Computational and Functional Characterization of Angiogenin Mutations,and Correlation with Amyotrophic Lateral Sclerosis

The Angiogenin (ANG) gene is frequently mutated in patients suffering from the neurodegenerative disease - amyotrophic lateral sclerosis (ALS). Most of the ALS-causing mutations in Angiogenin affect either its ribonucleolytic or nuclear translocation activity. Here we report the functional characterization of two previously uncharacterized missense mutations in Angiogenin - D22G and L35P. We predict the nature of loss-of-function(s) in these mutants through our previously established Molecular Dynamics (MD) simulation extended to 100 ns, and show that the predictions are entirely validated through biochemical studies with wild-type and mutated proteins. Based on our studies, we provide a biological explanation for the loss-of-function of D22G-Angiogenin leading to ALS, and suggest that the L35P-Angiogenin mutation would probably cause ALS symptoms in individuals harboring this mutation. Our study thus highlights the strength of MD simulation-based predictions, and suggests that this method can be used for correlating mutations in Angiogenin or other effector proteins with ALS symptoms.     

Conditionally activated E7 proteins of high-risk and low-risk human papillomaviruses induce S phase in postmitotic, differentiated human keratinocytes

The productive program of human papillomaviruses (HPVs) in epithelia is tightly linked to squamous differentiation. The E7 proteins of high-risk HPV genotypes efficiently inactivate the pRB family of proteins that control the cell cycle, triggering S phase in suprabasal keratinocytes. This ability has until now not been demonstrated for the low-risk HPV-6 or HPV-11 E7 proteins. An inducible system in which HPV-16 E7 is fused to the ligand binding domain of the human estrogen receptor (ER) was described by Smith-McCune et al. (K. Smith-McCune, D. Kalman, C. Robbins, S. Shivakumar, L. Yuschenkoff, and J. M. Bishop, Proc. Natl. Acad. Sci. USA 96:6999-7004, 1999). In the absence of hormone, E7ER is cytoplasmic, and upon addition of 17beta-estradiol, it translocates to the nucleus. Using organotypic epithelial raft cultures developed from primary human keratinocytes, we show that 16E7ER promotes either S-phase reentry or p21cip1 accumulation in differentiated keratinocytes in a stochastic manner as early as 6 h postinduction with 17beta-estradiol. A vector expressing the ER moiety alone had no effect. These observations prove unequivocally that the E7 protein drives S-phase reentry in postmitotic, differentiated keratinocytes rather than preventing S-phase exit while the cells ascend through the epithelium. HPV-11 E7ER and, much less efficiently, HPV-6 E7ER also promoted S-phase reentry by differentiated cells upon exposure to 17beta-estradiol. S-phase induction required the consensus pRB binding motif. We propose that the elevated nuclear levels of the low-risk HPV E7 protein afforded by the inducible system account for the positive results. These observations are entirely consistent with the fact that low-risk HPV genotypes replicate in the differentiated strata in patient specimens, as do the high-risk HPVs.     

Comparison of the CopB systems of plasmids R1 and Co1V2-K94: A single base alteration in CopB gene is responsible for the increased copy number of the low copy number plasmid CoIV2-K94

Summary We have isolated a deletion mutation and a point mutation in the copB gene of the replication region Repl of the IncFI plasmid Co1V2-K94. Subsequently, this copB gene with and without point mutation was cloned and sequenced, and the point mutation was mapped in the coding region of copB with a change of one amino acid from arginine to serine. Furthermore, this copB mutant had an approximately 10-fold increase in copy number. The CopB-phenotype of Co1V2-K94 could be complemented in trans by the copB gene of coresident IncFII plasmids such as R1 and R538, but not R100, suggesting that ColV2-K94 and R1 or R538 contain the same copB allele.     

Synthesis and evaluation of novel 2-pyridone derivatives as inhibitors of phosphodiesterase3 (PDE3): A target for heart failure and platelet aggregation

Twenty-six 2-pyridone derivatives (8a-8z), which are structurally analogous to amrinone and milrinone two important cardiotonic drugs, are synthesized and characterized. The synthesis of 2-pyridone derivatives involves addition, followed by cyclization between Baylis-Hillman acetates (7a-7k) and enamino esters or nitriles (3a-3e). Thus synthesized pyridones were subjected to PDE3 inhibitory activity, 14 pyridones were found to be hits out of 26 pyridones synthesized and out of 14 hits, there are 5 pyridones found to be lead compounds having excellent PDE3 inhibitory activity. Further we have carried out computational analysis to understand protein/enzyme and 2-pyridone derivative interactions to identify amino acid residues involved in the vicinity of binding and compared with milrinone drug.     

