oxidative stress in rat lung in vivo. Here, we report cigarette smoke as a source of serotonin (5-HT) to the airways and aim at investigating the effects of 5-HT on oxidative stress and inflammation in human bronchial epithelial cells (BEAS-2B). A 5-HT analog was identified to be present in aqueous phase cigarette smoke using the LC-MS/MS approach, which was later confirmed by a 5-HT enzyme-linked immune assay (EIA). Furthermore, exposure to 5-HT caused a time-dependent elevation of intracellular ROS level, which was blocked in the presence of apocynin (a NOX inhibitor). In support, the immunoblot analysis indicated that there was an increase in the expression of NOX2 time-dependently. 5-HT-induced elevation of IL-8 at both mRNA and protein levels was observed, which was inhibited by TEMPOL (a free radical scavenger), and inhibitors for p38 MAPK (SB203580) and ERK (U0126), in line with the time-dependent phosphorylation of p38 MAPK and ERK. In conclusion, our findings suggest that 5-HT presented in bronchial epithelium of smokers may be involved in cigarette smoke-induced oxidative stress and inflammation via activation of p38 MAPK and ERK pathway after the formation of free radicals.     

Conjugated linoleic acid inhibits proliferation and induces apoptosis of normal rat mammary epithelial cells in primary culture.

The trace fatty acid conjugated linoleic acid (CLA) inhibits rat mammary carcinogenesis when fed prior to carcinogen during pubertal mammary gland development or during the promotion phase of carcinogenesis. The following studies were done to investigate possible mechanisms of these effects. Using a physiological model for growth and differentiation of normal rat mammary epithelial cell organoids (MEO) in primary culture, we found that CLA, but not linoleic acid (LA), inhibited growth of MEO and that this growth inhibition was mediated both by a reduction in DNA synthesis and a stimulation of apoptosis. The effects of CLA did not appear to be mediated by changes in epithelial protein kinase C (PKC) since neither total activity nor expression nor localization of PKC isoenzymes alpha, beta II, delta, epsilon, eta, or zeta were altered in the epithelium of CLA-fed rats. In contrast, PKCs delta, epsilon, and eta were specifically upregulated and associated with a lipid-like, but acetone-insoluble, fibrillar material found exclusively in adipocytes from CLA-fed rats. Taken together, these observations demonstrate that CLA can act directly to inhibit growth and induce apoptosis of normal MEO and may thus prevent breast cancer by its ability to reduce mammary epithelial density and to inhibit the outgrowth of initiated MEO. Moreover, the changes in mammary adipocyte PKC expression and lipid composition suggest that the adipose stroma may play an important in vivo role in mediating the ability of CLA to inhibit mammary carcinogenesis.     

Molecular characteristics and interactions of the intermediate filament protein synemin. Interactions with alpha-actinin may anchor synemin-containing heterofilaments.

Synemin is a cytoskeletal protein originally identified as an intermediate filament (IF)-associated protein because of its colocalization and copurification with the IF proteins desmin and vimentin in muscle cells. Our sequencing studies have shown that synemin is an unusually large member (1,604 residues, 182,187 Da) of the IF protein superfamily, with the majority of the molecule consisting of a long C-terminal tail domain. Molecular interaction studies demonstrate that purified synemin interacts with desmin, the major IF protein in mature muscle cells, and with alpha-actinin, an integral myofibrillar Z-line protein. Furthermore, expressed synemin rod and tail domains interact, respectively, with desmin and alpha-actinin. Analysis of endogenous protein expression in SW13 clonal lines reveals that synemin is coexpressed and colocalized with vimentin IFs in SW13.C1 vim+ cells but is absent in SW13.C2 vim- cells. Transfection studies indicate that synemin requires the presence of another IF protein, such as vimentin, in order to assemble into IFs. Taken in toto, our results suggest synemin functions as a component of heteropolymeric IFs and plays an important cytoskeletal cross-linking role by linking these IFs to other components of the cytoskeleton. Synemin in striated muscle cells may enable these heterofilaments to help link Z-lines of adjacent myofibrils and, thereby, play an important role in cytoskeletal integrity.     

Lipid, ketone body and oxidative metabolism in the African lungfish, Protopterus dolloi following 60 days of fasting and aestivation

The potential importance of lipids and ketone bodies as fuels in the African lungfish, Protopterus dolloi, and the role of oxidative metabolism, were examined under control, fasted and aestivated conditions. In aestivating but not fasting lungfish, the activities of citrate synthase (CS) and cytochrome c oxidase (CCO) (enzymes of oxidative metabolism) showed tissue-specific changes. Significant reductions in CS activity occurred in the kidney, heart, gill and muscle, and in CCO in the liver and kidney tissues. Aestivation, but not fasting, also had a tissue-specific effect on mitochondrial state 3 respiration rates (using succinate as a substrate), with a >50% reduction in the liver, yet no change within muscle mitochondria. There is no indication that enzymes involved in lipid catabolism are up-regulated during periods of fasting or aestivation; however, both 3-hydroxyacyl CoA dehydrogenase (HOAD) and carnitine palmitoyl CoA transferase (CPT) activities were sustained in the liver despite the approximately 42% reduction in CCO activity, potentially indicating lipid metabolism is of importance during aestivation. Lungfish are able to utilize both the d- and l-stereoisomers of the ketone body beta-hydroxybutyrate (beta-HB); however, beta-HB does not appear to be an important fuel source during aestivation or fasting as no changes were observed in beta-HB tissue levels. This study demonstrates that an important aspect of metabolic depression during aestivation in lungfish is the tissue-specific down regulation of enzymes of aerobic metabolism while maintaining the activities of enzymes in pathways that supply substrates for aerobic metabolism.     

