Pre-fibrillar α-synuclein mutants cause Parkinson's disease-like non-motor symptoms in Drosophila

Parkinson's disease (PD) is linked to the formation of insoluble fibrillar aggregates of the presynaptic protein α-Synuclein (αS) in neurons. The appearance of such aggregates coincides with severe motor deficits in human patients. These deficits are often preceded by non-motor symptoms such as sleep-related problems in the patients. PD-like motor deficits can be recapitulated in model organisms such as Drosophila melanogaster when αS is pan-neurally expressed. Interestingly, both these deficits are more severe when αS mutants with reduced aggregation properties are expressed in flies. This indicates that that αS aggregation is not the primary cause of the PD-like motor symptoms. Here we describe a model for PD in Drosophila which utilizes the targeted expression of αS mutants in a subset of dopadecarboxylase expressing serotonergic and dopaminergic (DA) neurons. Our results show that targeted expression of pre-fibrillar αS mutants not only recapitulates PD-like motor symptoms but also the preceding non-motor symptoms such as an abnormal sleep-like behavior, altered locomotor activity and abnormal circadian periodicity. Further, the results suggest that the observed non-motor symptoms in flies are caused by an early impairment of neuronal functions rather than by the loss of neurons due to cell death.     

Characterization of the linker 2 region in human vimentin using site-directed spin labeling and electron paramagnetic resonance

Site-directed spin labeling and electron paramagnetic resonance were used to probe residues 281-304 of human vimentin, a region that has been predicted to be a non-alpha-helical linker and the beginning of coiled-coil domain 2B. Though no direct test of linker structure has ever been made, this region has been hypothesized to be flexible with the polypeptide chains looping away from one another. EPR analysis of spin-labeled mutants indicates that (a) several residues reside in close proximity, suggesting that adjacent linker regions in a dimer run in parallel, and that (b) the polypeptide backbone is relatively rigid and inflexible in this region. However, this region does not show the characteristics of a coiled-coil as has been identified elsewhere in the molecule. Within this region, spectra from positions 283 and 291 are unique from all others thus far examined. These positions, predicted to be in a noncoiled-coil structure, display a significantly stronger interaction than the a-d contact positions of coiled-coil regions. Analysis of the early stages of assembly by dialysis from 8 M urea and progressive thermal denaturation shows the close apposition and structural rigidity at residues 283 and 291 occurs very early in assembly and with a relatively sudden onset, well before coiled-coil formation in other parts of the molecule. These features are inconsistent with hypotheses that envision the linkers as flexible regions, or as looping away from one another, and raise the possibility that the linker may be the site at which dimer alignment and/or formation is initiated. Spin labels placed further downstream yield spectra suggesting that the first regular heptad of rod domain 2 begins at position 302. In conjunction with our previous characterization of region 305-336 and the solved structure of rod 2B from 328-405, the full extent of coiled-coil domain in rod 2B is now known, spanning from vimentin positions 302-405.     

Advanced glycosylated end products-mediated activation of polymorphonuclear neutrophils in diabetes mellitus and associated oxidative stress

Two important consequences of hyperglycemia in diabetes are development of oxidative stress and formation of advanced glycation end products (AGE) which are known to be associated with diabetic complications. Relationship between AGE formation and development of oxidative stress (OS) is yet to be established. In the present study, the involvement of AGE in PMN-mediated ROS generation and the associated OS were investigated in type 2 diabetic mellitus (DM) patients. We assessed OS parameters (serum MDA, FRAP and GSH), PMN oxidative functions (respiratory burst and superoxide production) and total serum AGE in 90 subjects divided equally in three groups--control group, Group I consisting of type 2 diabetic patients without microvascular complications and Group II consisting of type 2 diabetic patients with microvascular complications. PMNs isolated from both groups (I and II) exhibited higher level of respiratory burst (RB) and produced increased amount of superoxide anion as compared to the controls. The increase was more pronounced in diabetes with complications, as compared to those without. Serum malondialdehyde (MDA) level was elevated, whereas glutathione (GSH) and ferric reducing ability of plasma (FRAP) levels were significantly reduced in diabetes as compared to the controls, suggesting the presence of oxidative stress in DM. A positive correlation between PMN oxidative function and OS parameters suggested the involvement of PMN in the development of OS in DM. Serum AGE level was also elevated in diabetic groups as compared to the controls. Further, the positive correlation between serum AGE level and PMN oxidative function suggested the involvement of AGE in increased RB and generation of reactive oxygen species (ROS) by resting diabetic PMN. The results of the study indicate that AGE-PMN interaction possibly upregulates NADPH oxidase, leading to enhanced ROS generation and thus contributes to the pathogenesis in diabetes.     

