Origin and instability of GAA repeats: insights from Alu elements

Expansion of GAA repeats in the intron of the frataxin gene is involved in the autosomal recessive Friedreich's ataxia (FRDA). The GAA repeats arise from a stretch of adenine residues of an Alu element. These repeats have a size ranging from 7- 38 in the normal population, and expand to thousands in the affected individuals. The mechanism of origin of GAA repeats, their polymorphism and stability are not well understood. In this study, we have carried out an extensive analysis of GAA repeats at several loci in the humans. This analysis indicates the association of a majority of GAA repeats with the 3' end of an "A" stretch present in the Alu repeats. Further, the prevalence of GAA repeats correlates with the evolutionary age of Alu subfamilies as well as with their relative frequency in the genome. Our study on GAA repeat polymorphism at some loci in the normal population reveals that the length of the GAA repeats is determined by the relative length of the flanking A stretch. Based on these observations, a possible mechanism for origin of GAA repeats and modulatory effects of flanking sequences on repeat instability mediated by DNA triplex is proposed.     

Effect of endotoxin on the immunity of Indian major carp, Labeo rohita

Endotoxin, a lipopolysaccharide component of outer cell wall membrane of the Gram-negative bacteria is a factor responsible for a number of biological effects including immunostimulatory activities in different animal species including fish. In this study, L. rohita yearlings of weight ranging from 80 to 100g were injected intraperitoneally with 0.5, 1, 2, 5, 10 and 20 EU/fish dose of endotoxin to find out its effect on the immunity. The L. rohita yearlings were found to resist the endotoxin dose up to 20 EU/fish and at the lower doses, i.e., at 1 and 2 EU/fish; it acted as an immune potentiator. Different serum and immune parameters like protein, globulin, lysozyme, respiratory burst activity, myeloperoxidase activity, natural agglutination titre were found to be significantly high (p<0.01) at a dose of 1 EU/fish. While at 10 and 20 EU/fish, most of these parameters were lower thereby indicating the immuno-suppressive nature of the endotoxin at these higher doses.     

Slow-tight binding inhibition of xylanase by an aspartic protease inhibitor: kinetic parameters and conformational changes that determine the affinity and selectivity of the bifunctional nature of the inhibitor

The first report of slow-tight inhibition of xylanase by a bifunctional inhibitor alkalo-thermophilic Bacillus inhibitor (ATBI), from an extremophilic Bacillus sp. is described. ATBI inhibits aspartic protease (Dash, C., and Rao, M. (2001) J. Biol. Chem., 276, 2487-2493) and xylanase (Xyl I) from a Thermomonospora sp. The steady-state kinetics revealed time-dependent competitive inhibition of Xyl I by ATBI, consistent with two-step inhibition mechanism. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI*, and the dissociation of EI* were 13 +/- 1 x 10(-6) s(-1) and 5 +/- 0.5 x 10(-8) s(-1), respectively. The K(i) value for the formation of EI complex was 2.5 +/- 0.5 microm, whereas the overall inhibition constant K(i)* was 7 +/- 1 nm. The conformational changes induced in Xyl I by ATBI were monitored by fluorescence spectroscopy and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. ATBI binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled Xyl I. Our results revealed that the inactivation of Xyl I is due to the disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis and a model depicting the probable interaction between ATBI or OPTA with Xyl I has been proposed.     

Molecular docking of fisetin with AD associated AChE,ABAD and BACE1 proteins

Alzheimer׳s disease (AD) is one of the most common dementias showing slow progressive cognitive decline. Progression of intracerebralaccumulation of beta amyloid (Aβ) peptides by the action of amyloid binding alcohol dehydrogenase (ABAD), amitochondrial enzyme and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and the degradation ofAcetylcholinesterase (AChE) the main pathological characteristics of AD. Therefore, it is of interest to evaluate the importance offisetin (a flavonol that belongs to the flavonoid group of polyphenols) binding with AChE, ABAD and BACE1 proteins. Dockingexperiment of fisetin with these proteins using two different tools namely iGEMDOCK and FlexX show significant binding withacceptable binding values. Thus, the potential inhibitory role of fisetin with AD associated proteins is documented.     

Resistance of selected lines of Ostertagia circumcincta to thiabendazole, morantel tartrate and levamisole

Le Jambre I. F., Southcott W. H. and Dash K. M. 1977. Resistance of selected lines of Ostertagia circumcincta to thiabendazole, morantel tartrate and levamisole. International Journal for Parasitology7: 473–479. A strain of Ostertagia circumcincta was isolated from a field in which all sheep had been treated in sequence every 7–10 days from September 1970 to January 1974 with either thiabendazole, morantel tartrate or levamisole. Thiabendazole had not been used after the first 15 months. The LD₉₅ for this strain was 88 mg/kg thiabendazole, 6.9 mg/kg morantel tartrate and 5-4 mg/kg levamisole.Another strain of O. circumcincta isolated from an area where anthelmintics had been used much less frequently was divided into four lines for exposure to selection in the laboratory. The first line was selected with 50 mg/kg thiabendazole, the second with 5 mg/kg morantel tartrate, and the third with 3.2 mg/kg levamisole; the fourth line was not selected for drug resistance. After eight generations the three lines selected with thiabendazole, morantel tartrate and levamisole had (Spld)(in95) of > 200, 5.7 and 6.2 mg/kg for the selecting drugs respectively, compared with corresponding values of 20, 2.9, and 1.8 in the unselected line. That is, the field strain had about the same levels of resistance to morantel tartrate and levamisole as the respective laboratory strains selected with these individual drugs. However, the field strain, which had been exposed to thiabendazole for only 15 months, was less resistant to thiabendazole than the laboratory strain selected with this drug. These results show that giving of several drugs in sequence cannot be relied upon to prevent the development of resistance to the individual drugs.The dose responses of adult worms showed low, but significant resistances to morantel tartrate and levamisole and a relatively high resistance to thiabendazole. Levamisole was found to select for inhibition of development with approx. 8.0% of the inhibited larvae showing no dose response above 1.6 mg/kg. Levamisole was also associated with an increase from 0.1 % to 9.0% in the O. trifurcata component of an Ostertagia population.     