C nuclear magnetic resonance studies of the binding of alkyl isocyanides to soybean leghemoglobin; comparison with animal myoglobins

¹³C NMR of labelled alkyl isocyanide ligands has been used with a view to probe the protein environment around the heme site of Soybean leghemoglobin, and comparatively, those of sperm whale myoglobin and monomeric Glycera hemoglobin. The terminal carbon of the isocyanide, which is known to be highly sensitive to change in hybridization of the nitrogen, could be expected to reflect the movement of the alkyl group through steric interactions. Three alkyl isocyanides (alkyl = methyl, ethyl & n-butyl) have therefore been used and the ¹³C° chemical shift values were measured for each ligand bound to the various proteins studied.In all cases, the ¹³C° resonances of the bound ligand were shifted considerably downfield with respect to those of the free unbound species, but the pattern of these displacements revealed more pronounced steric hindrance in the case of some proteins compared to others. The modifications of the chemical shift values on binding Δδ = δ_bound — δ_free) were least in the case of leghemoglobin; moreover, the Δδ values were insensitive to the length of the alkyl chain (methyl to n-butyl) when bound to leghemoglobin, in contrast to the other proteins examined. The results are interpreted as arising from a diminished steric hindrance to isocyanide binding with leghemoglobin, in conformity with the recently published X-ray structure which reports the existence of a large heme pocket on the distal side.     

New furofurano lignans from Justicia simplex

A new 3,7-dioxabicyclo[3,3,O]octane lignan, named justisolin, and a new lignan O-glucoside, named simplexoside, were isolated from the whole plant of Justicia simplex D. Don. (Acanthaceae), collected at fruiting. The structure of the free lignan was established as 2e-(3,4-methylenedioxy-6-hydroxy)-phenyl-6e- Piperonyl-3,7-dioxabicyclo[3,3,0]octane (1) and that of the glucoside as 2e-(3-methoxy-4-O-β-d- glucopyranosyl)-phenyl-6e-piperonyl-3,7-dioxabicyclo[3,3,0]octane (2) on the basis of chemical transformation and spectral evidence. The biological functions of these and related lignans are appraised.     

Lipid profiles of Aspergillus niger and its unsaturated fatty acid auxotroph, UFA2

A comparative study of the mycelial lipid composition of a wild strain (V35) and one unsaturated fatty acid auxotroph (UFA2) of Aspergillus niger has been performed. The lipid composition of both strains are qualitatively the same but quantitatively different. All the strains contain the following phospholipids: cardiolipin, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylcholine, and phosphatidylserine; and triglycerides, diglycerides, monoglycerides, ergosterol, and sterol esters as the neutral lipids; mono- and di-galactosyl diglyceride as the major glycolipids along with small amounts of the corresponding mannose analogs. Phosphatidylethanolamine and phosphatidylcholine constitute the bulk of the phospholipids. The mutant (UFA2) contains a higher level of glycerides and lower levels of sterol (both free and esterified form), phospholipids, and glycolipids than the wild type. Aspergillus niger contains C16 to C18 saturated and unsaturated fatty acids. Small amounts of long-chain (C20 to C24) and short-chain (C10 to C14) saturated and unsaturated acids are also present. Linoleic, oleic, and palmitic are the major acids, stearic and linolenic acids being minor ones. UFA2 grows only in the presence of unsaturated fatty acid (C16 or C18) and accumulates a higher concentration of supplemented acid which influences its fatty acid profile.     

Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition

The stability of Watson–Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na⁺ or K⁺ in the presence or absence of Mg²⁺). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°₃₇ and T_m values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg²⁺, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR–Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.     

A review on interplay between small RNAs and oxidative stress in cancer progression

Molecular and Cellular Biochemistry - Oxidative stress has been known to be the underlying cause in many instances of cancer development. The new aspect of cancer genesis that has caught the...