Pharmacophore development and screening for discovery of potential inhibitors of ADAMTS-4 for osteoarthritis therapy

In the development of osteoarthritis, aggrecan degrades prior to cartilage destruction. Aggrecanase-1 (ADAMTS-4) is considered to be the major enzyme responsible for cleaving the Glu373–Ala374 bond in the interglobular domain of aggrecan in humans. Therefore, inhibitors of ADAMTS-4 have therapeutic potential in the treatment of osteoarthritis. In the present work, we developed a chemical feature based pharmacophore model of ADAMTS-4 inhibitors using the HipHop module within the Catalyst program package in order to elucidate the structure–activity relationship and to carry out in-silico screening. The Maybridge database was screened using Hypo1 as a 3D query, and the best-fit hits that followed Lipinski’s rule of five were subsequently screened to select the compounds. The hit compounds were then docked into the active site of ADAMTS-4, and interactions were visualized to determine the potential lead molecules. After subjecting all of the hits to various screening and filtering processes, 13 compounds were finally evaluated for their in vitro inhibitory activities. This study resulted in the identification of two lead compounds with potent inhibitory effects on ADAMTS-4 activity, with IC₅₀ values of 0.042 μM and 0.028 μM, respectively. These results provide insight into the pharmacophoric requirements for the development of more potent ADAMTS-4 inhibitors.

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Graphical Abstract The aggrecan-degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for osteoarthritis. In this work, we used HipHop-based pharmacophore modeling and virtual screening of the Maybridge database to identify novel ADAMTS-4 inhibitors. These novel lead compounds act as potent and specific inhibitors for the ADAMTS-4 enzyme and could have therapeutic potential in the treatment of OA

     

Extra thin alginate film: an efficient technique for protoplast culture

Summary. This paper reports an efficient protoplast culture technique, the “extra thin alginate film” technique. The development of this improved method of protoplast culture was an outcome of an assessment of the efficiency and shortcomings of various protoplast culture techniques. The efficiency of this technique was evaluated with two model plant systems, viz., Nicotiana tabacum and Lotus corniculatus, and a comparison was made with the “thin alginate layer” technique, another efficient protoplast culture system. Results indicate that the culture technique with extra thin alginate film is as efficient as the technique with thin alginate layer, with many additional advantages. The present innovation overcomes most of the limitations of protoplast culture techniques described so far and can now be applied to a wide variety of crops to check its general applicability. Correspondence and reprints: Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar 143 005, India.     

A novel method of estimation of lipophilicity using distance-based topological indices: dominating role of equalized electronegativity

Attempt is made to propose yet another method of estimating lipophilicity of a heterogeneous set of 223 compounds. The method is based on the use of equalized electronegativity along with topological indices. It was observed that excellent results are obtained in multiparametric regression upon introduction of indicator parameters. The results are discussed critically on the basis various statistical parameters.     

Isolation of mannose-binding C-type lectin from Heliothis virescens pupae

A mannose-binding C-type lectin (MBL) was isolated by affinity chromatography from Heliothis virescens immune pupal hemolymph. The immune pupal hemolymph was obtained after bacterial injection of live Enterobacter cloacae bacteria. MBL in mammals acts as an opsonin for phagocytosis and activates the lectin complement pathway of the innate immune response, which leads to killing of gram-negative bacteria and enveloped viruses. The affinity-purified and reduced pupal MBL showed a single band of 36 kDa by SDS-PAGE (12% gel). A dot-immunoblot ELISA (using guinea pig anti-MBL IgG as primary antibody) demonstrated specificity of the antibody for the affinity-purified pupal MBL. The immune pupal hemolymph contained 21 microg of MBL per ml of hemolymph. The amino acid composition of the purified pupal MBL was determined with high amounts of arginine and histidine detected. The presence of MBL in insect pupae has not before been reported and could be important in pupal innate immunity to bacterial infection.     

