TRITON: graphic software for rational engineering of enzymes

Engineering of the catalytic properties of enzymes requires knowledge about amino acid residues interacting with the transition state of the substrate. TRITON is a graphic software package for modelling enzymatic reactions for the analysis of essential interactions between the enzyme and its substrate and for in silico construction of protein mutants. The reactions are modelled using semi-empirical quantum-mechanic methods and the protein mutants are constructed by homology modelling. The users are guided through the calculation and data analysis by wizards.     

Expressions and localizations of Bax/Bcl-2 proteins during metamorphosis of Pelophylax ridibundus

Bcl-2 and Bax proteins are expressed in cells of the tails of Pelophylax ridibundus larvae. We investigated the levels of these proteins in tails undergoing apoptosis. Apoptotic cells were observed in the epidermis, muscle and notochord of tails of different lengths. The apoptotic cells in epidermis exhibited the typical features of apoptosis. Amorphous masses and irregularities in striated muscle tissue undergoing apoptosis and apoptotic remnants in the notochord also were observed. In general, Bax staining in the epidermis, subepidermal fibroblast layer, muscle and notochord cells increased, while Bcl-2 staining decreased as the tail regressed. Our results suggest that during tail regression due to metamorphosis, Bcl-2 and Bax proteins play key roles in the apoptosis of tail epidermis, subepidermal fibroblast layer, muscle and notochord cells.     

Dynamics and binding modes of free cdk2 and its two complexes with inhibitors studied by computer simulations

This article presents a molecular dynamics (MD) study of the cdk2 enzyme and its two complexes with the inhibitors isopentenyladenine and roscovitine using the Cornell et al. force field from the AMBER software package. The results show that inserting an inhibitor into the enzyme active site does not considerably change enzyme structure but it seemingly changes the distribution of internal motions. The inhibitor causes differences in the domain motions in free cdk2 and in its complexes. It was found out that repulsion of roscovitine N9 substituent causes conformational change on Lys 33 side chain. Isopentenyladenine forms with Lys 33 side chain terminal amino group a hydrogen bond. It implies that the cavity, where N9 substituent of roscovitine is buried, can adopt larger substituent due to Lys 33 side chain flexibility. The composition of electrostatic and van der Waals interactions between the inhibitor and the enzyme were also calculated along both cdk2/inhibitor MD trajectories together with MM-PB/GBSA analysis. These results show that isopentenyladenine-like inhibitors could be more effective after modifications leading to an increase in their van der Waals contact with the enzyme. We suggest that a way leading to better inhibitors occupying isopentenyladenine binding mode could be: to keep N9 and N7 purine positions free, to keep 3,3-dimethylallylamino group at C6 position, and to add, e.g., benzylamino group at C2 position. The results support the idea that the isopentenyladenine binding mode can be used for cdk2 inhibitors design and that all possibilities to improve this binding mode were not uncovered yet.     

Metal accumulation in two contiguous eutrophic peri-urban lakes,Chivero and Manyame,Zimbabwe

Concentrations of aluminium, cadmium, chromium, cobalt, copper, iron, lead, nickel and zinc were determined in surface water, benthic sediments, and the gills, liver and stomach muscle tissues of Oreochromis niloticus and Clarias gariepinus in peri-urban lakes Chivero and Manyame, Zimbabwe. Five sites were sampled in each lake once per month in November 2015, February, May, August and November 2016. Pollution load index detected no metal contamination, whereas the geo-accumulation index reflected heavy to extreme sediment pollution, with Fe, Cd, Zn, Cr, Ni and Cu present in both lakes. Significant spatial temporal variations were detected for Al, Cr, Cu and Pb across sites within and between the two lakes. High Fe, Al and Cr concentrations in water and sediments in lakes Chivero and Manyame derive from geogenic background sources in addition to anthropogenic loads and intensity. Elevated concentrations of Al, Pb, Cu, Cd, Fe and Zn detected in gills, liver and stomach tissue of catfish corroborate concentrations in water and sediments, and pose the highest ecological and health risk for hydrobionts in lakes Chivero and Manyame. Contiguity of peri-urban lakes exposes them to similar threats, necessitating creative water management strategies, which ensure ecological continuity.     

The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars

The effect of increasing NaCl concentrations was studied on two different cultivars (cv. Orhangazi and cv. Cumhuriyet) of Sesamum indicum. Seedlings were grown for 40 days in half strength Hoagland solution and after 40 days treated with different NaCl concentrations (0, 50 and 100 mM) for 21 days. Differences in growth parameters, lipid peroxidation, antioxidative enzyme activities and proline accumulation were tested in order to put forward the relative tolerance or sensitivity of the cultivars. Results indicated that both parameters differ according to the cultivar's ability in coping oxidative stress caused by salinity. Constitutive levels of antioxidative enzyme activities were almost the same between the cultivars; however, cv. Cumhuriyet was able to induce antioxidative enzyme activities more efficiently when subjected to salt stress. Growth parameters, lipid peroxidation and proline accumulation results are also in good correlation with supporting this cultivar's being relatively tolerant.     