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from seeds of chickpea (Cicer arietinum L.)

A novel lectin (CAA-II) was isolated and purified from the seeds of Cicer arietinum by ammonium sulphate fractionation and affinity chromatography on an N-acetyl-D-galactosamine-linked agarose column. The lectin is composed of four identical subunits of 30 kDa and the molecular mass of the native lectin was estimated to be 120 kDa by gel filtration chromatography and confirmed by mass spectrometry. The lectin showed agglutination activity against rabbit erythrocytes (trypsin-treated and untreated) as well as against human erythrocytes. Haemagglutination inhibition assays showed that the lectin is a galactose-specific protein having a high affinity for N-acetyl-D-galactosamine. The molecular weight, haemagglutination pattern, carbohydrate specificity and N-terminal amino acid sequence indicated that the lectin is clearly distinct from the previously reported chickpea lectin CAA-I.     

Impaired Expression of Type I and Type II Interferon Receptors in HCV-Associated Chronic Liver Disease and Liver Cirrhosis

Purpose

Chronic Hepatitis C Virus (HCV)-infected patients with liver cirrhosis (LC) respond poorly to interferon-alpha (IFN-α) and ribavirin (RBV) combination therapy, but the reason for this is unclear. We previously reported that HCV-infection induces endoplasmic reticulum (ER) stress and autophagy response that selectively down regulates the type I IFN-α receptor-1 (IFNAR1) and RBV transporters (CNT1 and ENT1), leading to IFN-α/RBV resistance. The goal of this study is to verify whether an increase in ER stress and autophagy response is also associated with the reduced expression of IFNAR1 and RBV transporters in chronic HCV-infected patients.

Methods

Primary human hepatocytes (PHH) were infected with cell culture grown HCV particles (JFH-ΔV3-Rluc). HCV replication was confirmed by the detection of viral RNA by RT-qPCR and HCV-core protein by Western blotting. The ER stress and autophagy response and expression of IFN receptors and RBV transporters in HCV infected PHH and liver tissues derived from patients were measured by Western blotting.

Result

HCV infection of PHH showed impaired expression of IFNAR1, IFNγR1 (Type II IFN receptor) and RBV transporters but not IL10Rβ (Type III IFN-λ receptor). ER stress markers (BiP, IRE1α and peIF2α) and autophagy response (LC3II, Beclin 1 and ATG5) were induced in HCV infected chronic liver disease (CLD) and LC patients. Liver biopsies (CLD) show a 50% reduced expression of IFNAR1 and RBV transporters. Furthermore, the expression of IFNAR1 and RBV transporters was impaired in almost all LC patients.

Conclusion

HCV infection induces ER stress and autophagy response in infected PHH and chronically infected liver tissues. The expression of IFNAR1, IFNγR1 and RBV transporters were significantly impaired in CLD and cirrhotic livers. Our study provides a potential explanation for the reduced response rate of IFN-α and RBV combination therapy in HCV infected patients with liver cirrhosis.     

Acetyl‐ed question in mitochondrial biology?

Lysine acetylation on numerous mitochondrial proteins, targeted by the sirtuin deacylase SIRT3, has been proposed to play a major role in regulating diverse mitochondrial functions, particularly in the liver. A new study by Weinert, Choudhary, and colleagues, in this issue of The EMBO Journal, finds that the absolute levels of hepatic mitochondrial protein acetylation in wild‐type mice are extremely low and may be insufficient to exert regulatory effects.     

Conformational analysis of invariant peptide sequences in bacterial genomes

The functional significance of evolutionarily conserved motifs/patterns of short regions in proteins is well documented. Although a large number of sequences are conserved, only a small fraction of these are invariant across several organisms. Here, we have examined the structural features of the functionally important peptide sequences, which have been found invariant across diverse bacterial genera. Ramachandran angles (phi,psi) have been used to analyze the conformation, folding patterns and geometrical location (buried/exposed) of these invariant peptides in different crystal structures harboring these sequences. The analysis indicates that the peptides preferred a single conformation in different protein structures, with the exception of only a few longer peptides that exhibited some conformational variability. In addition, it is noticed that the variability of conformation occurs mainly due to flipping of peptide units about the virtual C(alpha)...C(alpha) bond. However, for a given invariant peptide, the folding patterns are found to be similar in almost all the cases. Over and above, such peptides are found to be buried in the protein core. Thus, we can safely conclude that these invariant peptides are structurally important for the proteins, since they acquire unique structures across different proteins and can act as structural determinants (SD) of the proteins. The location of these SD peptides on the protein chain indicated that most of them are clustered towards the N-terminal and middle region of the protein with the C-terminal region exhibiting low preference. Another feature that emerges out of this study is that some of these SD peptides can also play the roles of "fold boundaries" or "hinge nucleus" in the protein structure. The study indicates that these SD peptides may act as chain-reversal signatures, guiding the proteins to adopt appropriate folds. In some cases the invariant signature peptides may also act as folding nuclei (FN) of the proteins.