4-Hydroxynonenal regulates mitochondrial function in human small airway epithelial cells

Prolonged exposure to oxidative stress causes Acute Lung Injury (ALI) and significantly impairs pulmonary function. Previously we have demonstrated that mitochondrial dysfunction is a key pathological factor in hyperoxic ALI. While it is known that hyperoxia induces the production of stable, but toxic 4-hydroxynonenal (4-HNE) molecule, it is unknown how the reactive aldehyde disrupts mitochondrial function. Our previous in vivo study indicated that exposure to hyperoxia significantly increases 4-HNE-Protein adducts, as well as levels of MDA in total lung homogenates. Based on the in vivo studies, we explored the effects of 4-HNE in human small airway epithelial cells (SAECs). Human SAECs treated with 25 μM of 4-HNE showed a significant decrease in cellular viability and increased caspase-3 activity. Moreover, 4-HNE treated SAECs showed impaired mitochondrial function and energy production indicated by reduced ATP levels, mitochondrial membrane potential, and aconitase activity. This was followed by a significant decrease in mitochondrial oxygen consumption and depletion of the reserve capacity. The direct effect of 4-HNE on the mitochondrial respiratory chain was confirmed using Rotenone. Furthermore, SAECs treated with 25 μM 4-HNE showed a time-dependent depletion of total Thioredoxin (Trx) proteins and Trx activity. Taken together, our results indicate that 4-HNE induces cellular and mitochondrial dysfunction in human SAECs, leading to an impaired endogenous antioxidant response.     

Resiniferatoxin-induced loss of plasma membrane in vanilloid receptor expressing cells

Resiniferatoxin (RTX), a potent analog of capsaicin, was evaluated electrophysiologically in dorsal root ganglion (DRG) cells and cell lines ectopically expressing the vanilloid receptor type 1 (VR1) to determine if cell phenotype influenced RTXs neurotoxic properties. Furthermore, capsaicin and heat activation of VR1 were evaluated in these cells to determine if cellular damage was unique to RTX activation of the receptors. RTX application to DRG cells identified as type 1, 2 or 5, cell types known to express VR1, induced large inward currents. RTX did not induce currents in DRG cells that do not express the receptor (type 4 cells). In cell lines ectopically expressing VR1, RTX-induced similar currents. RTX produced no effect in non-transfected cells. After exposure to RTX both DRG cells and transfected cells failed to respond to subsequent applications of the agonist. In addition, whole cell capacitance was reduced up to 70%. The decrease in capacitance was associated with the loss of plasma membrane, as determined by confocal microscopy. Cell phenotype, other than VR1 expression, did not influence the response to RTX. Interestingly, capsaicin and heat activation of vanilloid receptors also decreased cell capacitance, but the loss of membrane was not as great as with RTX and responses to these stimuli were not lost after the initial exposure. The loss of cell membrane required elevated intracellular levels of Ca2+. From these data it was concluded that the loss of cell membrane was dependent on the presence of both VR1 and intracellular Ca2+ accumulation, but not on cell phenotype.     

Exosome mediated differentiation of megakaryocytes: a study on TLR mediated effects

Journal of Thrombosis and Thrombolysis - Megakaryocytes are large polyploid bone marrow cells whose function is to produce circulatory platelets. Megakaryocytes are also known to release...     

Molecular investigations on the dilemma of nitrogen-fixing characteristics of Azotomonas

The ability of Azotomonas to reduce atmospheric nitrogen has been an issue of controversy since this genus was included in the family Azotobacteraceae. Here we report the investigations on the nitrogen fixation characteristics of Azotomonas fluorescens strain ATCC13544 and show that it can grow in both liquid medium and on solid agar plates made with modified BURK nitrogen-free medium supplemented with ammonium acetate as a nitrogen source. In contrast, when ammonium acetate was omitted from this medium, the cells did not show any noticeable growth. Southern blotting analysis of the chromosome of Azotomonas strain ATCC13544 did not show any hybridization with a probe made of DNA sequences corresponding to the nifH gene of Azotobacter vinelandii. Based on these results, we conclude that the Azotomonas fluorescens ATCC13544 does not contain the conventional genetic information on its chromosome that is needed to utilize atmospheric nitrogen as the sole source nitrogen to support its growth.     