Plasma non-esterified fatty acids of elasmobranchs: comparisons of temperate and tropical species and effects of environmental salinity.

We investigated the influence of environments with different average temperatures and different salinities on plasma NEFA in elasmobranchs by comparing species from tropical vs. cold temperate marine waters, and tropical freshwater vs. tropical marine waters. The influence of the environment on plasma NEFA is significant, especially with regard to essential fatty acids (EFA) and the n-3/n-6 ratio. n-3/n-6 ratios in tropical marine elasmobranchs were lower by two-fold or more compared with temperate marine elasmobranchs, because of higher levels of arachidonic acid (AA, 20:4n-6) and docosatetraenoic acid (22:4n-6), and less docosahexaenoic acid (DHA, 22:6n-3), in the tropical species. These results are similar to those in earlier studies on lipids in teleosts. n-3/n-6 ratios and levels of EFA were similar between tropical freshwater and tropical marine elasmobranchs. This suggests that the observation in temperate waters that marine fishes have higher levels of n-3 fatty acids and n-3/n-6 ratios than freshwater fishes may not hold true in tropical waters, at least in elasmobranchs. It also suggests that plasma NEFA are little affected by freshwater vs. seawater adaptation in elasmobranchs. Likewise, we found that plasma NEFA composition and levels were not markedly affected by salinity acclimation (2 weeks) in the euryhaline stingray Himantura signifer. However, in contrast to our comparisons of freshwater-adapted vs. marine species, the level of n-3 fatty acids and the n-3/n-6 ratio were observed to significantly decrease, indicating a potential role of n-3 fatty acids in salinity acclimation in H. signifer.     

Peony Glycosides Protect Against Corticosterone-Induced Neurotoxicity in PC12 Cells

Preclinical and clinical investigations have shown the involvement of dysregulation of hypothalamic–pituitary–adrenal (HPA) axis in the pathogenesis of depression. Hypercortisolemia and the associated hippocampal atrophy were observed in patients with depression, which could be ameliorated by the treatment with antidepressants. Therefore, neuroprotection has been proposed to be one of the acting mechanisms of antidepressant. Previous studies in our laboratory have demonstrated the antidepressant-like activity of total glycosides of peony (TGP) in mice. This study aimed to examine the effect of TGP treatment on corticosterone-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Treating the cells with corticosterone at 200 μM for 48 h caused apoptotic cell death. The cytotoxicity was associated with the activation of caspase-3 activity and the decrease in the mRNA ratio of bcl-2 to bax. TPG treatment at increasing doses (1–10 mg/l) protected against the corticosterone-induced toxicity in PC12 cells in a dose-dependent manner. The cytoprotection afforded by TGP treatment was associated with the inhibition of caspase-3 activity and the up-regulation of bcl-2/bax mRNA ratio. The anti-apoptotic effect of TGP is therefore likely mediated by the suppression of the mitochondrial pathway leading to apoptosis.     

Ionoregulatory physiology of two species of African lungfishes Protopterus dolloi and Protopterus annectens

Basic ionoregulatory physiology was characterized in two species of African lungfish, slender African lungfish Protopterus dolloi and West African lungfish Protopterus annectens , largely under aquatic conditions. There were no substantive differences between the two species. Plasma [Na], [Cl] and [Ca] were only 60–80% of those typical of freshwater teleosts, and plasma Ca activity was particularly low. Unidirectional Na and Cl influx rates from water were also very low, only c . 10% of teleost values, whereas unidirectional Ca influx rates were comparable with teleost rates. Protopterus spp. were fed a 3% ration of bloodworms every 48 h. The bloodworm diet provided similar amounts of Na and Ca as uptake from water, but almost no Cl. Efflux rates of Na and Cl through the urine were greater than via the faeces, whereas the opposite was true for Ca. Net ion flux measurements and ionic balance sheet calculations indicated that (1) both water and dietary uptake routes are important for Na and Ca acquisition; (2) the waterborne route predominates for Cl uptake; (3) unidirectional ion effluxes across the body surface (gills and skin) rather than urine and faeces are the major routes of loss for Na, Cl and Ca. Tissues (muscle, liver, lung, kidney, intestine and heart) and plasma ions were also examined in P. dolloi 'terrestrialized' in air for up to 5 months, during which plasma ion concentrations (Na, Cl, Ca and Mg) did not change and there were only a few alterations in tissue ions, that is, increased [Na] in intestine, decreased [Cl] in kidney and increased [Ca] in liver and kidney.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.