Suppression of tumor suppressor Tsc2 and DNA repair glycosylase Nth1 during spontaneous liver tumorigenesis in Long-Evans Cinnamon rats

Chronic inflammation and oxidative stress are arguably associated with an increased risk of cancer. Certain diseases that are characterized by oxyradical overload, such as Wilson’s disease (WD), have also been associated with a higher risk of liver cancer. The Long-Evans Cinnamon (LEC) rat, an animal model for WD, is genetically predisposed to the spontaneous development of liver cancer and has been shown to be very useful for studying the mechanisms of inflammation-mediated spontaneous carcinogenesis. Endonuclease III (Nth1) plays a significant role in the removal of oxidative DNA damage. Nth1 and a tumor suppressor gene Tuberous sclerosis 2 (Tsc2) are bi-directionally regulated in humans, mice, and rats by a common minimal promoter containing two Ets-binding sites (EBSs). In this study, we examined the expression of Nth1 and Tsc2 genes during disease progression in the LEC rat liver. During the period of acute hepatitis (16–17 weeks), we observed decreased Nth1 and Tsc2 mRNA levels and a continued decrease of the Tsc2 gene in 24 weeks in LEC rats, while the effect was minimal in Long-Evans Agouti (LEA) rats. This reduction in the mRNA levels was due to the reduced binding of EBSs in the Nth1/Tsc2 promoter. Increase in protein oxidation (carbonyl content) during the same time period (16–24 weeks) may have an effect on the promoter binding of regulatory proteins and consequent decrease in Nth1 and Tsc2 gene expressions during tumorigenesis.     

Multicolour flow cytometry analyses and autofluorescence in chlorophytes: lessons from programmed cell death studies in Chlamydomonas reinhardtii

Flow cytometry is a valuable tool in phycological studies. However, endogenous cellular compounds like nicotinamide adenine dinucleotide and chlorophyll a and b autofluoresce, potentially interfering with fluorescent markers. Furthermore, autofluorescent properties are not uniform across algae, nor are their effects consistent in different cytometers. The choice of instrument and fluorescent marker, therefore, requires careful consideration. We investigated the suitability of fluorescent markers by using standard four-colour and advanced multicolour flow cytometers in relation to the effects of autofluorescence over ranges of parameters including fluorophore excitation and emission spectra, band-pass filter configurations, voltage gains and the effects of growth in the light and dark. The unicellular chlorophyte and model organism, Chlamydomonas reinhardtii, was used and findings were correlated with investigations of programmed cell death. As previously found C. reinhardtii autofluoresces in the red, far-red and infrared spectra. This is independent of laser excitation wavelength, and autofluorescence emits and spills over into detection channels of both four-colour and multicolour instruments. Band-pass filter configurations capturing longer wavelength emissions or fluorophores excited or emitted in these longer wavelengths are generally unsuitable. Furthermore, neither dark nor light incubation impacted the autofluorescent signals. Consideration of these algal autofluorescent properties and their spillover effects is required to avoid erroneous results. Recommendations for the use of a range of fluorophores in programmed cell death and other studies in C. reinhardtii using four-colour and multicolour instruments are made.     

Phosphatidic acid binding inhibits RGS1 activity to affect specific signaling pathways in Arabidopsis

Modulation of the active versus inactive forms of the Gα protein is critical for the signaling processes mediated by the heterotrimeric G‐protein complex. We have recently established that in Arabidopsis, the regulator of G‐protein signaling (RGS1) protein and a lipid‐hydrolyzing enzyme, phospholipase Dα1 (PLDα1), both act as GTPase‐activity accelerating proteins (GAPs) for the Gα protein to attenuate its activity. RGS1 and PLDα1 interact with each other, and RGS1 inhibits the activity of PLDα1 during regulation of a subset of responses. In this study, we present evidence that this regulation is bidirectional. Phosphatidic acid (PA), a second messenger typically derived from the lipid‐hydrolyzing activity of PLDα1, is a molecular target of RGS1. PA binds and inhibits the GAP activity of RGS1. A conserved lysine residue in RGS1 (Lys²⁵⁹) is directly involved in RGS1–PA binding. Introduction of this RGS1 protein variant in the rgs1 mutant background makes plants hypersensitive to a subset of abscisic acid‐mediated responses. Our data point to the existence of negative feedback loops between these two regulatory proteins that precisely modulate the level of active Gα, consequently generating a highly controlled signal–response output.     

Purification and characterization of a human pancreatic adenocarcinoma mucin.

Pancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.     

Insecticidal pilin subunit from the insect pathogen Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.     

Rude mechanicals in brain haemodynamics: non-neural actors that influence blood flow

Fluctuations in blood oxygenation and flow are widely used to infer brain activity during resting-state functional magnetic resonance imaging (fMRI). However, there are strong systemic and vascular contributions to resting-state signals that are unrelated to ongoing neural activity. Importantly, these non-neural contributions to haemodynamic signals (or ‘rude mechanicals’) can be as large as or larger than the neurally evoked components. Here, we review the two broad classes of drivers of these signals. One is systemic and is tied to fluctuations in external drivers such as heart rate and breathing, and the robust autoregulatory mechanisms that try to maintain a constant milieu in the brain. The other class comprises local, active fluctuations that appear to be intrinsic to vascular tissue and are likely similar to active local fluctuations seen in vasculature all over the body. In this review, we describe these non-neural fluctuations and some of the tools developed to correct for them when interpreting fMRI recordings. However, we also emphasize the links between these vascular fluctuations and brain physiology and point to ways in which fMRI measurements can be used to exploit such links to gain valuable information about neurovascular health and about internal brain states.This article is part of the theme issue ‘Key relationships between non-invasive functional neuroimaging and the underlying neuronal activity’.     

Assessing Essential Trace Elements in Cave Nectar Bat (Eonycteris spelaea): A Study in Barak Valley of Assam,India

Biological Trace Element Research - This study investigated trace elements in the different organs of Eonycteris spelaea, a hill cave from the Bhuban Hills of Sonai Reserve Forest, Cachar, Assam...     

Enzyme elements involved in the interconversion of L-carbamylaspartate and L-dihydroorotate by dihydroorotase from Clostridium oroticum

Enzyme elements that are involved in the reversible cyclization of L-carbamylaspartate to L-dihdroorotate catalyzed by dihydroorotase (EC 3.5.2.3) from Clostridium oroticum (ATCC 25750) have been studied. Removal of Zn(II) from the enzyme by chelators followed by incubation of apoenzyme with Co(II) results in replacement of two to three of the four Zn(II) ions per molecule by Co(II). The catalytic properties of the Zn(II)Co(II) dihydroorotase are different from those of native enzyme. The Vmax is increased for both the synthesis and hydrolysis of L-dihydroorotate. The Km for L-dihydroorotate is unchanged, while the Km for L-carbamylaspartate is increased more than twofold. On the other hand, the kinetic properties of Zn(II)-reconstituted dihydroorotase are indistinguishable from those of native enzyme. The pH dependence of Vmax is also altered by the Co(II) substitution. For both Zn(II)- and Zn(II)Co(II)-dihydroorotase, this pH dependence is well described by a single ionization and the pK's for L-dihydroorotate synthesis and hydrolysis are different. Substitution with Co(II) increases the pK for both reaction directions to different extents. These results strongly support a role for the tightly bound metals in the catalytic mechanism. In addition, diethylpyrocarbonate rapidly inactivates the enzyme. The inactivation is prevented by L-dihydroorotate. This result is consistent with a role for at least one histidine in catalysis. The possibility that C. oroticum dihydroorotase may be useful model for the more complex mammalian enzyme is considered.