Investigations on the cell volumes of Azotobacter vinelandii by scanning electron microscopy

Previous experiments by other investigators on the DNA content of Azotobacter vinelandii have demonstrated that the DNA content in these cells is several folds higher than that of E. coli. On the basis of this observation, it was hypothesized that A. vinelandii has at least 40 to 80 identical chromosomes per cell. However, the gene dosage analysis in A. vinelandii cells suggested that many genetic operations can be performed in these cells without the constraints expected in a polyploid bacterium. In an attempt to explain this apparent discrepancy, we have done systematic analysis of the relationship between the DNA content and the cell volume of this bacterium. Since a linear correlation is observed between the DNA content and the cell size in many other cell types, we hypothesized that if A. vinelandii is polyploid in nature, it should have a much larger cell volume to accommodate such a large amount of DNA. Our scanning electron microscopic analysis revealed that the cell volume of the vegetative cells of A. vinelandii is about 16 times larger than the cell volume of E. coli. This result is apparently consistent with the concept that the A. vinelandii is a polyploid bacterium. It was also reported that the encysted cells of A. vinelandii contain about 25% of the DNA content of the vegetative cells. This would mean that an encysted cell of A. vinelandii could contain about 10 copies of its chromosome. Since the estimated molecular weight of A. vinelandii chromosome is very similar to that of E. coli chromosome, the DNA content of the encysted cells also should be about 10 times higher than that of E. coli cells. If we assume that the relationship between the DNA content and the cell size is linear, then the encysted cells should have a cell volume larger than that of E. coli and smaller than that of the vegetative cells of A. vinelandii. However our scanning electron microscopic analysis showed that the cell volume of the encysted cells of A. vinelandii is in fact very similar to the cell volume of E. coli.     

Noradrenaline is necessary for the hedonic properties of addictive drugs

To determine whether noradrenaline (NA) is an essential neurotransmitter for addictive and appetitive behaviors, we measured drug and food seeking in transgenic mice lacking dopamine beta-hydroxylase (Dbh), the enzyme responsible for synthesizing NA. Using the conditioned place preference test (CPP), we show that Dbh -/- mice do not exhibit rewarding behavior to morphine, cocaine, or the mixed reuptake inhibitor bupropion. In spite of their lack of preference for drugs, Dbh -/- mice had an unaltered preference for food. Drug seeking was induced when NA was restored to the central nervous system of Dbh -/- mice by administration of l-threo-3,4-dihydroxyphenylserine (DOPS) and carbidopa. When a NK1 receptor antagonist was co-administered with morphine or cocaine, it produced aversive behavior in Dbh -/- mice while it abolished place preference in the controls. NK1 antagonists alone did not have any rewarding or aversive effect in the CPP suggesting that substance P opposes some of the unpleasant effects of morphine and cocaine. Our results show that NAergic transmission is necessary for motivated behaviors, the dysregulation of which is a co-morbid factor of many depressive states. The reversibility of this phenomenon, by restoring NA, indicates that even when this behavioral deficit is genetically determined it can be reversed.     

Amygdalar neuronal responses to peripheral nociceptive stimuli in rats

Amygdala plays a very important role in the mediation of pain. In the present study the behaviour of the amygdalar neurons in response to various peripheral noxious stimuli was observed. Noxious mechanical, thermal, electrical, chemical and the non-noxious stimuli (touch) were applied individually to the animal and then the neuronal responses to these stimuli were recorded. Our results showed that the majority of amygdalar units recorded from medial, lateral and basolateral nuclei, responded to different peripheral noxious (thermal, electrical, chemical mechanical) and non-noxious stimuli by excitation. However few neurons decreased their activity on stimulation. Some of these neurons also exhibited after discharge following application of higher intensity of noxious stimuli.