Participation of chlorobiumquinone in the transplasma membrane electron transport system of Leishmania donovani promastigote: effect of near-ultraviolet light on the redox reaction of plasma membrane

The respiratory quinone composition of the parasitic protozoa Leishmania donovani promastigote was investigated. 1'-oxomenaquinone-7, a chlorobiumquinone was found to be the major isoprenoid quinone. Substantial level of ubiquinone-9 was also present. Isolation and identification of the quinone from the purified plasma membrane yielded mainly 1'-oxomenaquinone-7 and ubiquinone-9; menaquinone was not detected. Membrane bound 1'-oxomenaquinone-7 could be destroyed by near-ultraviolet irradiation, with a concomitant loss or stimulation of plasma membrane electron transport activities. The abilities of different quinones to restore alpha-lipoic acid and ferricyanide reductase activity in near UV-irradiated cell preparations were compared. The order was; conjugate of chlorobiumquinone and sphingosine base approximately conjugate of 2-methyl-3-(1'-oxooctadecyl)-1,4-napthoquinone and octadecylamine > chlorobiumquinone approximately 2-methyl-3-(1'-oxooctadecyl)-1,4-napthoquinone > menaquinone-4 approximately ubiquinone-10. After irradiation with near-UV light, transmembrane alpha-lipoic acid reduction was inhibited, while transmembrane ferricyanide reduction was stimulated. The result obtained indicates that chlorobiumquinone mediates the plasma membrane electron transport between cytosolic reductant and oxygen as well as alpha-lipoic acid. UV-inactivation of chlorobiumquinone shuts down the plasma membrane oxygen uptake and diverts the electron flux towards ferricyanide reduction via ubiquinone-9. Chlorobiumquinone is the only example of a polyisoprenoid quinone containing a side chain carbonyl group from photosynthetic green-sulphur bacteria. Recent work has revealed numerous genes of trypanosomatid sharing common ancestry with plants and/or bacteria. These observations pose some fascinating questions about the evolutionary biology of this important group of parasitic protozoa.     

Ceranib‐2‐induced suicidal erythrocyte death

Ceramide is known to trigger apoptosis of nucleated cells and eryptosis of erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Besides ceramide, stimulators of eryptosis include increase of cytosolic Ca²⁺‐activity ([Ca²⁺]_i) and oxidative stress. Ceramide is degraded by acid ceramidase and inhibition of the enzyme similarly triggers apoptosis. The present study explored, whether ceramidase inhibitor Ceranib‐2 induces eryptosis. Flow cytometry was employed to quantify phosphatidylserine‐exposure at the cell surface from annexin‐V‐binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3‐fluorescence, reactive oxygen species (ROS) from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. A 48 h exposure of human erythrocytes to Ceranib‐2 significantly increased the percentage of annexin‐V‐binding cells (≥50 μM) and the percentage of hemolytic cells (≥10 μM) without significantly modifying forward scatter. Ceranib‐2 significantly increased Fluo3‐fluorescence, DCF fluorescence and ceramide abundance. The effect of Ceranib‐2 on annexin‐V‐binding was not significantly blunted by removal of extracellular Ca²⁺. Ceranib‐2 triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to increase of ceramide abundance and induction of oxidative stress, but not dependent on Ca²⁺ entry. Copyright © 2016 John Wiley & Sons, Ltd.     

Isolation of colour components from native dye-bearing plants in northeastern India

Recently dyes derived from natural sources have emerged as important alternatives to synthetic dyes. A study was initiated in the year 2000 at the RRL (CSIR), Jorhat to extract dyes from parts of five different plant species indigenous to northeastern India. The colour components responsible for dyeing were isolated and their chemical constituents were established based on chemical and spectroscopic investigations. The principal colour components from the species Morinda angustifolia Roxb., Rubia cordifolia Linn. and Tectona grandis Linn. were found to contain mainly anthraquinone moieties in their molecules. Those from the species Mimusops elengi Linn. and Terminalia arjuna (Roxb.) Wight & Arn. contained flavonoid moieties in their molecules. The absorption of dye (%) on fibres increased with increasing concentrations of dye in the dye-bath. Maximum absorption of dyes on fibres was obtained at 3% concentration of dyes obtained from R. cordfolia (35.350%), M. angustifolia (31.580%) and T. grandis (25.888%) and at 4% concentration of the dyes from M. elengi (31.917%) and T. arjuna (12.246%). The K/S values were found to increase with the increase in concentration of mordants. The colour co-ordinates of dyed samples were found to lie in the yellow-red quadrant of the colour space diagram. The dyes obtained from the native plants may be alternative sources to synthetic dyes for the dyeing of natural silk and cotton.     

