Monitoring cholesterol organization in membranes at low concentrations utilizing the wavelength-selective fluorescence approach

We previously showed using a fluorescent analogue of cholesterol (NBD-cholesterol, or 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol), that cholesterol may exhibit local organization at low concentrations in membranes by the formation of transbilayer tail-to-tail dimers of cholesterol (Rukmini, R., Rawat, S.S., Biswas, S.C., Chattopadhyay, A., 2001. Biophys. J. 81, 2122-2134). In this report, we have monitored the microenvironmental features of cholesterol monomers and dimers utilizing wavelength-selective fluorescence spectroscopy. Our results utilizing red edge excitation shift (REES) and wavelength-dependent change in fluorescence anisotropy show that the microenvironment around the NBD moieties in the dimer form is more rigid possibly due to steric constraints imposed by the dimer conformation. These results provide new information and are relevant in understanding the organization of cholesterol in membranes at low concentrations.     

Computational identification of post-translational modification sites and functional families reveal possible moonlighting role of rotaviral proteins

Rotavirus (RV) diarrhoea causes huge number deaths in children less than 5 years of age. In spite of available vaccines, it has been difficult to combat RV due to large number of antigenically distinct genotypes, high mutation rates, generation of reassortant viruses due to segmented genome. RV is an eukaryotic virus which utilizes host cell machinery for its propagation. Since RV only encodes 12 proteins, post-translational modification (PTM) is important mechanism for modification, which consequently alters their function. A single protein exhibiting different functions in different locations or in different subcellular sites, are known to be 'moonlighting'. So there is a possibility that viral proteins moonlight in separate location and in different time to exhibit diverse cellular effects. Based on the primary sequence, the putative behaviour of proteins in cellular environment can be predicted, which helps to classify them into different functional families with high reliability score. In this study, sites for phosphorylation, glycosylation and SUMOylation of the six RV structural proteins (VP1, VP2, VP3, VP4, VP6 & VP7) & five non-structural proteins (NSP1, NSP2,NSP3,NSP4 & NSP5) and the functional families were predicted. As NSP6 is a very small protein and not required for virus growth & replication, it was not included in the study. Classification of RV proteins revealed multiple putative functions of each structural protein and varied number of PTM sites, indicating that RV proteins may also moonlight depending on requirements during viral life cycle. Targeting the crucial PTM sites on RV structural proteins may have implications in developing future anti-rotaviral strategies.     

Differential effect of cholesterol and its biosynthetic precursors on membrane dipole potential

Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. Cholesterol, a representative sterol in higher eukaryotic membranes, is known to increase membrane dipole potential. In this work, we explored the effects of immediate (7-DHC and desmosterol) and evolutionary (ergosterol) precursors of cholesterol on membrane dipole potential, monitored by the dual wavelength ratiometric approach utilizing the probe di-8-ANEPPS. Our results show that the effect of these precursors on membrane dipole potential is very different from that observed with cholesterol, although the structural differences among them are subtle. These results assume relevance, since accumulation of cholesterol precursors due to defective cholesterol biosynthesis has been reported to result in several inherited metabolic disorders such as the Smith-Lemli-Opitz syndrome. Interestingly, cholesterol (and its precursors) has a negligible effect on dipole potential in polyunsaturated membranes. We interpret these results in terms of noncanonical orientation of cholesterol in these membranes. Our results constitute the first report on the effect of biosynthetic and evolutionary precursors of cholesterol on dipole potential, and imply that a subtle change in sterol structure can significantly alter the dipolar field at the membrane interface.     

Glutathione signaling acts through NPR1-dependent SA-mediated pathway to mitigate biotic stress