Morphology, and muscle- and papilla-volume ratios, of the tongue of Laudakia stellio (Agamidae, Squamata): a histological and stereological study

We examined the histological structure of the tongue of Laudakia stellio, the starred agama lizard (Agamidae, Squamata), under light microscopy. We also investigated the muscle and papilla volume ratios, with volumes of each aspect of interest estimated according to the Cavalieri method. The macroscopically short, thick and muscle-rich front tip of the tongue of L. stellio does not show any bifurcation, and under light microscopy, the oval-shaped papilla-free front tip was seen to be covered by keratinized stratified epithelium. The dorsal and ventral parts were different, with the former partially covered by keratinized stratified epithelium and rich in secretory glands and secretory cells. The ventral part, which contained keratinized stratified cells, had a flat surface with no papillae. The dorsal surface of the anterior and posterior parts contained fungiform papillae, with the apical parts of these papillae containing minimal keratin; the interpapillar space was covered by keratin-free squamous stratified epithelium. The middle section of the tongue contained cylindrical-type papillae, with serous and mucous secretory glands and ducts at their base. Finally, the frontal and middle parts of the ventral and dorsal surfaces did not contain any taste buds, although there were some in the hind part of the dorsal surface. As morphometric estimates of volumes of the muscles and papillae, the mean volume ratios (relative to total tongue volume)+/-standard deviation were 0.66+/-0.03 and 0.33+/-0.03, with mean coefficients of error of 0.02 and 0.03, respectively.     

Hinge-like motions in RNA kink-turns: the role of the second a-minor motif and nominally unpaired bases

Kink-turn (K-turn) motifs are asymmetric internal loops found at conserved positions in diverse RNAs, with sharp bends in phosphodiester backbones producing V-shaped structures. Explicit-solvent molecular dynamics simulations were carried out for three K-turns from 23S rRNA, i.e., Kt-38 located at the base of the A-site finger, Kt-42 located at the base of the L7/L12 stalk, and Kt-58 located in domain III, and for the K-turn of human U4 snRNA. The simulations reveal hinge-like K-turn motions on the nanosecond timescale. The first conserved A-minor interaction between the K-turn stems is entirely stable in all simulations. The angle between the helical arms of Kt-38 and Kt-42 is regulated by local variations of the second A-minor (type I) interaction between the stems. Its variability ranges from closed geometries to open ones stabilized by insertion of long-residency waters between adenine and cytosine. The simulated A-minor geometries fully agree with x-ray data. Kt-58 and Kt-U4 exhibit similar elbow-like motions caused by conformational change of the adenosine from the nominally unpaired region. Despite the observed substantial dynamics of K-turns, key tertiary interactions are stable and no sign of unfolding is seen. We suggest that some K-turns are flexible elements mediating large-scale ribosomal motions during the protein synthesis cycle.     

THERAPEUTIC STRATEGIES FOR THE INHIBITION OF INDUCIBLE NITRIC OXIDE SYNTHASE—POTENTIAL FOR A NOVEL CLASS OF ANTI-INFLAMMATORY AGENTS

In recent years, NO, a gas previously considered a potentially toxic chemical, has become established as a diffusible universal messenger mediating cell—cell communication throughout the body. In mammals, NO is a recognized mediator of blood vessel relaxation that helps to maintain blood pressure. In the central nervous system NO acts as a non-conventional neurotransmitter and participates in the establishment of long-term plasticity required for memory formation. In addition, NO is responsible for some parts of the host response to sepsis and inflammation and contributes to certain disease states. A number of strategies have emerged with regard to a pharmacological control of pathological NO overproductions. This review will discuss these novel therapeutic approaches that may provide new means for clinical medicine.     

Effects of L-cysteine on lead acetate induced neurotoxicity in albino mice

Lead is a toxic heavy metal that adversely affects nervous tissues; it often occurs as an environmental pollutant. We investigated histological changes in the cerebral cortex, hippocampus and cerebellum of adult albino mice following exposure to lead acetate. We also studied the possible ameliorative effect of the chelating agent, L-cysteine, on lead-induced neurotoxicity. We divided albino mice into six groups: 1) vehicle-only control, 2) L-cysteine control, 3 and 4) treated for 7 days with 20 and 40 mg/kg lead acetate, respectively, and 5 and 6) treated for 7 days with 20 and 40 mg/kg lead acetate, respectively, followed by 50 mg/kg L-cysteine for 7 days. Lead acetate administration caused disorganization of cell layers, neuronal loss and degeneration, and neuropil vacuolization. Brain sections from lead-intoxicated mice treated with L-cysteine showed fewer pathological changes; the neuropil showed less vacuolization and the neurons appeared less damaged. L-cysteine at the dose we used only marginally alleviated lead-induced toxicity.     

Elastic properties of ribosomal RNA building blocks: molecular dynamics of the GTPase-associated center rRNA

Explicit solvent molecular dynamics (MD) was used to describe the intrinsic flexibility of the helix 42–44 portion of the 23S rRNA (abbreviated as Kt-42+rGAC; kink-turn 42 and GTPase-associated center rRNA). The bottom part of this molecule consists of alternating rigid and flexible segments. The first flexible segment (Hinge1) is the highly anharmonic kink of Kt-42. The second one (Hinge2) is localized at the junction between helix 42 and helices 43/44. The rigid segments are the two arms of helix 42 flanking the kink. The whole molecule ends up with compact helices 43/44 (Head) which appear to be modestly compressed towards the subunit in the Haloarcula marismortui X-ray structure. Overall, the helix 42–44rRNA is constructed as a sophisticated intrinsically flexible anisotropic molecular limb. The leading flexibility modes include bending at the hinges and twisting. The Head shows visible internal conformational plasticity, stemming from an intricate set of base pairing patterns including dynamical triads and tetrads. In summary, we demonstrate how rRNA building blocks with contrasting intrinsic flexibilities can form larger architectures with highly specific patterns of preferred low-energy motions and geometries.