Ultrafast events in the folding of ferrocytochrome c

Laser flash photolysis and stopped-flow methods have been used to study the dynamic events in the micro- to millisecond time bin in the refolding of horse ferrocytochrome c in the full range of guanidine hydrochloride concentration at pH 12.8 (+/-0.1), 22 degrees C. Under the absolute refolding condition, the earliest relaxation time of the unfolded protein chain is less than 1 micros. The chain then undergoes diffusive dynamics-mediated contraction and expansion, in which intrapolypeptide ligands make transient contacts with the heme iron, giving rise to two distinct kinetic phases of approximately 0.4 and approximately 3 micros. Under moderate to absolute refolding conditions, the rates of these processes show little dependence on the denaturant concentration, indicating the absence of structural element in the incipient or the relaxed state. Chain expansion and contraction events continue until the polypeptide finds a stable and supportive transition state. The crossing of this transition barrier, which rate-limits the folding of alkaline ferrocytochrome c, is characterized by a stopped-flow measured time constant of approximately 3 ms in aqueous solvent. Observed kinetics thus implicate no submillisecond folding structure. The folding kinetics is effectively two state in which the unfolded polypeptide first relaxes to an unstructured chain and then crosses over a late rate-limiting barrier to achieve the native conformation. The experimentally observed rates as a function of guanidine hydrochloride concentration have been simulated by numerically calculated microscopic rates of a simple kinetic model that captures the essential features of folding.     

Kinetic barriers to the folding of horse cytochrome C in the reduced state

To determine the kinetic barrier in the folding of horse cytochrome c, a CO-liganded derivative of cytochrome c, called carbonmonoxycytochrome c, has been prepared by exploiting the thermodynamic reversibility of ferrocytochrome c unfolding induced by guanidinium hydrochloride (GdnHCl), pH 7. The CO binding properties of unfolded ferrocytochrome c, studied by 13C NMR and optical spectroscopy, are remarkably similar to those of native myoglobin and isolated chains of human hemoglobin. Equilibrium unfolding transitions of ferrocytochrome c in the presence and the absence of CO observed by both excitation energy transfer from the lone tryptophan to the ferrous heme and far-UV circular dichroism (CD) indicate no accumulation of structural intermediates to a detectable level. Values of thermodynamic parameters obtained by two-state analysis of fluorescence transitions are DeltaG(H2O) = 11.65(+/-1.13) kcal x mol(-1) and C(m) = 3.9(+/-0.1) M GdnHCl in the presence of CO, and DeltaG(H2O)=19.3(+/-0.5) kcal x mol(-1) and C(m) = 5.1(+/-0.1) M GdnHCl in the absence of CO, indicating destabilization of ferrocytochrome c by approximately 7.65 kcal x mol(-1) due to CO binding. The native states of ferrocytochrome c and carbonmonoxycytochrome c are nearly identical in terms of structure and conformation except for the Fe2+-M80 --> Fe2+-CO replacement. Folding and unfolding kinetics as a function of GdnHCl, studied by stopped-flow fluorescence, are significantly different for the two proteins. Both refold fast, but carbonmonoxycytochrome c refolds 2-fold faster (tau = 1092 micros at 10 degrees C) than ferrocytochrome c. Linear extrapolation of the folding rates to the ordinate of the chevron plot projects this value of tau to 407 micros. The unfolding rate of the former in water, estimated by extrapolation, is faster by more than 10 orders of magnitude. Significant differences are also observed in rate-denaturant gradients in the chevron. Formation and disruption of the Fe2+-M80 coordination contact clearly impose high-energy kinetic barriers to folding and unfolding of ferrocytochrome c. The unfolding barrier due to the Fe2+-M80 bond provides sufficient kinetic stability to the native state of ferrocytochrome c to perform its physiological function as an electron donor.     

Statistical discovery of site inter-dependencies in sub-molecular hierarchical protein structuring

Background

Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families.

Results

The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function.

Conclusions

Our results demonstrate that the method we present here using a k- modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family.

     

Intramolecular cyclization of isothiocyanyl amino acids/peptide: arrival at unnatural thioxoimidazolidinyl/thioxooxazolidinyl amino acids

Amino Acids - A novel intramolecular cyclization of isothiocyanyl amino acids/peptide is reported to arrive at unnatural thioxoimidazolidinyl (TOI)/thioxooxazolidinyl (TOO) amino acids for the...