Evidences showing ultraviolet-B radiation-induced damage of DNA in cyanobacteria and its detection by PCR assay

Impact of ultraviolet-B radiation in causing the damages to the DNA of the cyanobacterium, Anabaena strain BT2 has been investigated. Exposure of genomic DNA (in vitro) to UV-B radiation for 1 h did not cause any shift in the absorption peak (lambda(max)) but more than 30% increase in absorbance was noticed in comparison to untreated control DNA (no exposure to UV-B). This increase in absorbance in a way may be comparable to typical hypochromic effect but there was no decrease in absorbance following transfer of UV-B-treated DNA to fluorescent light or in the dark. That the damaging effect of UV-B radiation on native structure of DNA is indeed real was also evident from the PCR-based assay such as RAPD, rDNA amplification, and ARDRA. Template activity of UV-B-treated genomic DNA was drastically inhibited, there was no amplification in RAPD assay after prior exposure of DNA to UV-B for 60 min. Only one band of approximately 400 bp was observed even after 60 min of exposure which suggests that certain segment of DNA strand is resistant to UV-B effects. Similar to the effects on RAPD profile, amplification of rDNA was significantly inhibited following exposure of genomic DNA to UV-B. Our findings clearly demonstrate that UV-B does affect the DNA of cyanobacteria and the killings of these microbes might be due to the irreversible damages caused to DNA by this high energy radiation. It is felt that PCR assay may be conveniently used for screening the damages caused to DNA by UV-B radiation in cyanobacteria and other microorganisms.     

Crystal structure of a highly acidic neurotoxin from scorpion Buthus tamulus at 2.2A resolution reveals novel structural features

The crystal structure of a highly acidic neurotoxin from the scorpion Buthus tamulus has been determined at 2.2A resolution. The amino acid sequence determination shows that the polypeptide chain has 64 amino acid residues. The pI measurement gave a value of 4.3 which is one of the lowest pI values reported so far for a scorpion toxin. As observed in other alpha-toxins, it contains four disulphide bridges, Cys12-Cys63, Cys16-Cys36, Cys22-Cys46, and Cys26-Cys48. The crystal structure reveals the presence of two crystallographically independent molecules in the asymmetric unit. The conformations of two molecules are identical with an r.m.s. value of 0.3A for their C(alpha) tracings. The overall fold of the toxin is very similar to other scorpion alpha-toxins. It is a betaalphabetabeta protein. The beta-sheet involves residues Glu2-Ile6 (strand beta1), Asp32-Trp39 (strand beta3) and Val45-Val55 (strand beta4). The single alpha-helix formed is by residues Asn19-Asp28 (alpha2). The structure shows a trans peptide bond between residues 9 and 10 in the five-membered reverse turn Asp8-Cys12. This suggests that this toxin belongs to classical alpha-toxin subfamily. The surface features of the present toxin are highly characteristic, the first (A-site) has residues, Phe18, Trp38 and Trp39 that protrude outwardly presumably to interact with its receptor. There is another novel face (N-site) of this neurotoxin that contains several negatively charged residues such as, Glu2, Asp3, Asp32, Glu49 and Asp50 which are clustered in a small region of the toxin structure. On yet another face (P-site) in a triangular arrangement, with respect to the above two faces there are several positively charged residues, Arg58, Lys62 and Arg64 that also protrude outwardly for a potentially potent interaction with other molecules. This toxin with three strong features appears to be one of the most toxic molecules reported so far. In this sense, it may be a new subclass of neurotoxins with the largest number of hot spots.     

MOLECULAR PHYLOGENY OF GRACILARIA SPECIES INFERRED FROM MOLECULAR MARKERS BELONGING TO THREE DIFFERENT GENOMES1

The nucleotide sequence data of molecular markers 18S rRNA, RUBISCO spacer, and cox2‐3 intergenic spacer were integrated to infer the phylogeny of Gracilaria species, collected from the western coast of India, reducing the possibility of misidentification and providing greater phylogenetic resolution. A phylogenetic tree was constructed using cox2‐3 and RUBISCO spacer sequences, exhibiting the same clustering but differing slightly from that of the rRNA‐based phylogenetic tree. The phylogeny inferred from the combined data set confers an analogous pattern of clustering, compared with those of trees constructed from individual data sets. The combined data set resulted in a phylogeny with better resolution, which supported the clade with higher consistency index, retention index, and bootstrap values. It was observed that Gracilaria foliifera (Forssk.) Børgesen is closer to G. corticata (J. Agardh) J. Agardh varieties, while G. salicornia (C. Agardh) E. Y. Dawson and G. fergusonii J. Agardh both originated from the same clade. The position of G. textorii (Suringar) De Toni faltered and toppled between G. salicornia and G. dura (C. Agardh) J. Agardh; however, G. gracilis (Stackh.) M. Steentoft, L. M. Irvine et W. F. Farnham was evidently distant from the rest of the species.