Glutathione (GSH) has widely been known to be a multifunctional molecule especially as an antioxidant up until now, but has found a new role in plant defense signaling. Research from the past three decades indicate that GSH is a player in pathogen defense in plants, but the mechanism underlying this has not been elucidated fully. We have recently shown that GSH acts as a signaling molecule and mitigates biotic stress through non-expressor of PR genes 1 (NPR1)-dependent salicylic acid (SA)-mediated pathway. Transgenic tobacco with enhanced level of GSH (NtGB lines) was found to synthesize more SA, was capable of enhanced expression of genes belonging to NPR1-dependent SA-mediated pathway, were resistant to Pseudomonas syringae, the biotrophic pathogen and many SA-related proteins were upregulated. These results gathered experimental evidence on the mechanism through which GSH combats biotic stress. In continuation with our previous investigation we show here that the expression of glutathione S-transferase (GST), the NPR1-independent SA-mediated gene was unchanged in transgenic tobacco with enhanced level of GSH as compared to wild-type plants. Additionally, the transgenic plants were barely resistant to Botrytis cinerea, the necrotrophic pathogen. SA-treatment led to enhanced level of expression of pathogenesis-related protein gene (PR1) and PR4 as against short-chain dehydrogenase/reductase family protein (SDRLP) and allene oxide synthase (AOS). These data provided significant insight into the involvement of GSH in NPR1-dependent SA-mediated pathway in mitigating biotic stress.Key words: GSH, signaling molecule, biotrophic pathogen, NPR-1, PR-1, PR-4, transgenic tobaccoPlant responses to different environmental stresses are achieved through integrating shared signaling networks and mediated by the synergistic or antagonistic interactions with the phytohormones viz. SA, jasmonic acid (JA), ethylene (ET), abscisic acid (ABA) and reactive oxygen species (ROS).¹ Previous studies have shown that in response to pathogen attack, plants produce a highly specific blend of SA, JA and ET, resulting in the activation of distinct sets of defense-related genes.^2,3 Regulatory functions for ROS in defense, with a focus on the response to pathogen infection occur in conjunction with other plant signaling molecules, particularly with SA and nitric oxide (NO).^4–6 Till date, numerous physiological functions have been attributed to GSH in plants.^7–11 In addition to previous studies, recent study has also shown that GSH acts as a signaling molecule in combating biotic stress through NPR1-dependent SA-mediated pathway.^12,13Our recent investigation involved raising of transgenic tobacco overexpressing gamma-glutamylcysteine synthetase (γ-ECS), the rate-limiting enzyme of the GSH biosynthetic pathway.¹² The stable integration and enhanced expression of the transgene at the mRNA as well as protein level was confirmed by Southern blot, quantitative RT-PCR and western blot analysis respectively. The transgenic plants of the T₂ generation (Fig. 1), the phenotype of which was similar to that of wild-type plants were found to be capable of synthesizing enhanced amount of GSH as confirmed by HPLC analysis.Open in a separate windowFigure 1Transgenic tobacco of T₂ generation, (A) three-week-old plant, (B) mature plant.In the present study, the expression profile of GST was analyzed in NtGB lines by quantitative RT-PCR (qRT-PCR) and found that the expression level of this gene is unchanged in NtGB lines as compared to wild-type plants (Fig. 2). GST is known to be a NPR1-independent SA-related gene.¹⁴ This suggests that GSH does not follow the NPR1-independent SA-mediated pathway in defense signaling.Open in a separate windowFigure 2Expression pattern of GST in wild-type and NtGB lines.Disease test assay with NtGB lines and wild-type plants was performed using B. cinerea and the NtGB lines showed negligible rate of resistance to this necrotrophic pathogen (Fig. 3). SA signaling has been known to control defense against biotrophic pathogen in contrast, JA/ET signaling controls defense against necrotrophic pathogen.^1,15 Thus it has again been proved that GSH is not an active member in the crosstalk of JA-mediated pathway, rather it follows the SA-mediated pathway as has been evidenced earlier.¹²Open in a separate windowFigure 3Resistance pattern of wild-type and NtGB lines against Botrytis cinerea.Additionally, the leaves of wild-type and NtGB lines were treated with 1 mM SA and the expression of PR1, SDRLP, AOS and PR4 genes were analyzed and compared to untreated plants to simulate pathogen infection. The expression of PR1 increased after exogenous application of SA. In case of PR4, the ET marker, the expression level increased in NtGB lines. On the other hand, the level of SDRLP was nearly the same. However, the expression of AOS was absent in SA-treated leaves (Fig. 4). PR1 has been known to be induced by SA-treatment¹⁶ which can be corroborated with our results. In addition, ET is known to enhance SA/NPR1-dependent defense responses,¹⁷ which was reflected in our study as well. AOS, the biosynthetic pathway gene of JA, further known to be the antagonist of SA, was downregulated in SA-treated plants.Open in a separate windowFigure 4Gene expression pattern of PR1, SDRLP, PR4 and AOS in untreated and SA-treated wildtype and NtGB lines.Taken together, it can be summarized that this study provided new evidence on the involvement of GSH with SA in NPR1-dependent manner in combating biotic stress. Additionally, it can be claimed that GSH is a signaling molecule which takes an active part in the cross-communication with other established signaling molecules like SA, JA, ET in induced defense responses and has an immense standpoint in plant defense signaling.     

Poxvirus uracil‐DNA glycosylase—An unusual member of the family I uracil‐DNA glycosylases

Uracil‐DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil‐DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non‐enzymatic function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus‐specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA‐binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. The adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three‐dimensional structure of D4. An overview of the current state of the knowledge on the structure‐function relationship of D4 is provided here.     

Impaired Glucose Tolerance or Newly Diagnosed Diabetes Mellitus Diagnosed during Admission Adversely Affects Prognosis after Myocardial Infarction: An Observational Study

Objective

To investigate the prognostic effect of newly diagnosed diabetes mellitus (NDM) and impaired glucose tolerance (IGT) post myocardial infarction (MI).

Research Design and Methods

Retrospective cohort study of 768 patients without preexisting diabetes mellitus post-MI at one centre in Yorkshire between November 2005 and October 2008. Patients were categorised as normal glucose tolerance (NGT n = 337), IGT (n = 279) and NDM (n = 152) on pre- discharge oral glucose tolerance test (OGTT). Primary end-point was the first occurrence of major adverse cardiovascular events (MACE) including cardiovascular death, non-fatal MI, severe heart failure (HF) or non-haemorrhagic stroke. Secondary end-points were all cause mortality and individual components of MACE.

Results

Prevalence of NGT, impaired fasting glucose (IFG), IGT and NDM changed from 90%, 6%, 0% and 4% on fasting plasma glucose (FPG) to 43%, 1%, 36% and 20% respectively after OGTT. 102 deaths from all causes (79 as first events of which 46 were cardiovascular), 95 non fatal MI, 18 HF and 9 non haemorrhagic strokes occurred during 47.2 ± 9.4 months follow up. Event free survival was lower in IGT and NDM groups. IGT (HR 1.54, 95% CI: 1.06–2.24, p = 0.024) and NDM (HR 2.15, 95% CI: 1.42–3.24, p = 0.003) independently predicted MACE free survival. IGT and NDM also independently predicted incidence of MACE. NDM but not IGT increased the risk of secondary end-points.

Conclusion

Presence of IGT and NDM in patients presenting post-MI, identified using OGTT, is associated with increased incidence of MACE and is associated with adverse outcomes despite adequate secondary prevention.